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MICROBIOLOGY

MARSHALL

MICROBIOLOGY

A TEXT-BOOK OF

MICROORGANISMS GENERAL AND APPLIED

CONTRIBUTORS

F. T. Bioletti, Berkeley, California.
R. E. Buchanan, Ames, Iowa.
M. Dorset, Washington, D. C.
S. F. Edwards, Guelph, Canada.
W. D. Frost, Madison, Wisconsin.
F. C. Harrison, Macdonald College,

Canada.

E. G. Hastings, Madison, Wisconsin.
H. W. Hill, Minneapolis, Minnesota.
W. E. King, Detroit, Michigan.

J. G. Lipman, New Brunswick, N. J,
W. J. MacNeal, New York City
E. F. McCampbell, Columbus, Ohio.
Earle B. Phelps, Boston, Massachusetts.
Otto Rahn, East Lansing, Michigan.
M. H. Reynolds, St. Paul, Minn.
W. G. Sackett, Fort Collins, Colo.
W. A. Stocking, Ithaca, New York.
Charles Thorn, Starrs, Connecticut.
J. L. To'dd, Montreal, Canada.

EDITI;D i;v

CHARLES E. MARSHAL'.
Amherst, Massachusetts

PROFESSOR OF MICROBIOLOGY AND DIRECTOR OF GRADUATE SCHOOL
MASSACHUSETTS AGRICULTURAL COLLEGE

WITH 128 ILLUSTRATIONS

PHILADELPHIA

P. BLAKISTON'S SON & CO.

1012 WALNUT STREET
1912

COPYRIGHT, 1911, BY P. BLAKISTON'S SON & Co.
Reprinted with Corrections, June 1912

THE. MAPLE. PRESS- YORK PA

CONTRIBUTORS.

BIOLETTI, FREDERIC T., M. S.

Associate Professor of Viticulture, University of California, Berkeley. Viti-

culturist of Experiment Station.
BUCHANAN, R. E., PH. D.

Professor of Bacteriology, State College, Ames, Iowa. Bacteriologist of Expe

ment Station.

DORSET, M., B. S., M. D.

Chief of Biochemic Division, U S. Bureau of Animal Industry, Washing-
ton, D. C.

EDWARDS, S. F., M. S.

Professor of Bacteriology, Ontario Agricultural College, Guelph, Canada.

FIDLAR, EDWARD, B. A., M. B.

Director of City Laboratories of Pathology and Public Health, City Hospital,

Hamilton, Ont., Canada.
FROST, W. D., PH. D.

Associate Professor of Bacteriology, University of Wisconsin, Madison.

HARRISON, F. C., D. Sc., F. R. S. C.

Principle, Professor of Bacteriology, Macdonald College (Faculty of Agricul-
ture, McGill University), Quebec, Canada.

HASTINGS, E. G., M. S.

Associate Professor of Agricultural Bacteriology, University of Wisconsin,
Madison. Bacteriologist of Experiment Station.

HILL, H. W., M. B., M. D, D. P. H.

Director: Division of Epidemiology, Minn. State Board of Health. Assistant
Professor of Bacteriology, University of Minnesota, Minneapolis.

KING, WALTER E., M. A.

Formerly Professor of Bacteriology, Agricultural College, Manhattan, Kansas.
Bacteriologist of the Experiment Station. Asst. Director, Research Laboratory,
Parke, Davis and Co., Detroit, Mich.

LIPMAN, JACOB G., PH. D.

Professor of Soil Fertility and Bacteriology, Rutgers College, New Brunswick,
New Jersey. Director, Soil Chemist and Bacteriologist of Experiment Station.

Note Edward Fidlar, B. A., M. B., Senior Demonstrator of Pathology and
Bacteriology in the University of Minnesota, cooperated with Dr. H. W. Hill in the
preparation of the account of specific diseases.

V

VI CONTRIBUTORS.

MACNEAL, W. J., M. D., PH. D.

Lecturer on Pathology and Bacteriology, New York Post-Graduate Medical

School.
MCCAMPBELL, E. F., S. B., Ph. D., M. D.

Secretary of State Board of Health of Ohio; Professor of Bacteriology, Ohio

State University.
PHELPS, EARLE B., S. B.

Assistant Professor of Research in Chemical Biology, Massachusetts Institute

of Technology, Boston.
RAHN, OTTO, PH. D.

Assistant Professor of Bacteriology, Michigan Agricultural College, East

Lansing. Assistant Bacteriologist, Experiment Station
REYNOLDS, M. H., B. S., M. D., D. V. M.

Professor of Veterinary Medicine, Agricultural College, University of Minnesota,

St. Paul, Minn. Veterinary Medicine and Surgery, Experiment Station.
SACKETT, WALTER G., B. S.

Bacteriologist of Experiment Station, Colorado Agricultural College, Fort

Collins.
STOCKING, W. A., M. S. A.,

Professor of Dairy Industry, Cornell University, Ithaca, N. Y. Dairy Bacteri-
ologist of Experiment Station.
THOM, CHARLES, PH. D.,

Mycologist, Dairy Division, Bureau of Animal Industry, U. S. Dept. of Agri-
culture, Stationed at Storrs, Conn.
TODD, J. L., M. D., D. Sc.

Associate Professor of Parasitology, McGill University, Montreal Canada
Dr. L. F. Rettger of Yale University kindly furnished a brief review of "Bacillary
White Diarrhoea of Chicks."

INTRODUCTION.

By a process of adaptation and growth, the branch of science commonly
recognized as "Bacteriology" has for many years included, besides
the bacterial forms, those microorganisms yielding to the same laboratory
methods of study and investigation. This is a policy or purpose instituted
by Pasteur. It is also the result of investigations and added knowledge,
more definite arrangements of available facts, and the highly specialized
training required for the work. In short, technic together with the eco-
nomic relations of the subject-matter has no little influence in placing
limitations. In the light of such circumstances, it appears more pertinent
to designate this text-book as "Microbiology," perhaps not the best term,
but one much in accord with French usage.

Agriculture, Domestic Science and certain other courses in scientific
schools and colleges call for the treatment of the subject in such a man-
ner as to make it basic to the interpretation of such subjects as air im-
purities, water supplies, sewage disposal, soils, dairying, fermentation
industries, food preservation and decomposition, manufacture of biolog-
ical products, transmission of disease, susceptibility and immunity, sani-
tation, and control of infectious or contagious diseases. A strong effort
has been made to provide the fundamental and guiding principles of the
subject and to show just how these principles fit into the subjects of a
more or less strictly professional or practical nature. Here the instruc-
tional work of the microbiologist stops in most educational institutions
and the instruction of the practical or professional man begins.

Because of the extreme massiveness and diversity of the subjects,
Agriculture and Domestic Science and Industrial Vocations in general,
a comprehensive consideration of the subject is demanded. Elimination
of many features not only becomes difficult but really precarious, because
so many avenues are open to the student that pertinency cannot always
be foreseen or determined. It is well to remember, too, that such aggre-
gate subjects as Agriculture and Domestic Science, unlike Engineering
and Medicine, because of their youth, have not developed to that stage

VII

Vlll INTRODUCTION.

in their educational history where practice and the science upon which
practice should be founded are amalgamated. The practical man in
Agriculture, and Applied Sciences generally, too frequently is so extremely
traditional in his practice that he utterly fails to separate the true from
the false, or, in other words, does not exercise his discriminative powers
at all, but depends entirely upon so-called haphazard methods and self-
willed processes. This factor operates against the proper development
and logical study of any branch of science in its relation to the farmer,
or manufacturer.

The plan of a text-book in Microbiology which seeks to furnish basic
principles, to train the mind in logical development and adjustment,
and to prepare the student to undertake an intelligent study of strictly
professional or practical subjects, must assume a definite and systematic
arrangement. With this in mind, the text has been divided into three
distinct parts: Morphological and Cultural, or that which deals with forms
and methods of handling; Physiological, or that which deals strictly with
functions, the key to the applied; Applied, or that which reaches into the
application of the facts developed to the problems met in the study of
professional or practical affairs.

In a text-book, the product of several hands, there is the most serious
difficulty in obtaining unity of thought and expression without repetition;
besides, that very conspicuous weakness of emphasizing some features
unduly while other features of importance are scarcely mentioned, con-
fronts us. A most earnest attempt has been made to overcome these
faults as far as possible, but a complete mastery of them cannot be ex-
pected in the first product. However, what is lacked in unity and con-
tinuity of expression and in balance, we sincerely hope will be made up,
in part at least, by the selection and the value of the material contributed.

Laboratory features of microbiology have been eliminated wher-
ever it has been practicable. Should any demonstrations be added
or needed, we have felt that they may be easily supplied by the instructor,
who, of course, will be governed by local facilities and conditions. Al-
though no space has been given to laboratory exercises, it should not be
gathered that the authors of this book are any the less earnest in urging
a well-organized laboratory course to supplement the general instruction
as an essential factor to a working appreciation of the subject.

In matters of spelling, new words, and phrases, conservatism has
controlled. Arbitrary decisions and selections have been forced in

INTRODUCTION. IX

several instances to secure clearness, consistency and definiteness. It
is painfully evident to anyone attempting to bring system out of the
confusion and chaos existing in many fields of microbiological action
that some rearrangement ought to be undertaken. As usual, however,
this will be very slow on account of the many almost insurmountable
difficulties.

We need and invite helpful suggestions and criticisms at all times,
for a valuable text-book of the nature of this is one of slow growth and
development and not of " sport evolution." The editor is certain that
each contributor will welcome suggestions and, further, will be in far
better position to judge his own contribution after the material appears
in book form and has been submitted to students for which it is designed.

No one better than the editor realizes fully the sympathetic part
played by the contributors. If any merit attaches to this book as it
finds its place in microbiological instruction, such merit should be recog-
nized as due the contributors whose unselfish aims have made it possible.

CHARLES E. MARSHALL, EDITOR.

EAST LANSING, MICHIGAN.

CONTENTS.

TITLE PAGE iii

CONTRIBUTORS v

INTRODUCTION (Editor) vii

CONTENTS (Editor) ' xi

HISTORICAL REVIEW (Harrison) i

PART I. MORPHOLOGY AND CULTURE OF MICROORGANISMS.

GENERAL (Editor). OUTLINE OF PLANT GROUPS (Thorn).
OUTLINE OF PROTOZOAL GROUPS (Todd).

CHAPTER I. MOLDS (Thom) 12

Fungi in general, Bacteria, Phycomycetes, Ascomycetes, Basidiomycetes, Imperfect
fungi. Molds, Cosmopolitan saprophytes, molds of fermentation, parasites and
facultative parasites. Consideration of groups, Mucor, Penicillium, Aspergillus,
Cladosporium, Alternaria and Fusarium, Oidium, Monilia, Dematium.

CHAPTER II. YEASTS (Bioletti) 28

Morphology of certain types, Definition and bases of classification, general morpho-
logical characteristics. The principal yeasts of importance to fermentation indus-
tries, True yeasts, pseudo-yeasts. Culture of yeasts.

CHAPTER III. BACTERIA (Frost) 37

Form, Fundamental form types, gradations, involution forms. Size. Motility,
Brownian movement, vital movement, organs of locomotion, character of move-
ment, rate. Reproduction, Vegetative multiplication, spore formation. Cell
grouping. Structure of the bacterial cell. Higher bacteria. Classification. Rela-
tionship of bacteria. Cultivation of bacteria.

CHAPTER IV. INVISIBLE MICROORGANISMS (Dorset) 64

A brief general discussion of the available knowledge of invisible microorganisms.

CHAPTER V. PROTOZOA (Todd) 68

Introduction. Structure of protozoa. Functions of protozoa, Locomotion and
reproduction, developmental cycle, encystment. Parasitism. Discussion of classi-
fication. Technic.

PART II. PHYSIOLOGY OF MICROORGANISMS (RAHN).

DIVISION I. NUTRITION AND METABOLISM. (A Few Paragraphs on
Protozoal Nutrition by Todd.)

INTRODUCTION, Principles of nutrition and metabolism, energy supply of
microorganisms.

xi

CONTENTS.

CHAPTER I. FOOD OF MICROORGANISMS 8

The composition of the cell Moisture, cell wall, cell contents.-Amount of food re-
Organic food materials,-Non-nitrogenous food compounds, nitrogenous
food compounds.-Mineral food.-Oxygen.-Additional remarks on microbial food -
Physiologic groups, synthetic media.

CHAPTER II. PRODUCTS OF METABOLISM

The chemical equations of fermentations. Physiological variations. Products' from
nitrogen-free compounds, Cellulose, starch, sugar, alcohols, organic acids, fats. Pro-
ducts from nitrogenous compounds Protein bodies, ptomains, urea, uric acid, hip-
puric acid. Products from mineral compounds, -Oxidations, reductions. -Unknown
products of physiological significance, Pigments, aromatic substances, enzymes and
-Rotation of elements in nature.-Carbon cycle, nitrogen cycle, sulphur cycle
phosphorus cycle.-Physical products of metabolism.-Production of heat produc-
tion of light.

CHAPTER III. MECHANISM OF METABOLISM x ,

General theory of metabolism, Fermentation (intra- and extra-cellular), katabo-
lism and anabolism. Intra- and extra-cellular fermentation.-Decomposition of in-
soluble food, properties of enzymes, mechanism of fermentation.-Classincation of
enzymes.-Hydrolytic enzymes (enzymes of carbohydrates, enzymes of fats, enzymes
of proteins), coagulating enzymes, zymases, oxidizing enzymes, reducing enzymes
Additional remarks on the relation of cells and enzymes.-Theory of katabolism -
Theory of anabolism Interaction of anabolism and intra-cellular fermentation,
reversibility of enzymic action.

DIVISION II. PHYSICAL INFLUENCES.
CHAPTER I. MOISTURE .

Osmotic pressure.-Plasmolysis.-Salt and sugar solutions, colloidal solu'tions. -Des-
iccation.

CHAPTER II. INFLUENCE OF TEMPERATURE ,

Optimum temperature. Minimum temperature. Maximum temperature. Bio-
logical significance of the cardinal points of temperature. -End-point of fermentation.
Freezing. Thermal death-point. Resistance of spores.

CHAPTER III. INFLUENCE OF LIGHT AND OTHER RAYS ... I ( >2

Phototaxis. X-rays. Radium rays.

CHAPTER IV. INFLUENCE OF ELECTRICITY l66

CHAPTER V. IHFLUENCE OF PHYSICAL STRUCTURE OF THE MEDIUM .... . 167

CHAPTER VI. INFLUENCE OF MECHANICAL EFFECTS .168

Pressure. Gravity. Agitation.

DIVISION III. CHEMICAL INFLUENCES.

CHAPTER I. STIMULATION OF GROWTH 17I

Chemotropism and chemotaxis.

CHAPTER II. INHIBITION OF GROWTH .173

Poisons, germicides, disinfectants, antiseptics, preservatives. Mode of action. Fac-
tors influencing disinfection. Classification of disinfectants.

DIVISION IV. MUTUAL INFLUENCES.
SYMBIOSIS. METABIOSIS. ANTIBIOSIS .!8i

CONTENTS. Xlll

PART III APPLIED MICROBIOLOGY.
DIVISION I. MICROBIOLOGY OF AIR (Buchanan).

CHAPTER I. THE MICROORGANISMS OF THE AIR AND THEIR DISTRIBUTION 185

Microorganisms present in the air. Occurrence in the air. How microorganisms
enter the air. Conditions for subsidence of bacteria. Determination of the number
of bacteria in the air. Number of bacteria in the air. Species of organisms in the air.

CHAPTER II. MICROBIAL AIR INFLUENCE IN FERMENTATION, DISEASES, ETC 19

Air as a carrier of contagion. Organisms of the air and fermentation. Freeing air
from bacteria.

DIVISION II. MICROBIOLOGY or WATER AND SEWAGE.

CHAPTER I. MICROORGANISMS IN WATER (Harrison) 192

Classes of bacteria found in water, Natural water bacteria, soil bacteria and surface
washings, intestinal bacteria usually of sewage origin. The number of bacteria in rain ,
snow, hail, etc., and in water from wells, up-land, surface waters, rivers, and lakes.
Causes affecting the increase and decrease of the number of bacteria in water, Tem-
perature, light, food supply, oxidation, vegetation and protozoa, dilution, sedimenta-
tion, other causes. Interpretation of the bacteriological analysis of water, Quantita-
tive standards, qualitative standards. Sedimentation, filtration and purification of
water, Sedimentation and filtration, coagulation basins and filtration, porous filters,
purification by ozone, purification by heat, purification by chemicals. Location
and construction of wells.

CHAPTER II. MICROBIOLOGY OF SEWAGE (Phelps) 212

Bacterial flora of sewage. Types of sewage bacteria, Putrefactive and anaerobic
bacteria (the liquefaction of protein, the fermentation of cellulose, the saponification
of fats, the fermentation of urea, the reduction of sulphates and nitrates), oxidizing
bacteria (the production of nitrates and nitrites, other oxidizing reactions) , patho-
genic bacteria (prevalence and longevity, life in septic tanks and filters). The culti-
vation of sewage bacteria, Filters, anaerobic tanks. The destruction of sewage
bacteria, By biological processes, by chemical processes.

DIVISION III. MICROBIOLOGY OF SOIL (Lipman).

CHAPTER I. MICROORGANISMS OF THE SOIL AS A FACTOR IN SOIL FERTILITY 226

Introduction. The soil as a culture medium; Moisture relations, The amount
and distribution of rain fall, range of soil moisture, effect of drouth and exces-
sive moisture. Aeration, Mechanical composition of soils, aerobic and anaerobic
activities, rate of oxidation of carbon, hydrogen and nitrogen, the mineralization
of organic matter. Temperature, Influence of climate and season, early and late
soils, production and assimilation of plant food. Reaction, Range of soil acidity,
causes of soil acidity, effect of reaction on number and species.- Food supply, Or-
ganic matter, the mineral portion of the soil. Biological factors, Molds, algae, proto-
zoa, higher plants, bacteria, (numbers and distribution, bacteria in productive and
unproductive soils, distribution at different depths, morphological and physiological
groups). Methods of study, Quantitative relations, qualitative reaction, transforma-
tion reactions, rate of oxidation of carbon, rate of oxidation of nitrogen, addition of
nitrogen, reactions concerning calcium, magnesium, sulphur, phosphorus.

CHAPTER II. DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 246

Carbohydrates, Origin, decomposition of cellulose, the production of methane and
hydrogen, oxidation of methane, hydrogen, and carbon monoxide, the cleavage and
fermentation of sugars, starches, and gums. Fats and waxes, Origin and decomposi-

XIV CONTENTS.

tion. Organic acids, Sources, transformation and accumulation. Protein bodies,
Amount and quality, carbon-nitrogen ratio. Transformation of nitrogen compounds,
Ammonification, nitrification, denitrification. Analytical and synthetical reac-
tions, Amount of bacterial substance in the soil, availability of bacterial matter,
transformation of peptone, ammonia, nitrate, nitrogen.

CHAPTER III. FIXATION OF ATMOSPHERIC NITROGEN. (Methods of Soil Inoculation

by Edwards.) 268

The source of nitrogen in soils, Early theories, chemical and biological relations.
Non-symbiotic fixation of nitrogen, Historical, anaerobic species, aerobic species,
energy relations. Symbiotic fixation, -Historical, modes of development, resistance,
immunity, and physiological efficiency, mechanism of fixation, variations and special-
ization, relation to environment. Soil inoculation, Methods of soil inoculation,
inoculation with legume earth, inoculation with pure cultures. (Edwards).

CHAPTER IV. CHANGES IN ORGANIC CONSTITUENTS 284

Weathering process, Origin and formation of soil, influence of biological factors.
Lime and magnesia, Removal and regeneration of carbonates, lime as a base, effect of
calcium, magnesium compounds upon bacterial activities. Phosphorous, Availa-
bility of phosphates, relation of phosphorus to decay and nitrogen-fixation. Sul-
phur, Sulphur compounds in the soil, sulphur bacteria, sulphate reduction. Potas-
sium, The transformation of potassium compounds in the soil. Other mineral con-
stituents, Iron, aluminum, manganese, and copper.

DIVISION IV. -MICROBIOLOGY OF MILK AND MILK PRODUCTS.

CHAPTER I. THE RELATION OP MICROORGANISMS TO MILK. (Stocking). (The acid-
forming bacteria by Hastings.) 292

Importance of milk as a food. Absorbed taints and odors. Changes due to micro-
organisms. Microbial, content of milk, Common milk, special milk, certified
milk. Sources of microorganisms in milk, Interior of cow's udder, (healthy udders,
diseased udders), exterior of cow's body, atmosphere of stable and milk house,
the milker, utensils, water supply. Methods of preventing contamination of
milk, Individual cows, care of the cow's body, dust in atmosphere, dairy utensils,
the milker. Groups or types or microorganisms found in milk, and their sources,
General significance of acid-forming bacteria, groups of acid-forming bacteria (char-
acteristics of the Bad, lactis acidi group, characteristics of the B. coli-aerogenes
group, characteristics of the Bact. bulgaricus group, characteristics of the coccus
group) (Hastings), bacteria having no perceptible effect upon milk, the digesting or
peptonizing, pathogenic organisms. Factors influencing the developing of microor-
ganisms in milk, Initial contamination, straining, aeration, centrifugal separation,
temperature, pasteurization, the use of chemicals. The normal development of
microorganisms in milk, Germicidal period, period from end of germicidal action
to time of curdling, period from time of curdling until acidity is neutralized, final
decomposition changes. Abnormal fermentations in milk, Gassy fermentation,
sweet curdling fermentation, ropy and slimy fermentation, bitter fermentation,
alcoholic fermentation, other fermentations. The commercial significance of mic-
roorganisms in milk, Relation of dirt contamination to germ content. Milk as a
carrier of disease organisms, Those microorganisms which are beneficial and detri-
mental to health, (acid forms, neutral forms, injurious organisms). Bacteriological
analyses of milk. Bacteriological milk standards. The value of bacteriological
milk standards and analyses.

CHAPTER II. THE RELATIONS OF MICROORGANISMS TO BUTTER (Hastings) 335

Types of butter, Sweet cream butter, sour cream butter. The flavor of butter,
Control of butter flavor, kinds and numbers of bacteria in cream, spontaneous ripen-
ing of cream, use of cultures in butter making, commercial cultures, use of pure cul-

CONTENTS. XV

tures in raw cream, use of pure cultures in pasteurized cream, pure cultures in
oleomargarine and renovated butter, abnormal flavors of butter. Decomposition
processes in butter. Pathogenic bacteria in butter.

CHAPTER III. RELATION OP MICROORGANISMS TO CHEESE (Hastings) 346

General. Types of cheese, Acid-curd cheese, rennet-curd cheese. Conditions affect-
ing the making of cheese, Quality of milk, tests for the quality of milk, ripening of
milk, curdling of milk, manipulation of the curd, ripening of cheese (theories of
cheese ripening, present knowledge of causal factors, causes of proteolysis, preven-
tion of putrefaction, flavor production in cheese) Abnormal cheese, Gassy cheese,
miscellaneous abnormalities of cheese (bitter cheese, colored cheese, putrid cheese,
moldy cheese). Specific kinds of cheese, Cheddar cheese, Emmenthaler cheese,
Roquefort cheese, Gorgonzola cheese, Stilton cheese, Camembert cheese.

CHAPTER IV. RELATION OP MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS

(Stocking) 363

General. Condensed milk, Sweetened condensed milk, unsweetened condensed milk,
concentrated milk, powdered milk. Canned butter and cheese. Special milk drinks
made by the action of microorganisms, Kumyss, kefir, leben, yoghurt, artificial
buttermilk. Frozen milk. Ice cream.

DIVISION V. MICROBIOLOGY OF SPECIAL INDUSTRIES.

CHAPTER I. DESICCATION, EVAPORATION, AND DRYING OF FOODS (Buchanan) 374

Factors that bring about changes in dried foods. Inhibition 'of growth of micro-
organisms in dried food. Methods of drying, Carbohydrate foods, as fruits,
macaroni, vermicelli, copra, syrups, molasses, jellies, jams; fats, as cotton seed,
olive, and other oils, etc. ; protein foods, as jerked meat, dried beef, dried fish,
pemmican, beef extract, gelatin, somatose, milk, eggs, etc.

CHAPTER II. HEAT IN THE PRESERVATION OF FOODS (Edwards) 381

Historical resume 1 . Economic importance, From the standpoint of health and die-
tetics, and from the standpoint of commerce. Alteration of foods, Physical changes
(appearance, mechanical disintegration), chemical changes (appearance, chemical
change, palatability and digestibility), biological changes (vital disorganization,
normal flora and fauna). Pasteurization, Economic consideration, specific applica-
tion (beer, fruit juices, milk and cream, condensed milk). Sterilization, Economic
considerations, specific application (meat, fish, vegetables, and fruits). Controlling
factors in successful canning, Cleanliness, soundness of raw material, receptacle,
water supply, degree of heat required, storage. Spoliation of pasteurized and ster-
ilized goods Chemical, microbiological, detection of spoiled goods. Disposal of
factory refuse.

CHAPTER III. THE PRESERVATION OF FOOD BY COLD (MacNeal) 395

Introduction. The effects of refrigeration upon foodstuffs in general, Changes
during chilling, changes during storage, changes after storage. Refrigeration of
certain foods, Meat, fish, poultry, eggs, milk, and butter, fruits and vegetables.
Legal control of the cold-storage industry.

CHAPTER IV. PRESERVATION OF FOOD BY CHEMICALS (MacNeal) 402

The effects of preservatives upon foods in general, The process of curing, the period of
storage, the after-storage changes. The chemical preservation of certain foods,
Meats, fish, butter, prepared vegetables, and fruits. The nutritive value of pre-
served foods. The effects of food preservatives, Substances which preserve by their
physical action, substances which preserve by their chemical action, inorganic food
preservatives, organic food preservatives, substances added to foods to improve
the apparent quality. The legal control of the preservation of foods by chemicals.

CONTENTS.

CHAPTER V. MICROBIAL FOOD POISONING (MacNeal) ................ 411

General considerations. Infections of food-producing animals transmissible to man.

Human infections transmitted in food. Food poisoning due to the growth of sapro-
phytic bacteria in the food, Poisonous meat, sausage, fish, shell fish, milk, cream,
cheese, and vegetable food. The chemical nature of food poisons.

CHAPTER VI. THE MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS (Bioletti) . 418
Wine: Grape juice, and wine as culture media. The microorganisms found on grapes,
(molds, yeasts, pseudo-yeasts, bacteria). Microorganisms found in wine, Aerobic
organisms (mycodermse, acetic bacteria) , Anaerobic organisms (slime-forming bac-
teria, propionic and lactic bacteria, mannitic bacteria, butyric bacteria). Control of
the microorganisms, Before fermentation, during fermentation, after fermentation.

Beer: The raw materials and microorganisms of brewing, Grains, yeasts of beer,
kinds of beer. Outline of the processes of brewing, Introduction, malting (produc-
tion of enzymes), work of enzymes and bacteria, fermentation (work of yeasts), after-
treatment. Diseases of beer. Miscellaneous alcoholic beverages: Cider, perry, fer-
mented beverages of various fruits, hydromel or mead pombe, ginger beer. Dis-
tilled alcohol: Introduction, Uses and sources of alcohol. Methods, Preparation
of the sugar solution, (saccharine raw materials, starchy raw materials), fermentation.

CHAPTER VII. MANUFACTURE OF VINEGAR (Bioletti) ............... 448

Acetic fermentation, Nature and origin of vinegar, vinegar bacteria. Processes of
manufacture, Raw materials, fermentation, starters and pure cultures, apparatus,
domestic method, Orleans method, Pasteur method, German method, rotating
barrels, after treatment. Diseases.

CHAPTER VIII. MANUFACTURE OF OTHER FERMENTED PRODUCTS (Bioletti) ..... 460
Preparation and conservation of food material, Compressed yeast, bread, vegetables,
starch, sugar, tobacco. Preservation and conservation of miscellaneous products,
Indigo, retting, tanning.

CHAPTER IX. MANUFACTURE OF VACCINES (King) ................ 467

Introduction. Active immunizing substances (vaccines), Attenuated viruses,
small-pox vaccine, blackleg vaccine, rabies vaccine, Dorset-Miles hog-cholera serum,
anthrax vaccines. Other vaccines, Asiatic cholera, bubonic plague, tuberculosis,
typhoid fever. Bacterial vaccines (bacterins).

CHAPTER X. THE MANUFACTURE OP ANTISERA, AND OTHER BIOLOGICAL PRODUCTS

RELATED TO SPECIFIC INFECTIOUS DISEASES (King) .............. 480

Antitoxins, Diphtheria antitoxin, tetanus antitoxin. Other antimicrobial sera,
Dorset-Niles anti-hog-cholera serum, antistreptococcic serum, antidysenteric serum,
antirabic serum, antigen ococcic serum. Tuberculins, Koch's tuberculin (old) , other
tuberculins. Mallein. Suspensions for the agglutination tests.

DIVISION VI. MICROBIAL DISEASES OF PLANTS (Sackett).
INTRODUCTION .............................. 49

CHAPTER I. BLIGHTS ............................. 492

Stem blight of alfalfa. Bacteriosis of beans. Blight of mulberry. Blight of oats.
Pear blight. Tomato blight. Walnut blight.

CHAPTER II. GALLS AND TUMORS ........... ....... Sz

Crown gall. Olive knot. Fingers and Toes of cabbages (Todd).

CHAPTER III. LEAF SPOTS .......................... So6

Spot of the larkspur. Bacterial spot of plum and peach. Leaf spot of sugar beet.

CONTENTS. XV11

CHAPTER IV ROTS 508

Black rot of cabbage. Wakker's hyacinth disease. Basal stem rot of potatoes. Soft
rot of calla lilly. Soft rot of carrot and other vegetables. Soft rot of hyacinth. Soft
rot of muskmelon. Soft rot of the sugar beet.

CHAPTER V. WILTS 515

Wilt of cucurbits. Leaf disease of nasturtium. Wilt of sweet corn. Wilt of
tomato, egg plant, Irish potato, and tobacco. Additional bacterial diseases.

DIVISION VII. MICROBIOLOGY OF THE DISEASES or MAN AND ANIMALS.

CHAPTER I. METHODS AND CHANNELS OF INFECTION (McCampbell) 520

Infection defined. Microorganisms of diseases considered and classified, Patho-
genic bacteria, pathogenic protozoa, ultra-microscopic microorganisms or viruses.
The distribution of pathogenic microbial agents in nature. The occurrence of
pathogenic microbial agents upon and in the bodies of healthy animals and man.
The manner in which infectious agents enter the body and their sources, Air-
borne infections, water-borne infections, infections from soil, infections from food,
animal carriers of infection, human carriers of infection, contact infection. The
routes by which infectious microorganisms enter the body. Variation in infec-
tions. The factors which influence the results of an infection, Virulence, num-
ber, avenue, resistance. The exact cause of infections, Soluble toxins, endo-
toxins, toxic bacterial proteins. The methods by which infectious microorgan-
isms are disseminated. The methods by which infectious microorganisms are
eliminated from the body. The effect of infectious microorganisms upon the
body, The period of incubation, local reactions, general reactions, metabolism,
blood-forming organs, parenchymatous tissues, epithelial and endothelial tissues,
erythrocytes and leucocytes, antibody formation.

CHAPTER II. IMMUNITY AND SUSCEPTIBILITY (McCampbell) 541

General, Definition, hypersusceptibility or anaphylaxis, predisposition and non-
inheritance of infectious diseases. Immunity, Natural immunity and susceptibility
(racial immunity and susceptibility, familial immunity and susceptibility, individual
immunity and susceptibility). Factors of natural immunity (the protection
afforded the body by the surfaces, skin and cutaneous orifices, subcutaneous
tissue, the exposed mucous membranes of the body, nasal cavity, mouth, lungs,
stomach, intestines, genito-urinary tract, conjunctiva, the protective nature of
inflammatory processes, natural antitoxins, natural antibacterial substances,
normal hemolysins, normal agglutinins, normal precipitins), acquired immu
nity, (active immunity, passive immunity). The origin and occurrance of anti-
bodies, Antitoxins, (the mechanism of the neutralization of toxin by antitoxin,
unit of antitoxin), lysins and bactericidal substances (the structure of lysins,
deviation of complement, the fixation of the complement as a test for anti-bodies),
cytotoxins and cytolysins, opsonins and phagocytosis (opsonic index, hemoopso-
nins), agglutinins (normal agglutinins, the production of agglutinins, the sub-
stances concerned in agglutination, structure of agglutinins and agglutinogens,
agglutinoids, the stages of agglutination, hemoagglutinins), precipitins (normal
precipitins, mechanism of the formation of precipitins, autoprecipitins and iso-
precipitins, the phenomena of specific inhibition, antiprecipitins, the precipitinogen,
precipitate, coprecipitins, the forensic use of precipitins). The theories of immu-
nity, Noxious retention theory, exhaustion theory, phagocytic theory, Ehrlich's
side-chain theory.

lHAPTER III. MICROBIAL DISEASES OF MAN AND ANIMALS 576

Diseases caused by molds and yeasts (various authors), Pneumomycosis (Thorn),
thrush (Thorn); Dermatomycoses, barbers itch, etc., (Thorn), favus (Thorn);
Miscellaneous fungus diseases (Thorn), actinomycosis (Reynolds), mycetoma,

XV111 CONTENTS.

(Fidlar), mycotic lymphangitis (Reynolds). Diseases caused by bacteria, Botryo-
mycosis (Reynolds), gonorrhoea (Fidlar), infectious mastitis (Reynolds), Malta
fever (Fidlar), staphylococcic infections (Fidlar), streptococcic infections (Fidlar),
pneumonia (Fidlar) , anthrax (Harrison) , bacillary white diarrhoea of young chicks
(Rettger), chicken cholera (Harrison), chronic bacterial enteritis (Reynolds), con-
tagious abortion (MacNeal), diphtheria (Fidlar), dysentery (Fidlar), fowl diphtheria
(Harrison), glanders (Reynolds), influenza (Fidlar), hsemorrhagic septicaemia
(Reynolds) , leprosy (Fidlar) , plague (Fidlar) , swine erysipelas (Dorset) , tuberculosis
(Reynolds), foot rot of sheep (Dorset), foul brood of bees (Harrison), malignant
cedema (Fidlar) , symptomatic anthrax (Reynolds) , tetanus (Fidlar) , typhoid fever
(Fidlar), Asiatic cholera (Fidlar). Diseases of unknown cause, (Scarlet fever,
measles, German measles, Duke's disease, smallpox, chickenpox, mumps) (Hill),
canine distemper (Dorset), cattle plague (Dorset), chicken pest (Dorset), contagious
bovine pleuro-pneumonia (Dorset), cowpox (King), horsepox (King), sheeppox
(King), dengue (Dorset), foot-and-mouth disease (Dorset), hog cholera (Dorset),
horse sickness (Dorset), infantile paralysis (Dorset), louping-ill (Dorset), pellagra
(MacNeal), rabies (MacNeal), swamp fever (Reynolds), typhus fever (Dorset),
whooping cough (Fidlar), yellow fever (Dorset). Diseases caused by protozoa
(Todd), Amcebic dysentery, entero-hepatitis of turkeys, African tick fever, relaps-
ing or recurrent fever, yaws, other spirochaetal diseases, syphilis, kala-azar, Delhi
boil, sleeping sickness, human trypanosomiases of South America, trypanosomiases,
of animals, coccidiosis of rabbits, white diarrhoea of chicks, malaria, red water,
miscellaneous protozoal diseases, pebrine,

CHAPTER IV. CONTROL OF INFECTIOUS DISEASES (Hill) 691

Principles. Practice. Disinfection. Carriage of infection by biological agents.

INDEX OF CONTRIBUTORS 701

INDEX OP ORGANISMS 703

INDEX OF SUBJECTS . 709

LIST OF ILLUSTRATIONS.

1. Jansen's microscope 2

2. Mucor, general 18

3. Mucor, zygospore 19

4. Penicilliiim expansum 26

5. Aspergillus glaucus 23

6. Aspergillus fumigatus, A.nidnlans 23

7. Cladosporium herbarum 24

8. Spores of Alternaria 24

9. Fusarium 24

10. Oidium lactis 26

11. Manilla Candida 27

12. Yeast cell 29

13. Spore-bearing yeast cells 30

14. Wine and beer yeasts 32

15. Wild and pseudo-yeasts 35

16. Types of micrococci 37

17. Types of bacilli 37

18. Types of spirilla 37

19. Involution forms 38

20. The division of bacterial cells 41

21. Mitosis in bacterial cells 41

22. Stages in division of bacterial cells 42

23. The formation of spores 44

24. Location of spores in bacterial cells 44

25. Spore germination 45

26. Division forms of micrococci 46

27. Division forms of bacilli 47

28. Threads of Bact. anthracis 47

29. Capsules (Bact. pneumoniae) 49

30. Plasmolytic changes 50

31. Nuclear division 52

32. Distribution of nuclear substance 51

33. Monotrichous bacteria (Msp. comma) 52

34. Monotrichous bacteria (Ps. pyocyanea) 52

35. Lophotrichous bacteria (Ps. syncyanea) 53

36. Lophotrichous bacteria (Sp. rubrum) 53

37. Peritrichous bacteria (B. typhosus] 53

38. Crenothrix polyspora 55

39. Chlamydothrix hyalina 58

40. Cladothrix dichotoma 59

41. Beggiatoa alba 60

42. Pasteur-Chamberland or Berkefeld filtering apparatus 65

43. Miescher's sac 68

44. Amoeba vespertilio 69

45. Stages in division of Amceba polypodia 72

46. Multiplication of Coccidium schubergi 73

47. Amxba proteus 83

xix

XX LIST OF ILLUSTRATIONS.

48. Crystals of bacteriopurpurin 121

49. Carbon cycle 125

50. Nitrogen cycle 126

51. Sulphur cycle 127

52. Action of light on bacteria 162

53. Action of light on molds 163

54. Action of light on mold colonies 164

55. Chemotaxis 171

56. Curve of disinfection 174

57. Influence of filtered water on typhoid fever and Asiatic cholera 197

58. Section of sand filter 205

59. Unglazed porcelain filters 207

60. 6 1, 62. Location of wells on farm 209

63. Construction of model well 210

64. Trickling filter, sand filter, dosing tank, septic tank 223

65. Septic tank . . 224

66. Non-symbiotic nitrogen-fixing organism (B. pasteurianus) 270

67. Non-symbiotic nitrogen-fixing organism (Azotobacter vinelandi) 271

68. Ps. radicicola 275

69. Section through root tubercle 276

70,71,72. Influence of Ps. radicicola 279-281

73. Section of cow's udder 297

74. Bacterial colonies in dust from udder 300

75. Bacterial colonies from cow's hair . 301

76. Bacterial colonies from dust of stable 302

77. Small-top milk pails . 305

78. Ropy cream 324

79. Ropy cream organisms 325

80. Chart of Rochester milk supply 330

81. Gassy cheese 348

82. Cheese from lactic starter 349

83. Influence of lactic organisms on casein degradation 354

84. Swiss cheese 360

85. Kepfir grain . 368

86. Bacteria of slimy wine 424

' 87. Bacteria of wine diseases 4 2 6

88. Vinegar bacteria 45

89. Vinegar barrel 454

90. Rapid process vinegar apparatus . 457

91. Ps. medicaginis 493

92. Pear blight 497

93. Crown gall 5 2

94. Plasmodiophora brassicce 54

95. 96. Oidium albicans 577

97. Trichophyton tonsurans

98, 99. Actinomyces boms 5^ J

100. Gonococci

101. Bact. anthracis, thread formation 600

102. Bact. anthracis, spores 600

103. Organisms of anthrax in capillaries 602

104. Bact. diphtheria 610

105. Westbrook's types of Bact. diphtherias .611

106. Bact. mallei 618

107. Bact. pestis 625

108. Bact. tuberculosis, branching forms '. 630

109. Bact. tuberculosis, from sputum 630

no. Bact. tuberculosis, in culture 631

LIST OF ILLUSTRATIONS. XXI

in. B. tetani, with spores 638

112. B. typhosus 642

113. Msp. comma 646

114. Msp. comma colonies in gelatin 647

115. Kidneys in hog cholera, hemorrhagic points 655

116. Negri bodies 663

117. Amoeba coli 668

118. Spirochceta dentium 670

119. Ornithodoros moubata 671

120. SpirochcEta duttoni 672

121. Treponema pallidum 673

122. Herpetomonas donovani 674

123. Structure of trypanosome 676

124. Trypanosoma gambiense . 676

125. Glossina palpalls 677

126. Malarial parasite in human and mosquito cycles 683

127. Longitudinal section of Anopheles 684

128. Babesia bigemina - 687

Colored Plate.

The Malarial parasites ... 683

HISTORY OF MICROBIOLOGY*

Geronimo Fracastorio, of Verona, was born in 1484, studied medicine
in Padua, and published a work in Venice in 1546, which contained the
first statement of the true nature of contagion, infection, or disease
organisms, and of the modes of transmission of infectious disease. He
divided diseases into those which infect by immediate contact, through
intermediate agents, and at a distance through the air. Organisms
which cause disease, called Seminaria contagionum, he supposed to be
of the nature of viscous or glutinous matter, similar to the colloidal
states of substances described by modern physical chemists. These
particles, too small to be seen, were capable of reproduction in ap-
propriate media, and became pathogenic through the action of animal
heat. Thus Fracastorius, in the middle of the sixteenth century, gave
us an outline of morbid processes in terms of microbiology.

Athanasius Kircher, in 1659, demonstrated the presence of "minute
living worms in putrid meat, milk, vinegar, etc."; but he did not describe
their form and character, and it is doubtful if he ever saw microorganisms.

In the year 1683 Antonius van Leeuwenhoek, a Dutch naturalist and
a maker of lenses, communicated to the English Royal Society the results
of observations which he had made with a simple microscope of his own
construction, magnifying from 100 to 1 50 times. He found in water, saliva,
dental tartar, etc., what he termed "animalcula." He described what he
saw, and by his drawings showed both rod-like and spiral forms, both of
which, he said, had motility. In all probability, the two species he saw
were those now recognized as Bacillus buccalis maximus and Spirillum
sputigenum. Leeuwenhoek's observations were purely objective and
in striking contrast with the speculative views of M. A. Plenciz, a Viennese
physician, who in 1762 published a germ theory of infectious diseases.
Plenciz maintained that there was a special organism by which each in-
fectious disease was produced, that microorganisms were capable of
reproduction outside of the body, and that they might be conveyed from
place to place by the air.

* Prepared by F. C. Harrison.

HISTORY OF MICROBIOLOGY.

The important role that the compound microscope has played in
microbiology calls for something regarding the invention of this instrument
an invention which antedates Leeuwenhoek's discovery by nearly 100

years.

The first compound microscope was made by Hans Jansen and his
son Zaccharias, in 1590, at Middelburg, in Holland. The instrument
was composed of two lenses mounted in tubes of iron; a representation
of it, made from the original and still kept at Middelburg, is shown
in Fig. i. From that date the microscope gradually improved. In
1844 the immersion lens was introduced by Dolland. In 1870 Abbe
brought out the substage condenser, which still bears his name. Apo-

6

FIG. i. Longitudinal section of a compound microscope made by Zacharias
Jansen (.1590). a, microscope tube; b, objective tube; c, ocular.

chromatic lenses and many minor improvements were introduced by the

firm of Zeiss about 1880.

In 1786 O. F. Miiller (a Dane) first attempted to classify, according
to the Linnean system, the various organisms previously discovered, and
characterized four or five genera among them, the genus Vibrio, in
which, under the terms bacillus, lineola, and spirillum, we recognize forms
that correspond with our "bacteria"

From the middle of the eighteenth century until well on into the nine-
teenth, the history of bacteriology is largely the story of a controversy
between those who believed that minute living organisms, such as those
above referred to, were produced from inanimate substances, and that
their formation was spontaneous. Philosophers, poets, and common
people of the most enlightened nations accepted this doctrine down to the
eighteenth century. The hypothesis regarding formation was known as
that of " spontaneous generation," "heterogenesis," and "abiogenesis."
The opponents of this theory denied the possibility of a transition from a
lifeless to a living condition, and contended that all life came from pre-
existing life a theory aphoristically summed up in the phrase "omne
vivum ex vivo." Such was the doctrine of Biogenesis, life only from life.

In 1668, Francisco Redi, an Italian, distinguished alike as scholar,

HISTORY OF MICROBIOLOGY. 3

poet, physician, and naturalist, expressed the idea that life in matter is
always produced through the agency of pre-existing living matter; but the
beginnings of the real controversy date from the publication of Needham's
experiments in 1745. The English divine boiled some meat extract in a
flask, made the flask air-tight, and left it for some days. When the flask
was opened, he found in it what he termed "infusoria." He naturally
concluded that all life had been killed by boiling; and, as the entrance of
fresh life from the outside was prevented by the closing of the flask, he
considered that the living infusoria must have originated spontaneously
from the inanimate constituents of the broth.

Twenty years later Abbe Spallanzani alleged that the development
of the infusoria "in an infusion maintained at boiling-point for three-quar-
ters of an hour was possible only, provided air, which had not been pre-
viously exposed to the influence of fire, had been admitted." Objections
were made to these experiments and the controversy went merrily on.
Gradually experimental evidence accumulated resulting largely from
the work of Franz Schulze, and the discovery by Schroeder and Dusch
in 1853, that putrescible fluids will not decay after boiling, if protected
from the bacteria of the air by means of a cotton- wool filter or plug; and
the epoch-making experiments of Pasteur in 1860, with the now well-
known Pasteur flask, showed conclusively that the hypothesis of spontane-
ous generation, or abiogenesis, could not be proved.

Liebig, the celebrated German chemist, strenuously opposed the
theories of Pasteur; his authority and the brilliancy of his expositions
influenced the scientific world during the period 1840-1860. To Liebig,
fermentation was a purely chemical phenomenon unassociated with any
vital process; and he treated Pasteur's results with disdain. : ' Those who
pretend to explain the putrefaction of animal substance by the presence of
microorganisms," he wrote, "reason very much like a child who would
explain the rapidity of the Rhine by attributing it to the violent motions
imparted to it in the direction of Bingen by the numerous wheels of the
mills of Mayence." Again and again Liebig formally denied the correct-
ness of Pasteur's assertions; finally Pasteur challenged him to appear
before the Academic Commission to which they would submit their
respective results. Liebig, however, did not accept the challenge; the
victory was with the French savant.

In 1841 Fuchs investigated some blue and yellow milk. He exam-
ined it with the microscope and discovered the presence of organisms.

4 HISTORY OF MICROBIOLOGY.

He succeeded in cultivating the "blue milk" microbe in mallow slime, and
re-developed the blue color in milk by introducing some of his culture.
The organisms obtained were sent to Ehrenberg, who named them Bac-
terium syncyaneum, now known as B. cyanogenus, Ps. syncyanea and
B. synxanthum, a name which is still retained in the literature.

Since 1860 the master mind of Louis Pasteur has dominated the realm
' of microbiology. His epoch-making discoveries were largely due to his in-
tuitive vision, his skill in device and in the adaptation of means to ends, his
prodigious industry, and the enthusiasm and love with which he inspired
his associates. Trained as a chemist, his first appointment was to a pro-
fessorship of chemistry, and his earliest research dealt with problems
in molecular chemistry and physics. On his being elected Dean of the
Faculty of Sciences at Lille, he commenced to study fermentation. His
work in this field was soon followed by important results: the discovery of
the organisms which produce lactic and butyric fermentation, and of
anaerobic life, or life which flourishes without free oxygen. He devised
an improved method of making vinegar, and demonstrated the presence
of the acetic organism which he named Mycoderma aceti. Later he
studied the diseases of wine, and discovered that bitterness or greasiness
was due to a special ferment, and suggested the heating of wines in closed
bottles to a temperature of 60, in order to kill the injurious micro-
organisms. This process, since called pasteurization, is now largely
used, and makes it possible for manufactures and merchants to keep and
export wine without losing its flavor or bouquet. It is interesting in this
connection to note that a French confectioner named Appert published,
in 1811, his method of preserving fruits, vegetables, and liquors by heating
and sealing, and hence may be looked upon as the founder of the packing
and canning industry.

In 1864-65 the silk districts of that region of France, known as the
Midi, suffered such serious losses that the yield of cocoons fell from twenty-
six million kilogrammes to four million, which entailed a loss of twenty
million dollars and caused widespread distress and poverty. An epidemic
had broken out among the silk-worms, the dread disease known as
Pebrine. Pasteur was induced to make an investigation as to the best
means of combating the epidemic; and, after several years of study, he
found the organism causing the disease, suggested remedies, and brought
back wealth to the ruined communities, but at the cost to himself of im-
paired health and partial paralysis.

HISTORY OF MICROBIOLOGY. 5

Pasteur's results were very suggestive; and one outcome of his work
was that between 1870 and 1880 several important discoveries were made
by other investigators. Prior to the dates mentioned, the mortality from
blood poisoning, gangrene, and other infections following operations
was extremely high. Surgeons regarded such a result as inevitable, and
many agreed with the saying of Velpeau, that "the prick of a pin is the
open door to death;" but, in 1860, Joseph Lister, an Edinburgh surgeon,
began to study the possible role of microbes in the infection of wounds. By
sterilizing his instruments, sponges, ligatures, etc., and using antiseptics,
he was able to obtain such a high percentage of recoveries that in two
years he saved thirty-four patients out of forty, a percentage unheard of
up to that time. Hence the origin of the antiseptic and aseptic methods
of surgery. Lister's methods, suggested by the ideas of Pasteur, have
rendered possible the marvelous surgery of the present day, banished hos-
pital gangrene, and robbed confinement of its terrors.

To Lister must also be given the honor of devising the first practical
way of obtaining a pure culture of bacteria by means of high dilutions.
By using this method, Lister obtained some idea of the different fermenta-
tions of milk, such as souring, curdling, etc. He also confirmed the con-
clusion of Robert Hall (1874), that milk could be obtained from the animal
in a sterile condition, thus proving that the souring of milk was caused by
organisms from some external source.

In 1872, F. Cohn's System of Classification, based on morphological
characters, appeared. He distinguished six genera, micrococcus, bac-
terium, bacillus, vibrio, spirillum, and spirochaete; four years later this
investigator made the important discovery of endospores (spores formed
within cells), and noticed that organisms in this state were more resistant
to heat than the rods from which they were derived. This fact was ob-
served in the well-known "hay bacillus."

In 1871, Weigert succeeded in staining bacteria with picro-carmine;
but it was not until 1876 that he used the aniline colors, or dyes, for this
purpose, and thus opened up a new field which was exploited with such
beautiful results by Ehrlich, Koch, Gram, and others. The staining
of microorganisms rendered it possible to obtain pictures of them by pho-
tographic methods; the art of photomicrography developed thus rapidly.

In 1879, Miquel discovered bacteria which grew or developed at tem-
peratures between 65* and 75. He isolated them first from the waters

* All temperatures are stated in Centigrade scale, unless otherwise indicated.

6 HISTORY OF MICROBIOLOGY.

of the Seine, and subsequently from dust, manure, and other substances.
Later researches have shown that these thermophilic organisms play im-
portant roles in various fermentations.

The ninth decade of the last century was prolific in important bac-
teriological events. Discovery followed discovery in rapid succession.
In 1880, Laveran, a French military surgeon, discovered the protozoon of
malaria; in 1881 Robert Koch introduced the poured gelatin and agar
plate, which made it possible to obtain pure cultures without difficulty.
Investigators were quick to take advantage of this method; and notable
results followed. Eberth and Gaffky discovered the bacillus of typhoid
fever, and succeeded in growing it in culture media. In 1882, Loeffler
and Schlitz discovered the bacterium which causes glanders; and in the
following year Koch isolated the vibrio of Asiatic cholera from the in-
testines of cholera patients. In 1883 Klebs described the diphtheria
bacterium; and, in 1884, LoefBer grew the organism in pure culture.

In 1884, Koch published his results on the etiology of tuberculosis,
in a paper which will remain as a classical master-piece of bacteriological
research, owing to the difficulty of the task and the thoroughness of the
work. Not only did Koch show the tubercle bacterium by appropriate
staining methods, but he succeeded in obtaining pure cultures of it and in
producing tuberculosis by inoculation with his isolated cultures.

In 1885, Nicolaier observed the tetanus bacillus in pus produced by
inoculating mice and rabbits with soil; later, in 1889, Kitasato isolated
this organism, and showed that the cause of the failure in earlier attempts
to isolate it were due to the fact that it could grow only in the absence of
free oxygen. The specific infecting agents in pneumonia were discovered
by Friedlander and Fraenkel about this time, as were also several organ-
isms associated with inflammation and suppuration, such as the Strep-
tococcus pyogenes and the Staphylococcus pyogenes, discovered by Rosen-
bach, and the green pus germ (Pseudomonas pyocyanea) by Gessard.

Whilst these discoveries were taking place, largely in Germany, Pas-
teur had been engrossed with his prophylactic studies. In 1880, he dis-
covered a method of vaccination against fowl cholera; and in 1881 he
published his method of vaccination against anthrax. On a farm at
Pouilly le Fort, sixty sheep were placed at Pasteur's disposal; ten of these
received no treatment, and twenty-five were vaccinated. Some days
afterward the latter were inoculated with virulent anthrax, and also
twenty-five which had received no vaccine. The twenty-five non-vacci-

HISTORY OF MICROBIOLOGY. 7

nated sheep died; and the twenty-five vaccinated ones remained healthy
and in the same state as the ten control animals. This convincing experi-
ment was followed by others; and, hi the twenty-five years immediately
following the introduction of the method, more than ten million animals
were vaccinated in France alone, with excellent results. In 1885, as the
result of much animal experimentation, Pasteur related to the Academy
of Sciences his discovery of a method of vaccination against rabies, or
hydrophobia; and six months after the successful treatment of the first
case, 350 persons bitten by rabid dogs were vaccinated. An institute for
the preparation of vaccines was built by public subscription and named
the Pasteur Institute; and since that date more than thirty similar estab-
lishments have been founded in different parts of the world.

This eighth decade, so pregnant with discoveries of the utmost im-
portance to medicine and surgery, was also notable for its discoveries in
agricultural bacteriology. The honor of having been the first to work out
the causal relation between a specific microbe and a plant disease belongs
to Burrill, who discovered the organism of Fire or Pear Blight; and in 1883
to 1888 Wakker discovered the bacillus which produces the "yellows" of
the hyacinth, a disease of considerable economic importance hi Holland.
To Beyerinck, Hellriegel, and Wilfarth we owe our earlier knowledge
of the development and morphology of the nitrogen-fixing organism which
produces the nodules or tubercles on the roots of legumes. In 1888
Winogradsky isolated from soils nitrifying microbes which grew in a
medium devoid of all traces of organic matter. During this period,
Hansen's investigations along the line of the fermentation industry were
most important. He devised methods for securing pure cultures of yeasts
starting from a single cell, showed that yeasts produced diseases in beer,
and established the method of identifying yeasts by observing their micro-
scopic appearance, the formation of ascospores, and the production of films.

The tenth decade of the nineteenth century was almost as prolific in
discovery as the ninth. In 1890 Behring discovered the antitoxin for
diphtheria, as a result of the pioneer work on toxins by Roux and Yersin. '
Five years later, this serum came into general use as a curative; and the
efficiency of the treatment is shown by a comparison of the death rate from
diphtheria before and after the introduction of the antitoxin. The average
annual death rate from diphtheria in eight large cities, during the period
1885-1894, was 9.74 per 10,000 of the population before the use of anti-
toxin; and during the antitoxin period of 1895-1904 it was 4.29.

8 HISTORY OF MICROBIOLOGY.

The subsequent researches on the constitution of toxins and antitoxins
by Ehrlich, Metchnikoff, Madsen, and others have been productive of
a better understanding of the problems of immunity.

In 1892 Pfeiffer discovered the organism of influenza or grippe; and
in 1894 Yersin and Kitasato independently discovered the bacterium of
bubonic plague.

The now well-known serum diagnosis of typhoid fever, whereby living
and motile typhoid bacilli are clumped and lose their motility when placed
in the diluted serum of a patient suffering from the fever, was due to the
work of Gruber and Durham, and the exploitation of the method by
Widal, and dates from 1896.

In 1898, Shiga discovered the bacterium of dysentery, and the pos-
sible cause of pleuro-pneumonia in cattle was found by Nocard. This
latter organism was so minute as to be at the extreme limit of micro-
scopic definition, and suggested that other well-known diseases, such as
foot-and-mouth disease, are probably caused by ultra-microscopic or-
ganisms.

This year, Ronald Ross worked out the relation between man, the
mosquito, and the malarial parasite, a discovery which at once sug-
gested the best means of controlling the disease.

In 1905, Schaudinn definitely established the causal agent of syphilis,
a spirochaete-shaped organism, which he named the Treponema pallidum,
and which had escaped earlier discovery on account of its being refractory
to the ordinary staining methods.

No one can deny that the progress of microbiology in the last forty
years has been extraordinary; but much still remains unknown. The
causes of some diseases have not been discovered. Smallpox, scarlet
fever, yellow fever, mumps, whooping cough, epidemic infantile paralysis,
hydrophobia, and others offer an inviting field to the medical micro-
biologist ; and the many problems of soil microbiology call for solution by
the agricultural microbiologist. Yet it cannot be said that the laborers
' are few.

The record of past achievements is an inspiration; and the knowledge
that each discovery was the result of persistent and concentrated effort,
may give us of the present day firmer faith and greater strength for
work in the broad and inviting field before us.

PART I.

THE MORPHOLOGY AND CULTURE OF MICRO-
ORGANISMS.

GENERAL.*

Microbiology includes some algcc, a few specific molds, which fall in the
realm of pathogenesis (disease production), zymogenesis (fermentation-
production), and laboratory manipulations; it deals mainly with yeasts,
bacteria, and invisible organisms; it dips deeply into the expanse of pro-
tozoa; in short, it is concerned almost wholly with the field of unicellular
life. On the one hand, the microbiologist meets the botanist and estab-
lishes reciprocal relations with him; on the other hand, he mixes with the
zoologist and delves into studies of mutual interest. Primarily, the tecli-
nic of the microbiologist together with, in part, the economic bearing of
the subject seems to be the determinant factor of limitation.

Assuming, therefore, that the province occupied by microbiologists
consists of the study of unicellular life-forms, because such limitations
have been established by actual studies and investigations, through the
instrumentality of microbiological technic, it will be pertinent and
clarifying to provide a general graphic outline at the start. By this means
the student will be able to locate himself, whether he is just launching or
has gotten far out on the troublesome and most fascinating sea of micro-
biology. The graphic outlines will always be his ready chart.

* Editor.

10

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

OUTLINE or PLANT GROUPS.*

The following is a diagram of plant groups, showing one scheme of placing the bacteria,
yeasts, and molds in relation to other groups. Only a few of the sub-groups can be shown
in such a scheme.

Schizophyta f Schizomycefes ( fission-fungi}, bacteria.

(fission-
plants)

Tballophyta

Phycomycetes j

Oomycetes

Schizophyceae (fission-algae), blue-green algae.

[ Chlorophyceae green algae.
Algae Phasophyceae brown algae.

[ Rhodophyceas red algae.
Characeae.

Myxomycetes.

f Chytridineae.

j Zygomycetes (Mucors).

Saprolegniaceae (water

fungi). .

Peronosporacese (downy
I ! mildews).

Hemiasci (Monascus).

Protoascineae (Saccharo-
Fungi i myces, Yeasts).

Protodiscineae.
Discomycetes.
Plectascineae (Aspergillus).
Pyrenomycetineae.
J Penicillium,Fusarium, Alternaria,
[ Oidium, Cladosporium, and others.
Rusts
Smuts
Mushrooms.

Ascomycetes

Euasci -

Imperfect Fungi,
Conidia only

Basidiomycetes

Bryophyta
(mosses and
liverworts)
Pteridophyta
(ferns, etc.)
Spermatophyta
(seed plants).

OUTLINE OF PROTOZOAL GROUPS, f

Limited on account of economic importance to " A CLASSIFICATION OF THE PATHOGENIC
PROTOZOA." For discussion of classification see p. 76.

Protozoa

Rhizopoda

I
* Charles Thorn.

f

Amoeba

Amoeba dysenteries
Amazba coli
A mceba meleagridis
A moeba buccalis
Plasmodiophora ^ Plasmodiophora brassica

t J. L. Todd.

OUTLINE OF PLANT AND PROTOZOAL GROUPS.

II

Spirochceta obermeieri

Spiroch<zta duttoni

Spirochata vincenti
Spirochaeta Spiroch(Eta p al udula

Spirochata theileri

Spirochccta gallinarum

Treponema < Treponema pallidum

( Herpetomonas donovani
Herpetomonas j Her p etomonas f unm culosa

Crithidia

Trypanosoma gambiense

Trypanosoma cruzi

Flagellata

Trypanosoma

Trypanosoma brucel

Trypanosoma evansi

Trypahosoma equinum

Trypanosoma dimorphon

Trypanosoma lewisi

Trypanosoma equiperdum

Trypanoplasma

Protozoa

Cercomonas

Trichomonas \ Trichomonas -vaginalis

Monas

Plagiomonas

Lamblia ^ Lamblia intestinalis

Gregarina Coccidium cuniculi

Coccidia \ Coccidium Coccidium hbminis

Coccidium avium

Plasmodium vivax

Plasmodium Plasmodium malaria

Plasmodium falciparum

Proteosoma

Sporozoa

Haemosporidia

Haemoproteus

Lankesterella (and other Haemogregarines)

Hepatozoon

f Babesia bieemina

Tiahpcio i

1 ><l 1 " ^lil N ,, ,

1 Babesia cams

Sarcosporidia ^ Sarcocystis \ Sarcocystis miescheriana

Haplosporidia \ Rhinosporidium \ Rhinosporidium kinealyi

Myxosporidia -j Myxobolus \ Myxobolus pfeifferi

Microsporidia <j Nosema \ Nosema bombycis

Infusoria \ Balantidium \ Balantidium coli

Histoplasma capsulatum

Parasites of uncertain position Chlamydozoa

Ultramicroscopic viruses

CHAPTER I.*
MOLDS.

FUNGI IN GENERAL.

A sharp line cannot be drawn between the bacteria and the fungi.
Certain border groups such as Leptothrix and Actinomyces, filamentous
forms in which branching and even the production of differentiated spores
occur, are sometimes described as bacteria and sometimes as fungi.
From the microscopic point of view, forms in which the cells can be
handled as bacteria by cover-glass staining may be conveniently treated
by bacteriologists. Forms in which the cells are larger, with definite
walls, vacuoles, and cell sap, in which the cells collapse when dried and
lose their distinguishing characters, may be better treated as fungi. No
rule holds for all groups.

With some exceptions, there is, among the cells of the true fungi,
a differentiation of function into vegetative or assimilative cells and re-
productive cells. .The fungous body is usually composed of threads
(technically called hypha, singular, hypha) . These hyphce usually branch
in more or less complex manner forming networks or webs, collectively
called mycelium. Hyphae may be one-celled or composed of many cells
placed end to end as shown by the cross walls, called septa, seen in them.
These threads grow either by the formation of new cells at the growing
tips (called apical growth) or by the division of cells in the hypha (inter-
calary growth) . The fungous cells rarely divide in three planes to produce
solid masses of cells. Both vegetative and reproductive masses are formed
in great variety from such hyphae. Often the thread-like character is
almost or quite obliterated in the ripe masses, which may be fleshy, woody,
carbonaceous, leathery and even horn-like in texture, as seen especially in
the mushrooms, bracket-fungi, etc., but even in such cases the early stages
show the structures to originate from masses of fungous threads.

The formation of differentiated reproductive cells is, in general,
characteristic of the fungi. The method of reproduction presents great

* Prepared by Charles Thorn.

12

MOLDS. 13

variety. In the simplest forms, the reproductive cells are scarcely if
at all distinguishable from the vegetative cells. In some species whole
hyphae break up so that each cell forms the starting-point of a new colony.
Other forms develop special branches bearing reproductive cells. From
these it is but a step to the production of fruiting branches, characteristic
in form, called conidiophores, bearing cells markedly specialized as re-
productive by form and frequently also by color, called conidia. These
conidia are entirely asexual in origin and capable of growing directly into
new colonies, although in many cases they are provided with resistant
walls which enable them to live for long periods if conditions are unfavor-
able to growth at once. In other species, specialized resting cells with
resistant walls are formed to enable the plant to survive unfavorable
conditions. These are called chlamydospores or sometimes cysts. The
name gemma is sometimes applied to similar structures, preferably to
such as grow at once. The same end is reached in still other groups by
the formation of sclerotia which are hard masses or balls of thick-walled
cells filled with concentrated food materials. These sclerotia are fre-
quently distinctive of the species producing them by size and appearance.
They sometimes resemble the sexual fruiting masses. Resting structures
of either type, especially when large, commonly produce typical spore-
bearing structures at once after germinating. Many very complex
fruit bodies such as the mushrooms appear to be entirely asexual in origin.
The systems of classification used are largely based upon the types of
sexual fruit bodies produced. Where such fruit bodies are not known, the
method of formation of the asexual spores furnishes the most satisfactory
basis for grouping. In classifying fungi, certain types of spore formation
are found to be characteristic of particular groups. Since within these
groups various accessory types of fruiting occur, so that some species
show three or even more forms of spores, that type of spore formation
which is regarded as characteristic of the group is known as the perfect
stage. If sexual fruits are found, these constitute the perfect stage of the
group; if no such fruit is found, the most characteristic asexual form is
used, as for example the common mushroom of commerce which is asex-
ually produced as far as we know, yet represents the most perfect and most
constant fruiting form produced by a very large group. Between the
typical forms are many gradations resulting in many families whose re-
lationship to one or the other group is difficult to determine. Probably
the ancestral history (phylogeny) of the fungi, if known, would show several

14 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

or many lines of descent rather than one. Certain of these groups may
be presented briefly.

BACTERIA. In the scheme of plant grouping presented (page 10),
which is only one of many attempts to show relationships, the bacteria
are placed with a group of single-celled green or blue-green forms as
Schizophyta or fission-plants because of reproduction only by the division
of the cells.

PHYCOMYCETES. The Phy corny cetes are called algal fungi because they
resemble certain groups of green filamentous forms in many particulars.
In this group two general types of sexual reproduction are met with,
zygospore formation and oospore formation. The first, found in the
Zygomycetes represented by the common mucors, consists of the fusion
of terminal cells of branches of the mycelium similar in appearance but
differentiated in sex. As a result of this fertilization large thick-walled
resting cells are produced, called zygospores, from a Greek root mean-
ing yoked (see Fig. 2). In oospore formation, found in the Oomycetes,
the conjugating cells differ in appearance as well as in function. The
oospore is large and is rich in food materials; the antheridium is much
smaller, penetrates and fertilizes the egg, which afterward develops
into a thick-walled resting spore. The very destructive downy mildews
belong to this group.

ASCOMYCETES. In this great group sexuality was denied until recent
years, but has been proved in cases enough to establish a presumption of
more general occurrence. The characteristic structure of the group is
the ascus, a sac containing, when ripe, typically eight spores, some-
times a less number by the failure of some to develop, sometimes a larger
number usually some multiple of eight. The ascus where sexuality is
known is developed subsequent to fertilization, not directly from an
egg cell. The group presents a great variety of fruiting masses produced
in connection with the asci. The simplest forms are loose webs of hyphse
enmeshing a few asci; other forms show clubs, cups, flask forms, crusted
areas, the type of mass in each case being characteristic of the family,
genus and species represented. Only a few of many thousands of these
forms are encountered in bacteriological work. One genus is, however,
constantly found. The commonest species of Aspergillus produces bright
yellow, globose fruiting bodies, called perithecia, filled with asci. These
are borne upon the surface of the substratum and often give a yellow color
to the colony by their abundance. Such perithecia consist of the ascog-

MOLDS. 15

enous cells and the asci produced by them, about which a more or less
completely closed sac or wall has been formed, by the development of the
sterile cells adjacent to the fruiting ones.

BASIDIOMYCETES. In the Basidiomycetes there is still further re-
duction of the evidences of sexuality. In one border group, the rusts,
sexual processes have been shown to be more or less developed. In the
typical Basidiomycetes sexuality is as yet undescribed if present at all.
The typical structure is the basidhtm, a spore-bearing cell characteristic-
ally producing at its apex four protuberances called sterigmata (singular,
sterigma), each bearing a single spore. These basidia are grouped into
many kinds of fruit bodies, from occurrence here and there upon a loose
web of hyphffi to dense columnar areas covering the gills of the mushrooms
or lining the cavities of the puffballs. Very few of these species occur in
bacteriological studies.

IMPERFECT FUNGI. A very large number of species are known which
have never been seen to produce the characteristic fruits of the great
groups. These are brought together and described as form-genera by
their method of asexual spore formation. From the lack of the organs
used in classifying the other groups, these are called the imperfect fungi
and their grouping regarded only as temporary, a convenience for the
identification of materials. These include many forms of economic im-
portance, and many of the species most frequently met in bacteriological
work. Sometimes one species of a large group produces a perfect form
while no other species can be induced to do so. Some of these species
undoubtedly represent stages of perfect fungi whose perfect forms
simply are not recognized as connected with these; others reproduce for
an indefinite number of generations by conidia. Such cases do not
appear to need the perfect form and hence apparently have, in some
cases, lost the power to produce it.

As found in nature all these forms are parasitic, saprophytic, or
capable of both modes of life. All depend more or less completely
upon organic matter for nourishment. Great diversity exists, how-
ever, in their adaptation to environment. Many of them are not only
parasitic but so closely adapted or parasitizing particular host-species
as not to be found elsewhere. Others attack several or many species,
usually related. Even among saprophytes many species are found only
upon particular forms of decaying animal or vegetable matter. The
great economic importance of these parasitic and closely adapted sapro-

1 6 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

phytic species has been recognized by the development in recent years
of the literature of plant pathology (phytopathology). These cannot
be considered in this work.

SPECIFIC CONSIDERATION OF MOLDS.

A few species are found to grow very constantly in the same situations
as bacteria. These are associated with forms of decay, fermentation,
or disease, either as primary or secondary causes. They thus become
important to the bacteriologist who studies them by the same methods
as bacteria. These species belong to widely scattered groups of fungi,
so that species found under the same conditions frequently differ greatly
in appearance. The common term, molds, is applied collectively to
these organisms, though no sharp limits can be set to the use of the term.
Physiologically these species can be considered in three series:

COSMOPOLITAN SAPROPHYTES. Certain species are capable of
growing within very wide limits of temperature and of composition of
substrata. Many of these have accompanied man everywhere and are
constantly found upon every kind of putrescible matter, especially as the
causes of fermentation or decay in food. Their spores (conidia) are
produced in countless numbers, and are so light that they float in air
currents and are carried by contact in every conceivable manner by
animals and by man. The life cycle from spore to spore is frequently
very short, often being completed in twenty-four hours or less. Many
of these forms are propagated for an indefinite number of generations by
asexual spores or conidia, while for some of them no sexual-fruiting form
is known. These species are the "weeds" of the bacterial culture-room,
since they cannot be entirely eliminated and will survive, as a rule,
conditions more severe than the bacteria themselves.

MOLDS OF FERMENTATION. A few species have acquired special
importance by their fermentative action. In most cases these forms
are widely distributed and able to utilize other media and conditions
also. They differ from closely related species of the same genera in the
ability to produce special enzymes or specially large amounts of such
enzymes as bring about particular forms of fermentation. Certain of
these species have been utilized in the manufacture of drinks, of citric
acid, in cheese ripening, etc. Others are so adapted to growth under
conditions of fermentation as to be found constantly in connection with

MOLDS. I?

such processes, in which their vigorous growth and fermenting power
seriously interferes with control of results.

PARASITES AND FACULTATIVE PARASITES. A few molds are found
as primary agents in causing diseases of man and animals. Some others
enter as secondary infections, but become pathogenic after entrance.
These comprise species of Aspergillus and Penicillium which produce
disease in the external ear of man, Aspergillus fumigatus, a cause of lung
disease of birds, and the series of forms causing skin diseases, dermatomy-
coses, of both man and animals.

GENERIC CONSIDERATION OF GROUPS.*

THE MUCORS OR BLACK MOLDS. The mucors or black molds con-
stitute a large group of species belonging to the Phycomycetes or algal fungi
whose general characters are a unicellar mycelium, at least in the vegetative
stage, and quite generally a well-developed form of sexual reproduction
(Figs, i and 2). In the mucors, the mycelium is usually richly developed
within and often also on the surface of the substratum; asexual reproduc-
tion is accomplished by spores borne as conidia or borne within sporangia;
and sexual reproduction is accomplished by the conjugation of special
branches from the mycelium forming zygos pores (Figs. 2 and 3) . The typical
mucors produce sporangia as capsule-like dilations at the ends of erect
fertile hyphse, each containing many spores. Septa are commonly de-
veloped in the mycelium when sporangia begin to appear. These fertile
hyphae may be microscopic or attain a length of several centimeters.

Important Species. Perhaps the commonest form is Rhizopus nigricans
(syn. Mucor stolonifer), the black mold of bread, a cosmopolitan species
associated with the decay of many kinds of food stored in wet condition
or in humid situations. Typical clusters of sporangiophores are borne
on stolons or runners, which are hyphse extending radially from the
center of the colony and fastened to the substratum or to the support at
intervals by root-like outgrowths above which several sporangiophores
are produced. Abundant growth of this species is found only under

* The series of forms presented contains representatives of the most common groups as
they occur in laboratory cultures, and such as have acquired importance to the worker in
bacteriology by participation in processes regularly studied by the bacteriologist. For more
complete discussion of the fungi, the student is referred to standard text-books of cryptogamic
botany. For discussions of species, Lafar's Technical Mycology includes the groups found
associated with the bacteria; for other groups, special botanical literature must be consulted.

i8

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

very moist conditions or in substrata with high water content. Rhizopus
is a very common contamination in laboratory cultures.

There are many common species of the genus Mucor, very few of
which are identifiable without critical study. The specific names as
commonly cited often designate groups of species or varieties rather than
sharply marked forms. Certain of these may be briefly considered.

Mucor mucedo L. is a common form upon dung, characterized by
heads (sporangia) upon long sporangiophores,* at first yellow then becom-

FlG. 2. Mucorinea. Mucor. From Tabulae Botaniccs, showing sporangia origi-
nating from mycelium, spores and spore germination, and the formation of zygospores
in a heterothallic species (diagrammatic). (Reduced one-half). (By permission of
A.F. Blaskeslee.)

ing dark brown or black and studded upon the surface with needles of
lime.

Mucor racemosus, Fresenius, is characterized by the production of
chlamydospores or cysts in the mycelium within the substratum, as
elliptical thick-walled cells. The sporangiophores typically branch to
make racemes of sporangia. The racemose mucors are active agents in

*The term sporangiophore is composed of the word sporangium combined with the suffix
phore, meaning bearer. In sympodial branching the first fruit is on the tip of the original hypha,
the first branch arises below this fruit and is terminated by the second fruit. Each successive
branch and fruit originates in similar manner.

MOLDS. IQ

changing starch to sugar and in the production of traces at least of
alcohol from sugars.

Mucor rouxii (Calm.), Wehmer, is the most important of a series of
forms with sporangiophores branching sympodially which are active in
changing starch to sugar and in producing traces at least of alcohol. The
mycelium of Mucor rouxii develops in fluid cultures as yeast-like cells
and groups of cells. The typical mucor fruits are produced only under
special cultural conditions.

FIG. 3. Mucorinece. Mucor, Rhizopus. A, B, C, D, formation of the zygospores
from conjugating branches; E, section of D; F, mature zygospore in section; G, germina-
tion of zygospore; H, diagram of fruiting stolons of Rhizopus nigricans; K, section of
sporangium during spore formation, highly magnified. From Tabula Botanies.
(Reduced one-half). (By permission of A. F. Blaskeslee).

Fermentation activity has been described for numerous species of
Mucor and Rhizopus. Many of these species have been found and
described as constituents of Chinese yeasts, or isolated in the study of the
fermentation industries of Japan, China, and other eastern countries.
Among them are Mucor circinelloides, Van Tieghem, Mucor javanicus,
Wehmer, Mucor plumbeus, Bonorden, Rhizopus oryza, Went, Rhizopus
javanieus. The fermenting power of mucors like that of yeasts varies

2O

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

greatly with the species or even with races used, approaching in some
species the efficiency of the more active yeasts.

THAMNIDIUM. Of related genera, Thamnidium differs from Mucor

FIG. 4. Penicillium expansum, Link, c, b, f, Branching and arrangement of
branches of conidial fructification (Xgoo); c, d, e, conidiiferous cells and conidial chains
(Xgoo); g, h, j, k, I, sketches of fructifications (Xi4o); m, n, o, germination of conidia
(XQOO); r, s, sketches from photographs showing in 5 loose aggregations of conidio-
phores beginning to develop into zonately arranged coremia, in r a coremium i mm. in
height. (From Bui. 118, Bureau of Animal Industry, U.S. Dept. Agriculture).

in the production of two kinds of sporangia. The terminal sporangium
of a fruiting hypha resembles that of Mucor; the secondary or accessory
sporangia which are borne upon side branches of the sporangiophores

MOLDS. 21

are smaller, lack the columella, and produce few to several spores within
an outer wall.

Thamnidium elegans, Link, produces primary and secondary spor-
angia on different hyphse, together making white colonies. The fertile
side branches are produced in whorls and bear whorls of branchlets
from their centers which in turn produce sporangioles from the tips of
short straight twigs or branchlets.

PENICILLIUM. The extremely abundant green molds most fre-
quently belong to the genus Penicillium, although some members of
other groups may be confused with them at times.

Characters. Colonies are composed of loosely woven hyphae, branched,
septate, colorless, or bright colored. The fertile hyphas (conidiophores)
are mostly erect, arising either from submerged hyphae, or as branches
of aerial hyphae, septate, usually branched only in the fruiting portion.
Conidial fructifications consist of more or less complex systems of
branches and branchlets, the ultimate fertile cells each producing a chain of
conidia (Fig. 3). The whole system is usually grouped near the end of the
conidiophore, giving the appearance of one or more brooms or brushes
(whence the name). Very few species are known to produce asci, hence
these are rarely encountered. The conidial form continues for an in-
definite number of generations, therefore all the activities of the genus
are associated with this form.

Cultural Considerations. Among the numerous species and races,
some of the green forms are widely distributed and almost omnivorous
in habit. Other species are closely restricted to particular substrata.
Starches and sugars appear to be especially favorable components of
nutrient media for members of the group. The larger number of the
species grows best at temperatures from 15 to 30; a very few of them
reach their optimum at 37, but many species are entirely inhibited
and some killed at blood-heat. Vegetative mycelium begins to be
produced at temperatures very close to freezing, but colored conidia
are produced slowly or not at all at low temperatures. The species of
Penicillium thrive through a wide range of concentration of culture
media, though perhaps the most characteristic growths are produced
in media high in water content. The common species of each genus will
grow in all the standard bacteriological media. With few exceptions
the species grow well in synthetic media composed of assimilable car-
bohydrates and inorganic salts. A few species require the presence of

22 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

some one of the higher nitrogenous compounds, but many species refuse
to produce typically colored fruit without some form of starch or sugar
in addition to ordinary peptone and beef-extract. Very few species
grow well in alkaline media, but most species are tolerant of organic
acids at the concentrations found in fruits and vegetables.

Some Common Species. Penicillium roqueforti, Thorn, is a green form
constantly found in pure culture in Roquefort cheese, frequently also in
ensilage. It is widely distributed and grows under many sets of conditions.

Penicillium camemberti, Thorn, is the chief organic agent in ripening
Camembert cheese. Cultures of this species are floccose or cottony, at
first white, later gray-green.

Penicillium expansum, Link, is a green form, always obtainable from
apples decaying in storage, upon which it frequently produces large
coremia. It is one of the most abundant species of the genus, widely
distributed in different countries. In cultures, colonies produce a
characteristic odor, suggestive of its common habitat, decaying apples.

Penicillium brevicaule, Saccardo, is a form with rough or spiny brown
spores which has been used physiologically to detect the presence of arsenic
by its ability to set free arsine from such substrata. Except species
associated with particular processes or substrata, the identification of
the green species of Penicillium requires special methods and greater
care than is possible aside from special study of the group.

ASPERGILLUS (AND STERIGMATOCYSTIS). The genus Aspergillus in-
cludes numerous species which develop under widely different conditions.
Many of these forms reach their typical development under drier condi-
tions than Penicillium and Mucor, such as stored grain, herbarium speci-
mens, dried flesh, or foods containing concentrated sugars, such as jams,
jellies, etc. Some excite processes of fermentation, and a few are asso-
ciated with diseases.

Characters. The vegetative hyphae are creeping, submerged in the sub-
stratum or sometimes aerial also, loose, floccose, branched, septate, usually
colorless, and sometimes bright colored. Conidiophores or fertile hyphae
are erect, unseptate, or few-septate, usually much larger in diameter
than the vegetative hyphae, and gradually enlarged upward, ending in a
more or less abrupt dilation or head which bears closely packed colum-
nar sterigmata or conidiifierous cells over the whole or a large part of its
surface (Fig. 6). Each of these cells bears, in one group of species, a
single chain of conidia, in other species (called by some authorities

MOLDS.

Sterigmatocystis) three or four secondary sterigmata which bear the
conidial chains. Part of the species produce also thin-walled perithecia
as yellow or brown spherical bodies upon the surface of the substrata.
These perithecia are filled with eight-spored asci (Fig. 5). A few species
produce sclerotia instead of perithecia, but many species are not known
to produce either perithecia or sclerotia.

Important Species.- Among the species constantly met with, Aspergittus
niger is recognizable by its black or very dark brown spores and in some

FIG. 5.

FIG. 5. Aspergittus glaucus. a, Con-
idiophore showing increased diameter over
the vegetative cells at its base (Xi28);
b, sterigmata (X45o); c, conidia, smooth
thick walled in this variety, other varieties
are spiny (X45o)i d, perithecium (XiaS);
e, ascus containing ascospores (X4So).
(Original.)

FIG. 6.

FIG. 6. Aspergillus. (i) A. fumi~
gatus, Fres; (2) A. nidulans. i and
2 show the simple sterigmata of A. fumi-
gatus and the secondary sterigmata of A .
nidulans. The conidia of these species
do not remain attached in ordinary fluid
mounts. (Original.)

strains by black sclerotia. Several black-spored forms are described, but
their separation is usually impossible from the data given. Aspergillus
niger ferments sugar solutions with the production of oxalic acid in con-
siderable quantity.

Of green forms, Aspergillus* glaucus, Link (Aspergillus herbariorum,
Wiggers), and Aspergillus repens, De Bary, both produce abundant
yellow perithecia. These abound upon herbarium specimens, hay,
grain, concentrated foods, such as jellies, preserves, and dried meats
upon which they produce green conidial areas which are later dotted with
bright yellow perithecia.

Aspergillus fumi gatus, Fresenius, is a green form characterized by

* Recent examination of a large number of American specimens shows that Aspergillus
repens is the usual green form in this country.

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

short conidiophores enlarging gradually into heads and bearing a single
set of sterigmata on the very apex, with chains of thin-walled green spores
about 3,* in diameter. This species produces a destructive disease of
birds known as aspergillosis. The same species is sometimes reported
as pathogenic to man.

Aspergillusnidulans differs by having two sets of sterigmata, but other-
wise frequently closely resembles Aspergillus fumigatus and is fre-
quently mentioned as pathogenic.

Aspergillus oryza has been used to produce " Takadiastase " from rice
in Japan. Other species produce amylase also, but in different degrees.

FIG. 7. FIG. 8.

FIG. 7. Cladosporium herba^um (r\ FIG. 8. Spores FIG. 9. Fusarium
showing the forms of conidiophores and of Alternaria sp. from decaying potato,
conidia which are very common upon (Original.) a, spores showing cur-

laboratory culture media. (Original.) vature and septa; b, ger-

mination of spores; c,
development of spores
in petri-dish culture; d,
mass of spores as found
in culture. (Original).

Aspergillus wentii, Wehmer, characterized by its long conidiophores
and coffee-colored heads of conidia, is found in the Soja preparation in
Java.

Of other forms constantly met, Aspergillus candidus has white or pale
cream fruiting surfaces, Aspergillus flavus produces several shades of
yellow and green, Aspergillus ochraceus, ocher or tan.

Much confusion is still found in the literature of this genus, so that
frequent references to the activities of particular species are difficult or
impossible to verify.

CLADOSPORIUM (AND HORMODENDRON). The species of Clado-
sporium occur frequently in cultures of decaying vegetable matter, of
milk and cream, or butter. The colonies liquefy gelatin. Both mycelium

*The unit of micro special measurement is the microm (p) or micro millimeter (.001 m.m.
or 1/25000 in.)

MOLDS. 25

and spores are at first colorless, but later dark colored to almost black,
with spores becoming two-celled in very old cultures.

Cladosporium herbarum is the commonest species encountered.* Col-
onies in culture media differ so greatly in structure from those upon natural
substrata as to make identification of species questionable. Fig. 6.
Much confusion is therefore found in the use of the names of species
of Cladosporium and the related genus, Hormodendron, which is sepa-
rated by some.

ALTERNARIA AND FUSARIUM. The frequent occurrence of species of
Allernaria and Fusarlum in cultures demands that the generic characters
be recognized. Both, as a rule, produce abundant growth with a tendency
to over-run cultures of other forms (Figs. 8 and 9). The spores of Alter -
naria are brown, Indian-club form, muriform (divided into several cells by
longitudinal as well as cross walls), and are connected together into
chains (Fig. 8). The spores of Fusarlum are colorless, either straight
or sickle- or crescent-shaped, divided into several cells by cross walls
occur singly or adhere into masses on the tips of the fertile branchlets
The morphology of colonies in culture varies widely from the descrip-
tions of the same species under natural conditions. Species of Fusa-
rium frequently produce bright colors in the mycelium and substrata;
colonies of Alternaria often become almost black. Identification of
species in cultures is thus far impossible, except for the specialist.

OIDIUM. Oidium (Oospora) lactis is universally found in cultures
from milk and milk-products and very frequently in decaying vege-
tables, manure, etc. Colonies of the species are colorless, have vegetative
mycelium entirely submerged, become powdery white with spores when
mature, liquefy gelatin, and produce a strong characteristic odor (Fig. 10).
Microscopically the species is recognized by dichotomous branching of
the hyphse at the margin of the colonies, and by the spores or oidia which
are abruptly cylindrical, varying with conditions in length and diameter
and produced both above and below the surface of the substratum in
long chains which break up readily. At times the whole mycelium ap-
pears to break up into oidia. Oidium lactis is a factor in the ripening
of many kinds of cheese: Limburg, Harz, Camembert, Gorgonzola, etc.
Its activity is associated with strong odor and taste.

MONILIA. Monilia Candida (Bonorden), Hansen. The line be-

* This species has been shown to be a conidial form of Sphcerella tulasnei Janczewski, but
the bacteriological student will meet only the conidial stage.

26

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

tween the Mycoderma group of yeasts, Oidium and Monilia, and the well-
fixed mold types shows a number of organisms which are found repeatedly
in the fermentation industries (Fig. n). One of these, Monilia Candida,
as described by Hansen, has been much studied. In morphology,
Monilia Candida appears as a yeast in young cultures in sugary fluids,

FIG. 10 Oidium lactis. a, b, dichotomous branching of growing hyphae; c, d, g,
simple chains of oidia breaking through substratum at dotted line x-y, dotted portions
submerged; e,f, chains of oidia from a branching out-growth of a submerged cell; h,
branching chain of oidia; k, I, m, n, o, p, s, types of germination of oidia under varying
conditions; t, diagram of a portion of a colony showing habit of Oidium lactis as seen
in culture media. (From Bui. 82, Bur. Animal Industry, U. S. Dept. Agr.).

but later develops a mycelium. It produces an alcoholic fermentation
which increases in vigor with the rise of temperature toward 40.

DEMATIUM. One species of Dematium, Dematium pullulans, has
been much studied. This is frequently found upon decaying fruit as
dark brown colonies. In culture, mycelium is sparingly produced, either
colorless or colored, and conidia are borne in clusters and chains all
along the hyphae submerged in the substratum. At first both mycelium

MOLDS. 27

and conidia are colorless, later some or all of the cells develop heavy
dark brown walls. Although not active as an agent of fermentation,
it occurs very frequently in the fermentation industries sometimes
discoloring the fermenting products. The conidia bud out from the cells

FlG. ii. A colony of Manilla Candida. (Photographed by Zae Northnip.)

of the mycelium in a manner resembling the yeasts. Its occurrence with
the yeasts has led to many careful descriptions of its several types of
spore production and its biological activities.

SAPROLEGNIACE^E AND ENTOMOPHTHORACE^;. These are two groups
of Phycomycetes which differ from the mucors in habit and in their
prominent development of sexual reproduction.

ENTOMOGENOUS FUNGI. Numerous species have been identified as
the destroyers of particular insects.

CHAPTER II.

YEASTS.*

MORPHOLOGY OF CERTAIN TYPES.

DEFINITION AND BASES OF CLASSIFICATION. If the cloudy freshly
expressed juice of grapes or other fruits be passed through a centrifuge,
the sediment will be found to consist principally of amorphous particles
of dirt and plant tissue. If the clear juice is now allowed to stand in a
warm place for a few days it will ferment and the sediment thrown
down by the centrifuge may be shown by the microscope to consist prin-
cipally of unicellular microorganisms.

These microscopic cells are called collectively "yeast" and belong
to various groups of fungi. Some of them are special vegetative forms
of Phycomycetes (Mucor), others of Ascomycetes (Saccharomyces, Asper-
gillus), while others are unknown in any other form and are classed as
Fungi imperfecti (Mycoderma, Toruld). They are widely distributed in
nature and some of them occur on all exposed surfaces and particularly
on moist organic substances containing sugar and acid. The true
yeasts (Saccharomycetes}, which are of the greatest importance indus-
trially, occur naturally on the raw material (S. ellipsoideus on grapes)
or are known only in the cultivated condition (S. cerevisia of beer).

The true yeasts occur in the form of spherical or more or less elongated
cells varying in normal width from 2.5/4 to 12 fj.. The first classifications
were based on shape and size alone but these vary and depend so much
on cultural conditions that they are of little value in differentiating
species or varieties.

The range of variation in shape and size, especially of the spores,
under given conditions of culture medium and temperature, is now used
only in conjunction with the reactions brought about in various solutions
to distinguish the various forms.

The true yeasts are characterized by the formation of endospores
and are classed with the Gymnoasceft. Each cell seems capable, under

* Prepared by F. T. Bioletti.

28

YEASTS. 29

favorable conditions, of developing into an ascus. Many unsuccessful
attempts have been made to connect the true yeasts genetically with
various forms of fungi such as Mucor, Ustilago and Dematium. At
present they must be considered as distinct species.

GENERAL MORPHOLOGICAL CHARACTERISTICS or THE TRUE YEASTS.
The yeast cell consists of an enclosing membrane and its contents. It
may be normally nearly spherical (S. ceremsice), ellipsoidal (S. ellipsoi-
deus) or sausage shaped (S. pasteurianus) .

The cell wall is thin at first, becoming thicker with age (o.5/z to i.o^)
Tt consists of two or more layers and may be shown distinctly by treat-

FIG. 12. Yeast cell. (Original.)

ment with glycerin, salt solution or dilute acids or alkalis. Under
certain conditions, it may give off a mucilage which forms a gelatinous
network, enclosing the cells and forming a kind of zooglea. This occurs
most noticeably in film or ring formation where oxygen has free access.
The cell contents consist of a network of protoplasm, containing a
single nucleus near one end. This nucleus can be seen only after harden-
ing and staining. In young cells the contents appear transparent and
homogeneous. As the cell enlarges, one or more vacuoles or cavities
filled with cell-sap appear. In each vacuole are usually one to three
highly refringent bodies, the vacuole- granules, which appear to be de-
composition products. They are extremely minute, show brownian
movement, and stain rapidly with a very dilute solution of neutral red.
In old cells, the contents become less transparent and numerous refrin-
gent granules appear. These granules are easily stained with methyl-

3

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

green and are slowly soluble in alcohol, ether, chloroform and caustic
potash. They appear to be of a fatty nature. Dead cells are usually
opaque and are distinguished by their staining more rapidly and deeply
than living cells.

Some yeasts have a tendency during fermentation to remain at the
bottom of the liquid; others form a thick foamy layer on top. These are
known respectively as bottom and top yeasts. No sharp distinction can be
made as there are intermediate forms.

O

FIG. 13. Spore-bearing cells. A . S. pasteurianus. (After Bioletti); B.Sch. octosporus.
(After Schidnning); C. S. anomalus. (After Kayser.)

The vegetative reproduction in the g&ausSaccharomyces takes place by
budding, in Schizosaccharomyces by fission.

The extreme temperatures for budding lie between i and 47, varying
with different species. The optimum temperature varies in the same way
between 25 and 35. The rate of multiplication under favorable condi-
tions will vary from one to several hours for the formation of a new cell.

When young, vigorous, well-nourished cells are supplied with abun-
dant air and moisture at a comparatively high temperature under conditions
that discourage budding (lack of nutriment) they form endospores.
These spores are usually about half the diameter of the mother cell and
from one to eight or more may occur in each cell. They may be formed

YEASTS. 31

by cells before or after budding and may even change to asci and form
new spores. They are generally spherical or slightly ellipsoidal, rarely
kidney-shaped (S. marxianus) or furnished with a zonal ring (S. anomalus]

(Fig. 13.)

In nutrient solutions they swell, burst the mother cell, become free
and germinate by budding, usually producing vegetative cells directly,
though occasionally producing first a short promycelium (S. ludwigii).

In Schizosaccharomyces octosporus the ascus is formed by the fusion
of two cells. Sometimes in other species, two or more spores in one cell
will fuse before germination.

Staining with warm carbol-fuchsin and partial decolorization with
weak acetic acid leaves the spores red and the cell colorless.

THE PRINCIPAL YEASTS OF IMPORTANCE TO FERMENTATION

INDUSTRIES.

TRUE YEASTS, SACCHAROMYCETES. The various yeasts used in brew-
ing and some of those used in producing distilling material are grouped
together as ,5. cerevisia. They are large and round or slightly oval.

They are divided into three main groups the bottom yeasts which are
used in the manufacture of German beer, and which, usually, are capable
of producing only a moderate amount of alcohol; the top yeasts, used in
English beers and compressed yeast, capable of producing more alcohol,
and the distillery yeasts, which have great fermentative power and produce
large amounts of alcohol.

Many forms of these yeasts have been described in great detail by
Hansen and others but the distinctions are based principally on physio-
logical pecularities such as the temperature and time limits of film and
spore formation, and the character of the fermented liquids. The various
forms seem to be fixed, and to retain their characteristics unchanged
under almost all forms of treatment.

The wine yeasts, S. ellipsoideus, seem to be even more diverse than the
beer yeasts, but have been less thoroughly studied. They are somewhat
smaller than the latter and usually slightly more elongated. They form
spores much more abundantly and easily than the beer yeasts and the
cells in film formation are often much elongated.

3 2

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

Their fermentative power is considerable, some of them being capable
of producing over 16 per cent by volume of alcohol. They differ in the
flavors and aromas which they produce in the fermented liquid, and especi-
ally in the rapidity with which they settle. Some yeasts, such as those of
Champagne and Burgundy, form a compact sediment which settles

FIG. 14. Wine and beer yeasts. A, S. ellipsoideus, young and vigorous; B, S.
ellipsoideus, (i) old, (2) dead; C, S. cerevisia, bottom yeast; D. S. cerevisice. top yeast.
(Original.)

quickly and leaves the liquid clear. Others remain suspended for a long
time and settle with difficulty.

Every region seems to have its own forms and the characteristics
of the various forms seem to be as well fixed as those of beer yeasts.

Wines are manufactured by the use of these yeasts. They are
also employed in distilleries. In breweries they are considered disease
yeasts and have a deleterious effect on the beer.

YEASTS. 33

5. pyriformis resembles in shape S. ellipsoideus, and in association
with Bacterium vermiforme produces ginger beer.

S. vordermanni is concerned in the manufacture of arrack. It ferments
the sugar produced from rice by the molds, Mucor oryzcs and Rhizopus
oryza.

S. fragilis and other yeasts have^ been found in kefir and other fer-
mented drinks made from milk. These yeasts working in conjunction
with bacteria produce alcoholic acid beverages.

Many true yeasts are more or less injurious. They do not, like
bacteria and pseudo yeasts, cause serious diseases, capable of completely
ruining the fermented product, but they may injure the quality more or
less. Some yeasts are useful in certain cases and injurious in others.
If beer yeasts become contaminated with wine yeast the resulting beer
may be persistently turbid. If any one attempts to ferment grapes with
beer yeast, a wine with a disagreeable beer aroma and of poor keeping
qualities is produced.

S. pasteurianus occurs in several forms as an injurious yeast in brew-
eries, causing bitterness and turbidity. Similar forms occur in wine but do
little harm except in the absence of the true wine yeast. The cells of this
species vary from oval to long ellipsoidal, often being much elongated and
in film formation sometimes producing a branching mycelium. Spores
are formed easily and abundantly.

The apiculate yeast, 5. apiailatus, is very abundant on grapes, and
most acid fruits. It is very variable and undoubtedly includes many va-
rieties. The cells are small, vary in shape from oval to cylindrical, most
of them having an apiculation at one or both ends, making them pear or
lemon shaped. According to Lindner they form spores in drop cultures,
one in a cell. Under favorable conditions this yeast increases with great
rapidity, but is checked by 3 to 5 per cent, of alcohol. It causes cloudi-
ness in wine, interferes with the growth of the proper yeast and injures the
flavor.

Many yeasts, mostly small and some of them rose-colored, have been
found on grapes and in wine, but they do not develop under ordinary con-
ditions of wine making sufficiently to be harmful.

Schizosaccharomyces pombe is a yeast found in pombe or millet beer,

made by negroes in Africa. It is cylindrical and large, though variable in

size. Both ends are rounded. It multiplies by forming a septum near

one end, the smaller division then growing into a normal cell. From one

3

34 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

to four spores are formed in a cell. These spores are often produced in
the fermenting liquid. The fermentative power is high and a large per-
centage of alcohol may be formed.

Several other species of this genus have been isolated from grapes and
from Jamaica rum.

PSEUDO YEASTS. Budding cells often occur in fermenting liquids
which have all the characteristics of yeast except that of producing endo-
spores. They are grouped together under the name of Torula. They
are usually small, spherical or slightly elongated. Some species produce
a little alcohol and some none. They seldom occur in sufficient quantities
to be harmful and one form is accredited with producing the special
flavor of some English beers.

The groups of forms included under Mycoderma resemble yeast in
form but produce little or no alcohol, are strongly aerobic and do not pro-
duce endospores. Their most noticeable characteristic is that they grow
only on the surface of the liquid, where they produce a thick film. They
cause complete combustion of the alcohol and other organic matters,
making beer and wine vapid and finally spoiling them.

CULTURE OF YEASTS.

PURE CULTURES. Yeast can be properly studied only in pure cultures. The
media used are either the liquids in which the yeasts are to be used as wort, cider,
grape juice, or a special medium devised for a special investigation. An example of
the latter is Laurent's medium:

Ammonium sulphate, 4 7 J 8-

Potassium phosphate, -758-

Magnesium sulphate, . 10 g.

Water, i L-

To this is to be added any carbohydrate to be studied. Media may be made solid
by the addition of gelatin or agar.

Pure cultures can be made, rarely, by inoculation from a naturally pure source,
such as the sporangium of a Mucor.

Physiological Separation. The first attempts at purifying mixed cultures were by
means of physiological differences. Pasteur freed yeast from bacteria by growing it
in a medium containing 2 per cent of tartaric acid. Effront used fluorides in the same
way. These methods may be made more effective by repeated transfers of the culture-
Each transfer will contain a larger proportion of the form most suited to the condi.

YEASTS.

35

tions, until finally a pure culture may be obtained. The principle of these methods is
of great use in practical fermentation, but is of little use in rigidly separating forms.
Methods of general application for the latter purpose must be such that a single
cell can be isolated in a sterile medium and a culture propagated from this single cell.
Separation by Dilution in Liquid Media. A mixed culture is diluted with sterilized

Q

J)

FIG. 15. Wild and pseudo-yeasts. A, S. pombe. (After Lindner}; B, Torulce.
(After Pasteur}; C, Mucor, (i) spores; (2) germinating spores and mycelium; D, S. apic-
ulatus; E, Mycoderma vini. (After Bioletti.)

water until each two drops contain one cell. A large number of flasks of a sterilized
nutrient medium is then inoculated from the dilution, one drop in each flask. If the
dilution has been properly made, about half of the flasks will remain sterile and half
will show growth. Many or most of the latter will contain pure cultures.

Separation by Dilution in Solid Media. If we dip a sterilized platinum wire in a

36 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

mixed culture and then draw it repeatedly over the surface of a solid culture medium
such as a slice of sterilized potato or a layer of nutrient gelatin in a Petri dish we will get
a series of streak cultures. The first of these will develop a strong growth of mixed
forms The last will show more and more isolated colonies until some of them will
show only a few, some of which may be pure cultures.

The most useful method of separation and one which is applicable to most cases
is that oi'plate cultures, first used by Koch and improved by others. In this method a
drop of the mixed culture is thoroughly distributed in 10 to 20 c.c. of liquefied nutrient
gelatin or agar. A drop of this mixture is then diluted in the same way in another
portion of the same medium. This process is continued until the requisite degree
of dilution is obtained. The various portions of nutrient gelatin are then poured, with
precautions against outside infection, on glass plates or more conveniently into petri
dishes. On cooling and solidifying, the gelatin imprisons every cell, each of which
on growing gives rise to a colony. It has been found that in practice a small per-
centage of these colonies may arise from two adhering cells and thus fail to be pure
culture.

Hansen's modification of the method is intended to obviate this uncertainty. By
making the dilutions in the way described for liquid media, a drop of gelatin containing
only one cell is obtained, placed on a cover-glass over a culture slide and, by direct
observation, the presence of a single cell verified. The development and multiplica-
tion of this cell can be watched.

DIFFERENTIATION OF YEASTS. With magnifications of 300 to 500, yeast
cells can be examined conveniently. Contamination with bacteria and molds of
special form can be detected, but otherwise a simple microscopic examination is of
little value in determining the purity of a culture. Some information regarding the
health, nutrition and vitality of the yeast may be obtained and the form of the spores
is of some value in distinguishing species. Yeast cells vary in size as much as in
form but under standard conditions each variety will show a certain normal range of
dimensions.

If a young, vigorous yeast, in a favorable liquid culture medium, is allowed to
remain at rest at a suitable temperature with full access to air and protection from con-
tamination, a growth of cells on the surface will usually take place. This growth may
extend over the whole surface (film formation) or may be restricted to the edges (ring
formation). This growth occurs at once with a few species (S. membrancefaciens) or
at the end of several days (S. ellipsoideus II) or may require several weeks. The time
and optimum temperature of film formation have been used as descriptive characters.

All the morphological and cultural characteristics of yeast are insufficient for
diagnostic purposes and must be supplemented by the physiological characteristics such
as their action on various sugars and other carbohydrates.

CHAPTER III.
BACTERIA.*

FORM.

FUNDAMENTAL FORM TYPES. The form of bacteria is exceedingly
simple. They are either spheres, straight rods, or bent rods (spiral).
In the spherical form they are known as cocci, or micrococci (sing, coccus
or micrococcus}. The straight rods are bacilli (sing, bacillus') and the
bent rods are spirilla (sing, spirillum').

ffi

**

,**

v-.- **:.;

FIG. 16. -Types of micrococci. (After Williams.)

FIG. 17. Types of bacilli (After Williams.')

FIG. 18 -Types of spirilla. (After Williams.)

GRADATIONS. The difference between these fundamental form
types is frequently very slight. It becomes a very difficult matter, for
instance, to distinguish at times between the micrococcus and the bacillus.
There is a number of bacteria, and among them the well-known example
of B. prodigiosus, that are described at one time by one investigator as

* Prepared by W. D. Frost.

37

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

micrococci and at another time, or, by another investigator, as bacilli.
The pneumonia germ is also another illustration of an organism that
occupies a dual position. Migula has suggested a method of differentiat-
ing these which will be discussed under a later head. The bacilli pass
almost imperceptibly into the spirilla. The cholera organism has been
described as a bacillus as well as a spirillum.

INVOLUTION FORMS. The form of bacteria is quite constant under
normal conditions, but very frequently they show abnormal or bizarre

ff

FIG. 19. Involution forms. Here are illustrated unusual forms of B. subtilis,
water bacteria, Bact. aceti, Bact. pasleurianum, bacteroids from root nodules, Bact.
tuberculosis^ Bact. diphtheria. (After Fischer from Frost and McCampbell.)

forms. These are known as involution forms (Fig. 19). It is some-
times suggested that these involution forms represent another stage in the
developmental history of the organism, and upon this supposition cer-
tain bacteria which very regularly show these involution forms have been
classified as belonging to quite a different order from that to which the
bacteria belong. The ordinary view of the involution forms is that they
are degeneration forms, that they correspond, in other words, to the halt

BACTERIA. 39

and maimed in society and are to be accounted for by the fact that they
are deformed by their own by-products. In fact, it is quite probable that
they are autogenic. Involution forms are very likely to occur in artificial
culture and are much more common with some species than with others.

SIZE.

The bacteria were formerly spoken of as the smallest of living things, but
since the recognition of the ultramicroscopic organisms it is necessary
to be somewhat more specific in characterizing their dimensions. The
unit of measurement in microscopy is the micron (/<), or micromillimeter.
This is .001 of a mm. or approximately 1/25000 of an inch. Applying
this unit to the bacteria we find that the micrococci and the short diameter
of the bacilli and spirilla average about i/*. The micrococci vary
in diameter from a small fraction of a micron to three or four microns
in diameter. The bacilli are sometimes very small, as the influenza
bacterium with a width of 0.2/1 and a length of 0.5^, and sometimes very
large as, for example, the Bad. anthracis with a width of 1.2/1 and a
length of 5.20 fi. The spirilla average about i.o/j in diameter but may be
as long as 3o//~4o / .

MOTILITY.

When bacteria are viewed under the microscope in a living condition
many of them are seen to move. This movement may be one of two
kinds. In some cases it is progressive, the individuals move about from
one part of the field of the microscope to another and change their relative
positions. In other cases the movement is vibratory, they move back and
forth but do not progress or change their relative positions to any extent.
This latter form of movement is known as brownian movement, because
it was first described by Brown.

BROWNIAN MOVEMENT. This movement is probably caused by the
impact of the molecules of the suspending medium and for this reason
is sometimes called molecular movement. It is not characteristic of
bacteria, or indeed of life, but is shared by all small microscopical
objects when suspended in a fluid medium. Most beautiful examples
of brownian movement can be seen by suspending granules of India
ink or carmine and examining them under the microscope. This

40 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

brownian movement is to be sharply differentiated from vital movement
which is possessed by some bacteria.

VITAL MOVEMENT. As already indicated, bacteria have the power of
independent movement due to inherent vital power. Only a few of the
micrococci are motile, while many of .the bacilli and spirilla are. This
movement is a change of position and is caused by certain protoplasmic
processes which these bacteria possess, known as cilia (sing, cilium} or
flagella (sing, flagellum). The fact of motility or non-motility of an
organism is of considerable value to the systematist. It is determined
by examination in a hanging drop. At times, however, it varies so little
from the brownian movement that it is difficult to tell whether a particular
organism or culture does or does not possess vital movement. An opinion
can be more definitely formed at times if some chemical producing an
anaesthetizing effect on the bacteria is introduced into the examining
medium. In case the organism is actually motile its position will be
changed or at least in case it is merely a brownian movement there will
be no change.

ORGANS OF LOCOMOTION. The protoplasmic threads referred to as
the organs of locomotion are known as flagella, or cilia. The difference
between the cilium and flagellum is the fact that a cilium has a simple
curve while a flagellum has a compound curve, like a whip lash. Most of
the bacteria possess flagella rather than cilia. The size, arrangement, etc.,
of these flagella are constant and characteristic of a particular organism.
Their structure and arrangement, therefore, will be discussed later.

CHARACTER OF MOVEMENT. Different bacteria present different
kinds of movement. Some dart forward with great rapidity, others move
slowly; some move in straight lines, others wobble, but any particular
character is quite constant and many of the bacteria may be recognized
by their characteristic movements.

RATE. The rate at which the bacteria travel when they possess
vital movement varies greatly. Some of them move very fast, others
very slowly. Many of them appear to move with wonderful rapidity.
Leeuwenhoek, when he first saw these moving bacteria, said that
they traveled with such great rapidity that they tore through one another,
but it must be borne in mind that under the high powers of the microscope
the rate of movement is magnified to the same extent as the object, and
that in reality the rate of movement is not excessive. When compared
to their size, the rate of movement is probably little greater than that of a

BACTERLA. 4!

trotting horse and considerably less than that of a speeding automobile
or a railroad train.

REPRODUCTION.

Reproduction among the bacteria is largely asexual and takes place
ordinarily bv what is known as binary fission. In addition to this a num-

FIG. 20. The division of bacterial cells (diagrammatic). (After Xovy.)

ber of bacteria go into a resting stage, or produce spores. The spore
formation is not, however, a method of multiplication, because usually
only a single spore is formed in a cell, but serves to tide the organism
through unfavorable conditions.

FIG. 21. i-n, Bacterium gammari, resting nuclei, and in various stages of mitosis.
12-16. a filamentous bacterium in the digestive tract of Bryodrilus. 13-14 and 15, ex-
tremities of the filaments with nuclei. 16, old filaments, two cells are without nuclei.
(After Vejdovsky from Guilliermond re-dew, Bull. Inst. Past.)

VEGETATIVE MULTIPLICATION. This is accomplished by means of
binary fission (Fig. 20). When a bacterium has reached maturity, fis-
sion begins. This change hi the cell is not customarily regarded as
preceded by any series of changes comparable to karyokinesis (mitosis)

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

in the higher cells, since no nucleus in the ordinary sense has been
demonstrated in the bacterial cell. The work of Butschli, Swellen-
grebel, and Schaudinn, however, indicates the existence of karyo-
kinetic division in bacteria (Fig. 21). Division begins by an invagina-
tion of the protoplasm in the middle of the cell, which proceeds until

.

FIG. 22. Bacillus biitschlii. 1-6, Stages in the division of the vegetative cell;
7-9, rudimentary sexuality; 10-14, formation of spores; 15-16, germination of spores.
(After Schaudinn from Guilliermond review, Bull. Inst. Past.)

the cell protoplasm is completely separated. The cell wall then grows
in and finally splits forming the two ends of the new cells. These new
cell walls are formed at right angles with the long axis of the cell in
the case of the bacilli and spirilla, except in rarest instances. In the
case of micrococci, the throwing of the cell wall across one diameter is

BACTERIA. 43

quite as economical as any other and may therefore proceed in any
direction. Migula makes a considerable point of the fact that bacilli
and spirilla elongate before division and micrococci divide before they
elongate, and this would be the criterion which he would use to separate
these two-form types. A generation among the bacteria is from one
division of the cell to another. This is sometimes very short, in fact,
only twenty to thirty minutes. Many of the bacteria after half-an-
hour's time have grown from newly formed cells to maturity and are
ready to divide again. This makes it possible for bacteria to multiply
with very great rapidity, and if we know the length of the generation in
a particular bacterium it would be easy enough to estimate the rate of
multiplication, at least theoretically. It would be only a matter of geo-
metrical progression. It is of course quite impossible for the bacteria
to maintain their theoretical rate of growth for any length of time, but,
practically, they grow with enormous rapidity, as is shown in cultures
and the changes which they bring about in nature, such as the produc-
tion of fermentation and the generation of toxin.

SPORE FORMATION. A considerable number of bacteria form spores
within the cell. Because they are formed within the cell they are
spoken of as endospores. Endospores are formed by the bacilli and the
spirilla, but not by the micrococci. Their chief value to the cell is their
ability to resist unusual conditions, and to enable the individuals of a
species to pass through unfavorable conditions which to the ordinary
vegetative form of the cell would prove disastrous. At the maturity
of the cell, spore formation may begin. It is an open question whether
spore formation occurs as a regular stage in the life history of an organism,
or is produced only under the stimulus of unfavorable environmental con-
ditions. Both theories have their advocates. The first evidence of
spore formation in the cell is a granulation of the protoplasm of the cell.
As spore formation proceeds the granules become larger and collect at
one portion of the cell. These granules then fuse to form the spore,
which soon surrounds itself with a spore wall. At times the spore is
smaller than the mother cell and is formed without changing the shape
of the cell. At other times it is larger than the mother cell and causes a
bulging of the latter. The position of the spore in the cell varies (Fig. 24).
In some species it is equatorial, in others it is polar, and in still others it has
an intermediate position between the equatorial and polar. When the spore
is larger than the mother cell and is situated equatorially it causes the cell

44

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

to bulge with the formation of a barrel-shaped organism, or a dostridmm.
If the spore is situated at the poles and is larger than the mother cell, a
capitate or drum-stick bacillus is produced. When the spore is smaller
than the mother cell and the cells form in chains, there is frequently a
tendency for the spore to be formed in opposite ends of contiguous cells
of the chain so that they appear in pairs. The reason for this is not
understood.

The endospores possess remarkable powers of resistance due to the
concentrated character of the protoplasm, or to the character of the
spore wall. The resistance here may be due to the structure of the wall
itself or to the chemical substances which it contains. It is readily con-

FIG. 23. The formation of
spores. (After Fischer from
Frost and McCampbell.)

^3

FIG. 24. Spores and their location in bacterial
cells. (After Frost and McCampbell.)

ceivable that the presence of certain fatty acids, or higher alcohols, might
give the spore its remarkable resistance. These spores are very resistant
to desiccation; they have been preserved in a dried condition for many
years. They are also very resistant to the action of heat; some forms are
known to withstand a temperature of boiling water for as long a time even
as sixteen hours. They are resistant also to chemicals and the action of
sunlight. Although in some cases, as pointed out by Marshall Ward,
the very chemical substances which furnish them the powers of resistance
towards environmental factors may be broken up under the influence
of sunlight, forming poisons so that the spore is killed more readily
than the cell would be.

When these spores are brought under favorable conditions of moisture,
temperature, and food supply, they germinate. There are several types of
germination (Fig. 25). In some cases the spore wall ruptures at the

BACTERIA. 45

pole and the young cell emerges so that its long axis is in the same direc-
tion as the long axis of the spore. In another type the spore ruptures
equatorially and the young cell emerges with its long axis at right angles
to the long axis of the spore. In still other types the spore swells and
the young cell absorbs the wall of the spore.

In the lower or true bacteria only a single spore is formed in a cell.
In the case of the higher bacteria, however, a number of spores may be
formed at the distal end of the filament. These are spoken of as gonidia,
and possess properties similar to those of the endospores.

a c

n ^

a

b

FIG. 25. Spare germination, a, direct conversion of a spore into a bacillus without
the shedding of a spore-wall (B. leptosporus); b, polar germination of Bact. anthracis; c,
equatorial germination of B. subtilis; d, same of B. megaterium; e, same with "horse-
shoe" presentation. (After Novy.)

In some cultures of bacteria, as for example in the micrococci, certain
cells seem to be larger and different from the other cells. In a strepto-
coccus filament, certain cells suggest to the observer the joint spores of
the algae and have therefore been spoken of as arthrospores or joint spores.
There is, however, no evidence of an experimental nature, which warrants
the belief that these cells are in reality spores, and it must be said that
at the present time the presence of arthrospores among the bacteria is
purely hypothetical.

CELL GROUPING.

Bacteria rarely occur singly but usually in groups. These cell
aggregates are frequently very constant and quite characteristic of the
organism possessing them. They are of sufficient definiteness and
constancy to be used by the systematists in chacterizing large groups.

CELL AGGREGATES AMONG THE MICROCOCCI. The grouping of
micrococci depends upon the plane of division and also upon the cohe-
sion of the cells. Since it is quite as economical for the micrococcus to

46 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

divide in one direction as another, it is possible for a number of different
cell groupings to occur. Whatever the direction of the dividing walls,
it is usually quite constant; if a particular species of micrococci has its
planes of division parallel, there will be formed chains of micrococci.
In some cases the cohesion is slight and only two cells remain attached
to each other, forming what are ordinarily known as diplococci. There
is a considerable number of very well known bacteria that are diplo-
cocci (Fig. 26). If the cohesion is stronger, we have chains of micro-
cocci or rosaries formed which are known as streptococci. Well known
and very important bacteria are grouped in this way. In other micro-
cocci the cell wall is not formed continuously in parallel planes but in

QO
GO

FlG. 26. Division forms of micrococci. a, diplococcus, perfect form with flattened
opposed surface (gonococcus), lanceolate from (pneumococcus'}; b, streptococcus; c, con-
secutive fission yielding a tetrad; d, sarcina form resulting from division of tetrad
c; e, staphylococcus. (After Novy.)

planes which alternate at right angles to each other. In this way cell
aggregates occupying two dimensions of space are formed. These are
known as tetracocci, or merismopedia. Still again, the planes of division
may proceed at right angles to each other in three dimensions of space.
In this case packets are formed which are known as packet cocci, or
sarcina. Another group of the micrococci occurs, known as the staphy-
lococci, so called because they are arranged in irregular bunches, like a
bunch of grapes. This arrangement may be due to the fact that these
micrococci divide in many different planes, or because during the course
of their growth their arrangement is changed.

CELL AGGREGATES AMONG THE BACILLI. In the case of the bacilli,
one diameter is usually considerably shorter than the other, so that nature
almost invariably throws the new cell wall across the bacilli at right angles
to their long axis (Fig. 27). There is, therefore, only one arrangement or
cell grouping possible, and that is end to end, so that streptobacilli
are formed. When arranged in pairs, the designation is diplobacilli.
The length of the chains appears to depend not only upon the cohesion
of the bacilli but also upon the shape of the end; those which have square

BACTERIA. 47

ends frequently have very long chains, while those with rounded ends
have short chains or occur singly. The same kind of arrangement is
maintained among the spirilla.

a

FlG. 27. Division forms of bacilli, a, single; b, pairs; c. in threads

(After Novy.)

ZOOGLGEA. Some of the bacteria secrete a mucilaginous substance
which causes the cohesion of the cells frequently in considerable number.
This aggregate of cells may assume some characteristic appearance and a
great many attempts have been made by systematists to make use of this

/

/if' -~--~~ "" C ^^X

$/'''*&**&**** ^\
Si t A $ ^ X

tf/^^ I \\

i"" ! /4 /'' -->- w VS

ft// N \ v . is

fl,V ' i'i il -^=--=--* xS x vv % "

/,',? /-'" \ \\ k

*E! I /n> // >V _v V .. * I .llll

/w / \x*

I W' / v\ \ T.

IP / ,"' I f

!iiii I *ii " *

A*? ' i/i 1 *'

i'!n j .!.'/ j '/

vv s

v \ \\

'4

/

//

Kit-

FIG. 28. Threads of Bact. anthracis. (After Migula.)

in the different cases of fission. These zooglceic masses usually assume
the forms of pellicles, but their value as diagnostic features is not great.
The formation of zooglcea is very frequently only a stage in the life history
of an organism.

48 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

MINUTE STRUCTURE OF THE BACTERIAL CELL.

The typical cell, such as that of a higher plant or animal, is made up of
cytoplasm surrounded by a cell wall. The cytoplasm contains a nucleus.
There are also frequently present other evidences of structure in the cy-
toplasm, such as nucleolus, polar bodies, etc. In addition to these there
may be appendages, such as the cilia or flagella. In the case of the bac-
terial cell, we find most of these structures present, such as cell wall,
cytoplasm, and appendages, but the nucleus is either wanting or is so
modified in form as not to be recognized as an ordinary nucleus.

BACTERIAL CELL WALL. Structure. All the bacteria have cell
walls and it is these that give rigidity to the cell. These walls are rigid
and elastic and are probably made up of two layers, the outer one is able
to deliquesce and form capsules, or perhaps zooglcea. The inner part re-
tains the elasticity and gives the form to the bacteria. These cell walls
are readily permeable to water and it is through them that all of the
nourishment of the cell is obtained; that is, there are no openings for
the entrance of food or the discharge of by-products, but the in-
take and output goes on through the cell wall which is entire. The
chemical nature of the cell wall is generally protein in nature and in this
respect perhaps resembles the animal cell wall more closely than it does
the plant cell wall, though many of the bacterial walls resemble those of
molds and some fungi. These cell walls take the ordinary stains with
difficulty, or not at all, and it is because they do not stain that they are
not seen. The ghost figures, frequently seen, are the walls of dead
mother cells from which spores have escaped.

Capsules. A considerable number of the bacteria regularly, or under cer-
tain conditions, form what are known as capsules (Fig. 29). These are mu-
cilaginous envelopes which in width frequently exceed that of the organism
itself. In microscopical preparations of bacteria it is important to differ-
entiate these from artifacts, since by ordinary staining methods the capsules
are not colored but appear as colorless areas surrounding the bacteria.
If, due to shrinkage of the bacteria, or other material on the preparation,
clear spaces are formed, it is readily seen that these might be confused with
the real capsule. It is possible to stain the capsules by special methods;
these must be used in order to determine positively the existence of the
capsules. The material of which the capsules are composed is derived
from the cell itself, probably the result of the deliquescence of the outer

BACTERIA.

49

portion of the cell wall. This material is mudn-like and is soluble in
water. The bacteria which grow in the bodies of animals frequently con-
tain these capsules but fail to show them when grown upon artificial cul-
ture media. It is difficult, therefore, to determine whether or not an
organism has a capsule by mere examination of cultures. Some culture
media, however, do cause a formation of capsules in the case of capsulated
bacteria. These are blood serum, sometimes, and milk, usually. Beau-
tiful capsules can be obtained by growing such bacteria as the Bact.
pneumonia, Bact. capsulation, and Bact. u'elchii in milk cultures. Strept.

FIG. 29. Capsules. Baft, pneumcnia (Friedlander). (After Weichselbaum

from Frost and McCampbell.)

mesenteroides is a bacterium which grows in the syrup of the sugar re-
fineries and forms abundant capsules. This organism changes the char-
acter of the syrup, and its entrance and growth is frequently the cause of
serious loss.

Sheath. Among the higher bacteria, such as Crenothrix, there is
present a thickened and hardened membrane which is spoken of as a
sheath. It forms a tube in which the different cells of the plant are con-
tained. This sheath is homologous to the capsule and in it are frequently
deposited certain by-products of the cell. In Crenothrix we frequently
have iron oxides (p. 55).

BACTERIAL CYTOPLASM. The cytoplasm of the bacterial cell is
similar to the cytoplasm of other cells except that chemical analyses seem
to show that it contains a higher percentage of nitrogen. As viewed under

4

50 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

the microscope, in either an unstained or stained condition, it appears as
a homogeneous mass filling the entire cell and rarely showing any evidence
of structure. Ordinary stains, such as are used in animal and plant histol-
ogy, fail to reveal the presence of the ordinary nucleus, the whole cell being
usually uniformly stained with those stains ordinarily characterized as
nuclear stains. When these stains are applied to some bacteria, particu-
larly at certain stages of their growth, certain parts stain more readily than
others, and we get either what is known as a bi-polar stain or polar gran-
ules. In the first case, the ends of bacilli are stained more deeply than the
center so that the cells appear very much as diplococci. This bi-polar
stain is characteristic of such organisms as the bacterium of chicken cholera
or the bacterium of bubonic plague. The polar granules are frequently
seen in the diphtheria bacterium and may be located at the poles and also

FIG. 30. Plasmolytic changes. (After A. Fischer.) a, cholera vibrio; b, typhoid
bacillus; c, Spirillum undula. (From Novy.)

at the center. In this germ and in some others it is possible, by special
staining, to give the granules a different color from the rest of the organism.
In this case these bodies are spoken of as metachromatic granules. Whether
or not the bi-polar stains or the polar granules are evidences of structure
or not is an open question, since the results obtained might be explained
upon the theory that the cells are plasmolyzed (Fig. 30). As a result of
plasmolysis the protoplasm of the cell is drawn away from the cell wall and
concentrated in areas which would very well explain the appearances.
And it seems likely also that the methods employed in staining might
lead to plasmolysis, but the metachromatic granules can hardly be ex-
plained upon this supposition. They must be either special protoplasmic
structures or reserve food material, and for each of these theories there
are able supporters.

The cytoplasm of the bacterial cell is slightly refractive. It is color-
less except in a few cases in which the green coloring matter, like chloro-
phyl, is present, as, for instance, Bact. viride and Bad. chlorinum. In the
purple sulphur bacteria, the coloring matter bacteriopurpurin is present.
The bacterial cytoplasm contains vacuoles at times.

BACTERIA. 51

Nucleus. The question of whether or not the bacteria possess a
nucleus is one that has engaged the attention of bacteriologists and biolo-
gists for some time. It is very certain that the bacteria do not possess a
nucleus in the ordinary sense in which the term is used in animal and plant
histology. There are several different views in regard to this matter.
One is that the bacterial cell is largely nucleus, and attention has already
been called to the fact that cytoplasm stains with what are known as nu-
clear stains. But this is not convincing proof that the material of the bac-

FIG. 31. i and 2, Bacillus mycoides; 3, B. megatherium; 4, B. radicosus; 5, B.
oxalaticus. (After Bohuslaw Rayman and Karel Kruis Jrom Guilliermowd review,
Bull. Inst. Past.)

terial cell is nucleus. Were this true, however, it would fit in very nicely
with the theory of evolution, since evolutionists are desirous of finding a
group of organisms with the cell reduced to its simplest form. Another
idea is that the nuclear material is widely distributed throughout the
bacterial cell and not concentrated into one structure (Fig. 31). Such a
view as this is in harmony with Biitschli's work. Zettnow appears to have
stained nuclei. Another class of thinkers, in which Alfred Fischer is
prominent, believe that the bacterial cell is without a nucleus. This is
borne out, to their minds, by the findings that the bacterial cell plasmo-
lyzes, that the nucleus of higher animals and plants does not; therefore
the bacterial cytoplasm is not nuclear.

FLAGELLA. The flagella are protoplasmic threads and, undoubtedly

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

are attached to the cytoplasm of the cell. Whether these flagella pass
through openings in the cell wall, or are attached in some way exteriorly

FIG. 32. i, Chromatium okenli; 2, B-actcrium lineola; 3, 4 and 5, sulpho-bacteria;
7, Ophidomonas jenensis; 8 and 9, Spirillum uiuhila; 10, Cladothrix dichotoma. (After
Biitschlifrom Guilliermond review, Bull. hist. Past.}

x\/

F IG - 33- Microspira comma.
Monotrichous bacteria. (After
Migula from Schmidt and Weiss.)

FIG. 34. Pseudomonas py ocy an ea.
Monotrichous bacteria. (After Migula
from Schmidt and Weiss.)

is a question that cannot be answered definitely. But in either case
these flagella possess the property of cytoplasm, i.e., that of irritability,
and are not to be considered as analogous to mere levers or oars to

BACTERIA.

53

FIG. 35. Pseudomonas syncyanea. FIG. 36. Spirillum nibritm. Lopho-

Lophotrichous bacteria. (After Migula trichous bacteria. (After Migula from
from Schmidt and Weiss.) Schmidt and Weiss.)

FIG. 37. Bacillus typhosus. Peritrichous bacteria. (After Migula from Schmidt

and Weiss, and Frost and McCampbell.)

54 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

a boat. They are very narrow threads, no one knows how narrow
since they cannot be seen without staining and they can only be
stained by precipitating some chemical which may add considerably
to their width. They are frequently longer than the organism which
possesses them and sometimes many times that length. B. symptp-
matici anthracis found in the soil has a flagellum sixty times its
own length. The arrangement of the flagella on the bacteria is quite
constant and is used by some authors to differentiate genera. Very few
of the micrococci are provided with flagella, as was indicated above, and
in the bacilli and spirilla they may be arranged at the, poles singly or in
brushes, or they may be arranged on the entire periphery of the cells.
When bacteria are provided with a single flagellum at one pole, the
arrangement is said to be monotrichous (Figs. 33 and 34). When they are
arranged in brushes, the arrangement is spoken of as lophotrichous
(Figs. 35 and 36) and when they are arranged on the entire periphery, the
arrangement is said to be peritrichous (Fig. 37). It frequently
happens that in the case of the monotrichous and lophotrichous the
flagella occur at both ends of the organism. This is explained by the
fact that the organism is just undergoing binary fission and that the
second group is on the newly forming cell. It is worth while in this con-
nection to call attention to the fact that the flagella on one end are new,
while those on the other end may be thousands of generations old.

THE HIGHER BACTERIA.

The higher or trlchobacteria are filamentous forms. The filaments
sometimes show true branching and frequently false branching. The cells
are similar in form throughout the filament and are capable of independent
existence, but when growing in the filament give evidence of differentia-
tion. Sometimes these filaments are attached to the substratum; in other
cases they are free. In the case of the sessile forms, the cells at the attached
end are smaller than those at the free end. In other forms the ends
may become. swollen or club-shaped. Frequently there is a difference
between the cells of the different parts of the filaments indicated by the
manner of reproduction. Certain cells are apparently set apart for the
purpose of reproduction, and, by a process of division, form spores, or
gonidia. Some of the free forms of the trlchobacteria move by undula-
tive movements of the protoplasm. The exact nature of this movement

BACTERIA.

55

is not understood. Many of the trick obaderia are surrounded by a mem-
brane homologous to the capsule in the lower bacteria and known as a
sheath.

The best-known member of this group is the water-pest bacterium
(Crenothrix polyspora), (Fig. 38) an iron bacterium, which has the power
of oxidizing certain forms of iron, causing a deposit to accumulate in the
water pipes of cities where it may cause considerable trouble. It is prob-

FlG. 38. Crenothrix polyspora Cohn, Brunnenfaden.

and Weiss.)

(After Migula from Schmidt

able also that this bacterium has had a very important part in the depo-
sition of our iron ores, such as those found on the Mesaba range. Another
member is the Actinomyces boms (Fig. 98) which is the cause of the
common disease in cattle known as lumpy jaw. This bacterium may
also infect man. Many other forms of trichobacteria are found in
nature and probably play important parts in the chemical transformation
of matter

56 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

CLASSIFICATION.

The classification of bacteria was early recognized by Mueller as a
matter of difficulty, since he says: "The difficulties that beset the in-
vestigation of these microscopic animals are complex; the sure and defi-
nite determination (of species) requires so much time, so much of acu-
men of eye and judgment, so much of perseverance and patience, that
there is hardly anything else so difficult." Early investigators found it
difficult to decide whether bacteria are plants or animals, and nowadays
we are finding it as difficult to decide upon a system of classification. A
great many systems have been proposed, but many of them are untenable
because those who proposed them were ignorant of or unconcerned by the
rules adopted by systematists in other lines. The only system that seems
worthy of continued life is that of Migula, who is a trained botanist. This
system, with slight modifications, is given below. In this system, the
characters which separate the genera are morphological ; while physiologi-
cal characters, including cultural, are used for the differentiation of
species and smaller groups. One of the rules adopted by systematists
in other lines is the binomial rule. In the violation of this rule, bacteri-
ologists have been great sinners, and some of the names proposed by
Migula and others following his system are quite different from those by
which well-known forms have been christened by their discoverers.

CLASSIFICATION OF MIGULA (MODIFIED).

The bacteria are phycochrome-free schizomycetous plants which divide in one
two, or three planes. Reproduction takes place by vegetative multiplication (fission).
Resting stages in the form of endospores are produced by many species. Motility is
noted in some genera, and this is due to flagella. In Beggiatoa and Spirochseta the
organs of locomotion are not definitely known.

I. Order: Eubacteria(true bacteria).

The cells are devoid of any nucleus (Zentralkorper) and free from sulphur and
bacteriopurpurin, colorless or faintly colored.

I. Suborder: Haplobacterinae (lower bacteria).

I. Family: Coccaceas (ZOPF) MIG.

The cells are globular when in a free state, but in the various stages of division
appear somewhat elliptical. A few species in this family are motile. Cell division
takes place in several directions of space. Frequently the cells remain attached to-
gether, and under these conditions usually show some flattening of the cell at the
point of junction with the cell next to it.

Genus : Streptococcus BILLROTH.

The cells are globular and do not possess any organs of locomotion. Cell division

BACTERIA. 57

takes place in only one plane. Usually the cells remain united together after division,
producing chains or diplococcus forms. No endospores have been noted.

Genus : Micrococcus (HALLIER) COHN.

The cells are globular and do not possess any organs of locomotion. Cell division
takes place in two planes at right angles. If the cells remain attached together after
cell division, merismopedia plates are formed. The plates give the appearance of a
regular flat mass of cells. No endospores have been noted in this genus.

Genus: Sarcina GOODSIR.

The cells are globular and do not possess any organs of locomotion. Cell division
takes place in three planes, all perpendicular to each other. Its cells remain attached
after division; cube-like packets are formed. The composition of the media some-
times prevents this typical cube formation.

Genus : Planococcus MIGULA.

The cells are globular. Cell division takes place in two planes at right angles
similar to genus Micrococcus. The cells of this genus are motile, possessing one or
two long flagella. No endospores are produced in this genus.

Genus : Planosarcina MIGULA.

The cells are globular. Cell division takes place in three planes as in Sarcina.
Cells are motile, having only one flagellum on each. Cells usually remain united in
twos and in tetrads and seldom form packets as Sarcina.

II. Family: Bacteriaceae MIGULA.

The cells are cylindrical in shape. They vary in length from short almost spherical
bodies to very long rods. Cell division takes place in one direction in a plane perpen-
dicular to the long axis of the cell. Some of the members of this family remain attached
together, forming threads, while others separate from each other soon after fission.

Genus: Bacterium EHRENBERG.

The cells are cylindrical, of longer or shorter length. Threads are frequently
formed. The cells do not possess any organs of locomotion. Endospores are produced
in some few species, but in the majority no such formation occurs. It is possible that
endospore formation occurs only under certain environmental conditions.

Genus : Bacillus COHN.

The cells are cylindrical, of longer or shorter length. The rods are sometimes
oval in shape. Cells are motile and possess flagella which are distributed over the
entire surface. Endospore formation occurs with marked regularity. The bacteria
in this genus are motile only during certain periods of their life. This period varies
greatly in length and occurs only in the vegetative stage.

Genus : Pseudomonas MIGULA.

The cells are cylindrical, of longer or shorter length. The cells are motile and
possess polar flagella. These flagella may vary from one to twelve in number. The
formation of endospores in this species is claimed by some. If they occur, it is ex-
tremely rare. Occasionally certain species in this genus form themselves into threads
or chains.

III. Family : Spirillaceae MIGULA.

The cells are wound in the form of a spiral or representing the portion of a turn of
a spiral. In the latter case, if the cells remain attached together in the form of a

58 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

FIG. 39. Chamydothrix hyalina Migula. (After Migula from Schmidt and Weiss.)

BACTERIA. 59

thread, a full spiral of several turns is produced. Cell division takes place in only one
direction of space, and this is transverse to the long axis of the cell.

Genus : Spirosoma MIGULA.

The cells are rigid and bent in the form of spirals. The members of this genus are
as a general rule quite large. The cells may be free or united together into small
gelatinous masses. Some of the cells individually are surrounded by a gelatinous enve-
lopes, while others are free.

Genus: Microspira SCHRO'TER.

The cells are rigid, short, and bent similar to a comma. When the cells are united
together, S-shaped threads are formed. The cells are motile, possessing usually one
flagellum and rarely two or three flagella. These flagella are about the same length as
the cell. No endospores are formed. Some writers make no distinction between
Microspira and Spirillum. The name Vibrio has also been applied by some writers
to this genus.

Genus : Spirillum EHRENBERG.

The cells are rigid, usually long and forming long, screw-like threads, or, in some
cases, only portions of a spiral turn. Cells are motile and possess a tuft of flagella at
}he pole. The flagella may occur at both ends of the spiral, and they vary greatly
in number. Endospore formation has been observed in some species.

Genus : Spirochaeta EHRENBERG.

The cells are flexible spirals, very thin and long. No flagella are present. These
bacteria move by rotation similar to a screw, and also by lateral motion similar to
a snake. The locomotive organs, if present, are not known. No endospores are
produced.

II. Suborder: Trichobacterinae (higher bacteria).

Family : Chlamydobacteriaceae MIGULA.

The cells are cylindrical, are united in threads, and surrounded by a sheath.
Reproduction takes place by means of motile and non-motile gonidia. These gonidia
arise directly from the vegetative cells and, without any resting stage, produce new
threads of cells,

Genus : Chlamydothrix MIGULA.

The cells are cylindrical, non-motile, and arranged in unbranched threads and
surrounded by a sheath of varying thickness in different species, being the same
diameter at apex and base (Fig. 39). Reproduction takes place by means of gonidia,
which are round and arise directly from the vegetative cell. This genus is called
Leptothrix by KUTZING and Streptohrix by COHN.

Genus : Crenothrix COHN.

The cells are united together into filaments which are unbranched. The filaments
gradually enlarge toward the free end, thus making a distinction between the apex and
base. The sheath which covers the filaments is thick and often becomes infiltrated
with the hydroxide of iron after being cast off in water in which there is a large amount
of iron. Reproduction takes place by the formation of round gonidia which are formed
in the beginning by division perpendicular to the long axis of the cell and later by divi-
sion in three directions of space. Only one or possibly two species can be placed in this
genus.

6o

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

Genus : Phragmidiothrix ENGLER.

The cells in the beginning form unbranched threads. Cell division takes place in
three directions of space, thus forming within the sheath a mass of cells. Later these

cells may burst through, multiply, and form branches after
acquiring sheaths. The sheath in this genus is quite thin and
can scarcely be seen.

Genus: Sphaerotilus KTJTZING, 1833, (Cladothrix COHN).
The cells are cylindrical and the threads are surrounded
by sheaths Dichotomous branching is present, and there is
no differentiation in size between the apex and base of the
thread (Fig. 40) . Reproduction takes place by means of gonidia
which swarm together within the cell. These gonidia burst out
of the cells, attach themselves to some object, and grow into
new threads. The gonidia are endowed with flagella which
are attached toward the end and below the pole.
II. Order: Thiobacteria (sulphur bacteria).
The cells do not possess any nucleus and contain sulphur.
The cells are colorless or pigmented rose, violet, or red by
bacteriopurpurin. The cells are never pigmented green.
I. Family: Beggiatoaceae TREVISAN.
Filamentous bacteria which do not contain bacteriopur-
purin. The cells contain sulphur granules. Reproduction
takes place in one direction of space.
Genus : Thiothrix WINOGRADSKY
The cells are non-motile
and the threads are attached to
some object. The threads are
surrounded by a delicate sheath

and the cells contain sulphur granules. Gonidia are pro-
duced at the end of the threads. These gonidia ear
motile and finally attach themselves to some object, and,
according to some authors, bend at right angles in the
middle and grow into new threads.
Genus : Beggiatoa TREVISAN.
The threads are not surrounded by a sheath and
are formed of flat cells. The cells are not attached
(Fig. 41). This genus moves by means of an undula-
ting membrane similar to Oscillaria. As the organism
moves, it rotates on its long axis and swings its free
ends. Gonidia are unknown and reproduction takes
place by a division and separation of the threads.

II. Family: Rhodobacteriacese (WINOGRADSKY'S
classification, artificial).

The cells contain bacteriopurpurin and on this account may be red, rose, or violet.
Sulphur granules may also be included within the cells.

FIG. 40. C I ad o-
thrixdichotomaCohn.
(After Fischer from
Schmidt and Weiss.)

FIG. 41. Beggiatoa alba.
Vaucher, Trevisan. (After
Winogradsky Jrom Schmidt
and Weiss.)

BACTERIA. 6l

I. Subfamily.

The cells are united into colonies. Cell division takes place in three directions of
space.

Genus : Thiocystis WINOGRADSKY.

The colonies are small, compact, and enveloped either singly or in groups by a
gelatinous cyst. The colonies are also capable of breaking up and the cells moving
about.

Genus : Thiocapsa WINOGRADSKY.

The cells are globular in shape and spread out on a substratum in flat colonies.
These colonies are surrounded by a common gelatinous secretion similar to a capsule.
The cells are non-motile.

Genus : Thiosarcina WINOGRADSKY.

The colonies form packets similar to the genus Sarcina of the Eubacteria. The
cells are non-motile.

II. Subfamily Lamprocystaceae.

The cells are formed into families. Cell division takes place first in three then in
two directions of space.

Genus : Lamprocystis SCHRO'TER.

The cells in the beginning are solid, then hollow, becoming perforated like a net.
They separate into small groups and become motile.

III. Subfamily Thiopediaceae.

The cells are united into colonies. Cell division takes place in two directions of
space.

Genus : Thiopedia WINOGRADSKY.

The families are formed similar to tubes and are composed of cells arranged in
fours and capable of motility.

IV. Subfamily Amcebobacteriacese.

The cells are united into colonies. Cell division takes place in one direction of
space.

Genus : Amcebobacter WINOGRADSKY.

The cells are united into colonies, and after division in one direction of space
remain attached together by threads of protoplasm. The colonies possess amoeboid
motility. The cells change form by contraction and the spreading out of the proto-
plasm.

Genus : Thiothece WINOGRADSKY.

The colonies are inclosed by a thick, gelatinous cyst. The cells are capable of
moving and are very loosely surrounded by a common gelatin.

Genus : Thiodictyon WINOGRADSKY.

The colonies are solid, non-motile, and consist of small cells which are pressed
together.

V. Subfamily Chromatiacese.

The cells are free and capable at all times of motility.

Genus : Chromatium PERTY.

The cells are moderately thick, elliptical or cylindric-elliptical in shape

Genus: Rhabdochromatium WINOGRADSKY.

62 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

The cells are free, rod-shaped, or spindle form; they possess flagella on the
poles and are motile at all times.
Genus: Thiospirillum.
The cells are free, continually motile, and spirally twisted.

RELATIONSHIP OF BACTERIA.

There has been a great deal of discussion as to whether bacteria are
plants or animals. They were first described as animalcula and to the
popular mind they are usually animals or "bugs." It is difficult to de-
termine their exact relation philogenetically. These difficulties are so
great that some scientists, as Haeckel, would create a new kingdom, call it
Protista, and put in it some of the lower plants and animals which are
difficult to classify, together with the bacteria. This view, however, is
not a very popular one, and the attempt is usually made to trace the re-
lationship of bacteria to well-known representatives of the plant and animal
kingdoms. The bacteria are undoubtedly more closely related to the
blue-green algae than to any other forms of life. They resemble these
organisms in form, method of reproduction, and absence of definite nu-
cleus. It is quite impossible to decide, furthermore, whether some forms,
such as Bad. viride and Bact. chlorimtm, are blue-green algae or bacteria.
On the other hand, there are some points of resemblance between the
bacteria and the protozoa. Spore formation, similar to that among the
bacteria, occurs among some of the protozoa. Another point of resem-
blance is the possession of flagella. Some of the flagellates quite closely
resemble the bacteria in many ways, and the spirochsetae, which are usu-
ally believed to be bacteria, have been classed as flagellates by eminent
proto-zoologists.

Physiologically the bacteria are quite closely related to the fungi, and
are frequently classed with them under the term Schizomycetes.

ARTIFICIAL CULTIVATION OF BACTERIA.

The introduction of methods of artificial cultivation marks the beginning of the
science of bacteriology. These methods were developed by Pasteur and Koch and
are depended upon by the bacteriologist of to-day as the foundation for most of
his work. It has been the aim of investigation to discover a more general culture
medium. So far it has been impossible to do this, but beef broth, made
after a formula suggested by LoefHer many years ago, forms the basis of nearly
all of our culture media. This beef broth, or nutrient bouillon, is made by
extracting meat free from fat in water, adding a small per cent of peptone,

BACTERIA. 63

correcting the chemical reaction, clarifying and sterilizing. To this broth various
substances are added for special purposes; gelatin and agar, in order to solidify the
media, and various sugars and other chemical substances for the purpose of determin-
ing the physiological characteristics of various bacteria. One of the difficulties with
the present methods of the artificial cultivation of bacteria is the inconstancy of the
composition of the media, due to the fact that the extract of beef, the peptone, and other
ingredients, cannot be obtained chemically pure. If it should prove possible to use
synthetic substances, such as the polypeptids, it would mark a great step in advance,
but it is probably quite impossible to devise a single medium upon which all bacteria
will grow. Some bacteria, such as those which produce nitrification, refuse to grow
on ordinary media containing organic material. The cultivation of bacteria in pure
culture is dependent upon isolation, and the method of isolation suggested by
Robert Koch in 1880, and known as the plate culture method, has given eminent
satisfaction. This method is dependent upon the use of a liquefiable solid medium,
such as gelatin or agar.

CHAPTER IV.

INVISIBLE MICROORGANISMS*

The term "invisible microorganism" is used interchangeably with such
expressions as "ultra-microscopic organism," "invisible virus" and
"filterable virus" to designate a group of microorganisms which cannot be
discerned with the most powerful lenses. Besides being invisible, these
microorganisms will pass through the ordinary "bacteria-proof" filters
and with one exception,! they have resisted all attempts at cultivation
outside of the animal body.

The virus of foot-and-mouth disease may be taken as a typical
example. In this disease vesicles form in the mouths and on the feet
of infected cattle. The virus is known to be present in the lymph which
forms in these vesicles because this lymph will produce typical attacks
of foot-and-mouth disease when inoculated into susceptible animals.
If now this infectious lymph be diluted with water and passed through a
Berkefeld filter the resulting filtrate will be found to be free from all
visible microorganisms and in addition the usual culture tests will give
negative results. Notwithstanding this apparent sterility, however, the
filtrate will produce disease in cattle in the same manner as the unfiltered
lymph. It is known that the symptoms produced by the filtrate are
caused by a living organism and not by a toxin, because by successive
filtrations and inoculations the disease can be transmitted through a long
series of animals, thus indicating clearly that there exists in the filtered
lymph a living organism which is capable of reproduction. Another proof
that the virulence of the filtered lymph is caused by the presence of living
corpuscular elements, and that it is not a mere solution of a toxin, is
found in the failure of the virus to pass through filters of finer grain than
the Berkefeld as, for example, the Kitasato filter.

The more important of the diseases which may be caused by invisible
microorganisms are yellow fever, infantile paralysis, hog cholera, bovine

* Prepared by M. Dorset,
t Bovine pleuro-pneumonia.

64

INVISIBLE MICROORGANISMS.

a

FIG. 42. Apparatus for fractional filtration, designed for use with Pasteur-Cham-
berland or Berkefeld filters, a, glass mantle surrounding filter; b, Chamberland filter;
c, paraffin joint; d and e, rubber stoppers;/, double side-arm suction flask; g, pinchcock
controlling outlet from suction flask; h, outlet tube surrounded by glass shield and
attached to lower end of suction flask by means of short rubber tubing; i, glass shield
fused to and surrounding outlet tube as a protection against contamination when the
filtrates are drawn off; j, glass inlet tube plugged with cotton, for admitting air into
suction flask; k, pinchcock governing the admission of air into flask; /, vacuum gauge;
m, stopcock connected with vacuum pump. (U. S. Dept. of Agriculture, Bureau of
Animal Industry, Bui. 113.)

66 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

pleuropneumonia, cattle plague, canine distemper, swamp fever or in-
fectious anaemia of horses, chicken pest, sheep pox, and horse sickness.

The invisibility of this group of microorganisms may depend upon
either their minute size or their peculiar structure. The most powerful
microscopes will not enable us to discern with distinctness objects which
are less than o.i/i in diameter. We know of bacteria which in size
approach this limit quite closely (M. progrediens, 0.15^ in diameter)
and there is no reason for believing that the size of organisms is limited
by our ability to see them. As already stated, invisibility may also
result from a peculiarity of structure, such as complete transparency
and failure to stain with the reagents ordinarily used for this purpose.

The ability of microorganisms to pass through niters is dependent
upon a variety of factors. The size and plasticity of the organism,
the fineness of the pores, and the thickness of the walls of the filter as
well as the conditions under which the filtration is performed, will all
influence the result.

The failure of the invisible microorganisms to develop under artificial
conditions is to be attributed to their strict parasitism and to our inability
to imitate exactly in the laboratory the conditions which exist in the animal
body.

While the invisible microorganisms possess certain qualities in com-
mon, in some respects they differ widely from one another. Some will pass
only through the coarsest of bacteria-proof filters, while others pass readily
through the densest filters, thus indicating wide differences in size or in
structure. Some are very susceptible to the action of germicidal agents,
whereas others are more resistant than the ordinary bacteria. Some
produce disease in only one species of animal, while others show little or
no limitation in this respect. The diseases produced by these microorgan-
isms likewise differ markedly, some being comparatively benign and local
in character, whereas others appear as the most profound septicaemias.
Some are extremely contagious, while others can be transferred from
one animal to another only by means of an intermediate host. In fact
these invisible microorganisms seem to differ among themselves quite
as widely as do those which are visible to us.

The existence of an invisible microorganism is determined as follows:

The infectious agent must pass through a bacteria-proof filter, which
is free from imperfections as shown by tests with visible organisms of
small size. Pressure exceeding one atmosphere should not be employed

INVISIBLE MICROORGANISMS. 67

during filtration. The time of filtration should not exceed one hour.
The filtrate should remain free from all visible bacteria as shown by
microscopic examination and cultural tests. The filtrate should
possess the specific disease-producing qualities of the unfiltered material.
Animals infected with the filtrate should yield material which, after
filtration, will in its turn possess the attributes of the original unfiltered
material.

CHAPTER V.

PROTOZOA.*

[Limited to the Study of Pathogenic Forms].

INTRODUCTION.

Most of those diseases which are known to be due to an infecting
agent are caused by bacteria; but some of them are caused by protozoa.
The bacteria belong to the vegetable kingdom. The protozoa are
minute animals; they are extremely numerous, and they
are very widely distributed throughout nature.

From a zoological point of view, the protozoa consti-
tute an important sub-kingdom. It is sometimes diffi-
cult to say whether one of these minute organisms is a
plant or an animal. For this reason, the unicellular
organisms are sometimes classified by themselves, as
Protista, in a kingdom which belongs to neither the
animal nor the vegetable kingdom; usually, however,
the protozoa are placed in the animal kingdom and
they are denned as organisms which have the following
characteristics: "They are unicellular; they reproduce
by various methods of division and, often, in addition,
by conjugation; they may be solitary or united in
colonies, free living or parasitic; with some exceptions,
they do not possess chlorophyl."

Many protozoa live in fresh water. Others live in
the sea; chalk is formed from the skeletons of myriads
of protozoa which once lived in the ocean. When they
died, their bones fell to the bottom and formed chalk.
Most of the protozoa are free-living; but others are parasitic on
animals and plants. Some of the parasitic protozoa are harmless and
do no injury to the hosts which support them; others produce severe
diseases. Before mentioning those which cause disease (see p. 667) it

* Prepared by J. L. Todd.

68

FIG. 43. Mie-
scher's sac from
the musculature
of a hog. X30
diameters. (After
Ostertag.)

PROTOZOA.

6 9

will be well to consider the protozoa as a class and to study the char-
racters which all have in common.

STRUCTURE OF THE PROTOZOA.

Most protozoa are microscopical; some of them are visible to the
naked eye as individuals, or as agglomerated masses of individuals. For
example, the Sarcosporidia, which occur in the muscles of mice and other
animals, can easily be seen without a microscope, and the huge plasmodial
masses of Amoeba, which are sometimes seen on rotting wood or in tan
pits, may measure several centimeters in breadth.

FIG. 44. A mceba vespertilio. (After Dqflein.)

Like all living things, the protozoa are composed of protoplasm.
Protoplasm is a complicated and, more or less, fluid mixture of albuminous
substances. A cell may be conceived of as a tiny drop of albuminous
and somewhat viscid fluid, like the white of an egg. By appropriate
methods, the protoplasm of a cell may be shown to have an alveolar or
foam-like structure; because the protoplasm is a mixture of two fluids,

70 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

one viscid and the other more labile. In such a mixture, the viscid fluid
forms tiny droplets, and each of them is surrounded by a layer of the less
coherent fluid (Fig. 44). The arrangement of the alveoli of the foam-like
cytoplasm of a living cell is the same as the arrangement of the bubbles
in a mass of foam which is artificially produced. The walls of the outer
layer of alveoli, or of alveoli which surround a resistant structure within
the cell, are perpendicular to the surface against which they lie. The
outline of the alveoli, which are not in contact with a firm structure, is
circular; an exactly similar arrangement of the alveoli may be seen in a
mass of soapsuds contained in a bottle; wherever the bubbles touch an
unyielding surface, their outline becomes rectangular.

The protoplasm of a protozoon may be divided into two main divisions:
the cytoplasm and the nucleus. The cytoplasm, as a whole, may be
divided, more or less easily, into a clearer, denser, more resistant outer
layer the ectoplasm; and a more fluid, granular, internal portion the
endoplasm. Denser, more resistant fibers sometimes run through the
cytoplasm and, like a skeleton, serve to fix the shape of the organism in
which they exist.

The nucleus, in its simplest form, is simply an area which is differ-
entiated from the remainder of the cell by being more refractile and
by being colored more deeply in specimens which have been stained
by dyes. It stains deeply because it contains a substance called chro-
matin. The chromatin usually occurs in granules; the granules may
vary considerably hi size and they are supported upon a linin frame-
work. This framework does not stain by ordinary methods and it is
probably continuous with, and of the same nature as the substance which
forms the alveoli of the cytoplasm. The interstices of the nucleus are
filled with nuclear sap. A limiting nuclear membrane may be present,
but it is not an essential part of the nucleus. The nuclear material may
be all gathered together in a single mass, or it may be distributed in
small granules so that, at the first glance, no nucleus seems to be present.
Such a nucleus is called a distributed nucleus.

FUNCTIONS OF THE PROTOZOA.

Most animals are composed of a great number of cells; a protozoon
consists of a single cell. In an animal which is composed of many cells,
the various functions of the body are each carried out by a special type

PROTOZOA. 7 1

of cell ; for example, movement is performed by the muscle cells, digestion
is partially provided for by the cells of the alimentary canal, and urine is
excreted by the kidney cells. A protozoon is a unicellular animal and each
of these functions must be performed within the single cell of which it
consists. Consequently, areas in every protozoon are differentiated so
as to form portions which are each devoted to special cell functions.
These portions are called organella, and by means of them all the ac-
tivities of a large animal go on in an organism which consists of a single
cell.

The functions of the nucleus are not completely understood but it
seems certain that the nucleus is a controlling center for the cell's activities.
Its functions, therefore, are, roughly, two-fold; they are either concerned
with the maintenance of the cell, or they are concerned with its reproduc-
tion; that is, they are either somatic or sexual. Usually, both functions
are subserved by a single nucleus; sometimes, however, as in the flagel-
lates, they are divided between two nuclei.

The activities of a protozoon may be divided into three classes:

LOCOMOTION, METABOLISM* and REPRODUCTION.

LOCOMOTION. The protozoa have several different methods of
moving themselves about. Some of them move by the formation of
pseudopodia; in this method of progression, the protoplasm flows out,
in finger-like processes, from the body of the parasite and, as the proto-
plasm flows into these processes, the whole organism progresses, literally,
by flowing along. Some of the gregarines move about by means of a
flowing of the protoplasm which always takes place in one direction; it
is probable that the control of the direction of the flow in these parasites
is effected by the contraction of myonemes. Myonemes are contractile
fibers, which usually lie near the surface of the organism possessing them.
Through their contraction, the form of the body of the parasite may be
altered and, in this way, motion may be produced. Cilia are small hair-
like multiple, often numerous processes, which may be placed either
in definite areas or over the whole surface of a protozoon. They produce
motion by waving; they usually act together and their motion has a strong
simultaneous stroke in one common direction. Flagella are larger than
cilia; they are whip-like processes which have a lashing movement. They
are usually few in number and are often placed at the ends of the organism.

REPRODUCTION. The protozoa reproduce in many different ways and

*For a consideration of metabolism, see p. 82, Part II, Physiology.

7 2

MORPHOLOGY AND CULTURE OF MICROORGANISMS.

several of these ways may occur in a single organism. For this reason,
their reproductive power is very great; in power of repeating their like,
they fall just short of the bacteria. The union of a male and a female
form does not always precede multiplication; sexual connection and re-
production, though now united in many animals, were originally two
entirely distinct phenomena and, in the protozoa, though sexual union
may be concerned with the production of new individuals, it is often
especially associated with the regeneration of the protoplasm of the
parasites taking part in it.

VK-:V>
r&alJ V;

) > "'* *ii- \
.^i^;.^:\ ^$%t$tiF$<

-:-";..; -i ^^j^ga*.

-^-^:,v

\

FIG. 45. Stages in the division of Amoeba polypodia. (After F. E. Schulze and Lange,

from Doftein.)

The simplest of the methods of reproduction is simple binary division,
in which the organism divides into two equal parts. A modification of
this process is gemmulation, in which a small protozoon buds off from a
larger parent; sometimes many buds are formed rapidly, one after the
other, until the parent protozoon disappears in a swarm of daughter cells
When a protozoon divides at a single division to produce a large number

PROTOZOA.

73

of daughter cells simultaneously, the process is called schizogony and the
young parasites are called merozoites, if a sexual fertilization has not
preceded the act of division; if such a division, in which the parent organ-
ism disappears, takes place after a fertilizing act, the process is called
sporogony and the young parasites are sporozoites.

FIG. 46. Coccidium schubergi. A-C, asexual multiplication; D-K, sexual multi-
plication; Z), microgametes; E, macrogamete; F, G, fertilization; H, I, K, division and
spore production. (After Schaudinn, from Dqflein.)

In protozoa, as in metazoa, the essential process in fertilization is the
union of two nuclei of opposite sex. Before union, these nuclei undergo
a series of divisions, in which the number of the chromosomes is reduced
and polar bodies are extruded. In dividing, almost all cells go through
a process called mitosis. In mitosis, the chromatin of the nucleus is

74 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

grouped into masses which are called chromosomes. The number of
chromosomes which are formed during mitosis is constant and character-
istic for each species. In the reproductive areas, during the two divisions
just preceding the maturity of cells which are to become ova or spermat-
ozoa, the number of chromosomes is reduced to exactly one-half of the
number which are formed during the division of cells outside of the re-
productive areas of the same animals. The process by which the number
of chromosomes is reduced is reduction, and the fragments of chromatin
which are unused and which are extruded from the cell during the process
are called polar bodies. Reduction and extrusion of polar bodies always
precede fertilization (Fig. 46).

The fertilizing processes which occur in the protozoa may be classed
under three heads: Copulation, Conjugation and Self-fertilization. In
copulation two whole cells unite. The cells taking part in this union are
called gametes and they are male micro gametes, or female macro gametes.
The cells which produce the gametes are called gametocytes. The product
of the union is called a copula or zygote. If the uniting cells be equal
in size the copulation is isogamous; if they be unequal, the copulation is
said to be anisogamous. Anisogamous copulation, the union of two
unequal cells, is most typically seen in the fertilization of a large macro-
gamete by a small microgamete. Copulation is a most important fertiliz-
ing process among the pathogenic protozoa. Conjugation, the second
method of fertilization, only occurs among the ciliata. In it, two adult
individuals are placed in apposition. The nucleus of each cell first re-
duces and then divides into two halves, one male, the other female.
Each organism retains its female half nucleus, while an exchange of the
male half nuclei is effected. Processes of self-fertilization, such as
autogamy and parthenogenesis, are included under the third heading. In
autogamy the nucleus of a single cell divides into two parts. Each of these
undergoes two further divisions, during which the chromosomes are re-
duced and polar bodies are extruded. The two resulting, reduced, half-
nuclei unite, always in the same cell, to form a new nucleus. Partheno-
genesis is the development of new individuals from a female cell without a
preceding fertilization; this process occurs in many protozoa, and through
it may be explained some of the acute outbreaks of malaria which may
occur in patients who once suffered from that disease and were thought
to have been cured of it.

It was said that fertilization and multiplication might be distinct

PROTOZOA. 75

processes. This is evident from a consideration of the phenomena
of autogamy and parthenogenesis, which have just been mentioned.
Reproduction occurs in both these processes without a preceding union
of two sexually differentiated cells.

The DEVELOPMENTAL CYCLE of a protozoon is the history of the
processes through which it may pass in the period intervening between
each fertilizing act. In many of the pathogenic protozoa, an alternation
of generations occurs; that is, states of being of the parasite in which an
asexual method of reproduction occurs, alternate with states of being
in which reproduction is effected by sexual methods. The developmental
cycle is often complicated by binary division, which may occur at any
point, by cyst formation, and by the intervention of a second host as a
necessary factor for the existence of a part of the cycle. An alternation
of generations occurs in the life cycle of the parasite which produces
malaria (Fig. 125) ; this parasite is one of the most important of the path-
ogenic protozoa. While it is in the body of its mammalian host, man,
it multiplies by binary division and by schizogony; it multiplies, sexually,
within the body of its invertebrate host, a mosquito. The host in which
the adult, sexual stages of the parasite occur, in this instance the mosquito,
is said to be the definitive host; hosts harboring the parasite while it is
in other stages are called intermediate hosts.

ENCYSTMENT. When unfavorable circumstances, such as drying,
occur, many protozoa are able to surround themselves by a resistant
cyst and to enter upon a resting stage of indefinite length. The cyst
protects them from hurtful influences and, surrounded by it, they remain
in a resting state until favorable circumstances come about once more.
The power of forming resistant cysts plays an important part in the
life-history of many parasitic protozoa; it is especially so with those
protozoa which have become so specialized that continued existence
outside of their appropriate host has become impossible for them. It
is often through the formation of cysts that an infection by a protozoon
is spread ; then, as in the coccidia, the presence of such a stage is abso-
lutely essential in the life history of the parasite.

PARASITISM.

A parasite is a living being which is, at some time, directly dependent
upon another, usually a stronger being.

Although the word parasite is often used as though it referred only

76 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

to organisms belonging to the animal kingdom, parasites may be either
animal or vegetable; bacteria and fungi, which live at the expense of
other living beings, are parasites just as the disease-producing protozoa,
and the biting insects which transmit them, are parasites.

Most parasites are simple organisms, low in the scale of life. They
nourish themselves without exertion, at the expense of their hosts, and,
as might be expected, their unemployed organs, such as the sensory,
locomotory and seizing appendages, through which food is usually ob-
tained, gradually disappear; degeneration always occurs in an organism
which assumes a parasitic mode of life.

Organisms, such as the malarial parasite, which are wholly depen-
dent for existence upon their hosts, are called obligatory parasites;
those which are not, such as the infusoria usually found in the stomach
of herbivorous animals, are facultative parasites. Facultative parasites
often feed upon dead material provided by the host, and not upon the
host itself; they are then said to be saprophytic.

If a parasite is attached to a host, and neither harms nor benefits it,
the parasite and host are said to be commensals. For example, the spiro-
chaetes found about the teeth of many persons are usually harmless;
they are commensals of their host. If the host of an obligatory parasite
dies, the parasite perishes also. Consequently, it is contrary to the
interest of such a parasite to destroy its host; yet parasites often do harm
their hosts. The harm done by a parasite to its host is recognized by the
disease caused by it. The pathogenic protozoa do harm to their hosts in
three main ways: They may feed upon, and destroy cells; they may
produce poisonous toxins; and their presence may do damage by me-
chanically obstructing some of the functions of its host. All three of
these ways are well exemplified by the action of the malarial parasite
on man (p. 685).

DISCUSSION OF THE CLASSIFICATION.*

This grouping of the protozoa gives a general idea of the position, in
zoological sequence, of the individual parasites which are spoken of in
the following pages. It simply divides the Protozoa into four classes:
the RHIZOPODA, the FLAGELLATA, the SPOROZOA, and the INFUSORIA; and
subdivides these classes directly into genera. This is by no means a

* (See page 10.)

PROTOZOA. 77

complete classification of the protozoa, for there are many orders and
genera which are unmentioned because they are parasitic neither in man
nor In animals.

The form of a protozoon may vary greatly at different stages of its
development; for example, the adult trypanosome is an active organism
moving by means of a flagellum, the spherical developmental form of the
same parasite may have no flagellum. Consequently, the whole life history
of a protozoon must be known before it can be classified with absolute
certainty. The whole of the life history is known of only a few protozoa;
and, though the organisms mentioned in this classification are placed in
the position usually given to them, it must be understood that this classi-
fication is not final, and that the discovery of new stages in the life history
of some of these protozoa might make it necessary to remove them
from the classes in which they have been placed; for example, before
its flagellate stage was known, Herpetomonas donovani was known as
Leishmania donovani, and it was classified with the sporozoa. Now
it is grouped with the herpetomonads.

The characteristics of the different genera and of the unimportant para-
sites are very briefly mentioned in the next few paragraphs; the important
parasites are treated more fully in the pages indicated by the references
given, in brackets, throughout the classification.

- The RHIZOPODA include the simplest forms of animal life. A
rhizopod, such as an amoeba, consists of a single cell, without a protective
covering, and without organs of locomotion; it moves and ingests food
by means of pseudopodia. Very few of the rhizopods are parasitic;
most of those which are parasitic belong to the genus Amoeba. Amoebae
of different species may occur in any part of the alimentary canals of
animals of many different species (p. 667).

The FLAGELLATA are distinguished by possessing one or more
flagella; they often have, also, a fin-like, undulating membrane. Most
flagellates are free-living. Comparatively few species are parasitic;
but some of these cause very serious diseases (p. 667).

The SPiROCELETjE, as their name signifies, are thread-like organisms,
whid seem to be coiled in a spiral. It is probable that the curves of
many spirochastes lie in one plane and, consequently, that their bodies are
really waved and not spiral. They often have an undulating membrane
and a flagellum at either end. A Herpetomonas is an elongated organism
which possesses a nucleus and a blepharoblast. The blepharoblast is

78 MORPHOLOGY AND CULTURE OF MICROORGANISMS.

usually situated near the blunter end of the parasite, and from it arises
a terminal flagellum. A Crithidia is an organism very much resembling
a Herpetomonas, with a pear-shaped body and, sometimes, a rudimentary
undulating membrane. A Trypanosoma is an elongated parasite which
has a nucleus, a kinetonucleus, an undulating membrane and a flagellum.
Species of Herpetomonas, Crithidia and Trypanosoma are frequently
found in the intestines of insects. One species of Herpetomonas is a
frequent and harmless parasite in the intestine of the house fly. The
genus Trypanoplasma contains organisms which have a flagellum at either
end, as well as an undulating membrane. They are parasitic in the blood
of fishes. The genera Cercomonas, Monas, and Plagiomonas include
small, unimportant flagellate organisms which have been found, occa-
sionally, to be parasitic in man; they have been found in the urine, and
in necrotic material from the lungs. A Trichomonas is a pear-shaped
organism which has four flagella attached to its blunt end, and an undu-
lating membrane. A species of it is sometimes found in the human
bladder. Other species are common, usually harmless, parasites in the
intestines of pigs, frogs and other animals. The most important species
of the genus Lamblia is Lamblia intestinalis. It also is a pear-shaped
organism. It has several flagella and is distinguished by possessing a
depressed sucker, by which it attaches itself to the wall of the intestine of
the animal in which it lives. It may cause severe diarrhoea in man, and
it may produce a fatal disease of the intestines in rabbits.

The SPOROZOA are protozoa which may multiply by the production
of spores; they are always parasitic at some stage of their life cycle. There
are very many sporozoa so, for convenience of classification, they are sub-
divided into seven orders. The GregarincB have a very distinctive
shape; the single cell, which composes them, is divided into two or more
divisions. The first of these divisions is furnished with hooks through
which the parasite attaches itself to its host. None of the gregarines are
parasitic on mammals ; worms are the hosts for some of them. The Coccidia
are usually parasitic within cells of their host, other than blood cells; for
example, Coccidium cuniculi (p. 68 1) enters the liver cells of the rabbit,
while Coccidium avium enters and destroys the cells lining the intestines
of the birds which it infects (p. 681). The Hamosporidia live, for a part
of their life cycle, within the red cells of the blood of their hosts. They
are a very important order. The genus Plasmodium causes malaria in
man (p. 682); while Proteosoma and Hamoproteus are malarial parasites

PROTOZOA. 79

of birds (p. 682). The H&mogregarince are usually harmless parasites
of reptiles and batrachians (frogs) ; a part of their life is passed within
the red cells of their host, but they have a slowly-moving stage, somewhat
resembling a gregarine, which occurs free in the blood. Hepatozoon
perniciosum is the best known of a group of haemogregarine-like parasites
which are parasitic, often within the white cells of the blood, in dogs,
in rats, and in other rodents; so far as is known, they do not cause disease.
The genus Babesia ( p. 686) includes parasites which cause important
diseases in cattle, sheep, horses and dogs. Similar parasites have been
found in the blood of monkeys, of dogs, of rats and other rodents. The
Sarcosporidia are tube-like in shape and filled with spores. They are
found within the cells of the voluntary muscles. The Haplosporidia
are an important group of very small sporozoa. Some of them are
parasitic in fish; one of them, Rhinosporidium kinealyi, has been found
in a tumor of the nose of a native of India. The Myxosporidia (p. 688)
are recognized by the peculiar form of their spores; each spore has a
capsule and is furnished with one or more threads. Members of this
order are parasitic in various parts of fishes and they often produce
disease in their hosts. The spores of the Microsporidia (p. 688)
are exceedingly small; a member of this order is the cause of pebrine
(p. 688).

The INFUSORIA (p. 689) are a large class. Most of them are not
parasitic. They are the most highly developed of the protozoa and
their bodies are more or less covered with cilia, by which they move"
themselves through the liquids in which they live.

In the last class, under the heading Parasites of Uncertain Position,
are grouped a number of organisms which cannot be classified because
so little is known of them at present. Histo plasma capsulatum (p. 689),
the Chlamydozoa (p. 689) and the Ultramicroscopic "viruses (p. 64, 690)
are all associated with important diseases in men and in animals.

Those parasites which are important enough to require special
consideration are described in the order in which they are mentioned in
the classification (p. 10). Whenever it is possible to do so, a single
species is taken as the type of each genus and that species, with the
disease it produces, is described; if the remaining species of the genus
are mentioned, they are spoken of only to indicate how they differ from
the description of the type species.

8o MORPHOLOGY AND CULTURE OF MICROORGANISMS.

TECHNIC.

The methods employed in studying the pathogenic protozoa are very similar to
those used in bacteriology. Microscopes, with the highest magnifications, are used in
order that the appearance of the protozoa may be observed. The observation, with
the microscope, of living specimens is a most important means of studying organisms
which undergo such wide changes of form as do many of the pathogenic protozoa.
Some parts of a protozoon cannot be seen in a fresh, living specimen; in order to make
these structures visible, preparations must often be fixed and stained by appropriate
methods; one of the methods most widely used is fixation in absolute alcohol and stain-
ing by some modification of Romanowsky's stain, such as Giemsa's method.

Much has been learned concerning bacteria and fungi by cultivating them on
artificially-prepared culture media. Some of the protozoa can be cultivated during a
part, or all, of their life cycle on similar media; but culture methods have not been so
important in the study of the protozoa as they have been in bacteriology. One reason
for this is, probably, that many of the parasitic protozoa live, naturally, upon certain
very highly specialized cells; it is difficult to prepare a culture medium sufficiently
resembling the substance of those cells to enable it to be substituted for them. Another
reason is that many of the protozoa exist under widely differing conditions at different
stages of their life history. Even though a culture medium might be devised suitable
for one of the stages of a protozoon, it is scarcely probable that the same medium would
be suitable for its growth at all stages of its development. Nevertheless, much has been
learned concerning the life histories of protozoa through the employment of culture
methods. Culture methods are sometimes useful in diagnosing diseases, caused by
protozoa; for the multiplication of the parasites, in a suitable medium, may reveal the
presence of pathogenic protozoa, when they are too few to be detected by the micro-
scopical examination of the infected material from which the culture was made; for
example, the flagellated stage of Leishmania furunculosa may develop in cultures of
material taken from a Delhi boil in which the resting stage of the parasite cannot be
found (p. 675).

PART II.

PHYSIOLOGY OF MICROORGANISMS*

DIVISION I.

NUTRITION AND METABOLISM.
INTRODUCTION.

PRINCIPLES OF NUTRITION AND METABOLISM. The nutrition and
metabolism of microorganisms is based on the same principles that regu-
late animal and plant metabolism. Only in a few instances, i.e., in the
anaerobiosis and in nitrogen fixation, we have processes unparalleled
in the more highly developed organisms. Since it will be necessary fre-
quently to refer to plant and animal nutrition in the course of this discus-
sion, these principles, therefore, are briefly discussed in the following
paragraph.

Green plants feed only on inorganic substances. They assimilate car-
bon dioxide (CO 2 ) from the air which unites with water, nitrates, potassium,
calcium, and other salts of the soil and form the body substances of the
plant. The cellulose, starch, sugar, protein and all other compounds
constituting the plant cells are produced from the above mentioned
inorganic substances. This formation of organic compounds requires a
certain amount of energy. If cellulose is burned to carbon dioxide (CO 2 )
and to water (H 2 O), a certain amount of energy is liberated in the form
of heat. Consequently the same amount of energy will be needed to
produce cellulose from carbon dioxide and water; for the law of the
conservation of energy requires that if a certain process liberates a certain
quantity of energy, the reverse process will require the same quantity of
energy. Green plants get their energy from the sunlight. The radiant
energy of light is transformed by the chlorophyl granules of the plant
leaves into chemical energy which causes the formation of organic com-

* Prepared by Otto Rahn.

6 8l

82 NUTRITION AND METABOLISM.

pounds from the inorganic or mineral matter. Chlorophyl is the green
coloring substance of plants, and only green plants can use the energy
of sunlight for their growth. The growth of green plants is a storing of
the energy of light in the form of organic matter; their metabolism is
largely synthetic, i.e., building up. Plants without chlorophyl, like mush-
rooms, molds, yeasts and bacteria, have to provide for their energy by
some other means.

Animals feed mainly on organic matter. Their body substances as
protein, fat, etc., are derived from the protein, fat, cellulose, etc., of plants
or of animals. Nevertheless, a certain amount of energy is required
in this assimilation process, since the animal protein and fat are different
from the plant protein and fat. Consequently, complex chemical
changes, which require energy, are necessary for growth. Energy is also
lost by radiation of heat and by locomotion. Animals, being entirely
unable to use the sunlight as a source of energy, obtain their energy from
the digestion of organic food. The larger part of this food is oxidized
completely; this part provides for the energy. Part of the food is used for
building the tissue of the body; it becomes part of the animal itself.
Animal metabolism is largely analytic, i.e., destructive. More organic
matter is decomposed than is formed. Often the same substance can
serve both purposes: the meat eaten by a dog furnishes to it energy as
well as material for growth. In other cases, certain food compounds
execute only one function and not the other. The distinction between
food for energy and food for growth will be of value in the interpretation
of microbial metabolism.

In the first part of this book, microorganisms have been divided into
plants and animals, but attention has been called in various places to the
fact that it is often hard to determine whether the plant characters or the
animal characters prevail. This holds true not only with the morphology,
but also with the physiology of microorganisms. Since none of the plants
discussed in this text-book possesses chlorophyl, none of them can use
light as a source of energy, therefore they depend entirely upon chemical
energy obtained by the digestion of food. This means that they require
organic food almost entirely, since inorganic food furnishes energy only in
exceptional cases. In this respect they resemble the animals very much.

The metabolism of protozoa is furnished by Todd (p. 7 1) as follows:
'The ingestion of food is accomplished in some protozoa by pseudopodia;
the protozoon simply flows around and so encloses a food particle. In the

INTRODUCTION. 3

same way, these protozoa flow away from waste particles which are to be
excreted. Other protozoa have definite mouth areas for the ingestion of
food, and definite anal areas for its excretion. Those protozoa which
ingest solid food, digest it within gastric vacuoles; the food is digested in
these vacuoles, as it is in many-celled animals, by the aid of enzymes and
of acids. The most important of the disease-producing protozoa live
within nutrient fluids, for example the blood, and they obtain their nour-

A

FIG. 47. A, Amoeba proteus; Na, a food particle; Cv, contractile vacuole; N, nucleus.

(After Doflein.)

ishment from the fluid in which they live, by osmosis; consequently,
they have no definite mouth area, nor gastric vacuoles, Fig. 47.

"Some of the protozoa, for example, the ciliata, possess contractile
vacuoles. A contractile vacuole is a clear area which appears, grows
slowly, empties itself, by a rapid contraction, of the water which has
drained to it, and forms again. The water which it ejects contains the
soluble waste products resulting from the metabolism of the protozoon.
One function of the contractile vacuoles is, therefore, excretion; in some
protozoa, they are probably also concerned with respiration. Contractile

84 NUTRITION AND METABOLISM.

vacuoles are usually absent in protozoa which are parasitic within other
animals."

A plant character which is found in most molds and yeasts, and in
some bacteria, is the assimilation of nitrates and ammonium salts, but
this distinction is not at all definite, for a large number of bacteria
are exceptions. It will be unnecessary, therefore, always to make a
distinction between microbial plants and animals.

ENERGY SUPPLY OF MICROORGANISMS. Microorganisms, like animals,
require food for energy as well as food for growth, and, as with animals,
one compound may often serve for both these functions. It is not always
possible to state whether or not a certain compound is used for growth as
well as for energy production, because the amount required for growth is
always very insignificant, but the distinction exists and can be indicated
by a few examples.

Certain bacteria are known to live entirely on mineral matter; they use
minerals exclusively for building their cell substances, resembling in
this respect the green plants, and provide for the necessary energy by
oxidizing mineral compounds. Two typical examples are the nitrifying
organisms in soil which oxidize ammonia to nitrates. This process, ac-
cording to Winogradski, is divided distinctly into two phases: the
Nitrosomonas oxidizes the ammonia to nitrous acid,

NH 3 + 3 O=HNO 2 +H 2 O

and the Nitromonas oxidizes the nitrous acid to nitric acid,

HNO 2 +O = HNO 3

These oxidation processes yield a certain amount of energy which enables
the bacteria to build their cells from carbon dioxide, ammonia, and certain
mineral salts. Without ammonia or without nitrous acid, respectively,
these bacteria cannot grow for lack of energy; they would be like a plant
without light. It is evident in this case that the food for energy is also
used to some extent as food for growth. The nitrogen necessary to the
bacteria is supplied by the ammonia or the nitrous acid.

As an example distinguishing between the food for growth and the
food for energy may be mentioned the hyposulphite bacterium studied
by Nathanson. This organism oxidizes hyposulphites to sulphates and
sulphur, largely following the formula

Na 2 S 2 O 3 + O = Na 2 SO 4 +S

Hyposulphite Sulphate Sulphur

INTRODUCTION. 85

Besides, some more complex compounds, like sodium tetrathionate
(Na 2 S 4 O 6 ), are formed. The bacterium builds its cells exclusively from
nitrates, carbon dioxide, and mineral salts; organic food is rejected. The
hyposulphite can hardly be used for the construction of the cell, and must
be considered entirely a food for energy.

This distinction is not confined to mineral decomposition only. The
urea bacteria get their energy from the decomposition of urea into
ammonium carbonate.

(NH 2 ) 2 CO + 2H 2 O - (NH f ) 2 CO 3

Urea Ammonium carbonate

But the urea and mineral salts are not sufficient for the development of
the urea bacteria. They cannot use urea as a material for building the
cells, nor can they use carbon dioxide or carbonates; they cannot grow
unless a suitable material for cell construction is added. Sohngen
demonstrated that a few milligrams of malic acid allow a good develop-
ment of the bacteria. The malic acid was used entirely for the formation
of cell substances. The energy for this formation came from the urea
fermentation. This example shows clearly the different requirements
for cell growth and for the energy supply.

This difference exists also in the alcoholic fermentation by yeasts.
Recently Lindner and Saito have shown that some yeasts ferment certain
sugars, and thus can obtain energy, but they cannot use them for growth.
They depend upon other organic substances for cell building material.
Other sugars can be used for growth, but cannot be fermented. As a
general rule, the monoses of the formula C 6 H 12 O 6 are most easily fer-
mented, while the bioses with the formula C 12 H 22 O n , especially maltose,
are much better adapted for growth.

Generally speaking, microorganisms obtain their energy by causing
chemical decompositions which may be either oxidations (nitrification,
vinegar fermentation) or intramolecular changes (urea fermentation,
alcoholic fermentation). Many organisms are able to cause various
decompositions. Yeasts will ferment sugar to alcohol and carbon dioxide
if air is completely or largely excluded, but if the yeast culture is well
aerated, the larger part of the sugar is oxidized completely to carbon
dioxide and water, and the alcoholic fermentation is retarded. This
double action upon sugar is still more pronounced with many representa-
tives of the mucor family. They oxidize in presence of air and cause

86 NUTRITION AND METABOLISM.

alcoholic fermentation when submerged in the liquid. A number of
bacteria can grow without sugar if oxygen is present, because they can ob-
tain their energy by oxidation. In the absence of oxygen, however, they
can multiply only if some fermentable material like sugar is present,
because this is the only source of energy available under anaerobic
conditions.

The amount of energy produced by the microbial cells is generally
larger than the amount required for growth. The excess energy is trans-
formed to heat which causes a rise of temperature in the culture medium.
The formation of heat will be discussed more in detail in one of the follow-
ing paragraphs.

CHAPTER I.

FOOD OF MICROORGANISMS.

THE COMPOSITION or THE CELL.

For the study of the amounts and kinds of different food substances
used by microorganisms, it will be necessary to know first the material
that constitutes the cells. Naturally, the main constituent of the cell
is water.

MOISTURE. The amount of water in the cells of microorganisms
will vary with the species as well as with the cultural conditions. The
total solids of " mother-of-vinegar " are only 1.7 per cent. This should
be considered as an extreme and very unusual case, owing to the spongy
nature of the jelly-like cell membrane. The average water content of
bacteria seems to be about 85 per cent; it varies more with yeasts and
still more with higher fungi. It seems reasonable to suppose that organ-
isms grown in concentrated solutions as the organisms of salted meat
and the molds growing in strong sugar solutions contain more solids.
Spores of molds contain much more solid matter than the mycelium;
the water content in two analyses of spores amounted to about 39 and
44 per cent respectively. Bacterial spores have not been analyzed, but
probably are much the same.

CELL WALL. The membrane of microorganisms does not generally
consist of true cellulose (C 6 H 10 O 5 ) X , though it is found in some cases.
Other compounds, related to cellulose, are more common; chitin*
(C 18 H 30 N 2 O 12 ), or another very similar nitrogenous compound is
also found. The slime surrounding some bacteria, and the capsules,
consist largely of carbohydrates, but often contain some protein.

CELL CONTENTS. The main portion of the cell is the protoplasm, a
mixture of protein substances, each of which has a very complex nature.
Enzymes which play an important role in metabolism (page 135) ar

C

* Chitin when hydrolized yields glucosamine and acetic acid.

C 18 H 30 N.,O 12 + 4H 2 O=2CH 2 OH-CHOH"CHOH'CHOH-CHNH-. ! CHO + 3CH- 3

37

88 NUTRITION AND METABOLISM.

produced in the protoplasm and are either secreted or retained. All pro-
ducts of metabolism will be found in the protoplasm of the cell in small
quantities. Among other substances frequently found in microorganisms
may be mentioned glycogen (C 6 H 10 O 5 ) D which can be readily detected
by the brown color it gives when acted upon by iodine. Glycogen may
be considered as a reserve substance stored by the organism. In the
same way, fats exist in microorganisms, but are more generally found;
their presence can be detected by microscopical examinations as well
as by chemical tests. The amount of fat in some bacteria is surprisingly
high. In the tubercle bacterium 26. to 39.29 per cent of the total
solids is fat. All acid-fast bacterial cells have a very high fat content.
Other bacteria also contain occasionally as much as 8 per cent fat.
Yeasts seem to have a lower fat content, while in molds it has been found
to vary from 0.5 to 50.5 per cent. Many other products of organic
nature are found occasionally, but their importance is not determined.
The minerals of the microbial cell are very essential, and like the
organic materials, necessary for the life of the cell. The total ash of
bacteria, yeasts, and molds, is small, about 1.5 per cent to 8 per cent of
the dry cell. The important minerals which seem necessary for the con-
struction of the cell are potassium, calcium, magnesium, iron, manganese,
and, of the metalloids, nitrogen, phosphorus, and sulphur. Some other
minerals are usually, found, but are unnecessary to the cell, as sodium
and silicon.

AMOUNT OF FOOD REQUIRED.

The amount of food that is ordinarily decomposed by microorganisms
and the amount that is absolutely necessary, differ widely. The quantity
of organic and inorganic matter just sufficient to support a very weak
growth is certainly very small, since a few species will multiply to some
extent in ordinary distilled water. Such water, after having stood for
some time, is found to contain several thousand bacteria per c.c. It
may seem to the layman that in such water it would be possible to detect
easily the organic and inorganic matter of the microorganisms and then
it would not be considered distilled water. An estimate of the weight
of bacteria demonstrates, however, that this is not the case. If we suppose
the average bacterial cell to be a cylinder whose base measures one
square micron and whose height is two microns (which is a high estimate)
the volume of such a cell would be 1X1X2 cubic microns = o.ooi X

FOOD OF MICROORGANISMS. 89

o.ooi Xo.oo2 mm. = 0.000, 000,002 cu. mm. The specific gravity of bac-
teria being very nearly i, the weight of one bacterium would be
0.000,000,002 mg. ; 100,000 cells per c.c. means 100,000,000 cells per
liter, which would weigh 0.2 mg. Of this total weight, at least four-
fifths is water and only one-fifth is solid matter. The total solid matter
in one liter of water containing 100,000 bacteria per c.c. amounts to the
unmeasurable quantity of 0.04 mg. Such water will pass the tests for
distilled water. How much food the bacteria in distilled water have
used is impossible to say, since besides the traces of minerals in the
water, they obtain some food from volatile compounds of the air like
carbon monoxide (CO), carbon dioxide (CO 2 ), ammonia (NH 3 ), hy-
drogen (H), and perhaps methane (CH 4 ). Under all circumstances
the amount of food used is very small.

On the other extreme, the maximum amount of food cannot be stated
very definitely. Usually bacteria cease to cause decomposition because
of the accumulation of noxious metabolic products. The ordinary
bacterium from sour milk will not form more than about i per cent of
lactic acid, because this is the highest acid concentration that this bacterium
can endure. If this acid is neutralized, the inhibiting cause is removed,
and the lactic fermentation starts anew until the maximum acidity is
reached again. The amount of food decomposed depends largely upon
the power of the organism to resist its own products. If the food is too
concentrated, however, physical influences may interfere with the meta-
bolism of the cell (p. 147).

ORGANIC FOOD MATERIALS.

The total weight of a large bacterial cell is estimated in the pre-
ceding paragraph to be about 0.000,000,002 mg., of which only about
one-fifth is dry matter. The smallest quantity that can be weighed
accurately on ordinary analytical balances is o.i mg. The solid matter
of 250,000,000 bacteria will amount to about o.i mg. MacNeal and
associates found that the dry matter of 550,000,000 cells of B. coli weigh
o.i mg. The amount of food that is used as the building material
for the cell is probably larger than the weight of the cell itself, since
there will be waste products, but it is of the same order of magni-
tude, i.e., very small and often hardly measurable. The example of
the urea fermentation (on p. 85) illustrates this point very well.

90 NUTRITION AND METABOLISM.

Quite large in comparison, though, is the amount of food used to provide
for the energy requirements of the cell. The quantities of protein matter
decomposed in soft cheese, of sugar destroyed by alcoholic and lactic
fermentations, are very large and easily determined analytically.

NON-NITROGENOUS FOOD COMPOUNDS. The simplest carbon com-
pound, carbon dioxide (CO 2 ), cannot possibly be used as a source of
energy, because it cannot be decomposed with liberation of energy. It is
used for cell construction by a few bacteria, e.g., the nitrifying and the
sulphur bacteria. This must be considered an unusual occurrence,
however, since nearly all other bacteria and all yeasts, molds and protozoa
depend on organic matter for their cell construction. Methane (CH 4 )
can be used by one or two bacteria for growth as well as for energy, and
even hydrogen gas serves as food, together with carbon dioxide, to one
bacterium. As a general rule, however, hydrocarbons are not attacked
by microorganisms. The compounds containing oxygen in addition to
carbon and hydrogen are better adapted for microbial food. The simple
alcohols can be used only by a few microorganisms, while the more com-
plex alcohols, like glycerin (C 3 H 5 (OH) 3 ), mannit (C 6 H 8 (OH) 6 ), etc.,
are very valuable as food for most molds, yeasts and bacteria. Apparently
the best nitrogen-free food compounds for microorganisms are the
carbohydrates, especially the hexoses, C 6 H 12 O 6 , and the bioses, C 12 H 22 O U .
They can be decomposed in many different ways, always yielding energy.
They are also very valuable as material for cell construction, and as
mentioned in the introduction to this chapter, the bioses are better adapted
for cell construction while the monoses are more easily fermented. The
insoluble carbohydrates like starch and cellulose are not as generally
decomposed by microorganisms as the soluble carbohydrates, though
many species have the ability to attack them.

Organic acids are excellent food for the microorganisms having strong
oxidizing properties, since oxidation is almost the only process of decom-
position that will yield energy from acids. Some of the dibasic organic
acids [succinic acid (CO 2 H' CH 2 'CH 2 -CO 2 H) and tartaric acid (CO 2 H'-
CH(OH)-CH(OH)'CO 2 H)] are quite commonly used as building mate-
rial ; they are often added to culture media for microbial plants. Ordinar-
ily, fats are not easily attacked. They are first split into their components,
glycerin and fatty acids, and the glycerin is decomposed readily while the
acids are used up very slowly. Other organic compounds may be used
occasionally by certain microorganisms, and probably there is no organic

FOOD OF MICROORGANISMS. 9!

compound that cannot be decomposed under certain conditions by certain
microorganisms. Even strong poisons like formaldehyde, and insoluble
compounds like paraffin, are known to serve as microbial food.

NITROGENOUS FOOD COMPOUNDS. Nitrogen is an absolutely neces-
sary constituent of the protoplasm, and therefore indispensable to the
life of the cell. The amount of nitrogen compounds required for cell
construction is necessarily very small, because of the small size of the cell.
Green plants assimilate nitrogen only in the form of nitrates or ammonia;
animals require proteins, or peptones, and may occasionally live on
amido-acids. Nothing can be said about microorganisms in general,
since some require proteins, while others can feed on ammonia, nitrates
and even on free nitrogen.

The few microorganisms assimilating the free nitrogen of the air are
very important since they increase the nitrogen content of the soil. They
must have some source of energy to form their protoplasm, and this is
supplied in the soil in the form of carbohydrates, organic acids, and
similar organic compounds. Unless some such compounds are available,
the nitrogen-fixing bacteria cannot grow. The amount of growth and
amount of nitrogen fixed depend mainly upon the amount of available food.
Nitrates are more commonly used by microorganisms as a source of nitro-
gen. They cannot serve as a source of energy, however, since they cannot
be decomposed in any way so as to yield energy. Many molds and some
bacteria can use nitrates. Ammonium salts are more readily used than
nitrates, and a large number of bacteria and yeasts and almost all molds
can use them in the formation of protoplasm. As a source of energy,
ammonium salts can be used only by the nitrate forming bacteria which
oxidize them to nitrates. The strictly organic nitrogen compounds may
be used as a nitrogen source and as an energy source at the same time.
This is true with the urea and with amido-acids. Many microorganisms
will thrive in a solution of asparagin [(CO 2 H.CH 2 .CH(NH 2 ).CO(NH 2 )].

With the amido-acids, we leave the chemically well-known compounds
and come to the very complex and chemically unknown peptones.
Peptones are a very good source of nitrogen and can be used as a source
of energy also by most organisms. Proteins are not quite so generally
used because many of them are insoluble. Many kinds of bacteria that
will grow in peptone solution do not liquefy gelatin. But a large num-
ber of microorganisms have very strong proteolytic (protein-dissolving)
qualities and use the protein to great advantage. Most molds and

92 NUTRITION AND METABOLISM.

many bacteria belong to this class of organisms, while only a few yeasts
have this ability. The absolutely insoluble compounds of protein
nature, like the keratin of hair and horn, can be decomposed by only a
very few organisms.

Though very little is known about the nutrition of protozoa, it may
be mentioned here that the saprozoic forms are not able to live on any
recognized soluble food media. They require solid food (bacteria, algae,
diatoms, other protozoa, etc.), and it is recorded that most of them
require living organisms for food and reject dead organisms.

MINERAL FOOD.

The minerals needed by bacteria are used (with very few exceptions)
entirely as structural food. The amount of mineral compounds is very
small since they make up not more than about 8 per cent of the solid
matter of bacterial cells. It is probable that the cells can exist and
multiply even with smaller amounts. The necessary elements are nitro-
gen, phosphorus, sulphur, chlorine, potassium, calcium, magnesium,
iron and manganese. There is still some doubt about the necessity of
certain other elements. Such investigations are extremely difficult
because of the mere traces of chemical compounds that enter into con-
sideration. These elements are assimilated usually in the form of inor-
ganic salts, with the exception of phosphorus and sulphur (and, of course,
nitrogen). The organic compounds of these elements may be absorbed
occasionally though the mineral sulphates and phosphates are by far
more commonly utilized.

The use of mineral compounds for the production of energy is limited
to a very few species of bacteria, while yeasts, molds, and protozoa cannot
possibly utilize them. The nitrite and nitrate bacteria and the hypo-
sulphite organisms have already been mentioned in connection with this.
Another group consisting of a number of bacteria which are morpho-
logically as well as physiologically different from the common species
and which are known as the sulphur bacteria or thiobacteria (p. 60),
oxidize hydrogen sulphide to sulphur, and sulphur to sulphates.

The sulphur formed by this process is stored in the bacterial cells which
are often found nearly filled with sulphur granules. Sulphur takes the

FOOD OF MICROORGANISMS. 93

place of fat, glycogen, or other stored substances. If the source of hydro-
gen sulphide ceases, the bacteria oxidize the sulphur within their cells to
sulphuric acid.

The second process of oxidation yields more energy than the one from
hydrogen sulphide to sulphur. The so-called "iron bacteria" are
another group of mineral-decomposing organisms, oxidizing ferrous
salts to ferric salts. They are commonly found in running brooks and
small rivers, and often develop in water pipes, sometimes forming in such
large quantities as to fill them entirely. It is not certain, however, that
they really obtain their energy by the oxidation of iron salts.

Organisms, feeding on marsh gas, hydrogen, and carbon monoxide,
have been mentioned hi the chapter on carbon supply. The very recent
discovery of bacteria which can oxidize carbon as such in its elementary
form may also be mentioned.

OXYGEN.

Oxygen is indispensable to the life of all highly developed organisms.
Animals especially need it to support the oxidation that takes place con-
tinuously in their cells and blood. Many animals die when the oxygen
supply is exhausted. Higher plants also cannot exist very long in any
atmosphere without oxygen.

The more simply organized forms of life are less sensitive, and micro-
organisms may grow without any free oxgyen. In the introduction to
this chapter, it has been stated. that some organisms obtain their energy
by decomposing organic compounds without oxidation. The fermenta-
tion of urea consists of a simple addition of water.

CO(NH 2 ) 2 + 2 H 2 = (NH 4 ) 2 C0 3

Ammonium
Urea carbonate

Alcoholic and lactic fermentations are other examples. The fermen-
tative change of alcohol to acetic acid, however, is a process of oxidation.

CH 3 CH 2 OH+ 2 O = CH 3 COOH+H 2 O

Alcohol Acetic acid

Oxygen is required for this process. Similar oxidation processes are
the formation of nitrates and decomposition of thiosulphate and of

94 NUTRITION AND METABOLISM.

hydrogen sulphide. Organisms which depend upon oxidizing processes
for their supply of energy will not be able to grow without oxygen. This
is the case with the acetic bacteria, with the nitrifying bacteria, and with
many molds which are generally noted for their strong oxidizing properties.

The question arises whether the organisms which provide for their
energy without oxidation will need oxygen for life processes other than
energy supply. The urea bacteria require no free oxygen for their
fermentation, but they require it for other life functions. Certain chem-
ical changes can take place within the cell of these bacteria only if oxygen
is present. They require very little oxygen, but they cease to grow if it
be removed completely. There are, however, certain other micro-
organisms which can live in a complete absence of oxygen and there are
some which die in the presence of oxygen.

The organisms which require free oxygen are called aerobes; those
which can live without free oxygen are called anaerobes. Among the
latter are the obligate anaerobes, which develop only in the absence of
free oxygen, and the facultative anaerobes which can grow either with or
without free oxygen. Even the aerobic microorganisms can tolerate the
absence of oxygen for a considerable length of time, perhaps for years.
They will not multiply but remain dormant until they come in contact
with free oxygen again.

The influence of free oxygen upon the obligate anaerobic bacteria is
remarkable; it does not only prevent their growth, but it kills them.
Some butyric bacilli die if exposed to air for fifteen hours, while the spores
of these bacilli are quite resistant. The study of these organisms is quite
difficult, since it takes considerable effort to remove the last traces of
oxygen from the culture media. Usually they are cultivated in an
atmosphere of hydrogen; carbon dioxide and coal gas do not give as good
results because these gases affect the growth of the bacteria. The best
gas for the cultivation of anaerobes is nitrogen, because it is absolutely
neutral, but it is difficult to obtain it free from oxygen. Anaerobic organ-
isms may also be grown in a vacuum.

Even the obligate anaerobic organisms can tolerate a certain amount
of oxygen. We can speak of a maximum concentration of oxygen for
the various organisms, and also of a minimum concentration. The
minimum of oxygen for the anaerobic bacteria is nil. This is the definition
of all anaerobic bacteria. The maximum concentration of oxygen varies
with the species. Certain bacteria are known to be killed by very small

FOOD OF MICROORGANISMS. 95

amounts of oxygen; others are less sensitive. There is enough free
oxygen in the atmosphere to prevent the development of certain bacteria
having purple pigment, but they multiply easily wherever there is a more
limited supply of oxgyen. Their maximum of oxygen is only a little
smaller than the concentration of oxygen in the atmosphere. Even the
aerobic bacteria have their maximum of oxygen which is many times the
amount of oxygen of the atmosphere. The following table gives the
maximum concentration of oxygen for a few species. The oxygen content
of the atmosphere is taken as 100.

Maximum Oxygen Tolerance

B. (Clost idium) butyricux, 1.35 per cent of atmospheric oxygen.

B. chauvei, . 5- 2

B. cedematis maligni, 3- 2 5

Purple bacteria (Molisch), about 90

Thiosulphate bacteria (Nathansson), about 400

Pink yeast, 9

Penicillium glancum, about 1700

B. prodigiosus, 3000

These numbers mean that B. (Clostridium) butyricus cannot live unless the
atmosphere is diluted to about i per cent of its original content of
oxygen, either by evacuation or by other gases. B. prodigiosus is able
to live even if the air is compressed thirty times, but not more. The
table demonstrates also that there is no natural line between anaerobic
and aerobic bacteria, and a classification founded upon oxygen supply
is as arbitrary as all classifications of organisms; but like all classifica-
tions, it is necessary for the designation of certain qualities of bacteria
though a few species merge into the well-defined groups at both
extremes.

The obligate anaerobic bacteria which tolerate only a very small
amount of oxygen can adapt themselves to higher oxygen concentrations
by a very slow increase of the oxygen pressure. It has also been claimed
that occasionally the obligate anaerobic bacteria in pure culture will grow
in air. It is known that these bacteria, if cultivated in an atmosphere
with small amounts of oxygen, will use up the oxygen. In what form
and how the oxygen is bound cannot be stated because the quantities in
question are too small to be traced in the various products of metabolism.

The chemical changes instituted by anaerobic organisms are partly
hydrolytic decompositions, partly intramolecular changes. Carbon

g6 NUTRITION AND METABOLISM.

dioxide, hydrogen, and methane are produced sometimes by the same
organism at the same time and from the same food compound. This
appears unusual because these gases represent the ultimate products of
oxidation and of reduction. The intramolecular changes caused by
anaerobic processes result mainly in a change of the oxygen atoms of
the food. One side of the molecule is reduced while the other side is
oxidized, as in the alcoholic fermentation, which can take place without
oxygen. In the sugar molecule, each carbon atom has one oxygen atom.
In the products of fermentation, carbon dioxide has two oxygen atoms to
one carbon atom, and in alcohol there is only one oxygen atom for two
carbon atoms. In the lactic fermentation, the oxygen, which is distrib-
uted evenly in the sugar, is shifted to one side of the molecule in lactic acid

H H H H H
O O O O O

Dextrose, H C C C C C C =O
H H H H H H

H

H O

Alcohol, HC CH Carbon dioxide, O=C=O

H H

H H
H O O

Lactic acid, H C C C = O
H H

These changes result in a liberation of energy which enables the organ-
isms to continue their life process.

Some bacteria provide for oxygen by taking it from mineral compounds.
Through the agency of certain microorganisms, nitrates are reduced to
nitrogen and sulphates are reduced to hydrogen sulphide by a complete
removal of the oxygen from the molecule.

Ca(NO 3 ) 2 -5O = CaO-(-2N
H 2 S0 4 - 4 = H 2 S

It seems that the opportunities for the development of obligate anaero-
bic organisms in nature are not very numerous. They may develop in the
animal body, in the deeper layers of soil, and at the bottom of waters.
They can also develop, however, on the surface of the soil and in other
places where the air has free access, provided that some aerobic organisms

FOOD OF MICROORGANISMS. 97

grow there at the same time. This peculiar association of aerobic and
anaerobic organisms cannot be explained simply by complete exhaustion
of the oxygen in the medium by the aerobic species; the latter do not
remove the oxygen completely. No other completely satisfactory explan-
ation can be given.

Of the facultative organisms, some prefer to grow in the presence of
oxygen, like the yeasts, while others thrive better without air, as certain
lactic bacteria. It has been doubted whether facultative organisms
can multiply continuously without oxygen. The latest experiments with
yeasts indicate that there is a limit to their anaerobic multiplication. They
will develop for about 20 to 30 generations; they then need free oxygen
again. With bacteria, however, the multiplication seems to have no
limit in this particular.

Facultative organisms thrive without air only in the presence of certain
food compounds, preferably carbohydrates. If they cannot get their energy
by oxidation, they depend upon anaerobic fermentations, and their ability
to ferment is naturally limited to a few compounds. B. coli can grow
without air only in the presence of sugars; it is customary to test for the
absence of sugar in broth by inoculating with B. coli a fermentation
tube which has been filled with the broth. Growth in the closed arm
indicates sugar; if there is growth only in the open arm, the broth is sugar-
free. Other facultative bacteria may be able to destroy protein under
anaerobic conditions, though experiments show that some of them lose the
power of liquefying gelatin if grown without oxygen. Some of the facul-
tative bacteria can provide for their oxygen by taking it from nitrates or
sulphates.

It is interesting to compare the energy liberated by an aerobic and an
anaerobic process. The total energy stored in an organic compound is
measured by the amount of heat liberated by its complete combustion.
One gram of dextrose produces 3750 calories, if burned to carbon dioxide
and water. The plant producing this dextrose from carbon dioxide and
water needs, therefore, 3750 calories for every gram made, which are
taken from the radiant energy of the sun. The mold oxidizing i g.
of dextrose completely gains in this process 3750 calories which may be
used in form of chemical energy for its growth and for building up the
complex compounds of cell life from small simply constructed molecules.
If the mold has ceased growing, these 3750 calories will not be used but will
produce a slight rise in the temperature of the nutrient medium. A yeast
7

98 NUTRITION AND METABOLISM.

fermenting the dextrose to alcohol and carbon dioxide does not use the
entire energy of the dextrose; there is considerable available energy
left in the alcohol; carbon dioxide contains no available energy. One
g. of dextrose gives about 0.48 g. of alcohol and 0.52 g. of carbon
dioxide. The heat of combustion of alcohol is 7183 calories per g. or
3476 calories for 0.48 g. The heat of combustion of carbon dioxide
is nil. The dextrose before fermentation represents 3750 calories, the
products of fermentation contain 3476 calories; the yeast therefore gains
by the fermentative process only 247 calories from i g. of dextrose.
This is a very small gain, compared with that of oxidation; the ratio is
274:3750 or 1:14. This means that a yeast, in order to get the same
supply of energy as an oxidizing mold, has to ferment fourteen times as
much sugar as the mold oxidizes.

A very good example of this is the different power of nitrogen fixation
with aerobic and anaerobic bacteria. The aerobic Azotobacter fixes about
15 mg. of nitrogen for each g. of sugar, while the anaerobic B. (Clostridiutri)
butyricus fixes only 2 mg. for the same amount of sugar, when this sugar is
fermented to butyric and acetic acid, carbon dioxide and hydrogen.
The nitrogen numbers indicate the rate of growth of aerobic and
anaerobic organisms with the same amount of food.

Occasionally, attention is called to the enormous destructive power
of microorganisms, the proportion of food to growth being entirely
different from that of animals. One reason for this difference can be
plainly seen from the above discussion. The higher animals oxidize
their food almost completely and consequently need only a fraction of
what a fermenting organism would require.

The oxygen requirements of protozoa have not been investigated
until recently. It is probable that some parasitic protozoa can live and
multiply without free oxygen for a considerable time.

ADDITIONAL REMARKS ON MICROBIAL FOOD.

PHYSIOLOGICAL GROUPS. It is customary to divide the microorgan-
isms into physiological groups according to the kind of food and the
metabolic products formed. This is convenient and helpful in describing
certain characters though the classification and nomenclature has been
accomplished as far as possible with strictly morphological characteristics,
as is the custom in all classification of plants and animals. The so-

FOOD OF MICROORGANISMS. 99

called "sulphur bacteria" certainly belong to one physiological group
which is very plainly defined by their ability to oxidize hydrogen sulphide
to sulphur and sulphuric acid. The official nomenclature distributes
them, however, into several genera. The "nitrate bacteria" are another
group having a very definite physiological character. In some groups
the morphological and physiological characters agree very largely, e.g.,
in the butyric bacteria which are all spore-forming rods with a tendency
to show spindle form, and in the Azotobacter group which is morpho-
logically distinctly different from any other bacteria.

With some other more general terms like "lactic bacterium," "putre-
fying organism," "acid producer," the quality indicated is very vaguely
expressed and therefore of little significance.

Another feature should be mentioned in discussing the relation of
microorganisms and their food, namely the fact that some organisms
prefer certain foods and live exclusively on a very few chemical com-
pounds, being unable to assimilate any others, while other organisms are
able to feed on nearly every organic substance. The nitrifying bacteria are
unable to use anything but ammonium salts. Certain pathogenic bacteria
require a very special medium. The invisible organisms cannot be culti-
vated on any medium except the living tissue. The fermenting yeasts can
grow on protein media without sugar, though their development is meager.
Some protozoa feed on many kinds of living bacteria, but cannot eat
dead bacteria. Of the scavengers which can live on all kinds of food,
the molds have many examples, living not only on protein, sugars, starch,
cellulose, fat, but also able to exist on inferior food like alcohol, acetic
or oxalic acids, with often no other nitrogen source than ammonium salt.
Such omnivorous species are also found among bacteria, especially among
sewage and soil bacteria.

SYNTHETIC MEDIA. Recent investigations, especially by Gorham and
his associates, of the physiology of microorganisms have shown that
most organisms do not necessarily require media made from meat extracts,
peptone and similar unknown compounds. A large number even of
pathogenic bacteria which had been believed to specialize very particularly
in their food requirements can be grown on synthetic media containing
only compounds of well-known chemical composition, as amino-acids
(asparagin, glycocoll), urea, ammonium salts of tartaric, succinic or lactic
acids, dextrose, glycerin, and perhaps other organic substances, besides
the necessary mineral salts. The great advantage of these media is that

100 NUTRITION AND METABOLISM.

they can always be made exactly alike, while meat extracts, peptones of
various manufactures, and milk, vary in composition and cannot be con-
sidered as standard media. Besides, the microbiologist can follow the
metabolic processes in synthetic media very easily, while it is impossible
even to enumerate all the compounds contained in the ordinary nutrient
gelatin, not to speak of a quantitative determination.

CHAPTER II.
PRODUCTS OF METABOLISM.

THE CHEMICAL EQUATIONS OF FERMENTATIONS.

The metabolism of all organisms is considered to be a chemical process
which follows in all respects the laws of chemistry. That we are not
familiar with all the changes taking place in the cell is not because we
are dealing with unknown forces, but simply because we do not know
all the factors involved in the process. Some of the chemical changes
caused by the living cell can be imitated exactly by the chemist in a test-
tube. This may be illustrated by the oxidation of alcohol to acetic acid,
the decomposition of urea to ammonium carbonate and of ammonia to
nitrate. Some other processes are not as fully understood and not as
easily imitated. The alcoholic and acid fermentations of sugars are of
such nature. There is no reason to suppose, however, that these processes
are other than chemical changes. A chemical process can always be
expressed by a chemical equation, consequently the various known
fermentations and decompositions caused by microorganisms should be
represented by chemical equations.

This formulation is not always simple, because the greater number
of microorganisms decompose organic substances in more than one way.
Also, certain compounds may be produced in such small quantities as to
escape the chemical analysis entirely, since the determination of many or-
ganic compounds is a very difficult task. Again, part of the decomposed
material will usually be assimilated in the growth of the cells; hence more
material disappears than can be accounted for by the fermentation pro-
ducts. There are several possibilities for discrepancies; accurate equa-
tions can be given only for the simplest fermentations, the products of
which can be analyzed more or less exactly.

The best studied microbial process is the alcoholic fermentation,
which is not only the classical example of fermentation, but also of great
commercial importance. The simplest equation for the decomposition
of dextrose into alcohol and carbon dioxide by yeast is

C 6 H 12 O 6 = 2C 2 H 5 OH+ 2CO 2
180 92 88

101

IO2 NUTRITION AND METABOLISM.

According to this formula, 100 parts of dextrose should give 51.11 parts
of alcohol and 48.89 parts of carbon dioxide. The actual yield comes
very close to these numbers, but does not reach them ; the largest amounts
found were 46-47.5 per cent of carbon dioxide and 47.5-48.67 per cent
of alcohol. Under the most favorable conditions, the total yield of
the products of fermentation ,was only 95 per cent of the theoretical
yield.

Other products are formed besides the alcohol and carbon dioxide.
The amount of glycerin found in fermented liquids varies very much with
the conditions of fermentation; it reaches from 1.6 to 13.8 per cent of
the alcohol or from 0.8 to 6.9 per cent of the fermented sugar. A small
quantity of succinic acid is also formed, usually about 0.6 to 0.7 per cent
of the fermented sugar. Traces of acetic acid and of lactic acid seem to
be normal products of the process of fermentation. All of these com-
pounds have been regarded as products resulting from the regular fermen-
tation, but the latest investigations seem to indicate that glycerin and
succinic acid are produced by yeast cells even in the absence of sugar.
This discovery makes it probable that the glycerin and succinic acid are
derived from the reserve substances of the yeast cells, such as lecithin,
and are not direct products of fermentation. This accounts also for
the variation of the proportion between alcohol and glycerin.

Similar are the experiences with the lactic fermentation which has been
studied almost as extensively as alcoholic fermentation. If it is supposed
that the formation of lactic acid follows the equation

C 12 H 22 11 +H 2 = 4 C 3 H 6 3

342 18 360

Lactose Lactic acid

the actual yield of acid is found to be between 90 per cent and 98 per cent
of the theoretical. The other 2-10 per cent are either used for cell-
growth or for products which thus far have escaped chemical determina-
tion. Small discrepancies will also be found in the fermentation of urea
and in the nitrifying process, where small amounts of the nitrogenous
material are used for the cell-growth.

Another difficulty in finding the chemical equation of a microbial
fermentation is the fact that this process may change with the age of the
culture. In those fermentations where several gases, as carbon dioxide
and hydrogen, are produced, the relative proportion of the two is not at
all constant. In the butyric fermentation of dextrose by B. amylozyma,

PRODUCTS OF METABOLISM. 103

Perdrix found the relation of hydrogen to carbon dioxide to be at first
65:35, later 52:48, and the relation of butyric to acetic acid 26:74, and
later 85:15. Perdrix tries to account for this change by assuming three
different phases of the process at various ages of the cultures, represented
by the following equations:

First stage: 56C 6 H 12 O 6 + 42H 2 O= n6H 2 + ii4CO 2 + 3oCH 3 COOH+

Dextrose Acetic acid

3 6CH 3 CH 2 CH,COOH.

Butyric acid

Second stage: 4 6C 6 H 12 O 6 + i8H 2 O= ii2H 2 +94CO 2 + i5CH 3 COOH+

38CH 3 CH 2 CH 2 COOH.
Third stage: C 6 H 12 O 6 -2H 2 +2CO 2 +CH 3 CH 2 CH 2 COOH.

It does not seem very probable that the protoplasm of the butyric
organism takes up 56 dextrose molecules at once and changes their
molecular structures. It is possible that the hydrogen, the carbonic acid,
and the organic acids are produced by three or four different processes
taking place at once in the same cell independent of each other, and
that by certain influences the one is favored, the other checked. But
there is no proof for such a theory.

In fermentations where acid is produced, it will naturally make a very
great difference whether this acid is neutralized or not. All organisms are
retarded by their own products, and the acid-producing bacteria often
give rise to so much acid that it kills them. The neutralization of the
free acid will permit them to make more acid. Oxalic acid is produced
in large quantities by Aspergillus niger if calcium carbonate is present
to neutralize the acid. Without calcium carbonate, only about one-sixth
of the amount of acid is produced. In some instances the acidity or
alkalinity' of the medium may also have a decided influence upon the
nature of the products of fermentation. The proportion of acids and
alcohols in butyric fermentation is said to be altered materially by the
addition of calcium carbonate.

Other complications occur when an organism is able to use its own
products as food, as is the case with some acetic bacteria. They will
at first produce considerable amounts of acetic acid and after a while
they oxidize the acid completely. It becomes impossible to account for
microbial activity by a chemical equation when several organic compounds
are decomposed at the same time as is found to occur in some foods, as
butter, cheese, ensilage and in sewage. It is also impossible to formulate

104 NUTRITION AND METABOLISM.

exactly decompositions which are caused by mixed cultures. The
complications become so great and the relations between different
organisms are so little known that it is useless to make the attempt.

PHYSIOLOGICAL VARIATIONS.

The great variability of microorganisms in morphological respects has
already been pointed out in Part I of this book. A similar variation and
adaptation are noticed in their physiology, especially with the food sub-
stances of bacteria and consequently with their metabolic products.
Microorganisms change their physiological properties very readily with
the environment; the new variety may keep its acquired properties for
some time even if brought back to the original conditions. It is stated
frequently that microorganisms tend more toward variations than the
more complex organisms. It should be considered, however, that the
experiences in the variations of green plants and animals are based on
individuals, while in the case of microorganisms these experiences are
gained almost always from millions of cells. A simple illustration is the
development of bacteria in salt solutions. If a broth culture of B. coli
is transferred into broth containing 8 per cent of salt, a large number of
cells will die, often more than 99 per cent. The surviving bacteria begin
to multiply after a certain length of time and a new variety is created
which can tolerate the salt. At first, only about one out of one hundred
cells had the power to tolerate salt, but, since the dying cells are not usually
counted or considered at all, it is customary to say that bacteria easily
adapt themselves to an 8 per cent salt solution. If only one single plant
out of one hundred could be adapted to a certain high temperature, it
could not be said that it adapts itself easily. This mistake is quite com-
monly made with microorganisms.

The best illustration for the variability of cultivated microorganisms
is the enormous number of varieties of Saccharomyces cerevisia. Nearly
every large brewery has a yeast type of its own which differs from others
by the amount of alcohol and aromatic substances produced, by time and
optimum temperature of spore-production, by the appearance of the
budding yeast in the hanging drop, and also in other respects. The cul-
tivated organisms are not alone in showing this tendency toward variation.
The transferring of a soil or water bacterium into the ordinary laboratory
media is a complete change of conditions; the different cells of the same

PRODUCTS OF METABOLISM. 105

species may react differently and give several varieties. A lactic bac-
terium on meat media without sugar does not thrive well in the first
generations, but it gradually becomes able to grow on this medium. By
this treatment, it loses gradually the power of producing acid and does
not thrive as well in milk. The attenuation of pathogenic bacteria by
cultivation on media, as potato, very different from the blood and muscle
upon which they grow most naturally, or by growing them at low tem-
perature, or above the maximum, furnishes another example. The
decrease and finally the entire loss of pathogenicity is caused by a change
of metabolism, by a loss of the power to produce toxin in many cases.

As by certain diet the metabolism can be changed, so certain physio-
logical properties of bacteria can, by proper cultivation, be increased. By
the frequent transferring of an organism on gelatin, its liquefying qualities
can be increased, provided it had some at the start. By continued passing
of a bacterium through an animal, its virulence can be increased. Strains
of bacteria which will produce a very high acidity can be bred ; this is il-
lustrated by the quick-vinegar process and by the strong alcohol-produc-
ing yeasts of the distillery process. It is also possible to accustom micro-
organisms to certain chemicals which are antiseptic for the ordinary
strains. Distillery yeast is by careful breeding acclimatized to tolerate
a very high degree of acidity which would kill an ordinarily bred yeast.
By continued cultivation of an organism upon a certain medium, it will be-
come so acclimatized that it degenerates readily when the conditions become
unfavorable. Such high-bred strains of microorganisms are used in
alcoholic and lactic fermentation, in pathogenic bacteriology and in the
inoculation of leguminous plants with nitrogen-fixing bacteria.

PRODUCTS FROM NITROGEN-FREE COMPOUNDS.

The decomposition products of nitrogen-free organic compounds will
be taken up in the same order as was done with the food substances,
beginning with the most complex carbohydrates.

CELLULOSE is attacked by many molds, but little is known about the
products. Probably they oxidize it completely to carbon dioxide and
water.

(C 6 H 10 O B )n+ i2nO = 6nCO 2 + 5nH 2 O

Some bacteria, as B. ferrugineus (van Iterson) and a few denitrifying
bacteria, can use cellulose as organic food material ; cellulose in soils is

106 NUTRITION AND METABOLISM.

known to favor denitrification. The best studied decompositions of
cellulose are the two anaerobic fermentations, generally distinguished as
methane and hydrogen fermentation. The ' 'methane" and the "hydrogen"
bacillus usually are found together; pure cultures have not been obtained
as yet though the two organisms can be separated from each other. The
so-called hydrogen fermentation of cellulose is caused by a thin, slender
rod, producing spores at the end of the cell, some of which have drum-
stick forms. The products of this destruction of cellulose are acetic and
butyric acid, probably a little valeric acid, carbon dioxide, and hy-
drogen. The mixture of gases contains at first more than 80 per cent of
hydrogen, at a later stage it contains only 4.5 per cent hydrogen, and the
remainder carbon dioxide^

The "methane" bacillus, which looks very much like the "hydrogen"
bacillus, gives mainly acetic acid with a little butyric acid, while carbon
dioxide and methane escape. The amount of methane is at first about
75 per cent of the total gas, but soon drops to about 30 per cent. The
marsh gas in the mud of marshes, lakes, and other waters is formed by
this organism.

STARCH is decomposed in many different ways. It may be oxidized
completely like cellulose to carbon dioxide and water. The same chem-
ical equation applies to this process. Some molds and some bacteria
will change the starch to various acids (lactic, acetic, oxalic, etc.) or to
alcohol and carbon dioxide. In every instance, the starch is converted
first into sugar, and then the sugar is fermented. The starch is not
fermented directly, since it is insoluble. (The fermentations of sugar
are dealt with in the next paragraph.) Dextrins are in all respects
similar to starch and are also converted into soluble sugars before being
fermented. Those organisms which cannot break up the starch or dextrin
molecule to sugar cannot use it as food.

SUGARS can be decomposed in several different ways. Some fermenta-
tions have been studied much in detail, especially those of commercial
importance; others are known to some extent, but no special effort has
been made to determine all products quantitatively, consequently the
equations of these fermentations cannot be exactly given.

The alcoholic fermentation by yeasts with its by-products has been
discussed so extensively in the paragraph above that there is little to be
added. Among the molds, the species of Mucor are the best-known
alcohol producers; nearly all mucorshave this quality and some of them,

PRODUCTS OF METABOLISM. IOy

imported from eastern Asia, in such a high degree that they are used for
the production of industrial alcohol since they have also, unlike the
yeasts, the quality of converting starch. These molds were first called
"Amylomyces" though the more modern classification put them in the
genus Rhizopus. Among the Aspergillacca, Aspergillus oryza is the only
species which is materially concerned in the production of alcohol. It is
found in the fermentation of Japanese rice-brandy, called "Sake," and
has been tried in Europe as a substitute for malt to change starch into
fermentable sugars. Since these molds make alcohol mainly in the absence
of oxygen, when the cells are spherical (gemmae) and look like budding
yeasts, it was believed for some time that yeasts were only a certain stage
of development of the mucor molds. Alcohol, along with other com-
pounds, is also formed by certain bacteria, though in small quantities and
not as the only product; this is true in the butyric fermentation, and in
gassy fermentations.

Alcohols other than ethyl alcohol are produced by several organisms.
Methyl alcohol (HCH 2 OH) is found to be produced by B. boocopricus from
cow-dung. Butyl alcohol (CH 3 (CH 2 ) 2 CH,OH) is a common product
in the butyric fermentation. B. amylozyma makes amyl alcohol [CH 3 -
(CH 2 ) 3 CH 2 OH] under certain conditions, and it is probable that the
fusel oil, the by-product of alcoholic fermentation in distilleries, is not
formed by the yeast, but by some anaerobic organisms developing in the
fermenting pulps. The use of pure cultures for the production of fusel
oil, especially amyl alcohol, has been suggested.

Besides alcohol, organic acids are very commonly produced from
sugar by many bacteria, yeasts and molds. The acid fermentations have
wide practical application, and much attention has been paid to their
study. If, however, our knowledge, especially of the quantitative
relations between food and products, is limited, this is largely due to
the great variability of the organisms and the difficulty of analysis.

The best known, because the simplest, fermentation, is the pure
lactic fermentation which has already been discussed on page 102; it is
very nearly represented by the equation

C 6 H 12 O 6 = 2CH 3 CH(OH)COOH

Dextrose Lactic acid

The bacteria which cause this fermentation are called true lactic
bacteria. They are the main acid producers in milk. Other acid
producers form gas besides lactic acid or other acids or alcohol. In

IO8 NUTRITION AND METABOLISM.

the group of gas-producing lactics, B. coli and Bad. aerogenes are the
main representatives. The fermentation of dextrose by B. coll is very
nearly described by the equation

2 C 6 H 12 O 6 + H 2 O = 2 C 3 H 6 O 3 + CH 3 COOH+ C 2 H 5 OH+ 2 CO 2 + 2 H 2

Dextrose Lactic acid Acetic acid Alcohol

A certain group of bacteria, including the Bad. aerogenes, the Strept.
pneumonia, and B. typhosus, cause fermentations which are very similar to
the one above. The products depend not only upon the species but also
upon the fermented sugar. Grimbert found that B. coli makes acetic
acid from all fermentable sugars, but lactic acid only from dextrose,
while with all the other sugars, succinic acid is formed instead of lactic
acid. B. typhosus gives no succinic acid, but always lactic and acetic
acid, often also butyric acid. The mechanism of these fermentations is
apparently not as simple as that of the alcoholic and the true lactic fermen-
tation. The greatest difficulty for the physiologist is the change of
products during the fermentation; at first one, later another product
predominates, consequently an attempt to give a certain definite equation
of the fermentation is frustrated. It is more convenient merely to enumer-
ate all products than to give an equation which may hold true only with
a certain stage of growth of a single variety under one set of conditions,
without giving any guarantee that other strains under different conditions
will give even approximately the same kinds and quantities of products.

In this group of gas- and acid-forming bacteria, we find a number
of organisms of practical importance. They are the organisms of the
fermentation of sauerkraut, dill pickles, brine pickles, ensilage. There
are further B. coli and a number of pathogenic bacteria, B. typhosus,
and the varieties of the Strept. pneumonia. The standard products are
acetic acid and alcohol, besides carbon dioxide and hydrogen; lactic
acid is usually, succinic acid often, produced. The products depend upon
the fermented sugar, the amount of oxygen present, temperature, the
presence of neutralizing agents and possibly some unknown factors.

Acetic acid is, besides the lactic acid, the most common acid formed
by microorganisms. Formic acid (HCOOH) is produced by a very
few bacteria (B. ethaceticus, page 1 1 1) and occasionally by some molds,
while acetic acid is a very common product of fermentation. In the
decomposition of sugars, it is never the only product; other acids, gases
or alcohols are formed besides acetic acid. Pure acetic acid is produced

PRODUCTS OF METABOLISM.

only in acetic fermentations, not from sugar, but from alcohol, by a very
simple process of oxidation.

CH 3 CH 2 OH+ 2 0=CH 3 COOH+H 2 O

Alcohol Acetic acid

If there is not plenty of air present, the oxidation may not become
complete and small amounts of acetaldehyde may form.

CH 3 CH 2 OH+O=CH 3 COH+H 2 O

Alcohol Aldehyde

Some acetic bacteria can destroy acetic acid by oxidizing it completely
CH 3 COOH+ 4 O = 2CO 2 + 2H 2 O

Molds frequently form acetic acid from sugar or starch; the peculiar
acid taste of moldy fruit preserves is due partly to this acid.

Another product of fermentation of sugars which has been mentioned
already in various places, is butyric acid (CH 3 CH 2 CH 2 COOH). It
has the very pungent odor of rancid butter. Butyric acid is formed not
only from carbohydrates, but also from butter fat and from proteins.
The production from cellulose has been discussed at the beginning of
this chapter. The difficulty of giving an equation for butyric fermentation
of dextrose has been pointed out in the chapter on chemical equations.
Butyric acid is, like acetic acid, not the only product of fermentation.
It is always accompanied by other acids and alcohols. The butyric
bacteria are distinguished and named according to the predominating
products of fermentation. They were known as B. (Clostridium) buty-
ricus, B. butylicus, B. lactobutyricus and B. lactopropylbutyricus. Recent
investigations of Bredemann have made it probable, however, that most
of these bacteria are only varieties of the same species. The amounts of
the different acids and alcohols formed by one strain vary with varying con-
ditions so much that a dominance of the one or the other product cannot be
used for diagnostic purposes. Butyric acid is also produced from lactic
acid and from glycerin. These fermentations have a theoretical interest,
because they represent a synthesis by bacteria; glycerin or lactic acid,
with three carbon atoms, are changed to butyric acid with four carbon
atoms.

In the decomposition of sugars by bacteria, mainly monobasic acids
are formed; lactic, acetic and butyric acids are predominant, though
occasionally succinic acid is also found. Yeasts change the sugar to
alcohol and carbon dioxide almost exclusively. The mycoderma

110 NUTRITION AND METABOLISM.

species are able to produce a little acid. The most characteristic quality
of molds is their great oxidizing power which enables them to use many
different kinds of organic substances for food. Besides this oxidizing
quality, they have also the power of causing acid fermentation or alco-
holic fermentation. The acids produced by molds are mostly di- and
tribasic. Besides the acetic acid, oxalic and citric acids are produced
by molds. Oxalic acid is formed, especially by the Aspergillacea, in
large quantities, if some alkali, like calcium carbonate, is present to
neutralize the free acid. Aspergillus niger, the typical oxalic-acid mold,
is supposed to oxidize dextrose according to the following equation:

C 6 H 12 6 + 9 = 3(COOH) 2 + 3 H 2

The yield of oxalic acid is, however, only one-half of what could be
expected from the above equation, because of the very pronounced
oxidizing qualities of this mold; it will oxidize part of the dextrose, or of
the acid, completely to carbon dioxide and water. If the acid is not
neutralized, the mold will finally oxidize all the oxalic acid to carbon
dioxide and water.

Citric acid (CH 2 (COOH)COH(COOH)CH 2 COOH) is formed by
many molds. Some of them produce it in such quantities as to render
possible the manufacture of citric acid from glucose by the use of molds.
The process can be expressed by the following equation:

C 6 H 12 6 + 3 0= C 6 H 8 7 + 2 H 2

The mold uses, however, about twice as much sugar as would be
expected from this formula, because of its strong oxidizing power.

This discussion involving the products of the decomposition of sugars
is far from complete, but aims to touch the most common fermentations.

ALCOHOLS. Besides the carbohydrates, another group of food sub-
stances has to be mentioned which is closely related to the sugars, namely
the higher alcohols. The two main representatives are mannit (CH 2 OH.-
CHOH.CHOH.CHOH.CHOH.CH 2 OH) and glycerin (CH 2 OH.CHOH.-
CH 2 OH). Mannit differs from the simplest sugars only by having two
additional hydrogen atoms which indicates that the aldehyde or ketone
group of the sugar is changed into an alcohol group in the mannit. The
products of mannit fermentation are very similar to those of sugar fer-
mentations. Different organisms give different products among which
alcohol and acetic acid prevail. The Strept. pneumonia decomposes it
in the following way:

PRODUCTS OF METABOLISM. Ill

6C 6 H 14 O 6 + H 2 O = 9C 2 H 5 OH+ 4 CH 3 COOH+ ioCO 2 + 8H 2

Mannit Alcohol Acetic acid

while the B. ethaceticus makes formic acid instead of hydrogen.

14 O 6 + H 2 O = 5C 2 H 5 OH+CH 3 COOH+CO 2 +5HCOOH

Glycerin is decomposed in many ways. It may be oxidized com-
pletely or it is fermented into the same products which are derived from
sugars. The butyric fermentation of glycerin has already been mentioned.
There are also some compounds formed from glycerin which are not
found in carbohydrate decomposition. These are the products of partial
oxidation, glyceric aldehyde and glyceric acid

CH 2 OH CH 2 OH CH 2 OH

I I I

CHOH CHOH CHOH

i r i

CH 2 OH COH COOH

Glycerin Aldehyde Acid

The only monovalent alcohol of general occurrence, ethyl alcohol,
undergoes only one method of decomposition, namely that of oxidation.
This may be incomplete resulting in aldehyde or acetic acid (p. 109) or
if the combustion is total, in carbon dioxide and water. The acetic-acid
formation is the result of the acetic bacteria. Total combustion is
characteristic of molds and Mycodermce.

ORGANIC ACIDS are valuable food for many microorganisms, in the
free form as well as in the form of salts. On acid liquids like fruit juices
and jellies, sour milk, sauerkraut, pickle brine, a flora of acidophile organ-
isms develops, among which the molds of the Oidium type and the Myco-
dermce predominate, while bacteria are found in insignificant numbers.
These acidophile organisms destroy organic acids, especially lactic and
acetic acid, by total combustion. The oxidation process for lactic acid
would be

For all other acids, the equation is very similar. Some acids are
fermented by bacteria by processes more or less similar to sugar fermen-
tations. There are so many different possibilities that only a few exam-
ples can be mentioned. B. tartar icus decomposes tartaric acid to acetic
and succinic acid. The same organism can also ferment succinic acid,

112 NUTRITION AND METABOLISM.

the products of which have not yet been analyzed. The calcium salt of
lactic acid gives with B. bulylicus butyric acid with a little acetic acid.

FATS are esters of glycerin with fatty acids. They can be hydrolyzed,
like all esters, and thus give free acids and glycerin. The following
equation shows the hydrolyzation (saponification) of tristearin:

(C 18 H 35 O 2 ) CH 2 + H 2 O C 18 H 35 O 2 H CH 2 OH

I I

(C 18 H 35 2 ) - CH + H 2 = C 18 H 35 2 H+ CHOH

I I

(C 18 H 35 2 )-CH 2 +H 2 C 18 H 35 2 H CH 2 OH

Tristearin Stearic acid Glycerin

Only a few microogranisms are able to decompose fat to glycerin and
fatty acids, and these appear to be the only organisms which can destroy
fat. This saponification is the only known method of fat decomposition.
After this first step is accomplished, the glycerin and acids are open to
decomposition by the methods described above, either by complete
combustion or otherwise. The most significant change, perhaps, in
the decomposition of fat is a production of free acids from the neutral
fat molecule.

There are still a few compounds occurring in nature which should be
considered, inasmuch as they are products of bacterial activity, and are
more or less constantly forming. The most important ones are methane
and hydrogen. Both of these gases are used by certain bacteria in a
very unusual metabolism as a source of energy.

B. methanicus (or B. oligocarbophilus) oxidizes methane completely.

This organism requires no other organic food. It is retarded or inhibited
by organic substances since it takes the carbon only hi form of methane
or carbon dioxide and the nitrogen only in mineral form as nitrate or
ammonia. In its life requirements, it resembles very much the nitrate and
sulphur bacteria. This bacillus is also able to oxidize carbon monoxide
to dioxide and to get all the necessary food and energy supply: in this way.
The "hydrogen" bacterium is called B. pantothropus by its discoverer
because it is able to live on ordinary laboratory media as well. If no
other substance is available, it will oxidize hydrogen. This oxidation
does not occur directly, as would be expected, following the equation

PRODUCTS OF METABOLISM. 113

but it requires the presence of carbon dioxide, which is reduced by the
hydrogen to formaldehyde.

CO 2 + 4 H=HCOH+H 2 O.

The formaldehyde is then used by this bacillus as a food and oxidized.

The presence of traces of formaldehyde in the cultures can easily be
proved.

PRODUCTS FROM NITROGENOUS COMPOUNDS.

The products resulting from microbial action upon nitrogenous
compounds are largely unknown or poorly defined. They are very num-
erous and many of them are of such complex nature that they cannot be
determined accurately; even the testing for their presence or absence is
often very difficult. It is therefore utterly impossible and quite unneces-
sary to mention even all the known products and the equations of their
formation. The aim is to give a thorough understanding of the gradual
degradation of the protein molecules to smaller molecules of less com-
plex nature, until finally crystallizing bodies are reached. These are
well-defined chemical bodies and the further decompositions can be
followed more exactly by chemical methods.

As in the preceding chapter, the most complex compounds will be
discussed first. The keratin bodies of hair, epidermis, horn, are absolutely
insoluble and only very few organisms can attack them. The products
have been studied but very little and do not seem to differ essentially
from the cleavage products of protein bodies.

PROTEIN BODIES are as numerous as plants and animals. Each
species of organism seems to have its particular protein which differs
from that of other species. With the more highly developed organisms,
there are several distinctly different proteins found in the same individual
in different parts of the body. The chemical structure of the protein mole-
cule is not known. The constituents, carbon, oxygen, hydrogen, nitrogen,
and sometimes sulphur and phosphorus can be determined in their
relative amounts without, however, furnishing any knowledge of the
structure of the molecule. The molecular weight of proteins is estimated
to be at least 10,000, while the weight of the very large molecule of sac-
charose is only 342. The protein molecule can be broken up into smaller
8

114 NUTRITION AND METABOLISM.

molecules. This is generally believed to be a hydrolytic process similar
to the decomposition of starch to maltose, or of saccharose into dextrose
and levulose. The first products of protein decomposition do not differ
essentially from the original protein, but they can be hydrolyzed again and
again, until finally products of crystalline nature are found which are well-
defined chemical bodies. Among the very first products of protein
degradation it is usually impossible to determine single compounds, but
several groups of compounds may be separated by certain precipitants, as
acetic acid, ammonium sulphate, zinc sulphate, copper sulphate, tannic
acid and others. In order to determine the degree of protein degradation,
e.g., in the analysis of cheese, it is customary to determine the nitrogen of
compounds precipitated by these various reagents, and state it in per-
centage of the total nitrogen. Thus the terms "water-soluble nitrogen,"
"acid-soluble nitrogen" and others originated, meaning the nitrogen of
the compounds soluble hi water or in acid respectively. Some of these
groups of degradation products have been named and defined more
accurately, of which the albumoses and peptones are the most common
and best described compounds. Their chemical nature and structure is,
however, just as little known as that of the protein bodies.

The amino-acids are the first well-known compounds of protein de-
composition. They are organic acids, in which a hydrogen atom is
substituted by a NH 2 radical. Some of them are simple compounds,
as the amino acetic acid NH 2 CH 2 COOH and also the amino-capronic
acid, usually called leucin, (CH 3 ) 2 CH CH 2 CH(NH 2 ) CO OH. Others
are of a more complex nature, such as the tyrosin or hydroxy-phenyl-
amino-propionic acid, C 6 H 4 (OH) CH 2 CH (NH 2 ) COOH, and the
tryptophan or indol-amino-propionic acid, C 8 H 6 N CH 2 CH(NH 2 ) COOH.

Of other nitrogenous products which are not amino-acids, a few
are of striking significance. The very disagreeable odor of putrefying
proteins and of excreta is due to indol (C 8 H 7 N) and methyl-indol or skatol
(C 8 H 6 N CH 3 ). Indol gives a rose color with nitrites in acid solution,
and this convenient reagent is used in the identification of bacteria.
Another group are the amins, hydrocarbons in which one or several
hydrogen atoms are replaced by an NH 2 radical. The simplest amins
are the methyl-amins, of which the tri-methylamin (CH 3 ) 3 N is produced
by several bacteria. The fishy odor of the brine of salted herring is
largely due to this compound. In this group belong also a large number
of the so-called ptomains.

PRODUCTS OF METABOLISM. 1 15

The ptomains (p. 417) are alkaloid-like bodies of basic character and
of more or less well-known structure. Some of them are notorious for
being very strong poisons, while others are quite harmless. These bodies
are usually called ptomains because they were first discovered in putrefy-
ing corpses. The best-known compounds of this character are the
putrescin or tetra-methylen diamin [NH 2 (CH 2 ) 4 NH 2 ] and the cadaverin
or penta-methylen diamin [NH 2 (CH 2 ) 5 NH 2 ], which can scarcely be
considered poisonous. The methyl-guanidin

/
HN=C<

X NHCH 3

may be mentioned as an example of a very poisonous ptomain. Another
poisonous ptomain is the neurin CH 2 =CH N(CH 3 ) 3 OH which has
been found frequently as a product of putrefaction.

All the products of protein degradation mentioned so far may be
formed in the absence of oxygen. The decomposition of protein to
amino-acids is partly hydrolytic, and probably it consists also of intra-
molecular changes comparable to the alcoholic and lactic fermentations.
None of the processes mentioned so far requires oxygen. Even the
amino-acids can be hydrolyzed further under anaerobic conditions, form-
ing ammonia and hydroxy-acids.

HOH+NH 2 CH 2 COOH=NH 3 +HOCH 2 COOH

Amino-acetic acid Hydroxy-acetic acid

In this way, ammonia is formed quite commonly from amino-acids.
In the products of protein degradation mentioned above only those
compounds have been considered which contain nitrogen. It is quite
evident, however, that in the cleavage of the large and complex protein
molecules, certain pieces of the broken molecule will contain no nitrogen.
An example of how such nitrogen-free compounds might be formed is
given in the hydrolysis of amino-acetic acid to hydroxy- acetic acid. Many
organic acids, like acetic, butyric, capronic, benzoic and phenylacetic
acids are quite generally found among the products of putrefaction.
Alcohols too, especially benzene derivatives like phenol and cresol, are
not unusual at all. Gas is often formed in putrefaction, especially
carbon dioxide and hydrogen; occasionally these gases are mixed with
traces of nitrogen and methane. Carbon dioxide is formed to some
extent by the hydrolysis of organic acids, as the following example shows:

Il6 NUTRITION AND METABOLISM.

Hydroxy-phenyl-acetic acid Cresol

The formation of hydrogen, methane, and nitrogen is not as easily
explained and probably means a more complicated change of the molecule
than a simple hydrolysis.

Many protein compounds contain, besides the organic elements,
carbon, oxygen, hydrogen and nitrogen, larger or smaller amounts of
phosphorus and sulphur. The phosphorus compounds may be changed
to phosphine (PH 3 ), which is a gas of a strong disagreeable garlic odor.
Generally, however, the phosphorus of protein after its degradation is
found as phosphoric acid (H 3 PO 4 ). Very little is known about the
phosphorus of organic compounds and the changes it may undergo in
the putrefaction process.

The sulphur of proteins is commonly changed to hydrogen sulphide
(H 2 S). This may be the result of a hydrolytic cleavage or of a reducing
process. Some microorganisms are able to form mercaptan (CH 3 SH),
a compound of very foul penetrating odor.

The production of all these compounds may take place under strictly
anaerobic conditions. Hydrolysis and intramolecular rearrangements
of the atoms within the molecule suffice to account for these changes.
The products of this anaerobic decomposition are mainly ammonia,
amins, amino-acids, alcohols (phenol), acids (acetic, butyric, capronic),
hydroxy-acids, phosphoric acid, hydrogen sulphide. Many micro-
biologists emphasize the distinction between the putrefaction as an
anaerobic process, and the decay as an aerobic process. The anaerobic
putrefaction produces offensive odors caused by indol, skatol, butyric
and capronic acids, amins, phenol, and hydrogen sulphide, while the
aerobic decay does not show such products. This distinction between
putrefaction and decay is an artificial one, however, and cannot always
be carried through. Facultative anaerobes will decompose proteins
in different ways with air and without, and in nature both processes
usually take place at once. It has been mentioned in the chapter on
oxygen requirements that many obligate anaerobes can grow exposed
to the air if at the same time some aerobic organisms develop in the same
medium. This is the case in most of the decompositions occurring in
soil, in the dead leaves of the forests, in manure piles, in spoiling foods.

If oxygen has free admittance to the decomposing protein, the above

PRODUCTS OF METABOLISM. IIJ

mentioned products will be readily oxidized to simpler compounds and
finally to carbon dioxide, water, ammonia; the ammonia may be oxidized
further to nitrous or nitric acid, as has been mentioned already in several
chapters. The final oxidation of the carbon compounds is the complete
combustion, accomplished at once, or in successive steps by different
organisms. Hydrogen sulphide is oxidized to sulphur or sulphuric acid.
Thus we obtain as the final products of protein degradation carbon
dioxide, water, ammonia, nitrates, nitrogen, hydrogen, hydrogen sulphide,
sulphuric acid, phosphoric acid. The protein is completely mineralized.
UREA, URIC ACID, HIPPURIC ACID are the products of protein digestion
in the animal body. Urea is a normal product in the urine of many
animals, especially the carnivora, while hippuric acid is produced
mainly by herbivora and uric acid is one of the main constituents in the
excreta of birds and snakes. The fermentation of urea to ammonium
carbonate has been discussed extensively in the chapter on metabolism,
page 85. It is a simple hydrolysis.

CO(NH 2 ) 2 + 2 H 2 O = CO 3 (NH 4 ) 2 .

Only one group of bacteria, the urea bacteria, can perform this change,
a few molds are also said to cause this fermentation. Hippuric acid is of
much more complex nature, benzoyl-amino-acetic acid. It is probably
split up by bacteria into benzole acid and amino-acetic acid

C 6 H 5 CO. NH CH 2 COOH +HOH=C 6 H 5 COOH+NH 2 CH 2 COOH

Hippuric acid Benzoic acid Amino-acetic acid

Uric acid, also a complex organic body, can be oxidized through the
agency of bacteria by several processes which yield ammonium carbonate
and carbon dioxide. Often urea is an intermediate product forming in
the following manner:

Uric acid Urea

The decomposition of these three compounds takes place continuously
in manure piles and in sewage. The final result is, as with proteins, a
complete mineralization.

PRODUCTS FROM MINERAL COMPOUNDS.

Minerals are used by microorganisms for cell construction almost
exclusively; consequently, they do not leave the living cell like fermenta-
tion products. But a few organisms can actually decompose mineral

Il8 NUTRITION AND METABOLISM.

matter and when this takes place mineral products are secreted. Two
main processes can be distinguished, oxidation and reduction.

OXIDATIONS are the result of the organisms seeking a supply of energy.
Several oxidations of minerals have been indicated previously, as the
oxidation of ammonia to nitrites, of nitrites to nitrates, of hyposulphites
to sulphates, of hydrogen sulphide to sulphur and of sulphur to sulphuric
acid, of ferrous salts to ferric salts. All these microbial changes are simple
processes and can be followed by chemical analyses much more easily
than organic fermentations. The organisms which cause these changes
do not thrive in organic substances and for this reason pure cultures
can be obtained only with difficulty. Their activity is of great impor-
tance in soil fertility.

REDUCTIONS of minerals by organisms have not been discussed.
They, too, are of great significance. As a typical example, nitrates may
be reduced to nitrites, to ammonia, to nitrogen gas, and, rarely, to nitro-
gen oxides. The reduction may be performed either by the direct removal
of oxygen, or by the formation of hydrogen. The reduction of nitrates
to nitrites can be written in the following three ways:

KNO 3 - O = KNO 2
KNO 3 = KNO 2 +O
KNO 3 + 2H= KNO 2 +H 2 O .

The result in all three cases is the same. Many bacteria can reduce
nitrates to nitrites or to ammonia. A few can reduce them to nitrogen.
These "true denitrifiers" are found in soil and in old manure. Their
reducing process is as follows:

Ca(NO 3 ) 2 - 50= CaO+ 2N.

Nitrates are reduced through the efforts of the organism to secure a
supply of oxygen. The denitrifying bacteria have strong oxidizing prop-
erties; they take oxygen from all sources possible. If cultures of denitri-
fying bacteria are well aerated, as in soils with a proper moisture content,
they scarcely attack the nitrates, while they will reduce them in ordinary
liquid cultures so fast that the escaping nitrogen gas forms a froth on top
of the nitrate solution. Denitrifying bacteria need the oxygen to oxidize
organic matter. They cannot live without organic food. It has been
stated previously that the oxidation of ammonia to nitrates liberates
energy, consequently the reduction of nitrates must absorb energy, and

PRODUCTS OF METABOLISM. 119

nitrates can be reduced by the denitrifying bacteria only if they can use the
oxygen to advantage; that is, if they can obtain by the oxidation of organic
bodies more energy than they expend in the reduction of the nitrates.
The same rule holds true with other reductions.

Sulphates are reduced in a very similar way to hydrogen sulphide

H 2 SO 4 - 4 O = H 2 S.

Tap-water, containing calcium sulphate, often forms hydrogen sulphide
if shut off from the air for some time.

While only a few bacteria reduce sulphates, many reduce sulphites or
sulphur to hydrogen sulphide. The potassium and sodium salts of selenic
and telluric acid (H 2 SeO 4 and H 2 TeO 4 ) are reduced by certain organisms
and not by others. The reduction results in a colored precipitate; this
reaction has been suggested as a diagnostic means to distinguish different
species. The reduction of arsenious oxide to arsin (AsH 3 ) is used as a
very delicate test for arsenic; it is applied in. the detection of arsenical
poisoning. The contents of the stomach are sterilized and inoculated
with Penicillium brevicaule (p. 22), the "arsenic mold." This will re-
duce most arsenious compounds to arsin (As H 3 ) or to diethyl arsin,
As H(C 2 H 5 ) 2 , both of which are easily recognized by their very pro-
nounced garlic odor.

UNKNOWN PRODUCTS OF PHYSIOLOGICAL SIGNIFICANCE.

Among the products of microbial action, there are certain substances
which must be mentioned because of their importance, though their
quantity is insignificant compared with the ordinary products of fermen-
tation. These substances can be divided into four groups: pigments,
aromatic compounds, enzymes, and toxins. The chemical structure of
pigments and of many aromatic substances is scarcely known; and as
far as enzymes and toxins are concerned, it is not even determined
whether or not they are of protein nature. The last two groups are
known only by their actions, while the pigments are very conspicuous
and cannot possibly be overlooked.

PIGMENTS have naturally attracted the attention of microbiologists
ever since pure cultures were known, and many investigators have tried
to explain the nature and the meaning of pigments. All experiments
concerning the purpose of pigment-formation by microorganisms have been

120 NUTRITION AND METABOLISM.

without results. It is not known that the pigment is of any material
advantage to bacteria; for it is possible to cultivate colorless strains of
pigment bacteria which grow apparently as well as the orginal pigmented
culture. Again, pigments cannot take the place of the chlorophyl in
plants except perhaps the bacteriopurpurin of the purple bacteria. It
does not even protect the cells against intense light, because the pigmented
organisms are not more resistant than the corresponding colorless
"sports." The only exception are the colored spores of the molds,
especially Penicillium and Aspergillus, which are very resistant to light,
while the spores of Oidium are killed just as easily as the mycelium.
Pigments cannot be considered as reserve substances, since many pig-
ments are excreted and remain outside the colorless cells. Pigment
production may be incidental. It is possible that the waste products
of certain organisms happen to be colored.

After Beyerinck, the chromogenic bacteria may be divided into three
classes:

1. Chromophorous bacteria, in which the pigment is placed in the cell
and has a certain biological significance analogous to the chlorophyl
of higher plants. In this division belong the green bacteria discovered by
Van Tieghem and Engelmann and the red sulphur bacteria or purple
bacteria.

2. Chromoparous or true pigment-forming bacteria, which set free the
pigment as a useless excretion, either as a color-body or as a leuco-body
which becomes colored through the action of atmospheric oxygen. The
individuals themselves are colorless and may under certain conditions
cease to form pigments. To this class belong B. prodigiosus, Ps. Syn-
cyanea, Ps. pyocyanea, and others.

3. Parachrome bacteria, which form the pigment as an excretory pro-
duct but retain it within their bodies, as B. janthinus and B. violaceus

When the pigment is soluble in water, as those produced by Ps. pyo-
cyanea and the fluorescent bacteria, it diffuses through the medium.
When the pigment is not soluble, it either lies within the cell wall or be-
tween the individuals.

This classification furnishes some details concerning the methods of
pigment production, which depends upon the presence of certain media;
according to Sullivan, sometimes certain mineral salts, sometimes sugar
will stimulate chromogenesis. The same is true with molds. Very
brilliant colors appear with certain species of molds if grown on cellu-

PRODUCTS OF METABOLISM. 121

lose or on fat, while on gelatin the pigment is not produced. The tem-
perature is an important factor. A large number of chromogens produce
no pigment when grown in the incubator. It is possible to obtain non-
pigmentation with many species by propagating them through many
generations at high temperatures. Oxygen also is necessary for the
chromogenesis of many bacteria. Some need a short exposure to day-
light in order to produce their pigment, while cultures grown in absolute
darkness may remain colorless. Strong sunlight, however, will check
pigment production in the same degree as do antiseptics and other
harmful influences.

The chemical nature of microbial pigments is little known. They are
distinguished according to the solubility in various liquids, water, alcohol,

FIG. 48. Bacteriopurpurin, from a Rhodospirillum, crystallized from a chloroform

solution. (After Molisch.)

ether, chloroform, benzol, and other solvents, and according to the change
of color caused by acid and alkali (Fig. 48) . A group of carotin bodies,
named because of their similarity to the pigment of carrots, the prodigiosin
bodies, named after B. prodigiosus, the fluorescent pigments and per-
haps a few other groups are distinguished, but their chemical nature is
rather vague as yet. The absorption of distinct lines of the spectrum
by solutions of these pigments is claimed to be a very reliable means of
distinguishing the pigments of different species.

AROMATIC SUBSTANCES constitute another group of metabolic prod-
ucts. The chemical analysis accomplishes more with these compounds
than with pigments, since they are frequently well-known compounds.

122 NUTRITION AND METABOLISM.

The main difficulty arising in their identification is in the very minute
quantities of the products available. Some substances with strong,
mostly very disagreeable odors have already been mentioned: indol,
skatol, hydrogen sulphide, mercaptan, the amins and ammonia, butyric
acid, and some of the higher alcohols. There remain to be mentioned cer-
tain oils and esters giving rise largely to pleasant aromas. The formation
of aromatic oils has been established although their nature is entirely
unknown. The same is true with the esters. The substance causing the
fishy flavor in butter is volatile with steam and is neither of an alkaline
nor acid nature. The strong odor of freshly plowed earth is caused by
an Actinomyces; the odor can be traced to a very volatile oil the nature
of which has not been determined. The aroma of fermented liquids-
wines, beers, and many others is partly due to compounds constituting
the fermenting material, and partly to the fermenting agent. Some
yeasts are known to produce fruit-esters, as succinic-acid-ethylester
and the corresponding esters of malic and other acids. Besides,
some glucosides may be split and traces of hydrocyanic acid and benzoic
acid may be liberated. The change of flavor with the aging of wines is
probably more a chemical than a biochemical change.

ENZYMES AND TOXINS. Among the most interesting and least
understood products of microbial action are the enzymes and the toxins.
These two groups are related in many respects. The enzymes will be
discussed extensively in the following chapter and toxins are treated
more extensively in Pt. Ill, Div. VII, Chap. II. Toxins and enzymes are
formed by the cells in such small quantities that they would never have
been discovered by ordinary chemical means were it not for the unusual
effects which they produce, the enzymes acting upon food substances,
and the toxins acting physiologically upon organisms. Toxins and
enzymes are chemically unknown. It is assumed that they are chemical
bodies, but even this has not been proved. A pure toxin has never been
obtained and we have no criterion for its purity. The presence of a
toxin is recognized only by an animal test, and in this way the com-
parative concentration can be determined approximately. Such standard-
ization of toxin solutions is only comparative, however, and gives no clue
as to the actual amount of toxin present. Not all animals are sensitive
to all toxins. It is quite possible that all bacteria produce compounds
with chemical qualities similar to toxins, and only a few of them happen
to react upon men or animals.

PRODUCTS OF METABOLISM. 123

Toxins are not always the product of microbial action. Vegetable
toxins or phytotoxins are known, among which the ricin of the castor-
oil bean is perhaps the most studied representative. The best-known
zootoxin is the rattlesnake poison. These non-microbial compounds
have the same quality as the microbial toxins they are extremely poison-
ous. Toxins are largely responsible for contagious diseases as diphtheria,
tetanus and perhaps many others. If a culture of these organisms is
filtered through a porcelain filter which removes all bacterial cells, the
filtrate injected into an animal will cause the disease with all its accompa-
nying symptoms though there are no microorganisms introduced into the
animal body. If the filtrate is heated, however, no effect will take
place after the injection, because heat destroys the toxin. The amount
of toxin that will kill an animal is extremely small; 0.000005 mg. of the
purest tetanus toxin will kill a mouse, 0.0007 m - f r i cm w i^ kill a rabbit,
less than 0.23 mg. of tetanus toxin will kill an adult man. The body
of an animal or man forms an anti-body against the toxin which neutralizes
its poisonous action. Anti-bodies are also formed against enzymes
injected into an animal.

Toxins are very sensitive to heat. A short exposure to temperatures
between 80 and 100 will inactivate them. They are also very sensitive
to light. While some toxins are secreted, others are retained within the
cells of microorganisms, and never leave them until the cells die or disin-
tegrate. Ptomains, which are also metabolic products of microorganisms
and sometimes cause poisoning, differ from the toxins in their resistance
to heat and light (p. 115). Ptomains differ in no way essentially
from ordinary organic compounds; the animal or human body produces
no anti-ptomains to counteract their poisonous effects. There is no
chemical relation whatever between toxins and ptomains, and the
physiological effects are also quite different, though they both cause
poisoning.

Toxins are not essential products of the metabolism of pathogens.
Strains of pathogenic bacteria can be bred which do not produce toxins
as chromogens can be bred without pigment, or lactic bacteria which
do not produce acid. The strains which lose their pathogenicity grow
better on artificial media, but are less able to produce disease in the
animal. They may regain the power of producing toxin if passed through
the body of the animal. The real object of toxin production by micro-
organisms is not known; the microorganisms derive no apparent benefit

124 NUTRITION AND METABOLISM.

from the death of the animal ; the toxin may be an incidental waste product
like the pigment.

ROTATION OF ELEMENTS IN NATURE.

All organic matter on earth is undergoing continuous change. Or-
ganisms grow and decay. The same carbon and nitrogen atoms which
constitute the organic world of to-day constituted it thousands of years
ago. The amount of carbon, nitrogen, hydrogen and of all other elements
of life on earth is limited, and the same atoms will be used for the future
generations of life that constitute the present. There are two mam fea-
tures in organic life, construction (anabolism) and destruction (katabolism) .
Construction is mainly the task of green plants, enabled by the chlorophyl
to use the energy of sunlight in building up organic substances from
minerals, water and carbon dioxide. Destruction is caused mainly by
animals and other organisms which have to break down organic matter
in order to exist. These two factors keep the atoms of the organic world
in perpetual rotation.

In this circulation of the elements it is necessary that all compounds
of organic nature be decomposed finally to a form available for plant
food. If this were not the case, the indestructible compound would
sooner or later accumulate in such enormous quantities that the elements
constituting this body would be removed entirely from general circulation.
Let us suppose, as an illustration, that for some unknown reason, all urea
bacteria on earth would die. Urea could be decomposed no more, and
the plants, unable to use urea as a source of nitrogen in place of nitrates,
would get but little benefit out of stable manure. All urea would pass
gradually undecomposed into rivers, lakes, and finally into the ocean
where it would accumulate continuously. The enormous quantities
of nitrogen taken out of circulation would cause a decreasing growth of
plants, and life would soon cease because of lack of nitrogen. For this
reason all products of living organisms must be further broken up by
some other organisms, and we find that the destructive work is to a large
extent the task of microorganisms. Many products of organic life
cannot be broken down by organisms other than bacteria, and therefore
bacteria are absolutely necessary for the circulation of the elements and
for life on earth. Bacteria and green plants are an absolute necessity
for the maintenance of life, the one breaking down, the other building up,

PRODUCTS OF METABOLISM.

I2 5

one dependent upon the products of the other; animals, however, could
be excluded from the circle without interfering with a continuation of
life on earth.

CARBON CYCLE. Carbon is the main element in organic nature, and
so dominant that the term carbon compounds is practically identical with
organic compounds.

Carbon is contained in the carbon dioxide of the air which is usually
considered a mineral or inorganic compound. It is absorbed in this
condition by the green plants, and is changed by the chlorophyl gran-

anisms

Carbondioxicle

Carbohydrates
fat, Protein

FIG. 49. Carbon cycle.

ules of the leaves to organic compounds of various types, either to car-
bohydrates (cellulose, starch, sugars) or to fats, or to protein substances,
occasionally to organic acids or other compounds. The plants will either
die and decay, or will be eaten by animals. In the first case, the decay
will be caused exclusively by microorganisms; if the plants are eaten,
they will be digested; part may be used to build up the animal body or
stored as reserve substances, largely fat and protein. If the animal
dies, a decomposition process will take place, which breaks down the
organic compounds to simpler products and finally the carbon will be
completely oxidized to carbon dioxide. Even the marsh-gas which
might be liberated in this process will find organisms that oxidize it to

126

NUTRITION AND METABOLISM.

carbon dioxide and water. Every product will find an organism to break
it up further until it is completely disorganized and the carbon atoms
can start the same circulation anew. Undoubtedly as long as organic
life has existed on earth, microorganisms have been present, in order
to render the dead organic matter again available for plant and animal
life. Figure 49 gives a schematic illustration of the carbon cycle;
the microbial activity is marked by heavy lines.

NITROGEN CYCLE. Nitrogen shows the same continuous change as
carbon. Plants take up nitrogen in mineral form usually as nitrates.

Dead
Organisms

//2t rates

Pro tein

FIG. 50. Nitrogen cycle.

The plants change this mineral nitrogen to the most complex bodies,
proteins, where it is combined with the other elements of organic nature.
The plants may be eaten by animals; part of the protein is then digested
to urea or hippuric or uric acid, which in turn are readily decomposed
by microorganisms to ammonia (Fig. 50). Part of the protein will be
stored in the growing animals, and if the animal dies, the body will
decay or putrefy, and the nitrogenous compounds of that body will pass
through the various stages of decomposition to the final product, am-
monia. Ammonia is then oxidized to nitrites and nitrates, when the nit-
rogen cycle is completed.

There is, however, one discrepancy in this cycle. It has been men-
tioned already that some organisms are able to reduce nitrates to nitrogen
gas. This is one of the "leaks" in the rotation of elements which would

PRODUCTS OF METABOLISM. 127

be disastrous to organic life on earth if there were no means to compensate
for the loss of nitrogen in circulation. Imagine what would happen if
there were no such compensation! Part of the nitrate in the soil is
destroyed, the nitrogen gas escapes into the air and is as indifferent as the
nitrogen of the atmosphere, lost to organic life forever. More nitrate
would be produced from decaying organic matter and would be destroyed.
After a certain time, this continuous loss of nitrogen would become quite
noticeable in the growth of plants; there would be a scarcity of nitrogen
in soil, since part of it is lost continuously. Finally, the plants would
cease to grow because the nitrogen in the soil would be exhausted.

Sulnidt.

n

U

rgan/3/nj

FIG. 51. Sulphur cycle.

The compensation for this destruction of available nitrogen is found
in the nitrogen-fixing bacteria, which either living in symbiosis with
leguminous plants or growing independently in the soil, have the power
to use the atmospheric nitrogen for the formation of their own protoplasm.
Thus, organic nitrogen is produced from nitrogen gas and the constancy
of organic life is guaranteed.

SULPHUR CYCLE. Little more can be said about sulphur, since the
rotation is quite similar to that of nitrogen. Plants will take sulphur
usually in the form of 'sulphates and make protein compounds containing
a certain amount of sulphur (Fig. 51). These bodies are either di-
gested by higher animals or broken down by putrefaction to the final

128 NUTRITION AND METABOLISM.

product, hydrogen sulphide, which is oxidized by the sulphur bacteria
first to sulphur, then later to sulphates.

PHOSPHORUS CYCLE. The cycle of phosphorus has not been worked
out completely, but from the discussion in the last pages, it is plainly seen
that a simple cycle very much like the ones above must exist. It is
probably much simpler because phosphorus does not enter as easily into
organic compounds as nitrogen.

PHYSICAL PRODUCTS or METABOLISM.

PRODUCTION or HEAT. It has long been known that fermentation
produces heat. The rise of temperature is usually not very great. In
lactic fermentation it amounts to about i, in alcoholic fermentation to
2 or 3, but in certain processes the heat liberated is considerable, as in
the fermentation of manure, of ensilage, of vinegar, and in others.

The cause of heat formation is quite evident from the discussion on
page 98. Organisms decompose organic matter which means a liber-
ation of energy. Part of this energy is used for the continuation of life-
processes; the rest, usually the larger part, is not employed and appears
in the form of heat. The amount of heat produced can be measured
directly with the thermometer if great care is taken that no heat is lost
by radiation or by evaporation of water.

The best known fermentation of this character is the vinegar fermen-
tation. In the quick-vinegar process (page 456) the temperature rises
sometimes as high as 10 to 15 above the temperature of the room and
the vinegar manufacturer uses the heat produced by the bacteria to keep
the generators at the optimum temperature. If the process is not con-
trolled carefully, the vinegar bacteria are likely to produce sufficient heat
to kill themselves.

The heat produced in the fermentation of manure, especially horse
manure, is used in the hot-beds to cultivate and force young plants. In
the manure pile, great heat production is not desirable because high
temperatures will volatilize the ammonia; the tight packing of manure
which keeps out the oxygen will prevent too strong bacterial action. The
highest temperature in silos which has been recorded is about 70, but
the best silage is secured by keeping the temperature below 50. Ensilage
fermentation is not thoroughly understood, however, and no accurate
statements can be made as to the cause of the increase in temperature.

PRODUCTS OF METABOLISM. I2Q

Sometimes the temperature in silos does not exceed 35. The curing of
hay is usually accompanied by a rise of temperature. For some time it
was believed that the spontaneous combustion of hay was mainly due to
microorganisms, but it has been shown recently that even sterile hay will
show a rise of temperature under certain conditions. This does not
exclude the formation of heat in hay by microorganisms under other
circumstances. The heating of tobacco, of green or moist grain or corn
is probably not of bacterial origin, but due to chemical oxidation or to
the respiration of the living plant tissue.

PRODUCTION OF LIGHT. The light-producing or photogenic organ-
isms are quite numerous and occur more frequently than is generally
believed. The phosphorescence of decaying tree stumps and leaves in
the woods and of meat and fish in the cellar are well-known phenomena.
The phosphorescence of wood and leaves is generally caused by Hypho-
mycetes; certain mushrooms have this quality in a very high degree.
The light of meat and fish is usually generated by bacteria, of which at
least twenty-six species have been described.

Many experiments have been carried on in order to discover the nature
and origin of the light, but, so far, few results have been obtained. The
phosphorescence is due to an oxidation process; all photogenic organisms
cease to generate light when the oxygen is removed. As soon as they
come in contact with oxygen again, they produce light immediately, and
this sudden flashing is used occasionally by physiologists as a very delicate
test for oxygen. The light appears to be produced always within the cell;
no cell product has ever been found to give rise to light outside the cell.
It is possible that a chemical compound is formed in the cell which gener-
ates light when in contact with oxygen.

The life processes of the photogenic microorganisms are not necessarily
connected with the formation of light. Photogenic bacteria are known
to lose the power of light production as the chromogenic bacteria may
lose the power of pigment production. Phosphorescence has, like pig-
mentation also, no bearing upon the development of the cell, and the light-
giving compounds may be regarded as incidental waste products. Cer-
tain chemical bodies stimulate light production, while others favor the
growth only. One of the most important factors in the production of light
is sodium chloride.

CHAPTER III.
MECHANISM OF METABOLISM.

GENERAL THEORY OF METABOLISM.

ANABOLISM AND KATABOLISM.- The study of the physiological
mechanism of the cell is the most interesting and most difficult problem
of biological research, for it tries to discover the secret of life processes.
The physiological mechanism of the cell consists of three parts generally
distinguished one from the other, being interdependent upon each other
Fermentation, intra- and extra-cellular, katabolism, the dissociation of
cell substances, and anabolism, the assimilation of food in the synthetic
processes of the cell. The katabolic processes have been studied suc-
cessfully for a long time, and our knowledge of the digestion of organic
and inorganic substances has a definite chemical basis. Digestive pro-
cesses can be accomplished in vitro, but the cell is required to furnish
the digestive agent which, after being removed from the cell, may be
treated as a definite chemical compound. The katabolism, consisting
of many destructive chemical processes, some of which are well-defined
and some wholly unknown, represents the degeneration of the cell. The
study of the anabolic or synthetic processes is to a large extent speculation.
Very meager is the positive knowledge of the chemical changes involved,
and only the very simplest of these reactions can be duplicated in vitro.

The problems of digestion have attracted the attention of scientists
long before microorganisms were known, and the knowledge of the digest-
ive processes of man and animals have helped the microbiologist in
many ways. Again, many results of microbiological research which
could be obtained only with single-celled organisms are employed now
to great advantage in general physiology.

INTRA- AND EXTRA-CELLULAR FERMENTATION.

DECOMPOSITION OF INSOLUBLE FOOD. It has been stated before that
many microorganisms feed upon cellulose, starch, fat, gelatin, keratin
and other insoluble compounds. It has also been previously stated that
microorganisms, with the exception of some protozoa, depend upon

130

MECHANISM OF METABOLISM. 131

soluble food since they have no means of incorporating insoluble com-
pounds into their protoplasm. The protoplasm, however, must be con-
sidered the center of metabolism, and the digestion of food and the forma-
tion of energy must take place in the protoplasm if the cell is to profit by
it. Since the food cannot diffuse into the cell, and the protoplasm does
not diffuse out, the food must be dissolved. This is accomplished by
the cell itself, which secretes certain agents having peculiar qualities.
These agents, the so-called enzymes, act upon the insoluble foods,
changing them into soluble compounds which then can diffuse into
the cell where they are digested or fermented. The final digestion or
fermentation of the food must take place within the cell. Energy
production outside the cell serves the same purpose as a stove
outside the house. The dissolution of insoluble compounds by cell
secretions must be considered a preparatory process which has no direct
relation to intra-cellular food digestion or fermentation. Enzymes are
not produced by microbial cells exclusively. All living cells produce
enzymes. They were known before the science of microbiology had been
established. In fact, microbial activity was considered for a long time
as an enzymic chemical process. Enzymes in the animal and plant body
may serve largely the purpose of metabolic changes. In the animal body,
many enzymes help to dissolve the insoluble food which cannot pass from
the alimentary canal into the body except by diffusion through the mucous
membrane. There is diastase in the saliva which acts upon starch, there
is pepsin in the stomach and trypsin in the intestine, both dissolving pro-
tein bodies; there is ereptase for the peptones, lipase for the fat, invertase
for the saccharose, and many other enzymes. The object of all these
enzymes is apparently to prepare the food for passing through the mem-
brane into the protoplasm of the cells, where the final changes which
liberate energy take place. The same processes occur with micro-
organisms but in a more simple manner. Surrounded by a liquid
medium, they secrete specific substances (enzymes) ; these dissolve certain
insoluble foods which then diffuse through the cell wall to be decomposed
further.

The food-preparing processes are all supposed to be simple hydrolytic
processes. For some of these changes the chemical equations are well
known. The hydrolyzation of starch to maltose by means of diastase is
represented by the equation

2 (C 6 H 10 5 ) n +nH 2 =

132 NUTRITION AND METABOLISM.

The splitting up of a fat molecule into glycerin and fatty acid is also a
well-known process

C 3 H 5 (C 18 H 35 2 ) 3 +3H 2 = C 3 H 5 (OH) 3 + 3 C 18 H 36 2

Tristearin Glycerin Stearic acid

The proteolysis is not so well known and the general supposition that the
first stages of protein degradation are hydrolytic is largely based upon
analogies. Some of these enzymes which are secreted by the microbial
cells act upon soluble compounds. Invertase decomposes saccharose
into dextrose and levulose:

C 12 H 22 11 + H 2 = C 6 H 12 6 +C 6 H 12 6

Other disaccharides are hydrolyzed in the same way by other enzymes;
glucosides are decomposed by emulsin; soluble proteins are changed to
peptones. It is not necessary that the enzymes act upon the soluble com-
pounds outside the cell since these compounds can diffuse into the cell;
these enzymes are found only occasionally within the cell. It may be said,
however, that the smaller molecules of the products of ^nzymic action
diffuse more readily than the larger molecules of the original food
compound.

PROPERTIES OF ENZYMES. These secretions of cells are treated in a
group by themselves because they differ distinctly in many respects
from any other chemical substance. Probably the most notable differ-
ence may be discovered in the fact that their action does not follow the
law of mass action which supposes that all substances reacting upon each
other diminish in quantity. This is not the case with enzymes. Rennet
will coagulate many hundred times its weight of casein, and still the whey
will contain rennet. Considering that part of the rennet is physically
absorbed by the coagulum, the amount of rennet is found to be the same
as before, though it has changed a comparatively enormous quantity
of casein. The same is true with other enzymes. The enzyme is not
destroyed by acting upon other substances. This exceptional quality
furnishes a reason for treating enzymes as a separate group or apart from
other chemical substances. But there are still other qualities which
distinctly separate them from the well-known chemical bodies, and show
at the same time their relation to proteins and toxins (page 122).
One of these is their sensibility to such outside influences as will destroy
life. Enzymes are inactivated by exposure to temperatures above 50 to
80, and can, like coagulated albumin, by no means be brought back to

MECHANISM OF METABOLISM. 133

their orginal state. They have, like organisms, a maximum, optimum
and minimum temperature of activity, and if heated above the maximum
they will be destroyed. In this respect they resemble albumin since the
maximum temperature for enzymes is very near the coagulating tempera-
ture of albumin. It is believed from this resemblance that enzymes
are of an albuminous nature. Another similarity is the fact that both
enzymes and albumins are precipitated by concentrated salt solutions.
Enzymes can further be inactivated by poisons. The same sub-
stances which kill living cells, like formaldehyde, hydrocyanic acid,
mercuric chloride, phenol, will also inactivate enzymes, though usually
stronger solutions are required for the destruction of the enzyme than
for killing the cell. It is the same with heat; a higher temperature is
generally required to destroy the enzyme than to kill the cell which se-
creted it. Light will also affect enzymes considerably. The great simi-
larity of enzymes and microorganisms in these respects, the similarity
of their reactions and the extreme minuteness of the bacteria render
it explicable why the chemists of eighty years ago could not deter-
mine the difference between microorganisms and enzymes.

With the toxins, the enzymes have in common the great sensibility
to heat, light, and chemicals. Both of these groups are resistant to
drying to a limited extent. So far as body reactions are concerned these
two groups seem to belong to one physiological group of compounds.
When toxins are injected, the body responds by the production of anti-
toxins which inactivate the toxin. In the same way the body responds
to enzymes by the production of anti-enzymes which prevent the action
of the enzymes. It may be mentioned that against protein compounds,
precipitins are produced by the body which precipitate only that protein
which was injected. This "specific" action is also true with toxins and
enzymes. The anti-body will inactivate only the specific kind of toxin
or enzyme that was injected.

What an enzyme really is cannot be defined. An enzyme is known only
by its reactions. Many chemists have tried to prepare pure enzymes
by continuously dissolving and precipitating, by dialyzing and other
means, but there are two great difficulties existing; there is no test for the
purity of enzymes, and they lose in activity if treated with chemicals.
The more they are freed from the protein bodies which always accompany
them, the more sensitive they are to injurious influences. The purest
enzymes that have been obtained do not give the reactions of protein

134 NUTRITION AND METABOLISM.

substances. Mineral salts seem essential for their action, because con-
tinued dialyzing weakens the activity which can be restored only by adding
salts.

MECHANISM OF FERMENTATION. It has been demonstrated in the
above paragraph that food is prepared for digestion or fermentation by
enzymes. The final decomposition, the process which yields the energy
for cell life, must take place within the cell. The investigations of recent
years have demonstrated that these processes also are caused by
enzymes. It has been proved beyond doubt that in the alcoholic, lactic,
acetic and urea fermentations the fermentation process may continue
after the death of the fermenting cells. In the case of alcoholic fermenta-
tion, the fermenting agent has been separated from the lacerated cells and
has been filtered through porcelain filters without losing its ability to act.
This proves the enzyme-nature of the fermenting agent which after once
being formed, remains and acts independent of the cell. These enzymes
are called zymases. They remain within the cell as long as it is alive.
They are much more sensitive to injurious influences than the above
mentioned food-preparing enzymes. Much skill and patience was
required to demonstrate their independence of the living cell. After
these enzymes were found in microorganisms, similar enzymes were
discovered in the cells of higher plants and animals. Many of the bio-
chemical changes taking place in the final dissociation of food within the
cell are now known to be the result of enzymic action; heretofore these
reactions were believed to be a part of the life-processes, inseparable
from the living cell. Even some of the oxidations and many reducing
processes have been recognized as caused by enzymes, and it is quite
possible that the whole process of intracellular food decomposition is
accomplished entirely by means of enzymes.

CLASSIFICATION OF ENZYMES.

Since the chemical nature of enzymes and of their action is largely
unknown, they can be classified only according to the compounds they
act upon. It is possible, however, to distinguish between the following
four groups: Hydrolyzing, zymatic, oxidizing, reducing enzymes. This
definition is not quite exact, since the urea fermenting enzyme is also a
hydrolyzing enzyme, and the acetic fermentation is caused by an oxidiz-
ing enzyme. The distinction between endo-enzymes (infra-cellular) and

MECHANISM OF METABOLISM. 135

exo-enzymes (secreted) is not exact, either, since invertase and lactase are
retained in the cells of some organisms and secreted by others.
The following classification is used in the further discussions:

I. Hydrolytic Enzymes.

1. of carbohydrates: cellulase (cytase), diastase (ptyalin, amylase), invertase,
lactase, maltase.

2. of fats: lipase (steapsin).

3. of proteins:

a. proteolytic (proteases): pepsin (peptase), trypsin (tryptase), erepsin
(ereptase).

b. coagulating: thrombase, rennet (chymosin).
II. Zymases.

1. of carbohydrates: alcoholase, lactacidase.

2. of other nitrogen -free bodies: vinegar-oxidase.

3. of proteins: endo-tryptase, autolytic enzymes, amidase, urease.

III. Oxidizing Enzymes.

Vinegar-oxidase, tyrosinase.

IV. Reducing Enzymes.

Katalase, reductases of nitrates, sulphur, sulphites, telluric salts, methy-
lene blue, litmus.

Several names have been given to some of the enzymes; these are
found in parentheses in the above classification.

The general action of enzymes being explained in the preceding pages,
it remains to describe more in detail the different enzymes of microbial
origin.

HYDROLYTIC ENZYMES.

ENZYMES OF CARBOHYDRATES. Enzymes which decompose carbo-
hydrates are very commonly found in nature, because carbohydrates
constitute a very extensive and common group of organic matter. By
far the largest part of the dry plant consists of cellulose, starch and sugar.
To decompose them, enzymes are necessary. The chemical reaction
of these enzymes is hydrolytic; in other words, the larger molecule is
broken into smaller ones by the simple addition of water. Thus, the
cellulose-destroying enzyme, called cellulase or cytase, decomposes the
cellulose into soluble sugars after the following formula:

C 6 H 10 5 +H 2 0=C 6 H 12 6

or, considering that the cellulose molecule is really many times C 6 H 10 O 5 ,
the formula will be more accurately written

(C 6 H 10 5 ) n +nH,0 =

136 NUTRITION AND METABOLISM.

which indicates at the same time that one cellulose, molecule gives many
sugar molecules.

Cellulase is an enzyme which is quite difficult to obtain. Though it
must be produced by all the cellulose destroying molds and bacteria,
experiments have failed in some instances to prove its presence. It is
found in some wood destroying fungi and in some of the bacteria causing
the rot of vegetables. The organisms of certain plant diseases force their
way into the cell by dissolving the cellulose membrane by an enzyme,
while certain molds are able to puncture the cell wall mechanically.

Diastase, or amylase, is the starch-dissolving enzyme which is one of
the most common enzymes in nature. It is found in all green plants, and
it forms during the sprouting of starchy seeds. Many molds and a few
bacteria produce this enzyme, while yeasts generally cannot decompose
starch for lack of diastase. Starch has the same formula as cellulose, and
it is broken up into soluble sugars in the same way. Much attention has
been paid to this process by the chemists, and it is found that the process
is a gradual one, giving first dextrins, and finally maltose (C^H^On).
The hydrolysis of starch expressed in chemical symbols may be presented
as follows:

2 (C 6 H 10 5 ) n + nH 2 = nC 12 H 22 n

Starch Maltose

The disaccharides or double sugars, having the chemical formula
C 12 H 22 O U are broken up into single sugars, monosaccharides, by the
following process:

C 12 H 22 U + H 2 0= C 6 H 12 6 + C 6 H 12 6

The two molecules of C 6 H 12 O 6 are different with different sugars. If
the disaccharide is saccharose, the two monosaccharide molecules are
dextrose and levulose. Lactose will yield dextrose and galactose, and
maltose will give two molecules of dextrose. For each of these sugars,
there is a special enzyme which can hydrolyze only its particular sugar and
none of the others; like a key, made for one lock, it will not open another
lock. Maltase will split only maltose molecules, not lactose, while the
lactase cannot attack the maltose. Invertase (or sucrase) will decompose
nothing but saccharose. This decomposition of the complex sugars into
the simple sugars is necessary because only sugars of the type C 6 H 12 O 8
can be fermented directly by the fermenting enzyme in the cell, be it an
alcoholic or lactic or gassy fermentation. This explains why beer yeast

MECHANISM OF METABOLISM. 137

cannot ferment lactose; it produces no lactase, and therefore cannot
attack the lactose molecules; they would be easily attacked, if besides the
yeast, some lactase were added. Certain lactic bacteria cannot ferment
saccharose, because they do not form invertase.

Invertase is, like diastase, a very common enzyme in green plants.
It is also produced by the larger number of molds and yeasts, and also of
bacteria. Maltase is not quite so common, and lactase is limited to a
few species of microorganisms. A few organisms are known which do
not secrete these enzymes but retain them within the cell. This is especi-
ally true of lactase, but is also known, in a few instances, of invertase.
The enzyme can be obtained from the broken cells. Such enzymes are
called endo-enzymes.

The decomposition of carbohydrates has been followed from the most
complex representatives to the simplest ones, the monosaccharides. If
these are decomposed further, the resulting product is no longer a carbohy-
drate. The simplest sugars are decomposed by zymases, inside the mi-
crobial cell, into compounds which are generally called fermentation
products; these may result from alcoholic, lactic, butyric fermentations or
some other.

Emulsin is an enzyme which is able to hydrolyze glucosides. Gluco-
sides occurring in plants are complex bodies which contain a sugar-radical.
Emulsin splits glucosides liberating the sugar, usually dextrose. The
typical example for emulsin action is the hydrolysis of amygdalin to
hydrocyanic acid, benzaldehyde and dextrose.

C 20 H 27 O n N+2H 2 O=C 6 H 5 COH+2C 6 H 12 O 6 +HCN.

Amygdalin Benzaldehyde Dextrose Hydrocyanic acid

Emulsin is found in many molds and bacteria, and recently has
been found in yeasts. Glucoside-splitting enzymes play an important
role in the fermentations of coffee-beans, cocoa, mustard and indigo.
In most of these fermentations, however, the emulsin is probably not
formed by microorganisms, but by the plant, from which the ferment-
ing material is derived.

ENZYMES OF FATS. All the enzymes acting on fat, decompose it
in the same manner; the fat molecule takes up three molecules of water,
breaking up into glycerin and three molecules of fatty acid, as indicated on
page 112. It is possible that there are several fat-splitting enzymes, but
the result of the cleavage process is always the same. The name formerly
assigned to enzymes of fat is steapsin, but this term is now almost exclu-

138 NUTRITION AND METABOLISM.

sively substituted by the more significant word lipase. Occasionally they
are called lipolytic enzymes which expression is analogous to the proteo-
lytic enzymes; in the same way, the term amylolytic enzyme is used for
diastase.

ENZYMES OF PROTEINS. The enzymes decomposing protein bodies,
generally called proteolytic enzymes or proteases, have been known for
nearly a century. Though the difficulty of analyzing protein bodies
accurately prevents an absolute knowledge of proteolysis, much effort has
been made to become acquainted with the very important group of en-
zymes which accomplish the digestion of protein food. Naturally,
most experimenting has been conducted with pepsin and trypsin of the
animal body, accordingly these are better understood than others, and
only little work has been done with microbial enzymes ; but there is so far
as can be determined little appreciable difference between the proteolytic
enzymes obtained from different organisms, whether low or high in the
plant or animal world, consequently many experiences with animal
pepsin and trypsin can be applied to microbial enzymes.

The specific chemical action of these enzymes is referable to hydro-
lysis; the large protein molecule is broken up into smaller molecules by
addition of water. Various proteolytic enzymes differ in the extent of
decomposition. While some, like pepsin, produce mainly peptones,
trypsin is able to split protein to amino-acids and even to ammonia.
Mavrojannis tested for the intensity of gelatin decomposition with
formaldehyde. The peptones of gelatin will solidify with formaldehyde
while amino-acids are not affected.

Proteolytic enzymes were first divided into two groups: pepsins, which
act best in slightly acid solutions, and trypsins, which act best in slightly
alkaline media. The names are derived from pepsin (peptase), the pro-
teolytic enzyme of the animal stomach, and from trypsin (tryptase) which
is found in the small intestine of animals. This classification cannot be
used for the enzymes of microorganisms because there is no definite line
established by the acidity. Some enzymes work in either acid or alkaline
media equally well, preferring a neutral reaction. Enzymes should be
classified according to the substances they act upon or perhaps according
to the nature of the products resulting from the fermentation. This
would bring pepsin and trypsin into one class, both acting upon protein
bodies as such; they, however, differ in the intensity of action as shown
by their products, the pepsin forming mainly peptones, the trypsin carrying

MECHANISM OF METABOLISM.

139

on the decomposition as far as amino-acids and traces of ammonia.
Another class recently recognized is ereptase (erepsin) which cannot
decompose protein, but readily attacks peptones, decomposing them much
in the same way as trypsin. Pepsin, trypsin and erepsin do not break up
amino-compounds.

The presence of proteolytic enzymes in microorganisms is readily
tested by cultivation on nutrient gelatin. The proteolytic enzyme
secreted by the cells will liquefy the gelatin. Generally, an organism that
liquefies gelatin will also decompose the casein of milk and the protein of
blood serum. There are some exceptions, however, as is shown in the
following table, after Frost and McCampbell. A + sign means proteo-
lysis, a sign means no action.

Organism.

Milk.

Coag. Digest.

Gelatin. Serum. Fibrin,

album.

Bact. anthracis

Microspira comma

M. pyogenes var. aureus.
Pseudomonas pyocyanea. .

B. violaceus

B. mycoides

B. prodigiosus

A spergillus niger

A spergillus oryztz

Apparently not all organisms which liquefy gelatin are able to de-
compose egg albumin, and we must conclude that the enzyme liquefying
gelatin is different from the proteolytic enzyme dissolving egg-white.

COAGULATING ENZYMES. The blood-clotting enzyme (thrombase)
does not occur in microorganisms. Rennet, however, is found in many
species. Rennet is extracted from the stomach of calves and pigs and
used to set the curd in milk for cheese making. The enzyme acts upon
the casein in milk, decomposing it into paracasein and some soluble
protein. The time of coagulation depends upon the temperature of the
milk and the concentration of the rennet. This coagulation of milk is
quite different from the acid curd, where the insoluble casein is precipi-
tated by the acid. If enough acid is added, the milk curdles immediately ;

14 NUTRITION AND METABOLISM.

if there is not enough acid, there will be no curd, not even after a long
time. An acid curd can be brought back to the original state by an ad-
dition of alkali, while a rennet curd by no means can be changed back to
casein. Rennet-forming bacteria are found in milk and dairy products,
in soil and other habitats. They will coagulate milk without causing
any appreciable increase of acidity. They all seem to digest the curd
after it is formed (see the above table). The relation between proteolytic
and rennet enzymes will be discussed in a later chapter.

Rennet is sometimes called chymosin; the Society of American Bac-
teriologists uses the German word "lab."

ZYMASES.

The zymases are the agents which furnish the energy for cell life
by causing fermentative decompositions. As has been stated before,
the processes which provide for energy must take place inside of the
cell. Consequently, all intracellular enzymes are endo-enzymes. The
difference between the soluble enzymes and the endo-enzymes is very
plainly shown in the following table, giving the energy liberated by the
various enzymes by acting upon i g. of substance.

Energy liberated from I g. of substance.

Soluble Enzymes. Endo-Enzymes.

Pepsin, trypsin o calories Lactacidase 82 calories

Lipase 4 calories Alcoholase 160 calories

Maltase, invertase. . . . 10 calories Urease 239 calories

Lactase. . . 23 calories Vinegar-oxidase 2,520 calories

The microbial cell does not lose much energy by the activity of the
soluble enzymes outside of the cell, because their energy yield is insignifi-
cant.

The first zymase recognized as such was urease, the enzyme which
changes urea to ammonium carbonate. The urease was not considered
an exceptional case, and no particular attention was paid to the fact
that it was the only zymase known at that time. The actual investiga-
gations of the zymases did not start until Buchner had demonstrated
that yeast can be ground with infusorial earth until all cells are lacer-
ated, and then can be pressed and the juice filtered without losing the
power of alcoholic fermentation. Such fermentation cannot be due to

MECHANISM OF METABOLISM. 141

anything but a soluble compound of the yeast cell. Thus the alcoholase
was recognized. It was found later that yeast may be killed by alcohol,
ether or acetone without losing its fermenting power.

This last method was applied later to lactic bacteria, and it was
proved that the lactic acid is also produced by an enzyme, lactacidase.
It is possible to kill the lactic bacteria cells so that they do not multiply
but still continue to form acid. It seems quite probable that other fer-
mentations of carbohydrates, like the butyric and the gassy fermentations,
are really due to enzymes. It is very difficult to give the experimental
proof, however. These enzymes are so sensitive that it requires much
experience to separate them from the cell, and it is also quite difficult to
obtain bacteria in quantities large enough for such experiments.

The vinegar oxidase is an enzyme which remains in the cell of the
acetic bacterium, oxidizing alcohol to acetic acid. Its independence of
the living cell has been demonstrated by killing the cells with acetone.

The PROTEOLYTIC ENDO-ENZYMES of yeasts, only, have been studied
extensively. That such enzymes exist is recognized by the observation
that certain microorganisms do not liquefy the gelatin until after they are
dead and the proteolytic enzymes diffuse out through the deteriorating
cell membranes. That yeast in the absence of sugar loses in weight, and
that leucin and other cleavage-products of protein are formed, was the
first indication of a proteolytic process in the yeast cells. By pressing
the juice out of the ground yeast cells, a liquid is obtained which liquefies
gelatin, digests casein, albumin and fibrin. The living yeast cell does
not attack these compounds, because they cannot diffuse into the cell
and the enzyme cannot diffuse out. The proteolytic endo-enzyme of yeast
is called endo-tryptase. Its object is apparently the regulation of the pro-
tein-content of the cell and perhaps it has some bearing on the formation
of cell plasma. The possible relation between enzymes and growth is
discussed in a following sub-chapter. "

If yeast is mixed with a weak antiseptic (chloroform, toluol) the pro-
teolytic process takes place quite rapidly. This process is called autolysis
(self-digestion). Similar autolytic enzymes are found in other micro-
organisms. Autolysis is a well-known process in the higher animals.
To this is due the ripening of meat.

Proteolytic endo-enzymes must be expected in all microorganisms
which depend upon protein as food material only. These organisms
will produce certain enzymes which diffuse out of the cell and decompose

142 NUTRITION AND METABOLISM.

the protein into bodies which diffuse easily into the cell. Here, proteoly-
tic endo-enzymes further decompose these products. Such an endo-
enzyme is the amidase discovered by Shibata in the mycelium of Aspergil-
lus niger which forms ammonia from urea, acetamid, oxamid, biuret.
Endo-erepsin and amidase were also found in Penicillium camemberti by
Box.

Similar to these proteolytic enzymes is the urease which is formed in
large quantities in the so-called urea bacteria, but it is also present in the
mycelium of some molds. An endo-enzyme, splitting hippuric acid into
benzoic acid and glycocoll, is found in the mycelium of a few molds.

OXIDIZING ENZYMES.

The most typical example of an oxidizing enzyme is the vinegar-
oxidase, because its chemical action is well known. Most of the oxidases
known act upon complex organic compounds, changing them to colored
bodies. Such an oxidase is the tyrosinase, which forms a black, insoluble
compound in tyrosin solutions. It is produced by several bacteria,
especially by chromogens, and its application in testing for small quantities
of tyrosin has been suggested. A number of oxidases are known to act
upon the leuco-bodies of certain organic dye-compounds, as aloin, guaiac,
phenolphthalein, and others. Hydrochinon is oxidized by the dead cells of
a few molds. Strange seems the oxidation of potassium iodide to iodine
by the endo-oxidase of a mold. Many other oxidations are supposed to
be of enzymic nature, but their independence of the living cell has not
been proved.

Many higher organisms are known to contain oxidases, the best
studied are those of certain mushrooms which change the white mush-
room meat into a bluish or brownish color as soon as it is exposed to the
air. Oxidases are very common in most of the tissues- of higher animals.

REDUCING ENZYMES.

Among the reductases, one enzyme stands apart from all the others,
that is the katalase or peroxidase which reduces the hydrogen peroxide
to water by liberation of oxygen.

H 2 O 2 + katalase =H 2 O+O

Katalase is one of the most commonly found enzymes; it is formed by
practically all plants and all animals and is contained by all but a few

' MECHANISM OF METABOLISM. 143

bacteria. Among these exceptions is the Bad. lactis acidi. The ab-
sence of katalase in this species has been recommended as a diagnos-
tic test. It is possible that this enzyme is necessary for intracellular
oxidations.

A number of other reductases are known. Nearly all of the reductions
mentioned in the paragraph on the products of mineral decomposition
are proved to be of enzymic nature; these processes will take place after
the cell is killed by a disinfectant or is ground to pieces. This can be
readily demonstrated by lacerating the cells with quartz sand. They
will then reduce nitrates to nitrites, sulphur to hydrogen sulphide. The
decolorization of litmus, methylene blue, indigo, and other organic dyes
is due in microbial cultures to enzymes which are almost exclusively
endo-enzymes.

ADDITIONAL REMARKS ON THE RELATION OF CELLS AND ENZYMES.

Enzymes are produced only by living cells. After they are once
formed, they act like chemical compounds, independent of the cell which
produced them. Even the endo-enzymes follow only the law of enzyme
action and are not influenced by the cell which contains them. The
enzymes are mostly influenced by their own products, and when a certain
yeast ceases to ferment sugar at the concentration of 8.5 per cent of
alchohol, this means that the alcoholase of this yeast cannot tolerate
more than 8.5 per cent of alcohol. The inability of the cell to regulate
enzymic action may account for the fact that often a culture produces an
amount of fermentation products sufficient to kill all cells. This is ob-
served in the lactic, acetic and alcoholic fermentations, and perhaps
occurs in many others.

Most cells produce more than one enzyme. Microorganisms feeding
upon various foods must form various enzymes. Frequently several
enzymes are necessary for the decomposition of one compound. Mucor
rouxii uses three enzymes in order to form alcohol from starch, first the
diastase to change starch to maltose, then maltase to change maltose to
dextrose and finally alcoholase to change dextrose to alcohol and carbon
dioxide. The number of enzymes formed by certain microorganisms is
surprising. Aspergillus niger has the reputation of forming almost all
enzymes which have ever been found in microorganisms. Penicillium
camemberti produces (after Dox) erepsin nuclease, amidase, lipase,

144 NUTRITION AND METABOLISM. '

emulsin, amylase, inulase, raffinase, invertase, maltase and lactase. It
has been believed for a long time that certain enzymes are regular products
of the cell while others are formed only if the substance upon which they
act is present. According to Dox's investigations with Penicillium
camemberti, there is no evidence that enzymes not normally formed by
the organism in demonstrable quantities can be developed by special
methods of nutrition. The addition of a particular food compound does
not develop an entirely new enzyme, but stimulates the production of the
corresponding enzyme which is normally formed, although in small
amounts, under all conditions.

THEORY or KATABOLISM.

Regarding katabolism as the sum of all destructive processes of the
living cell substance, i.e., of the protoplasm, and considering the cell
substance to be decomposed and renewed constantly as long as the cell
is performing the normal functions of life, then there must be a reno-
vating and a destructive process continuously going on in the proto-
plasmic molecules. If the food supply ceases, anabolism ceases with it,
but it has been demonstrated that katabolism may continue just the
same for some time. By this method, the products of katabolism can
be obtained free from the products of food digestion which would
obscure the results of experiment with katabolism in normally fed cells.

It is difficult to determine to what extent katabolism is controlled by
endo-enzymes, the so-called autolytic enzymes, which have been mentioned
in the above paragraph. In the autolysis of yeast cells, the only well-
studied example of microbial autolysis, have been found guanin, adenin,
xanthin, hypoxanthin and ammonia.

THEORY OF ANABOLISM.

INTERACTION OF ANABOLISM AND INTRA-CELLULAR FERMENTATION.
All changes discussed in the previous chapters are fermenting pro-
cesses in which organic or inorganic compounds are broken up to smaller
molecules. These processes are exothermic, i.e., liberating heat or
energy in other form. The opposite is true of the anabolic processes
which build up complex molecules from simple compounds. These
synthetic processes are endothermic, absorbing heat or energy in other

MECHANISM OF METABOLISM. 145

form. Growth is the typical manifestation of anabolism. It is the
formation of new cells from dead organic or inorganic matter, and it
means the formation of all the compounds necessary for cell life. Of
all the substances found in the cell, practically none are contained in the
food, and it is wonderful that in such a small unit as a microbial cell,
there are contained the powers of making protoplasm, enzymes, nuclear
bodies, chromatin-bodies, the substance of the cell wall and probably
many other unknown compounds. All these complex substances are
generally made from simple food compounds as amino-acids, carbo-
hydrates and others.

These synthetic processes of the cell will, like most endothermic
processes, take place only if energy is provided. This condition is usually
fulfilled in the living cell, due to the fermenting processes going on con-
tinuously. There is a strange interaction between anabolism and intra-
cellular fermentation proceeding in the protoplasm and this linking
together of destructive and constructive reaction is the basis of life
processes. The life processes decompose certain substances, the energy
liberated allows the formation of protoplasm, which again liberates
energy. Thus a continuous formation of protoplasm is secured.

An explanation of anabolism based upon chemical experiments is
not possible at the present time. In the study of intra- cellular destruction
it is possible to trace most processes back to enzymic action. There
our knowledge ceases because the nature and mode of action of
enzymes is unknown. In the study of anabolism our knowledge has
not even progressed so far. The most promising explanation at present
is based upon the reversibility of enzymic action.

REVERSIBILITY OF ENZYMIC ACTION. Chemical reactions between
organic compounds proceed quite rapidly at first, then become slower
and slower until the reaction stops entirely. The reaction is not com-
plete at the time it reaches an equilibrium. If the equilibrium is disturbed
by adding more of the reagents, the process will continue. If, however,
the products of reaction are added, the reverse process will take place.
Reactions between organic compounds can proceed either way, depending
upon the relative concentrations of the reacting substances. The stand-
ard exaniple is esterification. Acetic acid plus alcohol gives ester plus
water,

CH 3 COOH+ CH 3 CH 2 OH<=>CH 3 COO CH 2 CH 3 + H 2 O

Acetic acid Alcohol Ester

10

146 NUTRITION AND METABOLISM.

The process goes to a certain equilibrium and stops. If ester is mixed
with water, it gives acid plus alcohol, until the same equilibrium is
reached. If acid and alcohol are added to. a system in equilibrium, more
ester will be formed. If ester is added, more alcohol and acetic acid will
be formed. The same is true with enzymes, at least with some enzymes.
Maltase will decompose maltose into two molecules of dextrose. In a
concentrated solution of dextrose, however, maltase will form maltose, or
a similar sugar, isomaltose. Lipase is able to produce fat from glycerin
and fatty acids. A solution of albumose with trypsin or pepsin gives a
precipitate of a body which is more complex than albumose and which
gives the protein reactions. It is believed by many physiologists that
pepsin and rennet are the same body. Under certain conditions, it has
a dissolving power, under other conditions it has the power to coagulate.
The reversibility of enzymic action has given rise to much speculation
about assimilation and growth. It seems reasonable to suppose that the
cell forms its protoplasm from amino-acids by the reversed action of
proteolytic enzymes. In the same way, cellulose may be formed from
dextrose, fat from glycerin and fatty acids. Nearly all phases of growth
can be accounted for in this way. This is nothing but theoretical specu-
lation, and the only fact to support it is the reversibility of certain enzymes.
The conditions under which chemical reactions take place inside of the
cell are very largely unknown. There are so many processes going on at
the same time that it is absolutely impossible at the present time to obtain
a perfect knowledge of all these reactions. Our experience is limited to
the very simplest changes, and even these are not perfectly understood
because the detailed conditions under which the changes take place are
unknown. Information obtained by experiments in vitro is practically
all that is available, and these experiments are conducted under very
simple conditions, where complications as they might occur in the cell,
are excluded. Thus, our knowledge of the processes of growth is largely
based upon analogy and speculation.

DIVISION II.
PHYSICAL INFLUENCES.

CHAPTER I.
MOISTURE.

Moisture may be called the most important factor of life. Not only
bacteria, but every microscopic and macroscopic being requires a con-
siderable amount of moisture. Living organisms contain on the average
between 70 per cent and 90 per cent of water, and only 10 per cent to 30
per cent of solid matter. Microorganisms which live entirely submerged
in liquids need water not only within but without the cells. Bacteria,
yeasts, molds, and some protozoa obtain their food by diffusion through
the cell-membrane; their food-substances must be soluble and dissolved.
No other liquid can take the place of water.

The amount of water required by microorganisms cannot be stated
briefly. Several factors have to be taken into consideration, as the
osmotic pressure, the insoluble and the colloidal substances, the species
of organisms, temperature, and perhaps others.

OSMOTIC PRESSURE. In the organic world we find very commonly
membranes which will allow water to pass through but retain some
compounds dissolved in the water. Such so-called semi-permeable
membranes are found surrounding the protoplasm of cells. They are
not the cell wall, but separate the protoplasm from the cell wall. Similar
properties are found in parchment paper, pig's bladder, and other organic
membranes.

If a salt solution is poured in water, the two liquids will mix in a short
time and soon every smallest portion of the mixture will have the same
concentration. If a salt solution and water are separated by a membrane
which does not allow the salt to pass, the water will go through the
membrane toward the salt with a certain amount of pressure. This
pressure depends upon the nature of the dissolved substance as well as
upon its concentration.

147

148 PHYSICAL INFLUENCES.

The pressure increases in direct ratio with the number of molecules in
solution. Therefore, the pressure of solutions of equal concentration by
weight will be the smaller the larger the molecule, because the larger the
molecules the smaller the number required to make a certain weight.
The osmotic pressure of protein, starch and peptone solution can be
measured only with the finest instruments, while the pressure of a 30 per
cent dextrose solution is 22 atmospheres.* With acids, alkalies and
salts, the pressure is higher than would follow from the concentration,
because the molecules of these electrolytes are dissociated, thus increasing
the number of unit-particles in solution.

PLASMOLYSIS. If a cell is brought into a strong solution of a substance
which cannot pass the plasma-membrane, this substance will cause an
osmotic pressure and the concentration in the cell being lower than in the
medium, the water will pass out from the cell until the pressure inside and
outside is the same. This causes a shrinking of the protoplasm, while
the rigid cell wall keeps its shape. Such plasmolyzed organisms are
illustrated in figure 30, p. 50.

While plasmolysis is easily demonstrated with the cells of higher
plants, microorganisms do not show it so readily. In fact, many bacteria,
like B. subtilis, Bact. anthracis, cannot be plasmolyzed by any concentra-
tion of salt in solution. Others, as B. coli, B.fluorescens react promptly.
But even though many are killed, the rest recover from plasmolysis after a
few hours, and appear normal. This indicates that the salt passes slowly
through the plasma membrane and thus increases the pressure inside the
cell until finally the inside and outside pressure are the same again.

The fact that many microorganisms show no plasmolysis whatever is
explained in the same way. These organisms probably have plasma-
membranes so constructed that the salts diffuse through nearly as fast as
the water. An absolute exclusion of all soluble substances by the mem-
brane is impossible since the food can get into the cell only by diffusion
through the membrane.

The resistance of various microorganisms against concentrated
solutions depends upon the organism as well as upon the dissolved sub-
stance. The sodium and potassium salts of the common mineral acids
act upon a culture nearly in proportion to their osmotic pressure, but the
potassium salts always retard growth a little less than the sodium salts.

* One atmosphere equals the pressure of one kilogram per square centimeter or about 1 5
pounds per square inch.

MOISTURE. 149

The effect of salts upon microorganisms is therefore not due to the os-
motic pressure only; the chemical constitution of the salts also plays an
important role.

The different functions of life are influenced in different degrees by
concentrated solutions. Some bacteria will multiply but not form spores
in salt solutions. Molds will sometimes show a good growth in concen-
trated sugar solutions but fail to produce spores unless the medium is
diluted. Bact. anthracis loses its virulence in sea water. Often the
form of microorganisms is affected by concentrated solutions. Some
bacteria grow more spherical, others become elongated or distorted.
The deforming influence is not due to the osmotic pressure only, but
depends mainly upon the chemical character of the salt; magnesium
salts especially have a tendency to produce such involution-forms.

Salt and Sugar Solutions. Most experiments on the influence of
concentrated solutions have been carried on with sodium chloride, be-
cause of its wide application in the preservation of foods. Most micro-
organisms, especially the rod-shaped bacteria, are suppressed by a salt
concentration of 8-10 per cent. At 15 per cent only few cocci develop
slowly, while some species of torulae grow without a very noticeable re-
tardation. Above 20 per cent the torulae are practically the only organisms
which can develop. They are, therefore, found in all food products
which are preserved by salt, as salted pork, beef, fish, butter, and pickles,
often in nearly a pure culture. It seems that they are easily overpowered
by other organisms in the absence of salt, but this has a selective action,
preventing nearly all other organisms but the torulae.

The selective influence of salt is used in some fermented products to
prevent undesirable fermentations. This is true in sauerkraut and
brine pickles, where the desirable bacteria can grow in the presence
of salt while the undesirable ones are kept away. Possibly the salting
of butter has the same effects.

Another compound of great practical importance is cane sugar,
which is the standard preservative for fruits and condensed milk. Its
action has been studied mainly upon molds. Theoretically, dextrose
should be expected to have twice as strong a preserving action as saccha-
rose because it has only half the molecular weight and consequently
produces twice as strong an osmotic pressure in the same percentage of
concentration. But though its preserving action is a little higher than
that of saccharose, the proportion is not nearly 1:2. The common molds

150 PHYSICAL INFLUENCES.

are extremely resistant to strong sugar solutions, about 60-70 per cent
of cane sugar seems to be the limit of growth for Penicillium and Asper-
gillus species. Yeasts can also grow and ferment in very concentrated
solutions while bacteria in general do not tolerate solutions higher than
15 to 30 per cent, though many exceptions are known.

Colloidal Solutions. In order to determine the amount of water
which is absolutely necessary for microbial proliferation, only such
media can be used which do not cause osmotic pressure. If B. prodigiosus
does not develop in a 10 per cent salt solution, this is not due to lack of
moisture, because the same bacillus will grow in a 30 per cent sugar
solution which contains 20 per cent less moisture. Another factor be-
sides the water content enters, which can be avoided only in solutions
without osmotic pressure.

A few substances are known to give such solutions. It was stated
before that the osmotic pressure depends upon the number of molecules
dissolved in a liquid; the larger the molecule the lower the osmotic pres-
sure. There is a group of substances known as colloidal bodies which
have a very large molecular weight, between 1,000 and 1 0,000, it is estimated.
Their osmotic pressure even in very concentrated solutions would not be
high enough to interfere with microbial growth. Among these colloidal
bodies are found egg albumin, gelatin, peptones, all protein substances;
also starch, dextrin and gum arabic among the carbohydrates. None
of these substances has a retarding influence upon bacteria; some of them
can be mixed with water in all proportions; consequently, they are the
ideal medium to test the water requirements of microorganisms.

Experiments carried on with gelatin, powdered meat, crackers,
bread and potato, vary but little in results. A few bacteria cannot
grow in a medium with only 60 per cent water, but most organisms
develop slowly even with 50 per cent water and some may be able to
develop with only 40 per cent. Molds can grow very scantily in even
more concentrated media. Protozoa probably have to have a more
diluted medium for their development though no experiments bearing
upon their water requirements are known to the author.

The fact that in a colloidal solution growth will cease if the moisture
is below 30 to 40 per cent does not necessarily indicate the conclusion
that any substance with less than 30 per cent water cannot be decomposed.
The above statement refers only to solutions, while in natural media as
dried foods or soil, a combination of solid and dissolved substances is in-

MOISTURE. 151

volved. Butter is an excellent medium for many bacteria, yeasts, and
molds, though it contains only 12 to 15 per cent of moisture. If butter fat
were soluble in water, the concentration of 85 parts of solids in 15 parts of
liquid would certainly prevent any growth whatever, but fat is insoluble,
and the fat particles do not interfere at all with the growth of microorgan-
isms in the droplets of buttermilk distributed all through the butter.
The concentration in these small droplets is the deciding factor. If the
growth of microorganisms in butter is to be prevented by salt, it is un-
necessary to give any attention to the fat; the bacteria live only in the
water and not in the fat globules. In adding 3 per cent of salt to a butter
with 15 per cent of moisture, a brine of 3 parts of salt in 15 parts of water
is produced; in other words, a 20 per cent brine, because salt does not
dissolve in the fat. Similar considerations will come up in the preserva-
tion of fruit, vegetables, meat, milk, and other food substances by drying
or condensation.

DESICCATION. Microorganisms do not die immediately if water is not
present. The majority of bacteria will remain alive for a few days if
dried on glass or filter-paper. Some will live for a month or more, while a
few, like the B. carotamm, die within twenty-four hours after drying.
Spores of bacteria are extremely resistant to desiccation. Certain spores,
like those of Bad. anthracis and B. mesentericus, have been kept alive for
many years. The mycelium of molds is ordinarily killed if dried com-
pletely, while the spores survive. Yeasts show a varying resistance.

The resistance of microorganisms is influenced greatly by the medium
on which they are placed for drying. Hansen found that yeast cells
dried on cotton were still alive after two to three years, while if dried on
platinum wire some died in five days and others lived as long as 100 days.
Compressed beer yeast mixed and dried with powdered charcoal kept as
long as ten years; Pseudomonas radicicola dried on a cover-glass or filter-
paper died within twenty-four hours; on seeds, this same organism was
still alive after fourteen days and in the dried nodules of legumes a few
cells were able to reproduce after more than two years. Soil containing
an average number of 17,000,000 bacteria per g. was dried for two years;
the total number of organisms averaged then 3,250,000; 20 per cent of the
bacteria, therefore, could resist desiccation. Dried cultures of microorgan-
isms are commonly sold for several purposes, as dairy-starters and the so-
called "magic yeast" used for bread-making. Such cultures are dried on
milk, sugar, starch, flour or similar porous and absorbing material. Starters

152 PHYSICAL INFLUENCES.

are usually guaranteed only for a certain length of time, from one to
twelve months. The advantage of the dry culture is its better keeping
qualities. Liquid cultures produce substances harmful to themselves,
and die rapidly after a short time, while the dry cultures show little change.
The resistance of pathogenic bacteria to desiccation is of considerable
importance in the spreading of contagious diseases. Many pathogenic
bacteria die after desiccation of a few hours to a few days, and spreading
of such diseases by dust is highly improbable. Protozoa of soil decrease
in number 'by drying, but all are not killed.

CHAPTER II.
INFLUENCE OF TEMPERATURE.

Temperature, as well as moisture, is one of the most important factors
of life. It is so important that the most highly developed animals pro-
tect themselves by a very complicated mechanism of regulation against
changes of temperature; the life processes of such animals will take
place at a temperature nearly constant from birth to death. This neces-
sitates a distinct difference in the metabolism of warm-blooded animals
and all other organisms. The metabolism of the warm-blooded animals
takes place at a constant temperature. The required amount of food
is constant except for the part that is used for heating the body; at lower
temperatures, more heat-producing material is used and the result is that
warm-blooded animals require more food, the lower the temperature.
All other organisms, reptiles as well as bacteria, have the temperature
of their environment and the decrease of temperature will decrease the
intensity of metabolism as it retards any other chemical process. The
lower the temperature, the less food is required by all lower organisms.

There are, of course, limits to the favorable influence of high tempera-
ture. Growth and metabolism of microorganisms will increase with a
rising temperature to a certain point, called the optimum temperature,
and beyond this point the rate of growth will fall off rapidly and soon
cease entirely. The highest temperature at which growth can take place
is called the maximum temperature. Correspondingly, the minimum
temperature of an organism is the lowest point at which growth can take
place.

THE OPTIMUM TEMPERATURE which allows the fastest growth will be
quite different for different species. Groups of bacteria are known which
develop only at very high temperatures and others for which room tem-
perature is too high. The temperature requirement is largely dependent
upon the natural habitat of the organisms. The bacteria of the polar sea
and of a lagoon near the equator will very probably have different
optimum temperatures because of the acclimatization and selection
which has been taking place for centuries.

153

154

PHYSICAL INFLUENCES.

The great majority of bacteria and related organisms, in fact of all
living organisms, has its optimum temperature between 20 and 40.
The optimum temperature of an organism is generally somewhat higher
than the average temperature of its natural habitat. This will be in most
instances between 10 and 38. A few examples will illustrate this.
The bacteria of animal diseases are accustomed to body temperature and
grow best at 37 to 40. The organisms found in soil are of various
natures. Since good soil in summer under the direct radiation of the sun
quite often reaches high temperatures, a great number of soil bacteria will
have their optimum nearer 40 than 30. The ordinary lactic bacteria
of milk and many of the water bacteria have their optimum near 30.
Most of the common molds grow best between 20 and 30. Only very
few can grow at body temperature. The optimum temperature of some
water bacteria is quite low, the best-known example being perhaps the
photogenic bacteria (page 129).

The following table shows the data obtained for a few microorganisms.

Temperatures

Species

Minimum

Optimum

Maximum

Penicillium glaucum

i S

2C-27

, i-?6

A spergillus niger

7-io

f s * I

,,0-0

o- 1 o u

4O-41

Saccharomyces cerevisicz I

i-*

oo o/
28- 70

40

Saccharomyces pasteurianus I

S

2 e-o O

^4

Bacterium phosphoreum

"' J

below o

"j O u

i6-i8

OT-
28

Bacillus subtilis

6

30

r

Bacterium anthracis

10

o"
, o_, 7 o

O u
4 ,

Bacterium ludwigii

to

O" O/

rr-r7

^o
80

THE MINIMUM TEMPERATURE or the lowest limit of growth is usually
farther from the optimum than the maximum temperature. It will vary
with the organisms just as do the other cardinal points. But there is a
natural limit drawn by the freezing-point of the nutrient liquid. Not all
organisms can grow at such low temperatures, in fact the greater number
does not develop below 6 to 10. Those that can grow near the freezing-
point will be inhibited by the solidification of the water in the nutrient
medium, for if the water is frozen, food cannot diffuse into the cells and

INFLUENCE OF TEMPERATURE. 155

therefore all life processes are checked. If freezing is prevented by
adding salts or other soluble substances which lower the freezing-point,
growth may continue even below o. Milk freezes at about 0.5. Bac-
teria are found to multiply in it as long as it is not entirely solid. A
certain yeast multiplied slowly in salted butter kept at about 6.

The number of microorganisms that developed at the freezing-point
was found to be

in i c.c. of market milk, up to 1,000 germs.
in i c.c. of sewage, up to 2,000 germs,

in i g. of garden soil, up to 14,000 germs.

THE MAXIMUM TEMPERATURE is usually about 10 to 15 higher than
the optimum. The development of microorganisms above the optimum
temperature is not quite normal; there is a great tendency toward invo-
lution forms. The mycelium of molds grown near the maximum tem-
perature appears unhealthy and pathogenic bacteria lose part of their
virulence. This loss of virulence is made use of in the preparation of
attenuated cultures for vaccines.

The maximum temperature varies with different species of bacteria.
Most bacteria do not grow above 45, but with some the maximum
temperature is considerably lower. Bact. phosphoreum dies if exposed
for a few hours at 30; others may require still lower temperatures. The
average organisms found in water, soil, milk, and the body, which have
their optimum near 30 to 38, do not grow higher than about 45. There
are very noticeable exceptions to these, such as the physiological group
known as thermophile bacteria.

These extraordinary organisms have their maximum between 70 and
80, a temperature which coagulates albumin. Corresponding to the
high maximum the thermophiles have a very high optimum, and the
minimum lies with most of these species above 30. These organisms are
found in soil, sewage, ensilage and occasionally in milk. They find the
temperature suitable for their life only under extraordinary circum-
stances, as in fermenting manure piles, in silos, in self-heating hay and
similar organic material that develops a high temperature by fermenta-
tion. Some hot springs have a very remarkable flora of thermophile
bacteria.

BIOLOGICAL SIGNIFICANCE OF THE CARDINAL POINTS OF TEMPERA-
TURE. The importance of the temperature requirements of certain organ-

156 PHYSICAL INFLUENCES.

isms to the r61e they play in nature can be illustrated by a few examples.
Most molds cannot cause disease in man and warm-blooded animals
because their maximum temperature is below the body temperature.
Exceptions are some A spergilli and Mucors. Pathogenic microorganisms
must have their optimum temperature coincide with that of their host.

Organic substances may undergo a different change at different
temperatures. The biochemical changes in soil may not be the same in
northern Canada and near the Gulf of Mexico. Even the warm and
cold season of the same climate is apt to change not only the rate of
decomposition but possibly the products. Perhaps the most striking
example in this respect is the decomposition of ordinary market milk
kept at different temperatures. Such milk contains a great variety of
microorganisms; at various temperatures different types will predominate,
while the remainder are retarded or inhibited by unfavorable temperature
conditions and by the products of the dominant type of bacteria. If
milk is kept at about the freezing-point, only a few organisms will develop
slowly, but after a certain time their number will increase to many million
cells per c.c. There is, however, no apparent change ; no acid or deteriora-
tion can be discovered by the taste though chemical analysis proves the
presence of hydrogen sulphide and ammonia. Between 15 and 25,
milk will sour in about thirty-six to forty-eight hours, giving a firm curd
of an agreeable flavor without whey or gas; later Oldium lactis destroying
the acid develops on the surface. Near body temperature the milk will
lopper in twenty-four hours, the curd is usually contracted, a large quan-
tity of whey is extruded, and much gas is produced by Bad. aerogenes and
B. coll. The odor is disagreeable and later butyric acid is produced;
eventually the lactic acid increases further by the action of Bad. bulgaricum
If kept above 50 the milk usually keeps for twenty-four hours, but after
that a decomposition by thermophile bacteria begins which is either an
acid fermentation followed by digestion or a complete putrefaction
depending upon the species of thermophile organism that happens to be
in the milk sample. Thus there is in the same substance, containing the
same organisms at the start, four entirely different types of decomposition
induced only by the difference of temperature.

This indicates the importance of temperature regulation in the fer-
mentation industries. Even pure cultures may give different products
if working at different temperatures. Cream ripened with a pure culture
starter at too high a temperature will have a sharp acid flavor. The cold

INFLUENCE OF TEMPERATURE. 157

curing of cheese has become a very common practice because of the much
improved flavor. Bioletti claims that the value of the dry California
wines would be doubled if the fermentation were carried on generally at
a lower temperature.

In the chapter on enzymes it was stated that the fermentations were
due to enzymes which are independent of the cell after they are once
produced. The question arises whether the optimum temperature of
growth coincides with the optimum temperature of fermentation. Too
little is known as yet about the nature of enzymes to make definite
statements.

END-POINT OF FERMENTATION. Another question is the relation
between the end-point of fermentation and the temperature. Of the
few data existing, many indicate that at a lower temperature the final
fermentation goes farther than at a higher temperature. Miiller-
Thurgau found that under exactly the same conditions with the tempera-
ture as the only varying factor the following final amounts of alcohol
were produced by a pure culture of yeast.

at 36 3.8 per cent alcohol.

at 27 7.5 per cent alcohol.

at 18 8. 8 per cent alcohol.

at 9 9.5 per cent alcohol.

Concerning the lactic fermentation some investigators find no difference
in the end-point, while others obtained results similar to the results with
alcohol. With three strains of Bad. lactis acidi were obtained after thirty-
four days,

A. B. C.

at 37 0.89 per cent 0.87 per cent 0.60 per cent of lactic acid,

at 30 i. oo per cent 0.96 per cent o. 8 1 per cent of lactic acid,

at 18 i. 08 per cent 1.06 per cent 0.88 per cent of lactic acid,

at 6 0.70 per cent 0.73 per cent 0.62 per cent of lactic acid.

These results are quite logical and perhaps can be explained by the
recognized experience that all products of fermentation tend to check
the process of fermentation, and that any chemical product or substance
acts the more vigorously upon any life process the higher the temperature.
The same amount of alcohol that will still allow a slow fermentation at
10 may check the fermentation entirely at 20. Naturally the rate of

158 PHYSICAL INFLUENCES.

fermentation in the beginning will be higher at the higher temperature
but the end-point is lower. The end-point of the lactic cultures A, B,
and C at 6 is probably not final, because thirty-four days is a short time
of growth at so low a temperature. Above the optimum the rate of
decomposition will decrease rapidly with the rising temperature and the
end-point will also be lower. As already mentioned above, pathogenic
bacteria lose to some extent their virulence, that is, the power of producing
disease, if grown above their optimum.

FREEZING. The discussion of the relation of temperature to micro-
organisms has so far considered only the temperatures within the limits
of growth. However, the temperatures below the minimum and above
the maximum are also of greatest importance. If bacteria are cooled be-
low their minimum temperature they do not die immediately (except
a few thermophile bacteria) . They remain alive in a dormant condition
ready to multiply as soon as the temperature rises. Even the freezing
of a liquid will not kill them immediately. Of course, they cannot
multiply in ice, because they have no water, consequently no food, and
they cannot thaw the ice to get their water and food for lack of body
temperature of their own. As long as liquids are frozen solid the bacteria
in them will remain dormant much like dried organisms, and like them
their number will decrease very slowly. If ice has been kept frozen
many months, it may be sterile, while the fresh ice has nearly as many
bacteria as the water it came from. An example is given in the following
table relevant to the number of bacteria in frozen milk (after Bischoff).

Milk kept at 3 to 7.

Freshly frozen 200,000 bacteria per c.c.

After i day 105,500 bacteria per c.c.

After 2 days 7 2 ,3o bacteria per c.c.

After 3 days 62,000 bacteria per c.c.

After 4 days 46,400 bacteria per c.c.

After 7 days 44,000 bacteria per c.c.

After 14 days 40,50 bacteria per c.c.

After 21 days 30,300 bacteria per c.c.

After 35 days 22,500 bacteria per c.c.

After 49 days 14,200 bacteria per c.c.

The table shows plainly that it is impossible to sterilize milk by freez-
ing, but as long as it is frozen it will keep; there is no possibility of any
microorganisms decomposing a frozen liquid, for the organisms need

INFLUENCE OF TEMPERATURE. 159

water above all. If food substances change in cold storage (and
some food products do deteriorate), this must be either due to changes
other than microbial or the material was not completely frozen as is
probably the case with salted butter.

After bacteria are once frozen, they do not seem to be affected by any
lower temperature. Macfadyen and Rowland found that they tolerate
very low temperatures remarkably well. Many bacteria were not killed
by a twenty-hours' exposure to the temperature of liquid hydrogen
(-252). Yeasts are not quite so resistant and the mycelium of most
molds is easily destroyed by freezing, while the spores are hardier.

THERMAL DEATH-POINT. Heating above the maximum temperature
is decidedly harmful to microorganisms; if the exposure to such unsuit-
able conditions is prolonged for a certain length of time, it results in the
death of the organism. The length of time required to kill a certain
organism will depend mainly upon how high the temperature is above
the maximum. A few degrees above this point, it will take several or
even many hours to kill the organism; the higher the temperature the
shorter the time. An exposure to 10 to 20 above the maximum takes
generally but a few minutes. It can be stated generally that the lower
the maximum temperature the more easily is an organism destroyed
by heat. It is customary to speak of the thermal death-point of an
organism as the lowest temperature which will kill it within ten minutes.
(Instead of this time adopted by the Society of American Bacteriologists,
some investigators prefer one or five minutes as the standard time.)

The thermal death-point does not depend upon the species and the
temperature only. It varies with the age of the culture since older cells
are less resistant than younger ones especially if heated in their own pro-
ducts. The medium in which the organisms are heated is also of great
significance. The fact that acid liquids, as fruit juices, are more easily
sterilized than neutral meat or vegetables is largely due to a chemical
(poisonous) action of the acids upon bacteria. But the greater resistance
of tubercle bacteria in sputum compared with those suspended in salt
solution cannot be so readily accounted for.

A necessary factor for the prompt destruction of organisms by heat
is the presence of moisture. The resistance of dry organisms is remark-
ably higher than that of the same organisms in a liquid culture. The
following table shows the death-point of yeast cells and spores in a dry
and moist state.

i6o

PHYSICAL INFLUENCES.

Thermal Death-point of Dry and Moist Yeast.

<

^ells

SF

ores

Variety of yeast

Moist

Dry

Moist

Dry

Pale ale yeast.

6 <5

CK-IOS

6s-7o

Iii5 -i2<5

Hofbrau yeast

55

85- 00

65

II5-I20

Saccharomyces pasteurianus

5o-55

ioo-io5

60

H5

RESISTANCE OF SPORES. The most resistant organisms to heat are
the spores of certain bacteria. In the chapter on moisture requirements
attention has been called to the great resistance of spores to drying. We
find the same exceptional resistance to high temperatures. Boiling heat
will not kill spores readily. Some bacterial spores can stand the tem-
perature of 100 for several hours. In order to kill spores in one heating
the temperature must rise to about 110 for fifteen to thirty minutes;
this can be accomplished only by heating under pressure. This is not
always advisable for sterilizing food substances. While vegetables are
usually sterilized under pressure without losing much of their palata-
bility, other foods like milk are changed materially in taste and ap-
pearance. To prevent these changes, discontinuous sterilization is
sometimes used. This is based upon the following principle:

If milk or any other medium is heated to 100 for about fifteen min-
utes, all living cells of bacteria, yeasts and molds will be killed except a
few spores of bacteria. After cooling, these spores will germinate under
suitable conditions and the vegetative cells thus appearing instead of the
resistant spores are easily killed in a second heating. A third heating
is necessary in order to kill any vegetative cells which may have developed
from spores not yet germinated before the second heating. It is essential
to have the time between two heatings long enough to allow the germination
of spores, and not too long to permit formation of new spores. It is
customary to heat on three successive days for fifteen minutes each time.
In this case, sterilization is usually complete, while a forty-five minutes'
heating at once is not sufficient to guarantee sterilization. Among the
substances that are very easily sterilized are cider and other fruit juices,
while milk and soil are the most difficult materials to sterilize.

Dry spores will resist still higher temperatures than moist spores

INFLUENCE OF TEMPERATURE. l6l

Some dry spores survive an exposure to 140 to 150 for ten minutes. It
requires a very high temperature to sterilize glass, cotton, gauze, and
instruments with dry heat. A discontinuous sterilization of dry material
is useless since the spores will not germinate without moisture, therefore
their resistance remains unaltered.

The spores of molds are more resistant than the mycelium but, if
moist, they all die at 100. The dry mold spores can tolerate a some-
what higher temperature, but not as high as the spores of many bacteria.
Yeast spores and yeast cells are very much alike in their resistance to
heat. The table on page 160 shows hardly any difference between their
resistance.

ii

CHAPTER III.
INFLUENCE OF LIGHT AND OTHER RAYS.

Microorganisms in their natural environment are temporarily but not
usually exposed to light. The organisms of decay, living in soil, in
foods, in the intestines of animals, will only occasionally come in contact
with the direct rays of the sun. Water bacteria and the organisms on
the surface of plants and animals are more commonly exposed to the sun.

The influence of light varies with its intensity. Direct sunlight has a

FIG. 52. -These plates were heavily inoculated with B. coli and B. prodigiosus
respectively and then were exposed, bottom side up, to the direct rays of the January
sun, for four hours. On the instant of exposure, a figure O, cut from black paper was
pasted to the plate shading the bacteria underneath. After one, two and three hours,
the corresponding figures were pasted to the plates. The above picture was taken 24
hours after exposure, proving that three or four hours of direct sunlight weaken and
may even kill bacteria. B. prodigiosus proved more sensitive than B. coli. (Original.)

very harmful effect upon microorganisms. Most bacteria are killed by
direct sunlight in a few hours; the time depends upon the organism
as well as upon the intensity of light; this again varies with the amount of
moisture and dust in the atmosphere, with the time of the day and with
the season; an absolute measure for the action of light cannot be fixed,

162

INFLUENCE OF LIGHT AND OTHER RAYS.

163

therefore, as easily as with the action of heat in the thermal death-point.
The different colors of the spectrum do not act alike; the part of the spec-
trum from red to green is practically without influence upon micro-
organisms, while the blue light acts strongest and the intensity decreases
in the violet and ultra-violet. In carrying on experiments with the in-
fluence of light, it must be remembered that glass absorbs ultra-violet
rays, and further that the heating of the medium by direct radiation must
be avoided.

Yeasts, molds, and bacteria and probably protozoa are equally sensi-
tive to light. Even the spores of most bacteria do not show a greater
resistance to light, while the mold spores are an exception. The colored

FIG. 53. Phototropism of Rhizopus nigricans. The mold was grown on gelatin with
diffused light coming from right side. (Original.)

spores of the Penicillium, Aspergillus and Mucor species can be exposed to
light for a long time without being killed, but the colorless spores of
Oidium and Chalara show no increased resistance. It is supposed that
the pigment in mold spores is a protection against light. This is not true
with the pigment of bacteria. The colored and colorless strains of pig-
mented bacteria show no difference in their resistance to light. Only one
group, the so-called purple bacteria, is an exception. These peculiar
organisms, many of which feed on hydrogen sulphide, seem to thrive
better in light than without it. Direct sunlight does not kill them, it
rather attracts them and they move toward the light. This is called

164

PHYSICAL INFLUENCES.

phototaxis or heliotaxis. The pigment, bacteriopurpurin, does not take
the place of chlorophyl, however, since the bacteria do not produce oxygen
in light and always need organic food.

The effect of light upon microorganisms is mainly brought about by a
chemical change in the protoplasm, and also, to some extent, by a chemical
change in the medium, namely the formation of a peroxide or a similar
oxidizing agent.

The germicidal action of light is of importance in the purification of
rivers. It is applied also in curing diseases of the skin, as lupus and

FIG. 54. Two cultures of an Aspergillus, one grown in the dark, the other in diffused

light, showing rings. (Original.)

leprosy, by exposing the diseased parts to a very concentrated light of the
electric arc. This light contains plenty of blue and violet rays and is
preferable to sunlight because it is always ready for use and its com-
position and intensity can be controlled easily.

Diffuse light is not nearly as harmful to microorganisms as direct
sunlight. Long exposures to diffuse light will kill most bacteria, while
molds are not at all sensitive. They rather like a very dim light, and
many molds grown in a dark room with light only from one side will grow
toward the light. This property, which is characteristic for all green
plants, is called heliotropism or phototropism (Fig. 53). It has been
found that molds produce mycelium mostly in the dark, while in daylight
sporangia are produced mainly. This difference in the development

INFLUENCE OF LIGHT AND OTHER RAYS. 165

during the day and during the night accounts for the concentric rings
which are quite commonly found in older mold colonies, and which in-
dicate the age of the culture (Fig. 54). Similar rings are occasionally
found with yeast and bacterial colonies, and are possibly due to the same
influence of light.

X-RAYS. Of other rays, the invisible X-rays and the radium rays
have attracted the attention of bacteriologists and physiologists. It
is known that the X-rays will destroy living tissue by long exposures;
microorganisms cannot be considered less resistant. X-rays are used
in the treatment of microbial diseases of the scalp and skin.

RADIUM RAYS are not so well known, and their bactericidal action is
doubtful. The treatment of certain bacterial diseases has been attempted,
but it has not been applied as generally as yet as the X-ray method. The
sterilization of milk and possibly other foods by this method has been
suggested, but the practical application is at present quite improbable be-
cause of the cost and the uncertainty of the results.

CHAPTER IV.
INFLUENCE OF ELECTRICITY.

The influence of electricity upon microorganisms is much less than
one might perhaps expect, if the electricity as such is considered. A direct
electric current passing through a nutrient medium will, of course, cause
electrolysis which is usually manifested by the formation of acid on the
positive pole and of alkali on the negative pole. The acid and alkali will
kill microorganisms, as is discussed in the chapter on chemical influences.
In this case, it is not the electricity itself that destroys the bacteria. It is
also possible to kill bacterial cultures by passing an alternating current
through the medium for some time. No electrolysis takes place in this case,
still it is not the direct action of the current that acts upon the organisms,
but rather the heat produced by the current passing through a medium of
high resistance. If the culture is cooled properly the influence of the cur-
rent is insignificant if at all noticeable. Whenever electricity is applied
against microorganisms, the effect is considered electrochemical.

The electrical current is used in a very small way in the purification
of sewage. The sewage passes between two iron plates which represent
the two poles of a strong current. The electrical sterilization of milk has
been patented. Wines are improved by electricity. The sterilization of
drinking water by ozone is also an application of electricity, though of
course the ozone once formed by the current acts as a chemical com-
pound independent of its source.

166

CHAPTER V

INFLUENCE OF THE PHYSICAL STRUCTURE OF THE

MEDIUM.

The physical structure of the medium has often a remarkable in-
fluence upon the development of microorganisms. Very little attention
has been paid to this fact, as yet, and only a few instances are known where
the structure of the medium is a deciding factor in the life of microorgan-
isms. A few examples have already been mentioned. Saccharomyces
will only exceptionally form spores on the surface of liquids while they
are abundantly formed on the surface of a moist gypsum block. This
may be accounted for by the better aeration of the yeast cells on gypsum.
Another example, mentioned before, is the influence of the medium, on
which bacteria are dried, upon their resistance against desiccation.
Chemical action of the medium upon the bacteria cannot explain the
differences since the same stratum in different structures gives different
results. The great advantage of porous materials (soil, starch, charcoal,
etc.) over smooth surfaces may be due to the very rapid and complete dry-
ing in the porous material and possibly to the power of absorbing gases of
all kinds.

Recently, attention has been called to another example of considerable
importance, namely, the difference of development of bacteria in soil and
in solution. The processes of ammonia and nitrate formation do not run
parallel in solution and in soil; this is not due to certain chemical com-
pounds in the soil, since the result remains the same if the soil is sub-
stituted by the insoluble quartz sand; quartz has no absorptive powers, so
this cannot account for the difference. The most probable explanation
is the enormously increased surface of the water surrounding the soil
particles. The surface exposed to oxygen is many times larger in a soil
of a normal water content than in the same amount of water in a flask.
In agreement with this explanation, nitrification, as a strict oxidation
process, is greatly increased in soils with moderate moisture and is checked
if the soil is saturated with water. Ammonia formation is usually favored
in well-aerated soils, but occasionally it is decreased. This is probably
due to the different types of ammonia-producers, some of them growing
better with air, others growing better without it. Details will be given
in the division on Microbiology of Soil.

"167

CHAPTER VI.
INFLUENCE OF MECHANICAL EFFECTS.

PRESSURE. -The resistance of microorganisms to mechanical pressures
is very great. Pressures of 3,000 atmospheres* will not kill the majority
of bacteria in four hours. They are, however, weakened and some species
will die. A specific difference between the molds, yeasts, and bacteria in
this particular does not seem to exist. Of the organisms exposed to 2,000
atmospheres for ninety-six hours, Bad. anthracis, Bad. pseudodiphtherice, M.
pyo genes var. aureus, Oidium ladis and Saccharomyces cerevisice survived,
while seven other organisms lost the power of multiplication. Some of
these were not dead, however, since they retained their motility for several
days. It is noteworthy that high pressure will destroy one quality
(multiplication) and not affect another (motility). Pigment-production
and virulence of pathogenic bacteria were either diminished or lost com-
pletely. The resistance against high pressure is necessary for the
organisms which cause the decay of organic matter at the bottom of
the oceans. Vertebrates breathe oxygen in the form of gas or have at
least an organ filled with gas (fish bladder) ; the volume of gas is changed
considerably by slight changes of pressure; this will affect organisms
depending on gas. Microorganisms do not require gas as such. They
can absorb gases only in solution. A change of pressure therefore will
not cause a change of volume, since liquids have a very small coefficient
of compression.

The situation is entirely different if the liquid is not exposed to the
pressure directly, but to compressed gas. In this case, the chemical effect
of the gas is the deciding agent. The higher the pressure, the more gas
will be dissolved in the culture medium. The fatal pressure under these
conditions will vary as much as the fatal dose of an antiseptic ; it depends
upon the chemical qualities of the gas, upon the pressure (concentration) ,
upon the temperature, and upon the organism.

Some data have been given already in the chapter on oxygen require-

* One atmosphere is i kg. pressure per square centimeter (or about 15 pounds per square
inch).

1 68

INFLUENCE OF MECHANICAL EFFECTS. 169

ments. It was mentioned in that connection that Bact. butyricum cannot
tolerate more than 0.65 per cent of the total oxygen^o.2 atm.); content
in air in other words, an oxygen pressure higher than 0.0013 at-
mospheres will kill the organism. The maximum pressure for B.
prodigiosus was found to be about 5.4 to 6.3 atmospheres. Very few
experiments have been made with other gases. Carbon dioxide at a
pressure of 50 atmospheres retards the growth of bacteria in water and
will sterilize it in twenty-four hours. Suspensions of pure cultures of
B. lyphosus and Msp. comma are killed by 50 atmospheres carbon dioxide
pressure in three hours. Milk cannot be sterilized by this pressure but
bacteria do not multiply. Carbonated milk has been recommended as
a refreshing drink by several investigators. The ordinary market milk
will keep about two days longer under the pressure of 10 atmospheres
(150 pounds) than without pressure. If pasteurized it is said to keep
for a week.

GRAVITY. Gravity would have a great influence upon the growth of
microorganisms in liquids if their specific gravity were much greater than
that of water. This does not seem to be the case however. It has been
estimated by accurate weighing to vary between 1.038 and 1.065. Very
much higher results (1.3 to 1.5) have been obtained by centrifuging bac-
teria in salt solutions of varying specific gravity, but these data are not exact
since the salt solution will diffuse into the cells and thus increase their
weight. The specific gravity being very nearly that of the culture medium,
it is plainly seen that gravity has but little influence. The microorganisms
will live suspended in the liquid and sediment out very slowly. The
slightest current in the liquid will carry them around and distribute them
through the medium. The motility is of minor importance; the actual
distance covered by motile bacteria has been measured, and under the
most careful exclusion of currents in the liquid has been found to be about
a millimeter in a minute for B. subtilis. This is very slow compared with
the speed of the circulating water moved by changes of temperature or
other incidental agents.

Yeast cells and other gas producers use the carbon dioxide as a vehicle.
The gas bubbling up in the fermenting liquid keeps it constantly in motion
and moves the yeast cells against gravity toward the surface where the gas
escapes and lets the cells fall back to the bottom.

The production of scums and pellicles on the surface by organisms,
which are heavier than the liquid they float on, is often accomplished by

170 PHYSICAL INFLUENCES.

small gas bubbles between the cells. In other instances, it may be
just the floating of cells having oily surfaces.

The growth is influenced by gravity very little. The sporangia of
molds are the only exception, growing decidedly away from the center of
gravity (negative geotropism).

AGITATION. For the majority of microorganisms, the quiet, undis-
turbed growth of the laboratory culture is the normal or the ideal one.
Such cultures, if shaken for a considerable time, show a decrease of living
organisms, and it is possible to sterilize cultures by continued shaking.
The effect it not a simple mechanical breaking or tearing of the cells.
The bacteria break up into the finest particles. This is also the case if
cultures are exposed for several days to the trembling motion caused by
the working of very heavy machines. There is no grinding or tearing
effect but the cells break to pieces just the same.

A slight and slow agitation seems to be advantageous for many cul-
tures, only continuous heavy motion proves harmful. Different organisms
show wide variations in their resistance to agitation.

DIVISION III.

CHEMICAL INFLUENCES.

CHAPTER I.
STIMULATION OF GROWTH.

The influence of chemical substances upon microorganisms may be
helpful or harmful, or not noticeable. As helpful must be considered
above all the food compounds. Unless given in such large doses as to
cause a physical or osmotic effect (see chapter I, page 147) they will
stimulate the development. Other substances too, which are not food,
can also act as stimulants. It is a recognized fact of long standing that
many poisons in very small doses will stimulate. This applies to the
msot highly developed animals and plants as well as
to microorganisms. Raulin noticed in 1869 that
Aspergillus niger grew very much better in a nutrient
solution if a small amount of zinc salt was added. He
considered the zinc, therefore, as a necessary constit-
uent of the mold cells. Alcoholic fermentation can
be stimulated by metallic salts. It is believed by
some physiologists that, as a law of nature, every
substance that is injurious in a certain concentration
is a stimulant in a lower concentration. A similar
action of certain chemical compounds upon enzymes
has been noticed, retarding in high concentrations, FlG 55 _chemo-
stimulating in weaker solution. An explanation for taxis. (After Fischer.)
these facts cannot be given at the present time.

CHEMOTROPISM AND CHEMOTAXIS. Microorganisms manifest their
preference for certain foods not by a stimulated growth alone. They also
make efforts to obtain better food by growing or moving toward it, which
is not a manifestation of a rudimentary intellect. Such reactions of micro-
organisms, may be accounted for largely by chemical or osmotic forces.

171

172 CHEMICAL INFLUENCES.

In a solid medium the hyphae of molds will grow toward the best source of
food supply. This growth on account of chemical stimulation is called
chemotropism, analogous to the phototropism or growth toward light.
If some injurious compound is offered, the hyphae will grow away from it.
Thus we have to distinguish between positive and negative chemotropism.
The motile organisms, bacteria as well as protozoa, demonstrate their
preference for certain food compounds by swimming toward them. This
is called chemotaxis (Fig. 55). Here also a positive and negative
chemotaxis must be distinguished, the latter taking place if injurious
substances are present. These reactions do not seem to be of much im-
portance in the life of microorganisms.

CHAPTER II.
INHIBITION OF GROWTH.

POISONS, GERMICIDES, DISINFECTANTS, ANTISEPTICS, PRESERVA-
TIVES. A great number of inorganic and organic bodies will destroy life
in comparatively weak solutions. These substances are called poisons
if they are considered in their effect upon man and animals. In their
application to microorganisms they are generally called germicides (germ-
killers), or disinfectants if the emphasis is laid upon the prevention of
infection rather than upon the actual killing of the microorganisms.
Analogous to the general term germicides, the terms bactericide and
fungicide are used occasionally. The term antiseptic means a prevention
of sepsis which may be accomplished by checking the growth without
necessarily killing all microorganisms. The meaning of the word pre-
servative is practically the same, only the latter is used more commonly
in relation to foods, feeding stuffs and preparations of similar origin while
the word antiseptic is largely used in relation to microbial diseases. A
strict line cannot be drawn between any of these definitions. A disin-
fectant, if diluted, becomes an antiseptic. A strong salt solution is an
antiseptic for some organisms and a disinfectant for others. Of the above
expressions, germicide is the most definite, but is not so commonly used
as the others.

MODE OF ACTION. The action of a poison upon the cell is generally
considered an action upon the protoplasm. The poison is supposed to
combine chemically with the cell plasma producing compounds which
interfere with the continuation of the life processes and thus cause death.
If the cell has been subjected to the action of the poison only a short time,
it can be saved by removing the poison. Bacteria can be treated with
mercuric chloride (HgCl 2 ) so that they will no longer develop if trans-
ferred to a fresh medium. If the mercuric chloride is removed from the
cell by means of hydrogen sulphide, some of the organisms may be revived.

The action of a disinfectant upon a suspension of bacteria is not uni-
form. The largest percentage of cells will be killed in a short time,

173

174

CHEMICAL INFLUENCES.

\vhile a few will remain resistant for a considerable length of time. This
is the case with all disinfectants. The disinfecting process resembles in
many ways a slow chemical process, as the inversion of cane sugar. In
the beginning, the reaction is very rapid, because plenty of the cane
sugar is present; after a certain time, the action is much slower, because

3500

3000

Z500

9,000

I 500

1000

500

\
\

0\

i

f _A

10

20 30 HO

5-0

FIG. 56. Curve of disinfection. Spores of Bact. anthracis in mercuric chloride solu-
tion. (After Chick.)

most of the sugar is decomposed; finally it seems to nearly cease. It is
the same with disinfection; at first many bacteria are present, but they are
killed rapidly and only a few are left. These few die much more slowly
than the first ones, not because they were more resistant, but simply as a
consequence of the chemical law that the less there is of the react-
ing substances the slower is the rate of reaction. The curves in

INHIBITION OF GROWTH. 175

figure 56 show the numbers of living spores of Bad. anthracis suspended
in mercuric chloride solution. The continuous solid curve gives the
result in a o . 2 1 per cent solution, the dotted curve indicates a o . 1 1 per
cent solution. The number decreases rapidly in the first few minutes,
but the rate of dying is not constant, and even after a comparatively
long time, a few spores remain alive.

Similar curves are obtained in the destruction of microorganisms by
heat, by drying and by freezing.

FACTORS INFLUENCING DISINFECTION. The efficiency of a disin-
fectant depends upon several factors. Moisture is necessary a dry
poison has only a very slow action upon microorganisms. For this
reason, absolute alcohol has not nearly the same germicidal power upon
dry bacteria as diluted alcohol; the strongest poisonous effect is obtained
by a 40 to 50 per cent solution; bacteria suspended in water die the more
quickly the stronger the alcohol. The necessity of moisture is further
demonstrated in the sterilization with gases, as with formaldehyde. The
effect of formaldehyde gas without the provision of a very moist atmos-
phere is surprisingly weak.

The temperature is also quite an important factor in the study of dis-
infectants. Since poisoning is supposed to be a chemical effect, it must
be expected that the poisoning process like other chemical processes
will take place faster at a higher temperature. This is actually the case.
It is possible to cool a disinfectant solution to the extent that it is only an
antiseptic. On the other hand, it is easily seen that above the optimum
temperature, where the growth is not very vigorous, and when the dis-
infecting power of the poison is increased considerably by the higher
temperature, a very small amount of poison will have a very strong germi-
cidal effect. The combination of high temperatures with a disinfectant
has been suggested as a means of sterilizing foods. This has been tried
in the case of milk with hydrogen peroxide at 50 to 60.

It makes a considerable difference whether the organisms which are
tested with a certain disinfectant are in a culture with their food material,
or suspended in water or salt solution without any food. It is very pro-
bable that part of the disinfectant is acted upon by the food products
which are partly protein substances and are in many ways similar to the
protoplasm of the bacterial cells. It is especially difficult to poison bacteria
in blood, pus, or similar material. The sensibility of microorganisms
in pure water is remarkable. Very small doses which would not be

176 CHEMICAL INFLUENCES.

considered efficient under any other condition, will destroy microorganisms
in pure water.

The age of trie culture and the stage of development will naturally
change the resistance of a species materially. The old cultures which
are past the culmination of growth will be much more sensitive to any
poison unless a spore-producing organism is under test. In this case,
we find a greatly increased resistance, similar to the increased resistance
of spores against drying and heat.

THE CLASSIFICATION OF DISINFECTANTS is very difficult as long as we
cannot explain completely the process of poisoning. It is impossible to
arrange them according to the intensity of action, because the intensity
of influence depends not only upon the disinfectant, but also upon the
species of organisms. Some yeasts can resist ten times as much alcohol
as certain bacteria. Formaldehyde is not nearly as strong an agent with
molds as it is with bacteria. The disinfectant concentration of a
poisonous substance is not absolute. The simplest method of grouping
is by chemical structure and qualities. Of the following natural groups
can be distinguished acids (inorganic and organic), metallic salts, hydro-
carbons (aliphatic and cyclic), alcohols (aliphatic and cyclic), aldehydes,
anaesthetics, essential oils, oxidizing agents and reducing agents.

The first three groups, acids, alkalies and salts, can be distinguished
from the rest as electrolytes; the strength of acids and alkalies (chemically
speaking) is measured by the degree of electrolytic dissociation. The
disinfectant value follows largely the same law. The strongest acids in
the chemical sense are also the strongest disinfectants. There are ex-
ceptions, however, where, besides the poisonous effect due to the degree
of dissociation, there is a specific effect due to the chemical structure, as
in the case of nitrous, salicylic and hydrocyanic acids. The same is true
of alkalies. With metallic salts, the action will depend mainly upon the
metal in solution, but the electrolytic dissociation is also of importance.
NaCl will decrease the dissociation of mercuric chloride (HgCl 2 ) and de-
crease also its disinfectant power. Mercuric chloride dissolved in abso-
lute alcohol is not dissociated. In this case, it has almost no action upon
bacteria.

Acids are not commonly used as disinfectants, except in the household,
but they play a certain role in nature The common fruits contain so
much acid that bacteria cannot easily attack them; the decaying of fruit
is almost exclusively due to molds which have a preference for acid media;

INHIBITION OF GROWTH. 177

yeasts, too, are quite resistant to acid and can become accustomed to
quite strong acids; it is customary in the manufacture of industrial alcohol
from potatoes to prevent bacterial growth by adding about i per cent of
lactic acid or very active lactic bacteria together with an acid-tolerant
yeast, bred especially for this purpose. The acid in the stomach of man
and animals plays an important role as a sterilizing agent for the food.
Many microorganisms are killed in the stomach. In the household,
the natural acidity of fruit helps in keeping canned fruit, preserves and
jellies. Especially in heating, the acid together with the high temperature
has a very strong germicidal effect. Vinegar is often used to preserve
fruit and vegetables; in some parts of the country, meat is kept in butter-
milk. Benzoic and salicylic acids are often used in the preservation of
fruit and vegetables. Their poisonous influence is not so much due to
the acid reaction but to the specific chemical character of these
compounds.

Of the alkalies, only one is used extensively, namely, lime; quick-lime
(CaO) is considered a valuable disinfectant for excreta in privy vaults; it
is universally applied as a white-wash in stables, barns, poultry houses and
similar buildings. Quite commonly, it is used as "milk of lime" (one
part of slaked lime with four parts of water). It should be kept in mind
that the calcium oxide unites with the carbon dioxide of the air and thus
gradually loses its disinfecting power.

Of the metallic salts, many are well-known germicides. The most
powerful disinfectant is mercuric chloride (HgCl 2 ) which is one of the
standard disinfectants. It is generally used in a dilution 1:1000 which is
sufficient to kill all vegetative cells as well as spores in a few minutes.
Quite commonly, hydrochloric acid or salt is added, to prevent coagula-
tion or precipitation of slimy or albuminous matter which would protect
the enclosed bacteria from immediate contact with the poison. The
addition of hydrochloric acid or any chloride decreases somewhat the
disinfectant value for bacteria suspended in distilled water because it
decreases the electrolytic dissociation.

Another disinfectant of remarkable strength is silver nitrate; it is not
used commonly because of its high price. It also decomposes easily and
leaves dark spots on the skin and clothes. Of the other metallic salts
copper and iron sulphate are not used extensively, though recommended
for the disinfection of feces. Zinc sulphate may be applied to mucous
membrane the same as silver nitrate. Many other salts may be used

12

178 CHEMICAL INFLUENCES.

occasionally for disinfecting purposes, though the expense or undesirable
qualities prevent their common application.

The alcohols are well known for their poisonous effects, but the value
of ethyl alcohol as a disinfectant is usually overestimated. It takes quite
strong alcoholic solutions, more than 20 per cent, to kill certain yeasts and
the spores of some bacteria hi less than a day, and a complete sterilization
by alcohol hi a few minutes cannot always be guaranteed even with 50 to
60 per cent solution. It has already been mentioned that desiccated
organisms are very resistant to concentrated alcohol, more so than to a
50 per cent mixture. Methyl alcohol is weaker, the higher alcohols, es-
pecially amyl alcohol, are stronger disinfectants than ethyl alcohol. They
all give good results hi the presence of water while the absolute alcohols
have scarcely any effect upon desiccated bacteria. None of these alco-
hols in whatever concentration they may be used, can be relied upon to
kill bacterial spores.

Stronger germicidal effects can be obtained by the alcohols of the
benzene group, of which phenol or so-called carbolic acid (C 6 H 5 OH) is the
simplest representative. Phenol, like ethyl alcohol, is not as effective as
is commonly believed. It is applied in solutions from 0.5 per cent to 5
per cent ordinarily, but it usually takes a long time even for the 5 per cent
solution to kill vegetative cells as Bact. tuberculosis or B. coli; it is
inefficient against anthrax spores. More powerful are the higher cyclic
alcohols, of which the cresols are examples. They are used extensively
as disinfectants and antiseptics. They are, together with phenol, coal-tar
constituents and are sold commercially under many different names,
either pure or mixed with soap or other disinfectants which make
them emulsify readily in water. The cresols are almost insoluble in
water, and not as effective in solutions as they are in emulsions. The
disinfecting properties of tar come from the cresol contained in it.

Hydrocarbons are used only for laboratory experiments as very weak
antiseptics. The aliphatic bodies, as methane, etc., which constitute a
large part of coal gas, have but very little effect upon bacteria; the gas is
used occasionally in place of hydrogen for growing anaerobic bacteria.
Benzol, xylol, and toluol are antiseptics, if shaken frequently with the
liquid to be protected, but they are not reliable as disinfectants. The
same is true with the common anaesthetics, ether and chloroform. The high
prices of these agents forbid their general use, but they are sometimes used
for laboratory work.

INHIBITION OF GROWTH. 179

The essential oils have a little more practical importance. Some of these
are the main constituents of mouth washes, especially the oil of pepper-
mint (menthol) , of thyme (thymol) , and of eucalyptus (eucalyptol) . Their
action is very weak, however. The volatile oils of spices have to be con-
sidered in the preserving of fruit, pickles, catsups, and other food products.
Though the antiseptic value in general is insignificant, certain micro-
organisms are sensitive to certain spices. The bacteria of the mesen-
tericus group are said to be suppressed entirely by quite small quantities
of garlic, while others, like the lactic bacteria, are not affected at all.
Cloves, cinnamon and alspice are the most efficient spices, while the
disinfectant power of black and white pepper, and mustard is very small.

The most important disinfectant has not been mentioned, because
it does not belong to any of the above groups. This is formaldehyde.
Formaldehyde (HCOH) is a gas, soluble in water to the amount of 40
per cent at room temperature; it does not attack metal, clothing, wood-
work, and is, therefore, preferable to many other disinfectants for steriliz-
ing rooms. It kills spores of bacteria in a short time in a 1:1000 di-
lution. Its greatest importance lies, however, in its gaseous nature,
because it can be applied to rooms and buildings by simply evaporating it.
The saturated 40 per cent solution can be evaporated directly or by
generating steam which passes through the formaldehyde solution; this
latter method has the advantage of saturating the air with moisture,
which increases the power of the formaldehyde gas. Formaldehyde
can also be obtained in a dry form; it polymerizes to a white crystalline
substance, paraformaldehyde ((HCOH) 3 ) which can be changed back to
formaldehyde gas by gentle heating. This paraformaldehyde is com-
monly used instead of the liquid, because it is more easily handled and is
quite inoffensive in its solid form, while the formaldehyde solution has a
very penetrating odor and is exceedingly harmful to the mucous mem-
brane of the respiratory organs.

Of the oxidizing agents, oxygen itself has already been mentioned.
Though it is able to destroy certain anaerobic bacteria, it cannot be called
a disinfectant. For this purpose, oxygen must be activated; such oxygen
can be obtained in the form of ozone (O 3 ). It is formed in air under the
influence of electric discharges and can be produced at a price low enough
to allow its application for use in the sterilization of water. It has also
been recommended for preservation of milk.

Hydrogen peroxide (H 2 O 2 ) resembles ozone in its chemical reactions;

l8o CHEMICAL INFLUENCES.

it changes readily to H 3 O+ O, and this oxygen atom in the nascent state
is quite effective as an oxidizing agent. For an antiseptic, it must be used
in at least a i per cent solution, and for an absolutely reliable disinfectant
a still higher concentration is required. It loses its disinfecting property
easily because it is decomposed readily by the peroxidases of tissues and
organic liquids as blood, milk and pus. It is used in the preservation of
milk. Hydrogen peroxide is slowly decomposed by the katalase of milk
thus disappearing completely.

Chlorine in its gaseous form is not used as a disinfectant, though its
germicidal power is quite strong. The so-called "chloride of lime,"
manufactured by absorbing chlorine in slaked lime, gives in water
hypochlorite and free chlorine; these substances are good germicides
and chloride of lime is used in the disinfection of privy vaults, and other
places in which it may be employed without injury.

Potassium permanganate is only incidentally used as a disinfectant.
Its chemical qualities prevent an ordinary use.

Sulphurous acid, or sulphur dioxide (SO 2 ) was for a long time a
standard disinfectant and is still used occasionally for fumigating rooms,
stables, barns and out-buildings though it is substituted more and more by
formaldehyde which can be applied almost as easily. The burning of
sulphur is an extremely simple process, but it requires a moist air to dis-
infect properly, and under these circumstances it will attack metal, dyes
of clothing and even the fiber itself.

DIVISION IV.
MUTUAL INFLUENCES.

INTRODUCTION.

The biological relations of microorganisms are of the greatest im-
portance in nature. Pure cultures in nature are very rare and of exceptional
occurrence; they are hardly ever found except in certain diseases of man,
animals and plants. Generally, nature works with mixed cultures. All
natural fermentations, decompositions and putrefractions are accom-
plished by a number of different species among which perhaps one domi-
nates, but is influenced by the rest. The study of the mutual relations of
microorganisms is in the very first stage as yet; practically all laboratory
work is done with pure cultures. The experiences obtained with pure
cultures are not sufficient to explain all microbial activity in nature.

There are many possibilities of mutual influence between different
organisms. Generally three main cases are distinguished: symbiosis,
where two organisms profit by the combination; melabiosis, where one
profits by the other's action without benefiting the other in return, and
antibiosis, where one organism injures the other. These cases cannot be
separated strictly. The relations are not always constant through the
entire development of the cultures; an originally beneficial influence
may change to an injurious one in a few days. Many terms have been
coined to designate all these various possibilities, but in order to avoid
this multiplicity of more or less indefinite names for the various relations,
the general term "association" has come into use, especially when the
relationship is not well understood.

SYMBIOSIS.

Symbiosis is not very common among microorganisms, and it is
difficult to find examples where true symbiosis exists through the entire
development of both organisms. The association of lactic bacteria and
Oidium lactis in milk is, for a certain period at least, a symbiosis. The

181

l82 MUTUAL INFLUENCES.

bacterium will produce only a certain amount of acid, and then it can
grow no more because the acid is too strong; the mold will destroy the
acid and thus gives the bacterium a chance for continued activity. The
bacterium produces the acid which the mold likes; the mold in turn
removes the excess acid which otherwise would check the bacterial
activity.

True symbiosis is more common in the relation of microorganisms with
higher plants and animals. The standard example in the plant kingdom
is Ps. radicicola in the nodules of legumes, feeding on carbohydrates pro-
vided by the plant and furnishing the plant nitrogen from the air which
the plant cannot assimilate directly. The typical example in the animal
kingdom is B. coli in the intestine of animals, being nourished by the food
of the animal and rendering the food more easily digestible.

METABIOSIS.

Metabiosis may be considered a one-sided symbiosis; two organisms
live together, but only one is benefited the other remains uninfluenced or
later may be injured by the association; the latter case is the most common.
In this relation, one usually prepares the food for the other. It has pre-
viously been mentioned that the metabolic products of one species serve as
food for another species, thus breaking up the various organic compounds
step by step to smaller and simpler molecules. Quite commonly, each
step is accomplished by a different species of microorganism. Conse-
quently, metabiosis is a very common occurrence among microorganisms.

The classical example is the two nitrifying bacteria: the nitrate bac-
terium is unable to oxidize ammonia, and depends entirely upon the ni-
trite bacterium to oxidize the ammonia to nitrite; then, and only then, can
the nitrate bacterium grow.

The relation between yeasts and acetic bacteria is also very well known.
The yeast ferments the sugar to alcohol, and then the acetic organisms
oxidize the alcohol to acetic acid. The yeast is in no way helped by the
acetic bacteria, while these could not form acetic acid from sugar readily.
These bacteria depend upon the action of the alcohol-forming yeast.
Other cases of metabiosis are found in the association of lactic bacteria
with certain protein destroying organisms. The lactic bacteria often
develop much better if the protein bacteria grow together with them or
have grown previously in milk. Metabiosis does not require the growth

INTRODUCTION. 183

of the two associated organisms at the same time. The effect will be the
same if first the one and later the other develops, and even after the first
organism is killed or removed, its effect upon the pure culture of the second
will still be noticed. This does not occur in the case of symbiosis.

One species can favor the development of another by other means than
food provision or preparation. Certain bacteria cannot live in acid media,
and molds or Mycodermce destroying the acid will render possible the
growth of these bacteria though they do not provide them with food.
This is the case in the ripening of certain soft cheeses. Another example
is the production of heat by fermenting organisms in manure, hay, ensilage,
enabling the development of thermophile organisms. A very interesting
and important problem is the growth of strictly anaerobic bacteria near
the surface of liquids in association with some aerobic bacteria. How
this is really possible cannot be satisfactorily explained. Though the
aerobic bacteria continuously remove the oxygen from the water a certain
amount will remain, sufficient to prevent the growth of the anaerobic bac-
teria under ordinary conditions. There seems to be a certain protective
influence derived from the aerobic bacteria, the nature of which is un-
known.

ANTIBIOSIS.

The standard examples of antibiosis are the alcohol production by
yeast in sugar solutions and the acid production by lactic bacteria in milk.
Fresh cider contains a large number of bacteria, yeasts and molds; some
of these organisms cannot develop in the acid medium, but many will be-
gin to grow. Some of the bacteria will produce or destroy acid, others
may begin to work on the nitrogenous material of the cider, and the yeasts
produce alcohol and carbon dioxide. The carbon dioxide will soon satu-
rate the cider and begin to bubble up, thus removing the other gases.
The molds will stop growing if the oxygen is taken away, but some of the
bacteria may continue growing until the alcohol concentration checks their
further development. They first cease to grow, then cease to produce acid
and finally die, while the yeast is still continuing in the fermentation.

In the lactic fermentation of milk, Bact. lactis acidi combats all other
organisms by a rapid production of lactic acid. Though it is present in
fresh milk only in very small numbers, its rapid growth and the forma-
tion of acid which will check and even kill most other bacteria soon makes
it the dominant organism in the flora of milk, and at the time of curdling,

184 MUTUAL INFLUENCES.

it is often difficult to find any other organisms besides the lactic bacteria.
In the preceding chapter was mentioned the metabiosis of certain protein-
digesting bacteria with Bad. lactis acidi. This metabiosis can be con-
sidered as such only from the standpoint of the lactic organism. The
protein bacterium is killed by the acid formed by the rapidly growing
lactic bacteria. From the viewpoint of the protein bacteria, the relation is
antibiosis. Another illustration of antibiosis is the acetic fermentation.
The formation of acetic acid prevents the development of all bacteria and
of most yeasts and molds.

In all these cases, the deciding agent is a well-known chemical com-
pound. In other combinations, the principle is unknown. Bad lactis acidi
will check the growth of B. subtilis not only in milk where it forms acid,
but also in sugar-free broth where acid production is impossible. Acetic
bacteria act upon the yeast cells not only by means of the acetic acid pro-
duced, but also by some other, unknown agent, since vinegar is more
injurious than the corresponding amount of pure acetic acid in water.
A very remarkable organism is Ps. pyocyanea; it secretes a substance,
pyocyanase, which will kill and dissolve the cells of other bacteria rapidly.

Parasitism, which would be classified under antibiosis, has not been
found to exist among bacteria or yeasts; but we know of cases where one
mold grows on the other; this is especially true with the largest represen-
tatives of the mucor family, which are often attacked and sometimes
killed by smaller fungi.

PART III.

APPLIED MICROBIOLOGY.

DIVISION I*
MICROBIOLOGY OF AIR

CHAPTER I.

THE MICROORGANISMS OF THE AIR AND THEIR

DISTRIBUTION.

The atmosphere is not the normal habitat of bacteria, for growth and
multiplication cannot take place in it under ordinary conditions. The
phrase "microorganisms of the air" is therefore somewhat ambiguous.
The small size of microorganisms enables them to remain suspended for
considerable periods when physical forces have separated them from the
substrata on which they have developed.

MICROORGANISMS PRESENT IN THE AIR. Molds, bacteria, and yeasts
are all found in the air under certain conditions. The first two are usually
relatively abundant, the latter are less common.

The common molds have adapted themselves for the most part to
wind distribution. They bear spores that are small in size and with a
surface that is not readily moistened. These spores are resistant to
desiccation and light and remain viable for a considerable time even
under unfavorable conditions. Furthermore, the fruiting bodies of many,
though not all molds, show a distinct negative hydrotropism, i.e., the
mycelium remains in contact with the moist substratum while the threads
which bear the spores rise at right angles to it. These latter are so sensi-
tive that they can detect slight differences in the moisture content of the
air and grow in the direction which will bring the spores into the driest

* Prepared by R. E. Buchanan.

l86 MICROBIOLOGY OF AIR.

situations. A slight current of air will detach the spores from these
structures and carry them long distances.

Bacteria and yeasts lack the specific adaptations for wind distribution
found in molds. The material upon which they have been growing
must be dried and pulverized before they can be blown about. Many
species produce spores or other resistant cells, and physiologically, are as
well adapted for air distribution as are the molds.

OCCURRENCE IN THE AIR. Microorganisms are found free in the air,
attached to particles of dust, or enclosed in minute drops of water. Mold
spores are commonly free or in unattached clusters. Bacteria and yeasts
are usually associated with dust particles, frequently the pulverized sub-
stratum on which they have been growing. Not all dust particles have
living organisms attached. It has been computed that in the air of London
during a fog there is only one living organism for over thirty-eight millions
of dust particles. Microorganisms are sometimes sprayed into the air
with water. Droplets containing bacteria are thrown off in the saliva
in coughing or in speaking, and from the surface of fermenting liquids on
which bubbles are bursting. When the drop is small enough, the air
currents keep it in suspension and the water soon evaporates and frees
the organism. This brings about the condition first discussed, free
bacteria in the air. The decrease in weight and size incident to this
loss of water probably accounts for the fact that the so-called "infectious
droplets" are sometimes carried for considerable distances.

How MICROORGANISMS ENTER THE AIR. In comparatively few in-
stances do microorganisms possess mechanical devices for projecting the
spores or other cells into the air for wind distribution. Usually the or-
ganism is passive and is freed only by air currents or by mechanical
agitation. Some molds, as has been stated, release their spores even in the
presence of moisture, so that complete desiccation is unnecessary for
their dispersal. Bacteria and yeasts, on the other hand, are not usually
given off from moist surfaces. Only when dry and pulverized can the
bacterial medium be readily blown about. Hansen found that in the im-
mediate vicinity of a heap of decaying malt, the air was comparatively
free from bacteria. Winslow has shown that sewer air is frequently
practically free from bacteria although the surface with which it comes in
contact teems with bacterial life. Mechanical agitation often throws
large numbers of organisms into the air. Moving hay and straw, groom-
ing animals, sweeping a floor or carpet will multiply the dust and bacterial

MICROORGANISMS OF THE AIR. 187

content of the air many times. In a similar manner, tiny germ-holding
droplets may be scattered by the splashing of sewage or of fermenting or
putrefying liquids, and in speaking, sneezing or coughing.

CONDITIONS FOR SUBSIDENCE OF BACTERIA. The length of time
during which an organism may remain suspended in the air is dependent
upon several factors. Small particles settle out more slowly than large
for the reason that as the size of an object is decreased, the surface area
decreases less rapidly proportionately than the volume. The lifting
effect of air currents depends upon the ratio of surface area to volume
and specific gravity. The smaller the object, therefore, the greater is the
resistance to subsidence. Consequently, bacteria usually settle out of air
very slowly if free in a quiet atmosphere. The time of suspension is
determined also by the velocity of the air currents. While considerable
velocity may be necessary to dislodge microorganisms and bring them
into suspension, a very slight air current will sustain them. Winslow
has found that a current of seventeen inches per minute is sufficient to
sustain B. prodigiosus. The relative humidity of the air is also an im-
portant factor. In a supersaturated air solid particles, such as bacteria,
become nuclei of condensation for water and quickly settle out. When
dust is present in considerable quantities, and certain electrical or mois-
ture conditions exist, flocculation occurs and the larger bodies so formed
subside rapidly. The character and abundance of surfaces with which the
suspended particles may come in contact also play an important part.
Moist surfaces are much more effective in retaining particles than those
which are dry.

DETERMINATION OF THE NUMBER OF BACTERIA IN THE AIR. The
number of bacteria in the air is frequently determined by exposing open
petri dishes of gelatin or agar in different places for definite periods.
This is a comparative quantitative method only. The number of colonies
developing upon these plates will give the number of dust particles having
living spores or cells upon them that fall in the given area under the con-
ditions of the experiment. Evidently this is of value only for rough
comparative work as constantly shifting currents of air usually intro-
duce great errors. A somewhat more accurate method is to draw meas-
ured volumes of air into a flask, the bottom of which is covered with a
layer of gelatin or agar. The colonies which develop represent the
number of organisms which settle out from the given volume. More
accurate results still may be obtained by drawing measured volumes of

1 88 MICROBIOLOGY OF AIR.

air in small bubbles through liquid gelatin. Practically all of the particles
will be retained and the number of colonies which develop may be counted.
This method is sometimes modified by drawing the air through a definite
volume of water, care being taken to insure sufficient contact of air and
water to remove all dust particles. A proportionate part of the water
is then plated and the number of organisms estimated. Air is sometimes
drawn through a filter made of sugar, sodium sulphate, or sodium chloride,
and this material then dissolved in water and plated. Sand, asbestos,
glass, etc., are sometimes used as air filters, then thoroughly washed,
and the wash water plated.

Relative quantitative examination of the air is of more historical than
practical importance. It has been useful in the development of the germ
theories of fermentation and of disease and in overthrowing the theory
of spontaneous generation. There is so little ordinarily to be learned
by a study of the air flora that a comparison of plates exposed directly
will usually suffice. Where more accurate results are desired, one must
resort to one of the filtration methods discussed above.

Qualitative determinations of the species of air organisms are not
often made. When necessary it may be done by simple examination of
the colonies developed on the plates or by animal inoculations made from
the water used in the air filter. It is sometimes necessary to vary the
composition of the medium used in order to favor the development of
certain types of organisms desired, for example, a higher percentage of
molds will be found and a more luxuriant development will take place
if wort agar or acid gelatin is used.

NUMBER OF BACTERIA IN THE AIR. The number of bacteria in the
air is determined by a variety of conditions. The velocity of air currents
and the nature of the surface with which these currents will come in con-
tact, are probably most important. Bacteria are usually more abundant
on quiet days in the air of buildings than out of doors, but on windy
days the reverse is true. They are often more abundant in cities than in
the country. Fewer are found at high altitudes and over large bodies of
water. Frankland found that there are fewer in winter than in summer
They are washed from the air during rains. Bright sunlight destroys
many. The nature of the soil and the vegetation covering it has a marked
influence. The following figures from various authors are appended to
serve as an index to what may be expected in the air content of bacteria.

MICROORGANISMS OF THE AIR.

Locality

Number of organ-
isms per cubic meter

189

Observer

Outdoor air, Boston 100-150 bacteria. Sedgwick and Tucker

Open air

50-75 molds.
100-150 bacteria.

Fischer.

Open field

Uffelman

Seacoast . .

IOO

Uffelman

Mountain altitude 200 meters

o

Pasteur.

Mont Blanc . . *. . .

4-11

Ellis.

Spitzbergen (Arctic Regions)

o

Levin.

Middle of Paris

4,000

Ellis.

Paris Street . .

Fischer.

Tailor's Room in Whitechapel

17,000

Ellis.

Boot Workshop

2? .OOO

Ellis.

SPECIES OF ORGANISMS IN THE AIR. Penicillium is the most common
mold isolated from the air. Next in importance are Mucor, RJiizopus,
and Aspergillus in the order given. In addition to these a considerable
number of species of hyphomycetous molds are occasionally found.
TorulcB, but not true yeasts, are usually common. Bacteria are
either spore-bearing soil bacilli or cocci. Of the former, B. subtilis, B.
mycoides, and related forms are ubiquitous. Sarcina lutea and Sarcina
aurantiaca and certain other chromogenic cocci are to be found in almost
every plate exposed. Since the air does not have a true flora, the species
as well as the number of bacteria present must depend entirely upon the
character of the environment.

CHAPTER II.

MICROBIAL AIR INFLUENCE IN FERMENTATION,

DISEASES, ETC.

AIR AS A CARRIER OF CONTAGION. There are many popular mis-
conceptions of the influence of air upon health. Experience early taught
that exposure to the night air in certain localities or to swamp air during
certain seasons was generally followed by disease. Naturally, the air
itself was held responsible. We know now that certain fevers, malaria,
etc., are caused in every instance by infection with specific microorganisms
and that these organisms are not usually carried by the air but by insects,
such as the mosquito, in water and food. Nor can the emanations
from decaying organic matter or sewer gas itself be held to produce
disease directly. Before the establishment of the germ theory of disease,
leading sanitarians held that sickness was induced by the gases from
decaying organic matter, by the effluvia from cess-pools and by sewer
gas. However important the places named may be in harboring disease
microorganisms, we have learned that the air itself rarely acts as a carrier.
Sewer gas has been shown to be unusually free from bacteria. Hazen
says, "After many years of experience and long continued investigation,
there is not the slightest reason to believe that infectious diseases are
carried by the air of sewers."

Undoubtedly the air does play some part in the carrying of disease
germs. In certain diseases, as the exanthemata (smallpox, measles,
etc.), the infecting agent may be present on the dry skin and may be
blown about and inhaled. This means, however, is not established.
In certain nasal, tracheal, and pulmonary infections, the organisms
may be spread through speaking, sneezing, and coughing, for the infec-
tious droplets, as has been seen, remain suspended for a time in the
air. Pyogenic cocci are present in the mouth and care must be used in
surgical operations that the mouth is so protected that none of these organ-
isms gain entrance to wounds. Rarely, if ever, are intestinal infections,
as typhoid or cholera, spread through the air. We may therefore con-
clude that air is of secondary importance as a carrier of infection. It

190

MICROBIAL AIR INFLUENCE IN FERMENTATION, DISEASES, ETC. 19!

may be of importance in a crowded work-room, but even under these con-
ditions it is probable that transmission of infection comes about more
frequently through actual contact or through food and drink.

ORGANISMS or THE AIR AND FERMENTATIONS. A uniform inoculation
with soil bacteria such as produce the nodules on the roots of legumes is
obtained over considerable areas through the action of the wind in blowing
dust particles. The bacterial flora of milk is to some extent dependent
upon air currents as is also the development of the molds necessary to the
proper ripening of cheese, such as the Camembert. Acetic, butyric, and
other ferments are likewise distributed in this manner. The organisms
responsible for putrefaction and decay, the molding and spoiling of foods
are wind-borne.

FREEING AIR FROM BACTERIA. Air is most commonly freed from
bacteria by sedimentation, for this is the ultimate fate of most dust par-
ticles. We have seen that they gradually subside in a quiet atmosphere.
When large quantities of pure air are required, dust and bacteria may be
removed by passage through a spray of water or through various types
of niters, such as cotton, glass, wool, etc. A familiar example of this type
of filtration is the laboratory use of cotton plugs in test-tubes. It is
sometimes necessary to resort to fumigation to destroy the organisms of
the air when an undesirable species is present.

DIVISION II.
MICROBIOLOGY OF WATER AND SEWAGE.

CHAPTER I.*

MICROORGANISMS IN WATER.f

Water is necessary in the life of man. Besides its use as a beverage,
for cooking, and all domestic purposes, it is largely used in many manu-
facturing industries; therefore, the study of its chemical and biological
content is one of the most important features of modern hygiene. All
natural waters contain microorganisms, which gain entrance from many
sources.

Under the influence of the sun, sea water evaporates and forms a
water vapor, which we call clouds; and these, driven by the wind over the
land, are precipitated as rain and in the form of snow or hail.

Most of this water collects from vast areas into brooks, streams, rivers,
lakes, or in subterranean streams, and finally reaches the sea whence it
came.

The water vapor arising from the sea or land contains no organisms;
but as soon as the vapor is precipitated microorganisms find their way into
it. These come from the air and from the soil. Some of them find in
water sufficient nutriment for their life and growth; and, because of their
constant presence and evident ability to thrive in water, they are some-
times spoken of as belonging to the "water flora ." Others, such as the soil
bacteria, are found only at certain seasons, as after rain or during flood-

* Prepared by F. C. Harrison.

f For specific details regarding methods of analysis and a fuller presentation of the subject,
readers may consult any of the following excellent books:

1. Savage, W. G.: The Bacteriological Examination of "Water Supplies, London, H. K

Lewis, 1906.

2. Horrocks, W H. : An Introduction to the Bacteriological Examination of Water,

London, J. and H. Churchill, 1901.

3. Prescott and Winslow: Elements of Water Bacteriology, 2nd Ed. New York, Wiley

& Sons, 1908.

192

MICROORGANISMS IN WATER. U)J

time, and flourish only for a time; while some few, such as intestinal
organisms that find their way into water, survive for only a short period.

CLASSES OF BACTERIA FOUND IN WATER.

The bacteria found in water are here roughly divided into: (a) natural
water bacteria; (b) soil bacteria from surface washings; (c) intestinal
bacteria, usually of sewage origin. But there is no strict dividing line
between these three groups; for some organisms belonging to the water
flora are found in the soil, and vice versa. Water draining from manured
land frequently contains intestinal organisms. The division, however,
is sufficient for all practical purposes.

NATURAL WATER BACTERIA. The natural water bacteria are generally
regarded as harmless to man. These organisms are frequently numerous
in river, lake, and all surface waters; certain species predominate at
one season, and disappear at another. Some of the best known are
mentioned below. Several investigators have grouped the bacteria found
in water into classes according to their biochemical properties. Where
groups are subsequently referred to, the classification is that used by
Jordan and followed by many other workers.

B . fluorescens liquefaciens , Group V, together with some closely allied
varieties, is probably more frequently found in water than any other form,
and is easily recognized by the green fluorescence and liquefaction it pro-
duces in gelatin.

B. fluorescens non-liquefaciens, Group VI, as the name implies does
not liquefy gelatin, but produces characteristic colonies with a fluorescent
shimmer, is often very abundant in river waters, and is representative of
a group comprising B.f. longus, B.f. tennis, B.f. aureus, and B.f. crassus.

Certain organisms which liquefy gelatin and acidify milk classed by
Jordan in his Group VIII are quite common at certain seasons. Some
of these are soil organisms and are closely related to the proteus group;
and some of them are B. liquefaciens, B. punctatus, B. circulans.

Chromogenic bacilli and cocci (Groups XIII, and XI V] are often present
in water. Of those producing red coloring matter, the well-known
B. prodigiosus is the type of the group; others are B. ruber, B. indicus,
B. rubescens, and B. rubefaciens. Several yellow and orange organisms
are commonly found, such as B. aquatilis, B. ochraceus, B. aurantiacus,
B.fulvus, etc.

At certain times, particularly in river and brook waters, organisms

1 94 MICROBIOLOGY OF WATER AND SEWAGE.

producing violet pigment are quite common. B. violaceus or B. janthi-
nus, as it is sometimes called, is the prevailing type; others are B. lividus,
B. amethystinus, and B. coeruleus.

The chromogenic cocci produce either orange or yellow pigment, and
as a rule are not numerous in water. Sarcina lutea is the most common
species.

Non-chromogenic cocci (Group XV) are more frequent. M. candi-
cans, M. nivalis, M. aquatilis, are non-liquefying forms, and M . coronatus
is the type of those which liquefy gelatin.

SOIL BACTERIA FROM SURFACE WASHINGS. During times of flood,
high water, and after rains, numerous soil organisms are found in natural
waters; and occasionally certain species persist for a considerable time.
Among the commonest species is B. mycoides, with its characteristic
rhizoid colony; also B. sublilis, B. megatherium, and B. mesentericus vul-
gatus, with its allied varieties; likewise B. m.fuscus and B. m. rubera\\
belonging to Jordan's Group VII, and having many characters in common,
such as characteristic colonies, followed by liquefaction when growing in
gelatin, production of spores, etc.

Cladothrix dichotoma, one of the thread bacteria, easily recognized on
gelatin plates by the brown halo that surrounds the colony, is often found
in fresh and stagnant water, and in most soils. It seems to flourish where-
ever there is much organic matter.

These are the soil organisms most often found when beef peptone gel-
atin is used for isolating purposes; but if other media are used, a different
flora appears, and we find nitrifying organisms, yellow chromogens, etc.

INTESTINAL BACTERIA, USUALLY OF SEWAGE ORIGIN. Proteus Group.
-There are several groups of sewage organisms found in impure water;
some of these are very abundant in crude sewage, but are not found
in such relatively large numbers in contaminated water. Jordan's
Group III contains the organisms belonging to the large proteus group,
the principal species being B. mdgaris, B. zenkeri, B. mirabilis, B. zopfii,
the sewage proteus of Houston, and B. cloaca. All these are frequently
found in impure water, and in sewage. In the latter Houston has found
as many as 100,000 per c.c. All these organisms are motile, liquefy
gelatin, and produce gas in dextrose and saccharose broth, and little or none
in lactose; reduce nitrates, curdle milk, produce indol, and give a fecal,
disagreeable odor in broth or other media.

Sewage Streptococci. The streptococci found in sewage are probably

MICROORGANISMS IN WATER. 1 95

similar to those found elsewhere; but their appearance in contaminated
water may be regarded as indicative of recent sewage contamination,
because the bulk of the evidence available seems to show that they are
delicate organisms, which rapidly die outside of the body. While it is
easy to ascertain their presence in polluted water, it is almost impossible
to enumerate them; and they do not furnish such good evidence of sewage
pollution as the colon bacillus. They may be said to furnish valuable
confirmatory evidence of sewage contamination.

B. Enteritidis Sporogenes. This resistant, spore-bearing organism is
usually present in the intestinal tract of man ; is found in sewage, milk,
and dust; and occurs in foodstuffs, such as wheat, oatmeal, rice, etc.
On account of its ubiquity and the resistance of its spores, it cannot be
considered a good indicator of excretal pollution.

B. Coli. The presence of this organism in potable water is generally
accepted as the best bacterial indicator of sewage pollution. It must be
remembered, however, that there are many varieties of this organism, to
which certain investigators have given specific names, even when the
differences from the type organism have been very slight. It may be
well to mention some of these, to avoid confusion in the mind of the
reader. The true colon bacillus, B. coli, or B. coli communis, or B. coli
communis verus, is a short bacillus with rounded ends, motile, forms
no spores and is Gram negative, does not liquefy gelatin, produces
acidity and coagulation in litmus milk, gives rise to acid and gas in glucose
and lactose media, causes canary-yellow fluorescence in neutral red media,
and produces indol when grown in peptone water. The term "Excretal
B. coli" has been suggested as a convenient designation of an organism
which possesses the above characteristics.

A saccharose fermenting variety of B. coli has been named B. com-
munior; and we have a whole series of organisms which differ more or less
in various biochemical reactions, or lack some of their positive reactions.
To some of these the name "para-colon" has been given; and the name
"para typhoid" has been applied to those which more closely approximate
to the cultural peculiarities of the typhoid bacillus.

For practical purposes in the analysis of water, these distinctions are
unnecessary.

Bad. lactis aerogenes, a short, thick, capsulated, non-motile bacterium
related to B. coli, is also an intestinal organism, and must be regarded as
an indicator of sewage pollution.

ig6

MICROBIOLOGY OF WATER AND SEWAGE.

B. Typhosus (page 640). Very few instances are recorded in bac-
teriological literature of the direct isolation of the typhoid bacillus from
infected water. The organism is not long-lived, even in pure water
(eight to ten days); and when exposed to the action of sewage bacteria,
its longevity is greatly diminished (not more than five to six days). A
few resistant specimens may remain alive for longer periods of time.

Although the typhoid bacillus has been found so infrequently in
water, it is well understood at the present time that the purification of
the water supply of a town or city produces a marked decrease in the
number of cases and in the mortality from typhoid fever, as the following
table shows: (See also Fig. 57.)

Deaths from Typhoid Fever per 100,000 per Year.

Place

Purifica-
tion by

Date of
change

Five years
before
change

Five years
after
change.

Percentage
of
reduction

Hamburg

Filtration

1802 ?

47

7

8S

Ztirich

Filtration

1885

76

IO

87

Lawrence IVIass

Filtration

1803

121

26

70

Albany N. Y

Filtration

1800

IOA

28

77

Not only has such a marked improvement followed the purification of
public water supplies in the case of typhoid fever, but it has been shown
by statistics that "where one death from typhoid fever has been avoided
by the use of better water, a certain number of deaths, probably two or
three, from other causes have been avoided."

In the routine examination of water, no particular effort is made to
isolate this organism, owing to the difficulty of the task. The tests that
the present day investigator has to satisfy are extremely thorough; and
unless the suspected organism conforms to the whole of these necessary
tests it cannot be accepted as true B. typhosus.

Msp. Comma (page 645). The spirillum, or vibrio, of Asiatic cholera
is an intestinal organism; and the disease it produces is spread largely
by water. Epidemics of cholera are more easily traced to their source

MICROORGANISMS IN WATER.

197

Average annual death rate from typhoid fever per 100,000 of the population.

8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 DEATHS

SPRIN6FIELD
WILMINGTON

An instructive contrast between Altona'and Hamburg before the latter
filtered its water, having learnt its lesson from a sharp outbreak of cholera.

A FEW
SCATTERED CASES Of

ALTOHA^

HAMBURG.

POPULATION: 600.000
CHOLERA CASES'. 17.000

DEATHS: 8.600

ALTONA:

WATER FILTERED

HAMBURG:

WATER. UMFILTERED

FIG. 57. (After G. E. Armstrong.)

198

MICROBIOLOGY OF WATER AND SEWAGE.

than those of typhoid fever, owing to the "explosive" character of the
disease. At the time of the outbreak of cholera in Hamburg, in 1892, the
cholera vibrios were frequently isolated from the water of the river Elbe,
which was used to furnish the regular supply of the city. The adjoining
city of Altona also obtained its water from the same river, after it had
received some of the Hamburg sewage; yet it remained practically free
from the scourge, owing to the efficiency of sand niters which were
used to purify the water (Fig. 57). In times of epidemic, the organism
has been isolated from rivers, wells, and reservoirs in India, a country
in which the disease is endemic.

THE NUMBER OF BACTERIA IN RAIN, SNOW, HAIL, ETC., AND IN WATER
FROM WELLS, UPLAND SURFACE WATERS, RIVERS, AND LAKES.

RAIN. The number of bacteria found in rain depends upon the
month of the year and the dryness of the air. When considerable dust is
present in the air, the first rain beats it back to the soil; and at such time
rain water contains more organisms than usual. Rain falling in densely
inhabited cities always contains more microbes than rain falling on open
farm land or upland pastures. A few figures will be sufficient to illus-
trate:

NUMBER OF BACTERIA PER LITER or RAIN WATER.
Figures for Montsouris Park, Paris, France, and the average for two years.

Month

Number of organisms
per liter

Month

Number of organisms
per liter

January ....

8 ooo

July

c;,6oo

February

I 32O

Aueust .

8,300

March

2 O2O

September

C77O

April ....

2 1A.O

October

"?,22O

May

2 AA.O

November

3, 2 so

June ... ... .

56OO

December

4,^70

Yearly average 5,300 per liters per month.

The average for the interior of Paris corresponds with the larger
amount of dust in the air, and reaches a total of 19,000 organisms per 1.

MICROORGANISMS IN WATER.

With a yearly rainfall of 609. 6 mm. (24 in.), the rain washes down during
the year some five million organisms to the square yard.

SNOW. -The results obtained from snow are similar to those ob-
tained from rain; but as a rule the numbers are larger, a result doubtless
due to the larger particles of the snow flakes. One investigator has
found from 334 to 463 bacteria per c.c. of snow water. On the summit
of high mountains snow is practically sterile, Binot not finding a single
organism in 8 c.c. of water from mountain-top snow.

Water issuing from glaciers is of remarkable purity, containing only
from three to eight organisms per c.c.; but the numbers are larger as the
distance from the glacier increases.

HAIL. Hail stones usually contain large numbers of bacteria, vary-
ing from 628 to 21,000 per c.c. of water obtained from the melting hail.
Fluorescing bacteria have been found in some samples; and the presence
of these microorganisms suggests that surface water is sometimes carried
up by storms and congealed. The presence of many molds in hail is
due to contamination from the air.

DEEP WELLS. Deep well water and spring water contain as a rule
but few organisms, usually less than 50 per c.c. on gelatin at 20, and
less than 5 per c.c. on agar plates at blood heat. In a series of tests of
water taken direct from forty- three artesian wells, 152.4 M. (500 feet)
deep or more, the writer has found an average of 27 per c.c. for the
gelatin and 1.5 per c.c. for the agar counts. These tests have extended
over a period of several years; and water from deep springs has given
similar results.

SHALLOW WELLS. The bacterial content of shallow wells depends
greatly on their location and construction. Even in those well located
and constructed, the number varies with the amount of rain fall, and
is often large. In polluted wells, very high numbers of organisms are
found.

Sedgwick and Prescott found from 190 to 8,640 bacteria per c.c. in
unpolluted wells.

In the same class of wells, Savage found from 10 to 100 per c.c. by
the blood-heat count, and 100 to 20,000 or more by the gelatin count.

Sixty polluted wells examined by the writer gave an average gelatin
count of 740 bacteria per c.c.; and thirty-eight wells which were free of
contamination gave an average count of 400 per c.c.

Polluted wells often give counts approximating the higher numbers

2OO

MICROBIOLOGY OF WATER AND SEWAGE.

mentioned above; but, of course, the character of the bacterial flora is
quite different.

UPLAND SURFACE WATERS. There are few bacteria in upland sur-
face waters draining barren uplands. Cultivation, grazing of animals,
and human habitation produce other conditions. In pure waters, 50 to
300 per c.c. by the gelatin and i to 10 by the agar count are found.

RIVERS. The greatest variation in the number of bacteria exists in
river waters. Many factors, such as sewage contamination, temper-
ature, rain fall, vegetable debris, etc., influence the microbial population.
A few figures may be given for illustration.

Bacteriological Examination of Rivers at and below Large Sources of Pollution (Boyce

and Co-workers}.

Distance

Direction

Munich.
River Isar

Cologne,
River Rhine

About o

6 miles

above
below

35
o 387

4,786

About 2

7 miles

below

y>ow

13 CO3

About 6 .

o miles

below

"Os J^O

8,764

30,4^2

About 12 .

o miles . .

below

4. 706

I2,46o

About 15.

o miles

below

3 602

QCQC

About 26.

o miles

below

7,860

In the Chicago drainage canal, Jordan found 1,245,000 bacteria per
c.c. at Bridgeport; 650,000 at Lockport, twenty-nine miles below; and
3,660 at Averyville, 159 miles below. Below where the sewage of Peoria
enters, the numbers rise to 758,000 at Wesley City, and decrease to
4,800 at Kampsville, 123 miles from Peoria.

The river Rhone contains an average of 75 bacteria per c.c. above
Lyons and 800 below. The Dee, 88 above Braemar and 2,829 per c.c.
below. Many more similar results are found in the literature.

LAKES. The water of lakes is generally much purer than river water.
Near the shore, the bacterial content is higher than farther out, showing
the contaminating influence of habitation. Thus Lake Geneva contains
as many as 150,000 bacteria per c.c. near the shore, and further out only

MICROORGANISMS IN WATER. 2OI

38 per c.c. Other figures are as follows: Loch Katrine, 74 per c.c., Lake
Lucerne, 8 to 51 per c.c., Lake Champlain, 82 per c.c.

SEA WATER. There are few bacteria in sea water remote from the
coast; but near the shore and in the neighborhood of seaports there may
be large numbers.

Examples: 350 meters from Naples, sea water contained 26,000
bacteria per c.c. At a distance of 3 kilometers, only 10. Samples taken
from depths of 75 to 800 meters at distances from 4 to 15 kilometers from
shore were found to contain from 6 to 78 bacteria per c.c. in surface water,
and from 3 to 260 at various depths below.

CAUSES AFFECTING THE INCREASE AND DECREASE OF THE
NUMBER OF BACTERIA IN WATER.

There are a number of causes which influence the multiplication or
diminution of microorganisms in natural waters; and while it is necessary
to discuss each of these causes in detail, it must be remembered that a
number of them may be simultaneously influencing the increase or
decrease.

TEMPERATURE. In natural waters, a low temperature probably
acts injuriously on parasitic bacteria, reducing their numbers; but the
bacterial content of water during the hot summer months is generally
not so large as during the cooler seasons. Water collected for exami-
nation should be analyzed at once; otherwise, contradictory results as
to numbers will be found. Usually, in most waters, there is a reduction
in numbers for a few hours, followed by a large increase. Very much
polluted waters, however, show a marked decrease of intestinal organ-
isms, if the samples are kept cool.

LIGHT (page 162). Although the germicidal effect of sunlight is
well known, yet it has not such powerful effects on the bacteria in water.
Much depends, no doubt, on the turbidity and speed of the current,
the maximum killing effect being produced in shallow, clear and slow-
moving water. It has been found by experiment that the germ-killing
power of light extends to a depth of three meters (about 9.84 feet). As
a means of purifying water, direct light produces very little effect.

FOOD SUPPLY. The amount of organic matter in water directly
influences the growth of bacteria. Where a large amount of this is
present, the number of microorganisms is also large. Rivers containing

202 MICROBIOLOGY OF WATER AND SEWAGE.

considerable organic matter derived from vegetable debris, etc., contain,
as a rule, more organisms than rivers in which there is but little of such
material. Thus the Ottawa River, which drains a large area of forest
lands and is characterized as an upland peaty water carrying a rather
high percentage of organic and volatile matter, contains throughout the
year a larger number of organisms to the cubic centimeter than the water
of the river St. Lawrence, which is much clearer and contains much less
organic matter. Sewage water is rich in organic matter, and proportion-
ately rich in bacterial life; and bacterial purification is synchronous
with a diminution of organic matter.

Jordan remarks in this connection that "in the causes connected with
the insufficiency or unsuitability of the food supply is to be found the main
reason for the bacterial self-purification of streams."

OXIDATION. On the surface of waters, in rapids, falls, and tidal
rivers, much oxygen is absorbed, and much impure matter is thus oxidized.
Such oxidation is one of the minor agencies in the purification of water.

VEGETATION AND PROTOZOA. Low forms of plant and animal life,
like certain species of algae, river plants, and the numerous protozoan
forms, bring about a reduction of organic matter in water, and thus reduce
the amount of food available for bacteria. There is also the antagonism
between these forms and bacteria. The chemical products of the higher
forms are considered by some authorities to be injurious to bacterial
life; and many bacteria are ingested by predatory protozoa.

DILUTION. Sewage flowing into a river or lake is at once diluted
with quantities of pure water, and the amount of available food material
is thus diminished; the space occupied by a definite number of bacteria
is increased; and it is easy to see that the greater the dilution, the fewer
sewage bacteria will be found. An example will suffice to illustrate.
The sewage of the city of Ottawa amounts to about 454 1. (100 gal-
lons) per second; and the gelatin count from it gives an average in round
numbers of 3,000,000 bacteria per c.c. The yearly mean discharge of
the river is about 1,364,511 1. (300,000 gallons) a second; and thus the
sewage becomes diluted 3,000 times.

SEDIMENTATION. Impurities, suspended matter, and bacteria having
weight, naturally gravitate to the bottom; and the subsidence of these
matters is spoken of as sedimentation.

Lake water being still, sedimentation in it is more marked than in
moving water; and such water contains but few bacteria. In slow-

MICROORGANISMS IN WATER. 203

moving rivers the influence of this factor is also quite pronounced; and,
according to Jordan, "The influences summed up by the term sedi-
mentation are sufficiently powerful to obviate the necessity for summoning
another cause to explain the diminution in numbers of bacteria" in sewage
polluted rivers. The example already given (page 200) of the self-
purification of the Chicago drainage canal illustrates Jordan's contention.

OTHER CAUSES. There is a number of other causes, not well
known nor of sufficient practical importance for more detailed comment,
which may increase or decrease the number of bacteria in water, such
as the inhibiting action of microorganisms and their products on one
another, the effects of pressure, etc.

A peculiar fact, which has never been satisfactorily explained, is the
quick death (in three to five hours) of the cholera vibrio in the waters
of the Ganges and Jumna. When one remembers that these rivers are
grossly contaminated by sewage, by numerous corpses of natives (often
dead of cholera), and by the bathing of thousands of natives, it seems
remarkable that the belief of the Hindoos, the water of these rivers
is pure and cannot be defiled, and they can safely drink it and bathe
in it, should be confirmed by means of modern bacteriological research.
It is also a curious fact that the bactericidal power of Jumna water is lost
when it is boiled; and that the cholera vibrio propagates at once, if placed
in water taken from wells in the vicinity of these rivers.

INTERPRETATION or THE BACTERIOLOGICAL ANALYSIS OF WATER.

In making any analysis of water, all data, such as the kind of water
and the particulars regarding collection, transmission, sampling, rainfall,
etc., should be given, as these are a great help in interpreting the results.
One analysis is rarely sufficient; examinations should be regularly and
systematically made.

QUANTITATIVE STANDARDS. No absolute guide can be given to
determine the potable quality of water from the number of microorganisms
in it. It may, however, be safely assumed that high bacterial counts
indicate a large amount of organic matter. The number of organisms
growing in beef peptone gelatin at 20-22, and termed the "gelatin
count," should be given. For deep wells and springs, this should not
exceed 50 per c.c.; and for shallow wells and rivers, not over 500 per
c.c. After rains or floods, these figures might be exceeded, and would
not necessarily indicate dangerous pollution.

204 MICROBIOLOGY OF WATER AND SEWAGE.

The number of organisms which develop on beef peptone aga
incubated at blood heat, commonly termed the "agar" or "blood-heat"
count, is perhaps more important than the gelatin count, as many water
bacteria do not grow at blood heat, whereas sewage and soil organisms
grow readily at this temperature. The agar count eliminates the water
flora, but obscures the sanitary results by reason of the presence of soil
bacteria. For deep waters, the agar count should generally not exceed
10 per c.c. ; and for surface waters, not over 100 per c.c.

QUALITATIVE STANDARDS. The isolation and identification of
specific disease organisms, such as typhoid and cholera microbes from
water, is sufficient to condemn such a sample as unfit for use; but on
account of many technical difficulties it is practically impossible to make
such an examination. Apart from a few special cases, when it may be
necessary to attempt the isolation of these pathogenic bacteria, the pres-
ence of the colon bacillus (B. coli) in small amounts of water, is generally
looked upon as significant and indicative of sewage pollution. The
technical methods used in this isolation and numeration are many, and
may be found in the works cited; but there is considerable difference of
opinion as to the number of B, coli which should condemn a sample of
water. Prescott and Winslow state that if the colon bacillus is in "such
abundance as to be isolated in a large proportion of cases from i c.c. of
water, it is reasonable proof of the presence of serious pollution." Savage
suggests that B. coli should be absent from 100 c.c. in the case of water
from deep wells and springs, and should be absent from 10 c.c. in surface
waters, such as rivers used for drinking purposes, shallow wells, and
upland surface waters.

The streptococcus examination is next in importance as an indicator
of sewage. Streptococci should be absent from the amounts of water
mentioned above for B. coli; and B. enteritidis sporogenes should not be
present in 1,000 c.c. of water from deep wells, nor in 100 c.c. from sur-
face waters.

SEDIMENTATION, FILTRATION, AND PURIFICATION OF WATER.

As areas become more and more thickly settled and towns and cities
increase in population, the problem of obtaining sanitary control over
the water supply increases in importance. Very few towns and cities
are fortunate enough to obtain their water supply from an unpolluted

MICROORGANISMS IN WATER.

205

area. Consequently expensive installation must be made, in order to
purify a suspiciously contaminated water by freeing it from organisms
injurious to health. There are several methods of accomplishing such
purification; and these will be briefly mentioned.

SEDIMENTATION AND FILTRATION. This method of purifying water
has been used for nearly a hundred years; but the great impetus given to
this hygienic measure was due to Koch, who showed in 1893 that the

FIG. 58. Section of a sand filter.

proper filtration of Elbe water saved the town of Altona from an epidemic
of cholera which devastated Hamburg as a result of drinking unfiltered
water. In this system of purification, the water is first stored in large
reservoirs, where the effect of sedimentation and storage reduces con-
siderably the number of bacteria. From the reservoir, the water is
filtered through sand, gravel, and pebbles, etc., arranged as shown
in Fig. 58. This filtration removes from 97 to 99.5 per cent of the
microorganisms.

2O6

MICROBIOLOGY OF WATER AND SEWAGE.
Mean of Monthly Examinations for the Year.

Microorganisms per c.c.

At source After storage After filtration

London Lambeth Works. . .

16 138

7 820

7C

London, Chelsea Work.

16 138

i 067

/ D
24.

Berlin, Lake Miiggel

I,4OO

60

Paris, Marne

7O,OOO

6^O

Paris Seine ....

186 986

"0"
AQQ

The action of the filter bed is due to the mechanical obstruction of
impurities, to oxidation of the organic matter, and to nitrification due to
the living bacteria in the scum which forms on the top of the layer of sand.
Of these, the last is the most important; for until this gelatinous layer
forms, the filter does not act properly in fact, it has little filtering action,
as the following figures show:

'Bacterial Content of Water Before and After Cleaning the Sand Filter.
Before cleaning, i.e., before removing the scum layer .... 42 per c.c.

One day after cleaning ". 1880

Two days after cleaning 752

Three days after cleaning 208

Four days after cleaning 156

Five days after cleaning 102

Six days after cleaning 84

Thus provision must be made to permit the scum or film to form before
the filtered water is used for domestic purposes.

The rate of filtration must be regulated; for if the water is allowed to
exceed a certain rate (101.6 mm. or 4 inches per hour), inefficiency
follows.

COAGULATING BASINS AND FILTRATION. This method of purification
consists in adding a coagulant, such as basic sulphate of aluminum, by
means of a mechanical device which regulates the quantity, as the water
is pumped into the coagulating basins or reservoirs, where it remains for
six to twenty-four hours. The aluminium sulphate is decomposed by the
lime in the water and forms insoluble aluminium hydrate; and the sul-
phuric acid combines with the lime. The hydrate of aluminium is pre-

MICROORGANISMS IN WATER.

207

cipitated in large flocculent masses, entangling all particles of soil or
organic matter; and these, being deposited on the surface of the sand,
form the filtering layer. Such filters are very efficient; they remove from
97 to 99.8 per cent of the bacteria from the water.

POROUS FILTERS. (Fig. 59.) These filters are either made from
unglazed porcelain or baked diatomaceous earth; the former are known
as Chamberland, and the latter as Berkefeld filters. These filters
are usually candle-shaped, require considerable pressure to force water

FIG. 59. Unglazed porcelain niters. Chamberland system; A, without pressure;
B, fitted to main water supply; C, section of a porous porcelain filter.

through them, and can be used only when a small supply of water is
needed. Water which is forced through these filters is at first sterile; but
with repeated use they allow bacteria to pass through the pores and thus
the filtering efficiency is impaired and will remain so, until the filters
are cleaned and baked to red heat in a muffle-furnace. Unless this is done
regularly, no dependence should be placed on these filters, as they only
put those who use them off their guard against the danger to which they
are exposed.

PURIFICATION BY OZONE. The antiseptic properties of ozone are

208 MICROBIOLOGY OF WATER AND SEWAGE.

well known. It is used in the purification of the water supply of some
towns Nice, Chartres, etc. Ozone used for this purpose is usually
obtained by means of the electric current; and a flowing film of water is
brought into contact with an upward current of air charged with ozone,
which current makes the water almost completely sterile. This method
of purification is efficient, but rather expensive.

PURIFICATION BY HEAT. By bringing water to the boiling point, all
harmful bacteria are destroyed; a few spores may resist this treatment,
but they are harmless. Boiled water is of a flat, insipid taste, due to the
driving out of the contained gases. The taste may be improved by cooling
and shaking. The boiling of water is often resorted to as a hygienic
measure in times of epidemic, and for the supply of armies in the field.

PURIFICATION BY CHEMICALS. The addition of a small amount of
calcium hypochlorite, or potassium iodide, etc., purifies water; but these
methods are seldom used, except for the use of soldiers on campaign.

LOCATION AND CONSTRUCTION OF WELLS.

Farms in many sections of this country are practically all supplied
with surface water collected in shallow wells. Hence farmers should
understand the principles involved in the location and construction
of wells.

Many farm wells are badly located too near such sources of contami-
nation as out-houses, cess-pools, stables, or barn-yards; and those who
locate them give too little attention to the slope of the ground, and the
nature and slope of the subsoil. There should be at least 22 to
30 M. (75 to 100 feet) between the well and all probable sources of
contamination; and this distance is too small, if the soil is very porous,
or if the surface and subsoil drainage is toward the well, or if the well
is sunk in fissured rock as it is obvious that there are serious chances of
contamination in each of the above circumstances.

In all cases, the surface drainage should be away from the well; and,
as far as possible, the subsoil drainage also should be from the well.

Sketches 60, 61, and 62 illustrate these points, the upper part of each
drawing showing the plan and the lower portion a section through the dotted
line marked on the plan. Figure 60, shows that the surface drainage is
trom the house, privy, stables, and barnyard toward the well. The section
fhrough the line "A" shows the relation of the impervious subsoil "B" to

MICROORGANISMS IN WATER.

209

,

1

1

i

6

5

^ _

/

(_

FIG. 60.

/

---A

FIG. 61.

FIG. 62.

FIGS. 60, 6 1 and 62. In each figure plan above section through A B below.
S = Soil; B=impervious subsoil or strata, i, .House; 2, well; 3, outhouse; 4, piggery;
5, stables; 6, stable yard; 7, hen house; 8, sheep stable. Arrow heads indicate direction
of water flow. (Original.)
14

210

MICROBIOLOGY OF WATER AND SEWAGE.

the drainage. Water falling on the surface of the ground would pene-
trate through the soil to the upper portion of the subsoil, and then move
along it in the direction of the greatest slope. In this sketch, the subsoil
drainage is away from the well; and in this respect the well is located
properly; but, in respect to the surface drainage, improperly located. A
better place for the well would be at the letter "X."

In Fig. 61 the surface drainage including that from the adja-
cent outhouse at 3, which is too close to the well is toward the barn,
and away from the well; but the subsoil drainage from all the buildings,

Soil

FIG. 63. Construction of a model well. On the right is brick construction, on the left
stone construction, as illustrated. (Original.)

except the house, is in the direction of the well; and thus contamination
of the water supply is liable to occur.

Fig. 62 shows a well properly located as regards both surface and
subsoil drainage. Such a well will supply pure water, if it is properly
constructed.

Figure 63 shows the proper construction of a well with brick or stone.
Large vitrified drain pipes with cemented joints will answer equally well
when there is an abundant supply of water; but in case the supply of water
is limited, a large area is needed, and a stone or brick well is necessary.

MICROBIOLOGY OF WATER. 211

Reference to the illustrations will show that every endeavor is made
to prevent surface water from entering directly into the well. The walls
are impervious; and the earth or clay is well rammed against the outer
side of the wall. The curb is carried well above the surface of the ground.
The waste water is conducted by means of a sloping platform, trap, and
drain, away from the well; and the well opening is properly covered. All
water entering such a well must percolate through a considerable depth
of soil, and undergo purification by means of the aggregations of living
bacteria in the soil spaces. Thus the soil around a well fulfils the same
function in purifying the surface water as the scum layer that forms on
the surface of gravel filters.

CHAPTER II.*
MICROBIOLOGY OF SEWAGE.

THE BACTERIAL FLORA OF SEWAGE.

COMPLEXITY OF FLORA. Sewage is made up of the miscellaneous and
varied wastes of human life and activity, and the bacteria which are
found therein are the result of a haphazard and chance admixture of sub-
stances of diverse origin and character. The resulting flora is not only
of great diversity and variability, but it is with few exceptions non-
characteristic. In brief, the medium with which we have to deal has had
an origin too indefinite and a history too short to have permitted the
establishment of anything approaching a constant or characteristic
bacterial flora.

TYPICAL FORMS. Our interest in this sewage flora is a very practical
one, being confined to those organisms which carry on the work of biological
purification and to certain pathogens which for obvious reasons require
special treatment. We are interested chiefly in what these bacteria do
rather than in what they are, and our classification is influenced accordingly.
It is based, not upon the species or the genus nor even upon the group or
type, that proves so convenient in general bacterial classification, but
upon a sort of physiological or functional type, having to do solely with
the activities of the organisms in sewage and in its purification. Bacteria
performing a common function or producing a common result are members
of one type. Individuals may belong to several of our types and there
are doubtless a great many that belong to none. These latter simply
have no place assigned them as yet in the role of sewage purification,
because they possess none of the recognized typical functions.

Apparent exception may be taken to these general principles in
the case of such organisms as the B. coli, sewage streptococci and
B. enter itidis. These are, to a certain extent, characteristic sewage
bacteria. But interest in them as individuals is confined to water
bacteriology. If they have any functions in the bacterial changes of
sewage, they receive attention as members of a corresponding type, not

* Prepared by Earle B. Phelps.

212

MICROBIOLOGY OF SEWAGE. 213

as individuals. A study of these sewage types, therefore, is a study of
the chemical changes induced in the medium by the activities of one or
the other group of bacteria.

TYPES OF SEWAGE BACTERIA.

According to the general character of the changes which they bring
about, sewage bacteria are divided into two large groups, the anaerobic
or putrefactive bacteria, and the oxidizing bacteria. In regard to the
former, no attention is paid to the fine distinctions that have been made
in recent years in connection with the definition of putrefaction. In
sewage chemistry putrefaction is that change which takes pi ace naturally
in sewage after anaerobic conditions have become established. It in-
volves the reduction of urea, the hydrolysis of protein and of cellulose, the
emulsification of fats, the reduction of nitrates and sulphates and possibly
of phosphates, and those other changes which are characterized by the
withdrawal of oxygen and the hydrolysis of complex molecules. These
changes are always noted in sewage under anaerobic conditions and the
terms putrefactive and anaerobic change are for the present purposes
practically synonymous.

The oxidizing reactions on the other hand might be classed under
the general heading of aerobic reactions, except that they constitute only
a small portion of the group of reactions which take place normally under
aerobic conditions. They are distinguished by the fact that oxygen is
added to the molecule, the product always containing more oxygen than
the initial substance. Carbon dioxide, water and nitrates are produced,
in distinction from methane, hydrogen and ammonia, which characterize
the anaerobic reactions. A third type, possessing objective rather than
subjective functions, in sewage, is made up of pathogenic and other
harmful bacteria. These play no part in our theories of purification and
the proof of their presence is generally lacking. For the protection of the
public health, it is assumed that they are always present in sewage, and
our procedure in sewage disposal is modified throughout in accordance
with this assumption.

With these definitions in mind we may proceed to a more detailed
study of the bacterial types themselves.

PUTREFACTIVE AND ANAEROBIC BACTERIA. Putrefaction or anaerobic
fermentation involves the withdrawal of oxygen from one molecule or
part of a molecule and the subsequent oxidation of another molecule or

214 MICROBIOLOGY OF WATER AND SEWAGE.

part of the same molecule. The energy released in this process is utilized
in the vital functions of the organism. This action is neither oxidation
nor reduction, or more strictly, it is both taking place simultaneously.
A good example of such a process is the fermentation of urea. The
reaction takes place as follows:

CO(NH 2 ) 2 + 2 H 2 O=(NH 4 ) 2 CO 3 .

Carbon is oxidized at the expense of hydrogen, a process which, by itself,
is endothermic, that is, requires heat or energy for its maintenance. But
the heat of formation of the final product is greater than that of the initial
substances and the energy thus liberated becomes available for use by
the bacteria. It is in this way that hydrolytic changes of this character
play the same role in anaerobic reactions that is played by direct oxida-
tion under aerobic conditions.

The Liquefaction of Protein. One of the most clearly defined and
useful types of bacterial activity to be seen in the various sewage disposal
processes is that which we term liquefaction. This term is used to denote
broadly all those changes by which solid and insoluble organic matter is
converted into a soluble condition. The particular process known as
protein liquefaction is in the main analogous to gastric digestion. Its one
characteristic is the increased solubility of the product. The practical
importance of protein liquefaction in sewage disposal is very great and the
value of the liquefying bacteria correspondingly high. Nevertheless,

%

aside from our knowledge of analogous processes in digestion and in
bacterial putrefaction of albuminous substances, we know almost nothing
of the chemistry or the bacteriology of this process. An enormous
variety of bacteria are included in this group. The whole process is
doubtless the result of a very complicated symbiosis in which various sub-
groups of bacteria carry out the initial reaction, from which point other
groups carry it through successive stages. Absence of one or another of
these groups or of some important species of any group doubtless accounts
for the diverse results that are recorded. It is well known that the activi-
ties within a septic tank, for example, are seldom twice the same. Gross
differences readily apparent to the senses of one versed in such matters
certainly exist, and in actual results it is rare to find two tanks doing
exactly the same kind of work. Much depends of course upon the chem.
ical character of the sewage itself, but much, that is still unexplained, must
eventually be traced to the great diversity of the sewage flora and the com.

MICROBIOLOGY OF SEWAGE. 215

plex symbioses as well as bacterial antagonisms that are involved in the
reactions with which we are dealing.

During these reactions proteins and albumins are hydrolyzed by suc-
cessive stages to albumoses, peptones, amino-acids, amines, and finally to
ammonia, carbon dioxide, methane, hydrogen, etc. Simultaneously am-
monia, amines, and carbon dioxide are eliminated at each stage as side
products. The tendency then is toward simple, soluble and gaseous
products, and hence of value in the preliminary resolution of the
sewage.

The Fermentation of Cellulose. The fermentation of cellulose is, next
to protein hydrolysis, the most important work of the anaerobic bacteria in
sewage treatment. So far as is definitely known this action is usually
confined to anaerobic conditions. The fact that fence posts decay first at
the surface of the ground, or that wood in general decays more rapidly
when it is exposed to only a slight degree of moisture, than when it is
immersed in water is only an apparent contradiction. The conditions
are aerobic in both cases and aerobic bacteria would not be favored by
total immersion but the effect in both instances seems to be due to fun-
gus growths which are more active in the moist wood.

The anaerobic fermentation of cellulose is that which is found typically
in marshes and of which the chief products are carbon dioxide and
methane or "marsh gas." Nitrogenous food material is also requisite,
which accounts for the preserving property of reasonably pure water
upon wood.

In the septic tank the solution of cellulose is extremely rapid, and
large pieces of cotton cloth or rolls of paper are completely dissolved
within a few months. Wood itself is more resistant and withstands the
action of the tank for years. This is largely due to the fact that the wood
molecule is much more complicated than a simple cellulose molecule,
and, among the conifers at least, to the further fact that antiseptic
intercellular substances are present.

Chemically considered the action is hydrolytic and can be imitated by
prolonged boiling in dilute acids. Pectin substances, starches and finally
sugars are produced while butyric and other organic acids, carbon dioxide
and methane appear as by-products. Bacteriologically, although it
has variously been ascribed to one or another organism, it is probably the
result of the activities of many and is possibly not the principal activity of
any one of these. In other words, cellulose fermentation is probably a

2l6 MICROBIOLOGY OF WATER AND SEWAGE.

series of side reactions produced during the fermentation of the ni-
trogenous material rather than a definite reaction upon which the met-
abolism of any single species depends. This view is strengthened by the
general observations that this fermentation is in most cases due directly
to enzymes. Viewed in this light it is easy to understand the difficulty
that has surrounded the isolation of definite cellulose fermenting organ-
isms. Many have been described, chief of which are B. butyricus or B.
amylobacter, B. omelianski, Sp. rugula.

The Saponification of Fats. A third great group of type reactions
occurring under anaerobic conditions is the saponification or splitting of
fat. Our knowledge of this process is even less definite than of the
cellulose fermentations. It is a fact that there does take place in sewage a
gradual saponification and emulsification by which the fat loses its
identity and mingles with the liquid. This effect is most noticeable in the
case of long sewers in which considerable velocities are maintained. In
quiescent tanks there is a tendency for the fats to rise to the surface and
thus become removed from the influence of this action. Thus in small
installations enormously heavy scums form upon the tanks and analysis
shows a considerable percentage of fat in this material. In larger systems
on the other hand there is less and less evidence of fatty material as such.
It is true that there is a deposit upon the walls and tops of such sewers
and that small floating objects, like matches, rolling along such a wall will
accumulate layers of grease and become eventually the familiar "grease-
balls" found in the discharge, but in the main the fatty material has be-
come well disintegrated before the outlet is reached.

In this case also as in that previously discussed it is not believed that
the action is a direct result of the activity of any particular organism.
The proteolytic changes are accompanied by the freeing of alkaline
products, ammonia and amines, which leads to some saponification, and
which, in turn, leads to a further emulsification. Whether specific
enzymes are present which assist in this final process or not has never
been determined. It is significant to note, however, that where sewages
are slightly acid, unaltered fats are much more abundant, even though the
acidity is insufficient to prevent vigorous putrefactive changes in the
sewage itself.

The Fermentation of Urea. The fermentation of urea has already
been referred to as a typical and simple case of anaerobic decomposition.
This reaction has great significance in sewage chemistry since a consider-

MICROBIOLOGY OF SEWAGE. 21 7

able proportion of the nitrogen of sewage is present initially as urea.
Owing to the ease and rapidity with which the reaction takes place,
however, no special effort is necessary to bring it about in sewage
treatment and it therefore receives brief attention in discussions of the
chemistry of sewage. The change to ammonia takes place in the small
sewers of the system and it is difficult and generally impossible to detect
the presence of urea in sewage. It has even been suggested that certain
enzymes present in fecal matter are instrumental in bringing about this
change and that the bacteria are only indirectly concerned. It is known,
however, that a large number of bacteria of general occurrence have the
power to produce this fermentation. Of these the Bad. uretz (Miquel)
may be cited as an example.

The Reduction of Sulphates and Nitrates. The production of
sulphuretted hydrogen during the anaerobic decomposition of sewage
is commonly noted. This substance may arise in at least two
ways. Sulphur, being a constituent of most protein substances, is split
off from the molecule in this form during certain types of fermen-
tation. Its formation in these cases is analogous to that of ammonia
from protein. The amount so produced is small and is usually
neutralized and precipitated by the small amounts of iron and other
metals always present in sewage. There is therefore no liberation of the
gas itself and it is often said that sulphuretted hydrogen is not formed
normally in a septic tank. This conclusion is readily disproved by a
simple test of the black residue found at the bottom of such tanks.

A second and more important source of this substance is the sulphate
normally present in many sewages. Throughout many parts of the
country the water supply contains material quantities of magnesium or
calcium sulphate, and upon the sea coast the sewage generally receives
more or less salt water.

In these cases the reduction of sulphates to sulphuretted hydrogen is
not only of interest bacteriologically but probably exerts an influence upon
all the reactions that are going on simultaneously. In fact this example
serves excellently to illustrate the great complexity of these anaerobic
reactions and the mutual interdependence of each upon all the others.
Sulphates, under anaerobic conditions, are a source of oxygen and it is
upon oxygen that the course of all these reactions depends. Therefore
the presence of sulphates and the possibility of their yielding oxygen may

2l8 MICROBIOLOGY OF WATER AND SEWAGE.

alter the course of the other reactions involved. The products of the
protein hydrolysis for example may be profoundly modified by the pres-
ence of this additional source of oxygen.

The effect upon the bacteria themselves is also to be considered as a
factor quite distinct from the purely chemical effect just described. It
has frequently been observed, and in fact would be expected, that the
products of anaerobic putrefaction are themselves detrimental to the activ-
ity of the organism producing the changes in question. The nature of
sulphuretted hydrogen makes it appear quite probable that we are dealing
here with a toxic substance that would at least inhibit the activities of
certain bacteria and in this way further modify the final result.

The same might be said of almost all the reactions with which we
have to deal but this example is cited as a typical one.

It is known in practice that the presence of sulphates in a sewage does
lead to a distinct type of anaerobic change which is characterized by the
marked blackening of the sewage, the formation of secondary reaction
products which precipitate after the removal of the suspended matter of
the sewage, the evolution of hydrogen sulphide, an excessive amount of
mineral or non-volatile residue in the sludge and the formation of free
sulphur upon subsequent aeration of the sewage.

Here again, as in the other types of reaction, it is useless for the present
to attempt to ascribe this reaction to any particular species. Sp.
desulphuricans and B. sulpliurens have been isolated. A non-liquefying
anaerobic bacillus, which reduced sulphates strongly, was isolated from
Boston sewage in the writer's laboratory by G. R. Spaulding. -Others have
been described and there is undoubtedly a large group of organisms capable
of bringing about the reaction.

Just as the reduction of nitrates is a function performed by many,
perhaps most, anaerobes, so the reduction of sulphates, although a less
common function, is still common to many forms. In fact nitrates,
sulphates, and phosphates form a series in regard to their reducibility
and the effect of their presence upon the reaction as a whole. The phos-
phates so far as has been recorded are not ordinarily reduced.

OXIDIZING BACTERIA. The Production of Nitrate and Nitrite. A
long series of investigations upon the organisms which oxidize nitrogen
began with the Franklands and Winogradski, and has continued to the
present day. These have given us much information concerning the

MICROBIOLOGY OF SEWAGE. 2IQ

habits and functions of the nitrifying organisms. Winogradski 's original
types were Nitrosomonas and Nitrobacter, the former oxidizing ammonia
to nitrite, the latter completing the oxidation to nitrate. Work upon
these organisms constitutes such an important factor in soil bacteriology
to-day that more detailed discussion of this nitrifying function is left for
another place.

In the earlier days of sewage purification great stress was laid upon
the work of these organisms, which was believed to be fundamental.
The degree of nitrification was accepted as a measure of the work of the
filters and little thought was given to the possibility of oxidizing reactions
by other forms. With the development of modern sewage disposal
methods, the work of this latter type of bacteria has assumed a more
important role and the actual work of the nitrifying organism has been
found to be of only minor and incidental importance.

Other Oxidizing Reactions. The great groups of aerobic and facultative
bacteria are in general concerned in the oxidation of organic matter.
There is nothing specific in this reaction and very little that is character-
istic of any special or smaller groups. Under certain special and restricted
conditions, typical products are formed by particular species, as in the
manufacture of vinegar, and it is possible that a careful study of the com-
plex reactions involved in the oxidation of sewage would show a certain
sequence in the order of events and certain definite work being accom-
plished by definite groups. In other words, symbiosis and specializa-
tion doubtless take place to a limited extent. But the fundamental fact
remains that the metabolism of the organism demands that organic
matter be oxidized for the production of energy. Even though certain
food substances may be preferred and certain decompositions be norm-
ally produced there is necessarily a great latitude and great adaptability.

For this very reason a study of the individual organism and its action
upon specific materials throws no light upon the major problem, which is,
given fifty different types of organisms and fifty different fermentable
substances, in a mixture, what will be the course of the reaction. Here
the preferences, the adaptability and the antagonisms all come into play
and while it is impossible to say what has happened or how, it is readily
conceived and, in fact, almost apparent, that out of this heterogeneous
mixture there will come a homogeneous symbiotic family and an orderly
sequence of chemical events, in which metabolic needs and food supply
are all delicately adjusted.

220 MICROBIOLOGY OF WATER AND SEWAGE.

PATHOGENIC BACTERIA. Prevalence and Longevity. Owing to its
origin and nature, sewage may at any time contain infectious material and
for the purposes of the sanitarian it is assumed that at all times the germs
of disease are present. Such an assumption is possibly in excess of the
actual facts and is only justified because it supplies the only possible
hypothesis having an adequate margin of safety. The actual prevalence
of pathogenic bacteria obviously depends in the first instance upon the
amount of sickness in the contributing community. Furthermore, if,
as we are coming to believe, a definite proportion of the population are
perpetual carriers of typhoid infection then to just as definite an extent
is the bacterial population of the sewage made up of typhoid bacteria
from apparently well persons. In addition to these, about five one-
hundredths of i per cent of the population of American cities are suffer-
ing from the disease in acute form. Making due allowance for the extra
precautions that are, or should be taken in the care of the dejecta, these
persons constitute a definite and fairly constant source of infection.

In the case of the other infectious diseases of the alimentary tract,
and, possibly to a less extent in the case of tuberculosis, diphtheria, and
many others, these general statements are equally applicable, so that the
possibility of the occurrence of infectious material in sewage is not a
remote one, but definite and almost quantitatively determinable.

As to the persistence of active pathogenic bacteria in the sewage for
any length of time the data are less exact. In the case of typhoid fever,
which has been more carefully studied than any other disease, the germs
are more persistent in pure water than in impure, but whether this general-
ity can be extended to sewage is debatable. Our best information leads
to the belief that any reduction in numbers of typhoid bacteria which may
take place within the sewer before discharge is of minor importance and
of slight sanitary significance.

Discussion of other pathogens must be in even more general terms.
Information is almost wholly lacking and it can only be assumed for
purposes of safety that, in so far as organisms of these various types are
discharged into the sewer, they will persist to a certain extent in the sewage
until it is finally disposed of. If such disposal be by discharge into a
stream without purification, then the waters of that stream become pol-
luted with infectious material. Studies recently made by Sedgwick and
McNutt have indicated the possibility that many diseases, other than the
oft-quoted typhoid fever, may be transmitted in this way.

MICROBIOLOGY OF SEWAGE. 221

Life in Septic Tanks and Filters. With the introduction of the septic
tank at Exeter, England, in 1893, the question of the fate of pathogenic
bacteria in such a tank was raised. It was even suggested that bacteria,
such as the typhoid organism, might multiply in the tank. The question
was investigated by Professor Sims Woodhead, who concluded that no
organisms capable of setting up morbid changes in animals were dis-
charged from the tank. This negative evidence however has little weight
in the light of more recent experiments. Pickard introduced an emulsion
of typhoid bacteria into this same tank and noted only a gradual decrease.
After fourteen days he was able to detect i per cent of the initial number.
He also reported a removal of 90 per cent of the typhoid organisms in-
troduced into a contact filter. These data must be interpreted in the light
of two established facts. The typhoid organism tends to die at a rapid
but diminishing rate under any but the most favorable conditions. This
results in a rapid decrease at first, with a prolonged survival of a few
individuals. This process takes place in sewers, in streams, and, in
fact, under most artificial conditions. The second fact of importance
is the difficulty of recovering the typhoid organism under experimental
conditions like those described.

A thorough study of the bacteriology of sewage and of filter effluents
led Houston to conclude that the biological processes at work in a filter
or tank were not strongly inimical, if hostile at all, to the vitality of path-
ogenic germs.

A conservative study of all the evidence bearing upon this important
question including the vitality and fate of certain non-pathogenic species,
such as B. coli, leads to the conclusion that the removal of pathogenic
bacteria in purification methods is due to two allied causes, the efficiency
of which can be approximately determined. There is first the time
element and the known rapid decrease in the numbers of certain bacteria
such as B. typhosus when placed under conditions that preclude multipli-
cation. The rate of decrease varies but is roughly about 50 per cent in
twenty-four hours.

The second factor, acting in reality in conjunction with the first, is the
mechanical hindrance that is offered to the free passage of suspended
materials through the body of a filter. Even fine sand offers little strain-
ing action as such, since the open channels are thousands of times as
big as the bacterial cell, but surface tension phenomena tend to make
all solid material adhere to the medium and thus its passage is delayed.

222 MICROBIOLOGY OF WATER AND SEWAGE.

This action is prominent although of less importance in coarse-grained
filters. Actual experiments by the writer have indicated that while the
liquid may pass through a trickling filter in half an hour, small suspended
particles such as ultramarine and B. prodigiosus cells require an average
of over twenty-four hours. In this way the actual time of passage is
greatly delayed even when coarse broken stone is the filter medium, and
the times that are now known to be necessary for the passage are ample
in themselves to account for the reductions that have been noted.

It may therefore be stated as a conservative view of the efficiency of
purification processes in the removal of pathogenic bacteria, that there are
no strongly inimical processes at work in the tanks or filters, and that the
rate of decrease is not materially greater than would be observed in the
same period of time under the conditions of a running stream.

THE CULTIVATION OF SEWAGE BACTERIA.

There are two general methods employed for the cultivation of those
bacteria which are of assistance in sewage purification. They may be
cultivated in so-called filters of sand or coarser material, or in specially
constructed tanks such as the septic or the hydrolytic tank. In the for-
mer case the bacterial growth occurs upon the special medium provided,
the sand or stone; in the latter, it takes place in the liquid itself and a
continuous life history within such a tank is possible only when the rate
of flow is sufficiently slow to permit of the inoculation of the incoming
stream by the contents of the tank.

FILTERS. The filtering media most commonly employed are sand or
crushed stone or other coarse material. In natural sand beds a brief
period of treatment with sewage suffices to produce an active state of
"nitrification." By this term is indicated all the complex processes of
oxidation one index of which is the formation of nitrates. After such a
filter has once become active in this way it will continue, with proper care,
to oxidize sewage almost indefinitely. Improper care, such as an over-
dose of sewage or continued flooding of the surface due to poor drainage,
will soon destroy the activity of the filter. The addition of germicidal
substances has a similar effect and cold weather somewhat reduces the
efficiency. From all this it is apparent that a filter is a biological culture
medium upon which the various types of bacteria are growing and carry-
ing out their functions and that such a medium requires careful control
and is sensitive to unfavorable changes in environment (Fig. 64).

MICROBIOLOGY OF SEWAGE.

223

The other filters are similar to this and illustrate the true function of
filtration. In the case of the sand filter it might be maintained that
filtration or straining was an essential element in the process, but in the
case of these coarse-grained media straining action is eliminated. Here
there is nothing but a pile of stones, varying from one to three inches or
more in diameter, upon the surface of which the bacteria grow. The

FIG. 64. Sewage Experiment Station, Mass. Inst. Technology. Trickling filter in
front, sand filter just behind trickling filter, dosing tank just behind sand filter, and
septic tank just behind dosing tank.

sewage trickles slowly over the surfaces, or is held in contact with them
temporarily, according as we are dealing with trickling or contact filters.
Solids adhere to the stones or settle upon them, and soluble material is
"absorbed" by the surface growth and removed from solution. Within
these gelatinous growths to which the air also has free access, the pro-
cesses of oxidation take place and the products, the semi-oxidized organic

224

MICROBIOLOGY OF WATER AND SEWAGE.

material, are later "shed" from the stones appearing again in the effluent
as humus or stable organic matter.

ANAEROBIC TANKS. The cultivation of bacteria in anaerobic tanks is
not quite as simple a matter as that which has just been described. The
sewage is allowed to flow slowly through the tank and after some time,
from a few days to a month or more, a normal and constant flora will
have become resident there. This flora will soon have become so well
established that the incoming sewage laden with a flora of its own mingles
with a liquid in which the established flora is so greatly in excess that the

Siphon
Chamber-

Chamber

FIG. 65. Sketch of septic tank. (Original.)

former in large measure gives way to the latter. In this way, while the
sewage itself moves onward and is gone within a few hours, the flora is
constant and persistent. A further aid in preserving this constant flora
is the sludge at the bottom, in which the bacteria lodge and multiply and
from which they are carried upward by the ever moving eddies and con-
stantly re-inoculate the liquid above (Fig. 65).

THE DESTRUCTION OF SEWAGE BACTERIA.

BY BIOLOGICAL PROCESSES. Reference has already been made to the
effect of biological processes of purification upon pathogenic bacteria.
What was stated in regard to the pathogens is equally true of the sewage

MICROBIOLOGY OF SEWAGE. 225

bacteria as a whole. Their destruction is due to time and an environment
unfavorable to growth, rather than to any specific cause. Further evi-
dence of these facts may now be given. Bacteria as a whole do pass even
the fine-grained filters in large numbers. Careful analyses of their types
show them to be a haphazard mixture from the original sewage flora with
little or no observable selection. Houston pointed out the relative
abundance of the streptococci, supposedly delicate organisms, and found
on the whole that the relative abundance of the different kinds of bacteria
seemed to be much the same in the effluent as in the crude sewage.

On the whole we may conclude that the biological processes remove
bacteria not by any specific antagonistic action but by delaying their
passage and permitting the natural decrease that occurs when multiplica-
tion is prevented. The more efficient the mechanism of the filter in
producing this delay the more complete will be the removal.

BY CHEMICAL PROCESSES. A much more reliable and economical

*

method for bacterial destruction is now available in chemical disinfection
of sewage effluents. The writer's studies at Boston, Baltimore and
elsewhere have shown that the application of hypochlorite of calcium in
amounts depending upon the character of the effluent, and ranging from
one to five parts per million of available chlorine (25 to 125 pounds of
bleaching powder per million gallons), will produce a bacterial removal
amounting to 98 or 99 per cent. This disinfectant is the most efficient of
the known germicides, cost being considered. By this means it is possi-
ble to practically eliminate the bacteria, good and bad, from an effluent
and it is no longer necessary nor desirable to seek high bacterial removals in
the purification process proper. By thus dividing the work of purifica-
tion into its component parts each part can be carried out at a maximum
of efficiency and economy.

DIVISION HI.*
MICROBIOLOGY OF SOIL.

CHAPTER I.

*

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY.

INTRODUCTION.

Rational views on soil fertility were first presented, in a systematic way,
by Justus von Liebig in 1840. In his " Organic Chemistry in its Applica-
tions to Agriculture and Physiology" he developed important theories on
the circulation of carbon and nitrogen in nature, and on the function of
the so-called mineral constituents of plants.

When Liebig 's book appeared many of the leaders and students of
agriculture still believed that humus, the partly decomposed residues of
plants and animals in the soil, was the direct food of crops. They
believed that soils could yield poor or rich harvests in proportion to the
amount of humus present in them; they believed, in other words, that
plants, like animals, used organic substances as food.

Liebig rendered a great service to agriculture in emphasizing the sig-
nificance of decay processes. He made it evident that humus as such is
of no use to plants, and that it becomes valuable only in so far as it is
resolved into the simple compounds carbon dioxide, ammonia, nitric acid
and various mineral salts. To be sure, he regarded the decomposition
of organic matter as a phenomenon purely chemical, nevertheless he
succeeded in showing that decay, putrefaction and fermentation are funda-
mental facts, connecting links between the world of the living and the
world of the dead.

The research of the following decades brought to light the intimate
relation existing between microorganisms and the decomposition of

* Prepared by Jacob G. Lipman with exception of sub-chapter on " Soil Inoculation" which
has been prepared by S. F. Edwards.

226

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY. 227

organic matter. In the realm of soil fertility the new discoveries revealed
the vastness of the task assigned to soil microorganisms in providing
available food for crops. It was shown that under the attack of bacteria
and of other microorganisms the various organic debris in the soil are
split into relatively small chemical fragments; that the carbon is restored
to the air as carbon dioxide; that the nitrogen is changed into ammonia,
nitrites and nitrates. It was shown, further, that in this breaking down
of organic matter the various cleavage products, and, particularly, carbon
dioxide, hasten, to an amazing extent, the weathering of the rock particles
and make available thereby the mineral portion of plant food. It was
shown, likewise, that apart from accomplishing the transformation of
unavailable into available plant food, microorganisms are concerned
also in the addition of nitrogen compounds to the soil. The evidence
gathered slowly by many investigators made it plain, therefore, that
microbes are an important factor in the growing of cultivated and uncul-
tivated plants. Hence, the important place assigned to microorganisms
in the study of soil fertility problems.

THE SOIL AS A CULTURE MEDIUM.

Arable soils present so wide a range of conditions as to modify,
materially, the development and predominance of different species.
Variations as to moisture, temperature, aeration, reaction, food supply and
biological relations are important, in each case, in determining the survival
or disappearance of any particular species. For this reason, the study
of soil microorganisms must reckon with the mechanical composition
of soils, their ability to retain water and their content of inert and soluble
plant food.

MOISTURE RELATIONS IN THE SOIL.

AMOUNT AND DISTRIBUTION OF RAINFALL. Precipitation in different
regions of the earth's surface varies from practically nothing to more
than 1,524 cm. (600 in.) per annum. A portion of this water
runs off the surface into the nearest stream, another portion is rapidly
changed into vapor and is returned to the atmosphere, and the remainder
passes downward, into the soil and becomes the medium in which plant
food is dissolved. It is estimated that only about half the total rainfall
percolates through the soil. Where the soils are open and nearly level

228 MICROBIOLOGY OF SOIL.

the proportion of percolating water is relatively greater; where the soils
are fine-grained and more or less impervious, or the topography broken,
the proportion is relatively smaller.

Bacteria and other microorganisms, as well as the higher plants, are
directly influenced by the amount of moisture available for their various
needs. Hence soil microbial activities are affected not alone by the
amount of rainfall, but also by its distribution. It is obvious, for instance,
that an annual rainfall of 762 mm. (30 in.) distributed rather uniformly
throughout the year would produce different soil-moisture relations than
the same amount of precipitation confined to only two or three months.
As is pointed out by Abbe, a daily precipitation of 2 mm. (.079 in.)
distributed throughout the three summer months would be quickly
changed into vapor, and would hardly wet the soil; whereas the total
quantity of 180 mm. (7 in.) evenly divided into ten or twelve rains
would penetrate the soil to a considerable depth, and would furnish very
favorable conditions for microbial development. In a similar manner it
is pointed out by Hilgard that Central Montana, and the region in the
vicinity of the bay of San Francisco, have each a total precipitation of
610 mm. (24 in.). But while in Montana the rainfall is distributed over
the entire year and irrigation becomes necessary, the precipitation near
San Francisco is limited to the portion of the year that nearly coincides
with the growing season, and crops are enabled to mature without
irrigation.

RANGE OF SOIL MOISTURE. Any given volume of dry soil consists
of solid particles separated by empty spaces. The sum of these spaces
is known as the "pore-space." It varies from about one-third of the
entire volume in coarse sands to more than two-thirds in pipe clay. In
peat and muck it may amount to as much as 80 or 90 per cent, of the
entire volume. Under air-dry conditions each soil grain is surrounded
by a very thin film of moisture designated as hygroscopic water. When
air-dry soil is moistened the films around the soil particles become thicker
and finally cease to be isolated. A continuous liquid membrane, as it
were, is stretched from particle to particle, and the surface tension that
thus comes into play is capable of lifting large amounts of water to the
surface. The continuous film of soil water that can hold its own against
the pull of gravity is known as capillary water. Finally, when the liquid
films around the soil grains increase in thickness beyond a certain point,
the attraction between the molecules in the soil grains and the more

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY. 229

distant molecules of water is no longer great enough to overcome the
force of gravitation, and the excess of water percolates downward. The
water more or less readily moved by gravitation is called hydrostatic
water.

For any given conditions of the soils the amount of hydrostatic,
capillary and hygroscopic water is directly dependent on their mechanical
structure. It is evident that the aggregate surface of the particles in a
fine-grained soil is much greater than that in a coarse-grained soil. Actual
determinations have shown that the aggregate inner surface of one
cubic foot of coarse sand may be but a fraction of an acre; whereas the
same quantity of the finest clay may have an inner surface equivalent to
three or four acres. These differences are to be expected, since, as is
shown by Lyon and Pippin, i g. of fine gravel may contain 252 particles;
i g. of medium sand, 13,500 particles; i g. of very fine sand, 1,687,000
particles; i g. of silt, 65,100,000 particles, and i g. of clay, 45,500,000,000
particles.

Since the soil water is spread as a film over the solid particles and
varies in amount with the fineness or coarseness of the soil, and since the
quantity of plant food going into solution is determined largely by the
amount of water in contact with the soil particles, it follows that clay
soils will, under the same conditions, contain more plant food in solution
than loam soils and still more than sandy soils. From the standpoint of
soil microbiology this is important, for the microorganisms live and
multiply in the film water surrounding the soil particles. The concen-
tration of salts in this film water as well as their composition must of
necessity affect bacterial activities. In the same way, methods of tillage
and cropping affecting the concentration and composition of the film
water will modify the chemical changes caused by bacteria.

EFFECT OF DROUGHT AND OF EXCESSIVE MOISTURE. Optimum
conditions for plant growth and the development of many important soil
bacteria are furnished when about half of the entire pore space is filled
with water. In light sandy soils the optimum moisture content may be
reached when the wet material contains scarcely more than 8 to 10 per
cent of water by weight; while in silt and clay soils the optimum may
reach 16 to 20 per cent or even more.

Continued depletion of soil moisture by plant roots and evaporation
at the surface causes the film of capillary water to stretch more and more.
Finally it becomes very thin, breaks, and ceases to be continuous. The

230 MICROBIOLOGY OF SOIL.

soil then becomes air-dry and contains only hygroscopic water. It is
estimated by Lyon and Fippin that, under average conditions of humidity,
light sand will contain 0.5 to i per cent of hygroscopic moisture; silt
loam, 2 to 4 per cent; and clay, 8 to 12 per cent. The amount of water
present in air-dry muck or peat may range up to 40 per cent, or even
more. According to Hall the film of hygroscopic moisture is about
o.75/( (0.00003 in.) thick. As the soil dries out bacterial activity is sus-
pended and many vegetative cells undoubtedly perish. Nevertheless, it
will be seen that the moisture film even in air-dry material is deep enough
to allow the bacteria a reasonable degree of protection. This will account
for the survival of non-spore-bearing bacteria in dry soil for a long time.
Indeed, instances are on record of the isolation of Azotobacter and Nitro-
somonas from soils that had been kept in a dry state in the laboratory
for several years. It may be noted, in this connection, that in the pro-
cess of drying the soluble salts in the soil may be sufficiently concentrated
in the thin films to cause plasmolysis and the destruction of individual
cells.

On the other hand, excessive moisture in the soil is not only directly
unfavorable to aerobic species in that it limits their supply of oxygen,
but is objectionable because it encourages the formation of reduction
products that are toxic to these species. It is apparent, therefore, that
favorable conditions for the formation of available plant food by bacteria
are created when a certain relation is established between the volumes of
moisture and air in the soil. The shifting of this relation in one direction
or another is bound to react on species relationships and numbers.

AERATION.

MECHANICAL COMPOSITION or SOILS. Soil ventilation is an impor-
tant factor in crop production. It provides for the proper supply of
elementary oxygen so essential to decomposition processes in normal
soils; for the supply of elementary nitrogen required by nitrogen-fixing
species; for the removal of excessive amounts of carbon dioxide; and for
the destruction of various toxic substances. The intimate relation
existing between soil ventilation and the mechanical composition of the
soil material is bound to react on the microbial factors involved. It is
well known that the rate of flow of air through soils is inversely propor-
tional to the fineness of the material; in other words, the fine-grained

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY. 231

soils, notwithstanding their greater pore space, will not allow air to pass
through them as rapidly as coarse-grained soils. King shows, for in-
stance, that 5000 c.c. of air passed through a column of fine gravel in
thirty-seven seconds, whereas in similar columns of medium sand, fine
sand, loam and fine clay soil the same amount of air required for its
passage 1,178, 44,310, 282,200, and 2,057,000 seconds respectively.

AEROBIC AND ANAEROBIC ACTIVITIES. The more rapid diffusion of
gases from open soils naturally leads to a more frequent renewal of their
oxygen supply. In its turn, the latter affects the ratio of aerobes to an-
aerobes; it follows, therefore, that in clay soils and clay loam soils the
activities of aerobic species are retarded to a greater extent than they are
in sandy loams or sandy soils. It follows, also, that in fine grained soils
the activities of the aerobes are confined to a shallower soil layer than in
coarser grained soils. The reverse is true of anaerobic species. Methods
of soil treatment tending to improve soil ventilation react both on the
amount of chemical change produced by definite species, as well as the
numerical ratio of different species to one another. Among such
methods may be included drainage, liming, manuring and tillage.

RATE OF OXIDATION or CARBON, HYDROGEN AND NITROGEN. Ex-
periments carried out by Wollny proved conclusively that the production
of carbon dioxide in soils is directly affected by the amount of oxygen
supplied; that is, by the more or less thorough aeration of the soil. In one
of these experiments air containing varying proportions of oxygen and
nitrogen was passed through columns of soil. When this air contained
21 per cent of oxygen there were produced for every 1000 volumes of air
12.51 volumes of carbon dioxide; while with 2 per cent of oxygen in the
entering air there were produced only 3.62 volumes of carbon dioxide.
Similar observations were made by Schloesing in connection with the
formation of carbon dioxide and of nitric acid. Deherain and many
others have recorded the favorable influence of aeration on the rate of
nitrate formation, while Lipman and Koch have observed an increased
fixation of nitrogen by Azotobacter, consequent upon a better supply of
oxygen.

THE MINERALIZATION OF ORGANIC MATTER. Conditions that favor
the intense activities of decay bacteria lead to a relatively rapid restora-
tion of the phosphorus, sulphur, calcium, magnesium and potassium that
had been made fast in plant tissues, to the stock of available plant food in
the soil; indeed, in extremely well aerated soils the decomposition of or-

232

MICROBIOLOGY OF SOIL.

ganic matter and its ultimate mineralization proceed too fast. It often
happens that the farmer is unable to maintain a proper supply of humus
in these soils because of their openness and is forced to adopt measures
that will retard soil aeration. He resorts therefore, to rolling, marling,
manuring and green manuring.

On the other hand, heavy, fine-grained soils are not sufficiently well
aerated to allow a rapid mineralization of the organic matter. Under
extreme conditions the decomposition processes do not keep pace with
the process making toward the accumulation of organic matter, and a
more or less considerable increase in the amount of the latter takes place.
This occurs in low lying meadows, and, more particularly, in bogs and
swamps. Hence the farmer attempts to intensify aeration and the
resulting mineralization of the humus by more thorough tillage, drainage,
liming and manuring.

TEMPERATURE.

INFLUENCE OF CLIMATE AND SEASON. An illustration of the differ-
ences that may exist in the soil temperatures of different regions is given
by a comparison of the mean temperatures of 1901 recorded at Moscow,
Idaho, and New Brunswick, New Jersey. The soil temperatures were
taken to a depth of 152 mm. (6 inches).

Soil Temperature,* 1901.

Jan.

Feb.

Mch.

Apr.

May

June

July

Aug.

Sept.

Oct.

Nov.

Dec.

Moscow, Idaho

32

30

35

40

52

S8

68

72

S7

So

40

34

New Brunswick,

3i.S

28.6

35-3

47

57-9

72.1

76.4

73 .4

68.5

56.0 41.1 33.4

N. J.

Air Temperatures* 1901.

Moscow, Idaho

30

30.5

38.3

44.0

56.9

55-0

65.5

69.6

50.3

50.5

39-5

39-0

New Brunswick,

30.8

24.8

39-1

48.3

59- 2

70.9

77-4

74-6

67.6

54-6

38.6

32.6

N. J.

*Recorded in Fahrenheit scale.

It will be observed that in the months of November to March the soil
temperatures in the two places were nearly the same. On the other hand,
in April to October the average temperatures at New Brunswick were
for soil 14.5 (58 F.) and for air 22.5 (72 F.), respectively; and in July

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY.

233

they were 20.0 (68 F.) and 24.5 (76.4 F.) respectively. It will also
be observed that there is an unmistakable relation between the corre-
sponding air and soil temperatures.

As a further illustration of the relation of climate to temperature a
comparison may be made of the average daily mean temperatures at
Bismarck, North Dakota, for the period 1873-1895. and at Key West,
Florida, for the period 1872-1895.

Daily Mean Temperatures* (Air).

Jan.

Feb.

Mch.

Apr.

May

June

July

Aug.

Sept.

Oct.

Nov.

Dec.

Bismarck, N. D. 4.5

9.5

22 .6

42.1

54. 2

63.8

69.5

67-5

S7.o 43.8

25-9

14-7

Key West, Fla. 69.7

7i .4

72.7

76.1

79-4

82.5

83.9

83-9

82.5

78.5

74-2

70.0

*Recorded in Fahrenheit scale.

It is obvious from the figures given here that, because of the important
temperature variations of different soil regions, the microbiological activi-
ties must be profoundly modified. But apart from the climatic variations
already indicated there are seasonal variations in any particular locality
that are of great moment for soil microbiological activities. Such differ-
ences are demonstrated by the temperatures of 1898 and 1902, taken to a
depth of 152 mm. (6 inches), at New Brunswick, N. J.

Soil Temperatures.*

Jan.

Feb.

Mch.

Apr.

May

June

July

Aug.

Sept

Oct.

Nov.

Dec.

New Brunswick,

N J. (1898).

33-2

33-1

45-1

48.9

59-1

76.0

79-3

77-8

72.0

60. i

44-6

33-6

New Brunswick

N. J. (1902).

30.7

28.9

41-3

49-5

60.4

68.0

72 .6

70.5

65-9

56.4

48.6

34-1

* Recorded in Fahrenheit scale. '

In this instance, the season of 1898 was not only earlier, but the tem-
peratures of June to September were sufficiently higher to favor more
intense bacterial growth and activity.

EARLY AND LATE SOILS. Under any given climatic conditions the
warming up of soils in the spring will depend on their chemical and
mechanical composition, color, tillage and topography. Because of the

234

MICROBIOLOGY OF SOIL.

high specific heat of water, fine-grained soils containing a relatively
large amount of moisture will warm up more slowly than coarse-grained
soils containing a relatively small amount of moisture. The differences in
the specific heat of humus, sand, clay and chalk are less important, yet
they introduce appreciable variations in the soil temperature according to
the proportion of each present. The topography of the soil introduces a
factor of some importance for it affects the inclination toward the sun's
rays as well as the drainage conditions. Tillage operations are of con-
siderable moment, since they influence the rate of evaporation, that is,
the rate at which heat is lost from the soil by the transformation of liquid
water into vapor. Finally the color of soils exerts an influence on their
temperature in that it affects the absorption and reflection of heat.

Taking all of the factors together, it is found that sandy soils and sandy
loams are early soils, because they part readily with their excess of water.
Clay soils and clay loams are, on the other hand, late soils; it means,
therefore, that in the more open soils microbial activities become intense
earlier in the spring. Market gardeners usually attempt to improve
matters still further by the use of large quantities of readily fermentable
manure that develops enough heat to raise slightly the soil temperature.

PRODUCTION AND ASSIMILATION OF PLANT FOOD. It was already
observed by Moller that slight amounts of carbon dioxide may be evolved
from frozen soil. Kostychev could detect a considerable production of
carbon dioxide at o-5. In a series of experiments carried out by Wollny
the amounts of carbon dioxide produced were as follows:

COi in 1000 Vols. of Air.

Water in Soil

10

20

30

4

S

6.79 per cent

2 O3

^ 22

6 86

IA 60

2< 17

26.79 per cent . . .

18 38

CA 22

6^ so

80 06

81 52

46 79 per cent

2 c o7

82 12

91 86

The increased production of carbon dioxide at the higher temperatures,
as shown in the above table, correspond with the observations that had
already been made by Ebermayer, Schloesing and others, that carbon
dioxide production in the soil is greater in summer than it is in winter.
These facts, taken together with the early observations of Forster on the
multiplication of photo-bacteria at o, and the more recent observations

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY. 235

of numerous investigators on the multiplication of individual species,
or of mixtures of species in milk, water, soil, butter, etc., at o, or even
below that, make it evident that bacterial activities are not entirely
suspended at relatively low temperatures. As the latter rises these ac-
tivities become more intense as gauged by the formation of carbon dioxide.

Coming down to specific groups of soil bacteria, it may be noted that
at 12 nitrification is already quite perceptible; that urea bacteria grow
slowly at 5; Ps. radicicola at 4; members of the B. subtilis group at 6 10,
etc. At 15 the breaking down of organic matter is fairly rapid, and at
25 the optimum is reached for many species. It follows, thus, that
the production of plant food namely, ammonia, nitrates, sulphates,
phosphates, etc. gains rapid headway as the optimum temperatures are
approached. The organic matter itself, apart from serving as a source of
plant food, furnishes carbon dioxide and various organic acids that help
to attack the rock fragments and to render available compounds of
phosphorus, potassium, calcium and magnesium. It is likewise evident
that in warm countries bacterial activities are not only more intense at
any one time, but they continue through a longer period. For this rea-
son, the soils of the South can furnish both relatively and absolutely
a greater amount of available plant food than the soils of the North.

The production of plant food is necessarily followed by more vigorous
growth of bacteria and of higher plants. More food is, therefore, assimi-
lated and more moisture used up until the very rank growth of the crops
hastens the depletion of the soil moisture. In this manner the soil may
be dried out sufficiently to retard seriously the growth of soil bacteria
and to retard thereby the decomposition of organic matter; under such
conditions, moisture, rather than temperature, becomes the controlling
factor of growth.

REACTION.

RANGE OF SOIL ACIDITY. Acid soils are very common in humid
regions. The older soils of Europe include extensive areas whose lime
content has been restored repeatedly by the application of wood ashes,
marl, oyster and clam shells, and various grades of burned or crushed
limestone. In the United States acidity is becoming prevalent in many
of the cultivated soils, as is shown by the investigations of the Rhode
Island, Ohio, Illinois, Oregon and Florida experiment stations. These
investigations, confirmed by experiments in other states, show that there

236 MICROBIOLOGY OF SOIL.

is a marked removal of lime and of other basic materials from the soil
as cultivation and the use of commercial fertilizers become more thorough.
Knisley shows, for instance, that 38.75 per cent of the Oregon soils ex-
amined were acid, and that 16.25 per cent were strongly acid. Similarly,
Blair found that of 189 soil samples of different Florida soils and subsoils,
examined, 68.22 per cent of the former and 51.35 per cent of the latter
were acid. He also found that virgin soils were less acid than cultivated
soils.

CAUSES OF SOIL ACIDITY. Soil acidity may be due to acids or acid
salts, both inorganic and organic. ' Under ordinary conditions the latter
are of much greater importance than the former as a cause of soil acidity.
This is demonstrated by the extremely acid conditions of peat and muck
soils that are particularly rich in organic acids. In soils left to themselves
the formation of basic substances in the breaking down of silicates and
other compounds keeps pace with their neutralization by acid and their
removal in the drainage water. When soils are placed under cultivation,
lime and other bases are removed more rapidly and the inert humic acids
are left behind. The loss of bases is intensified by application of acid
phosphate, potash salts and ammonium sulphate, commonly used as
fertilizers. This accounts for the less extensive acidity in and among
virgin soils as compared with cultivated soils. Arid soils lose scarcely
any of their basic substances by leaching and are seldom acid. Residual
limestone soils may be alkaline, neutral or acid, according to the loss of
bases they have suffered by leaching. Low-lying soils, including meadows
and swamps may accumulate large amounts of organic acids because of
their imperfect aeration.

EFFECT OF REACTION ON NUMBERS AND SPECIES. Some of the
important groups of soil bacteria including nitro, azoto and ammonifying
species will develop slowly or not at all, when the amount of acid in the
medium is increased beyond a certain point. Hence it is realized by
progressive farmers that a proper supply of lime is essential for the satis-
factory decomposition of organic matter in the soil, and the abundant
supply of available nitrogen compounds, as well as of other constituents
of plant food to growing crops. The influence of lime on the multipli-
cation of soil bacteria is well illustrated, for instance, by the experiments
of Fabricius and von Feilitzen. These investigators found only 138,500
bacteria per g. in newly broken and unlimed peat soils; whereas in
similar soils that had been limed and cultivated for several years the

MICROORGANISMS AS A FACTOR IN SOIL FUTILITY. 237

numbers averaged about 7,000,000 per g. and reached a maximum
of 22,132,000 per g.

FOOD SUPPLY.

ORGANIC MATTER. It may be said truly that a soil devoid of organic
matter is practically devoid of bacteria. To the fresh and the partially
decomposed organic matter (humus) the soil organisms must look for
most of their food and energy. Being largely of plant origin this organic
matter contains starches, fats, organic acids, higher alcohols, proteins and
amino-compounds. Because of the different relations that these vege-
table substances bear to the several species of soil bacteria, a high or low
proportion of starch, of cellulose, or protein must necessarily modify both
numbers and species relationships. For instance, observations have
been made by Coleman and others that small amounts of dextrose favor
nitrification, whereas larger quantities retard it; similarly, it has been
noted that in the spontaneous decomposition of protein bodies bacteria
are prominent and molds absent or relatively few in numbers. But where
dextrose is added to the decomposing proteins molds soon appear in large
numbers. There may also be cited, in this connection, the observations
of Hilgard that humus should contain at least 4 per cent of nitrogen
if it is to furnish a sufficient quantity of available nitrogen compounds;
otherwise, the soil bacteria seem to be unable to decompose it, so as to
meet the needs of the growing plants. Many similar facts could be cited
to show that as a culture medium the soil is influenced by green manures,
barnyard manure, commercial fertilizers, lime, tillage and any other
treatment that will modify the quantity as well as the quality of its organic
matter.

THE MINERAL PORTION OF THE SOIL. The moisture films surround-
ing the soil grains contain in solution substances derived from these soil
grains. A particle of calcium carbonate will be surrounded by a moisture
film containing some calcium bicarbonate. In the same way particles of
feldspar may give rise to a solution of potassium bicarbonate; particles
of apatite to a solution of calcium phosphate; particles of selenite to a
solution of calcium sulphate; particles of protein to a solution of ammonia,
etc. In view of the fact that these reactions are more or less localized
and diffusion slow, there are, undoubtedly, in the soil minute zones where
individual species are more prominent than they are in others. For
example, Heinze has found it convenient to isolate Azotobacter by inocu

238 . MICROBIOLOGY OF SOIL.

lating suitable culture solutions with particles of calcium carbonate picked
out from the soil. Evidently these organisms were present in much
greater abundance on these particles than on others of non-calcareous
origin. Indeed, he occasionally obtained in this manner Azotobacter
membranes that constituted almost pure cultures. The more general
significance of this relation is apparent when it is remembered that nitro
bacteria are particularly favored by magnesium carbonate; tubercle
bacteria by gypsum and calcium carbonate; Azotobacter by cal-
cium phosphate and calcium carbonate; photo-bacteria by sodium
chloride, etc.

Considerable as must be the local differences in any one soil, they are
undoubtedly even more pronounced when different soils are compared.
Extreme conditions are met with in certain irrigated soils in which a
marked concentration of salts occurs. In so far as crop production is
concerned, it is stated by Hilgard that the upper limit is practically reached
when the concentration of soluble salts in the irrigation water is about
4-55 g- (7o gr.) per gallon. Nevertheless, in Egypt and the Sahara
region irrigation water is occasionally used that contains more than 13 g.
(200 gr.) of soluble salts per gallon. Further differences are introduced
by the quality of these salts, e.g., the proportion of sodium sulphate,
magnesium sulphate, sodium chloride, sodium carbonate, etc. Again,
instances are on record, as in the investigations of Headden in Colorado
and California, where the concentration of nitrates in the soil water is
so great as to kill even relatively resistant plants like alfalfa. It is to be
shown by future investigations what the effect of the concentration and
composition of such salts may be on the soil bacteria.

In humid soils conditions are less extreme, yet even here the variable
concentration and composition of the soil solution are of direct moment
for the different microorganisms. Granite soils, for instance, are fairly
well supplied with phosphoric acid and abundantly with potash, but
when hornblende is lacking they are apt to be deficient in lime. Ill-
ventilated clay soils may contain reduction products of iron salts, while
green sand, chalk, slate, shale, sandstone and other soils may have their
individual peculiarities from the standpoint of a culture medium.

BIOLOGICAL FACTORS.

MOLDS. Soil bacteria must not only compete with other micro-
organisms and higher plants for their food, but must contend with unf avor-

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY. 239

able changes caused in their medium by the competing organisms. In
the case of molds a sufficient degree of acidity is often produced in the
medium to retard or stop the growth of bacteria. This point is well
illustrated by the prominent development of molds in culture solutions
containing ammonium salts as the sole source of nitrogen. When the
ammonium salts are replaced by nitrates under certain conditions the
growth of molds is practically suppressed and bacteria come to the fore.
In the soil itself the rapid development of molds has been observed in
plots or pots receiving large additions of ammonium salts, a phenomenon
due to the acid residues of the ammonium salts. The nitrates, on the
other hand, give rise to alkaline residues since the "NO 3 " radical is used
up in greater proportion than the basic radical. Similarly, molds are
enabled to compete with bacteria when the proportion of sugar or of
starch to protein is increased, or where large amounts of fats are present.

ALG.E. At times the influence of algae in changing the character of
the soil as a culture medium for bacteria is quite considerable. As
chlorophyl-bearing organisms they are enabled to manufacture sugar and
starch with the aid of sunlight, and favor thus the development of Azoto-
bacter and of other microorganisms dependent for their energy on the
organic matter in the soil. Investigators both in France and in Germany
have found that the fixation of nitrogen in sand used for pot culture
experiments occurs in the surface layer possessing a growth of algae.
The advocates of bare fallows attribute the greater productivity of
fallowed land to the growth of alga?, the accumulation of nitrogen by them
and to other changes affecting the soil bacteria.

PROTOZOA. It has been known for a long time that certain species
of protozoa are common in soils and that their food consists of bacteria.
To what extent protozoa play a part in soil fertility has not yet been fully
explained, even though Russell and Hutchinson of the Rothamsted
Experiment Station have maintained that these minute animals are
extremely important in that they maintain a certain bacterial equilibrium
in the soil. Their claim is mainly based on the fact that partially sterilized
soils (either by means of heat or antiseptics) soon come to contain enor-
mous numbers of bacteria. It is, therefore, assumed by them that this
abnormal increase is made possible by the destruction of the protozoa
that normally check the increase beyond a certain point.

HIGHER PLANTS. Higher plants modify the soil as a culture medium
for bacteria in at least three ways. The root-hairs come in contact with

240

MICROBIOLOGY OF SOIL.

the moisture films surrounding the soil grains and not only modify the
composition of the film water, by withdrawing a portion of the dissolved
matter, but also change its character by secretions from the roots. The
changes thus effected must, necessarily, modify the character of the soil
and the soil solution as a culture medium. Again, the rapid removal of
water from the soil by growing crops causes the film water to become
more concentrated in so far, at least, as some salts are concerned. Modi-
fications are, also, introduced thereby in the proportions of oxygen,
nitrogen and carbon dioxide in the soil air. Finally, higher plants modify
the soil environment for bacteria by their root and stubble residues.
For example, residues of leguminous plants, being richer in nitrogen
and possessing a narrower carbon-nitrogen ratio than the corresponding
residues of non-legumes, will affect the soil somewhat differently than the
latter.

BACTERIA. Occupying, as they do, the leading role, bacteria demand
a more detailed consideration, in fact, most of the biological discussions
of soil are based upon a knowledge of these organisms.

Numbers and Distribution; (Bacteria in Productive and Unproductive
Soils] . Among the various factors that affect the numbers of bacteria in
the soil moisture, aeration, food supply and reaction have already been
discussed as important. Sandy soils because of their relative dryness and
poverty in organic matter contain a comparatively small number of bac-
teria, at times less than 100,000 and usually less than 1,000,000 per g.
But when such sandy soils receive deep and thorough cultivation and are
enriched in organic matter the numbers increase to several millions per g.
In most loam soils in a good state of cultivation the numbers of bac-
teria per g. range from about 1,000,000 to several millions. In heavy,
compact soils the aeration conditions are unsatisfactory, acid substances
and reduction products accumulate and the numbers are greatly reduced.
Again, in peat and muck soils the numbers of bacteria are small when
the reaction is markedly acid. It has been found that under such condi-
tions the molds are more numerous than the bacteria and that the latter
often occur to the extent of less than 100,000 per g. Generally speak-
ing, the nigher the state of cultivation the greater the number of bacteria
in the soil. It increases as we pass from forest and prairie to plowed
fields, and from extensive field practice to intensive garden practice.
Under special conditions, as in green-house soils generously supplied with
manure and moisture, in sewage polluted soils, and in soils partly steri-

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY. 24!

lized by heat or antiseptics the numbers may become enormous, 50,000,000
or even 100,000,000 per g. of material.

Distribution at Different Depths. Most of the soil bacteria are found
in the stratum in which the organic residues are concentrated, that is,
in the surface soil. Immediately at the surface the rapid evaporation
and the germicidal effect of direct sunshine act as disturbing factors,
hence the numbers in the uppermost 25-50 mm. (1-2 in.) are smaller
than in the layer of soil immediately below. Beyond the depth of 20
cm. or 22 cm. (8 or 9 in.) the numbers diminish rapidly. Material
from a depth of .6 M. to .9 M. (2 to 3 ft.) is nearly sterile in humid
regions. Differences occur, however, in keeping with the mechanical
composition of the soil. In light, open soils the bacteria are not only
carried down to greater depths by the percolating water, but can also
multiply there, thanks to better aeration. On the contrary, fine-grained
compact soils are more effective in holding back suspended material and
do not allow the bacteria to pass downward as readily. Moreover, the
less thorough aeration of these soils and the accumulation of toxic reduc-
tion products in the subsoil serve as an effective check on the increase of
bacteria in the deeper layers.

In irrigated soils of the arid and semi-arid regions bacteria are dis-
tributed at much greater depths. Their occurrence 2 M. to 3 M.
(8 or 10 ft.) below the surface is made possible not only by the better
aeration of these soils, but by the penetration of roots to great depths and
the accumulation there of considerable amounts of organic matter. The
practical significance of distribution appears, among other things, in the
use of soil for inoculation purposes; for instance, it is reported by Sals-
trom that in making peat soils arable the addition of small amounts of
fertile loam increases to a very marked extent their crop-producing power.
The efficiency of the inoculating material decreases as it is taken from the
deeper soil layers. Similarly, in the use of alfalfa soil for the inoculation
of new fields the most efficient material is secured at a depth between
7.62 cm. and 17.78 cm.( 3 and 7 in.).

Morphological and Physiological Groups. (Morphological Groups).
Rod-shaped organisms are numerically the most prominent among soil
bacteria. They occur at times to the extent of 80 or 90 per cent of the
total number. Spherical organisms usually constitute less than 25 per
cent of the bacterial flora. Spirilla and Sarcinoe are present in slight
numbers. Conditions may occur, however, when the proportion of
16

242 MICROBIOLOGY OF SOIL.

spherical organisms is markedly increased. This happens, particularly,
when large quantities of composted manure (rich in spherical organisms)
is added to the soil.

Among the rod-shaped species B. mycoides, B. stibtilis, B. mesentericus
B. tumescens and other members of the subtilis group are quite prominent
Members of the amylobacter group are seldom absent. Members of the
proteus group and various fluorescents are always present, while Bact.
arogenes and allied species are common inhabitants of the soil.

(Physiological Groups}. In the decomposition of organic matter in
the soil certain important changes in both nitrogenous and non-ni-
trogenous material are accomplished by definite groups of bacteria. The
breaking down of protein substances is accomplished by the formation of
ammonia, nitrites and nitrates. These in turn may be transformed back
into more complex amino- compounds, peptones, and proteins, or they may
be destroyed with the evolution of free nitrogen. Moreover, there are
groups of bacteria capable of joining non-nitrogenous organic matter to
elementary nitrogen and of fashioning thus nitrogen compounds. Again,
there are groups of bacteria bearing distinct and important relations to
the decomposition of cellulose, or the transformation of its cleavage pro-
ducts, methane and hydrogen. There are, likewise, definite groups of
bacteria concerned in the transformation of sulphur and its compounds,
and of ferrous compounds.

METHODS OF STUDY.

QUANTITATIVE RELATIONS. Since the early work of Koch in 1881
many investigators have determined the number of bacteria in soil
samples, by means of the plate method. It is well known, however, that
on ordinary gelatin or agar plates kept under aerobic conditions but a
fraction of the soil organisms produce visible colonies. The anaerobic
species do not appear, nor do aerobic Azotobacter, and nitro bacteria,
while other common soil organisms form colonies sparingly or not at
all. By employing synthetic agar media instead of beef broth gelatin
or agar, Lipman and Brown have succeeded in securing the growth of
a much larger number of colonies from any given quantity of soil, yet
even these larger numbers were incomplete for reasons mentioned above.
It is evident, therefore, that the results secured in the counting of soil
bacteria have a relative value only. With the same media and methods

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY. 243

some information may be secured concerning the influence of fertilization,
tillage, liming, etc., on certain of the soil bacteria. But even this infor-
mation must be properly discounted, since equal numbers do not neces-
sarily mean equal amounts of chemical work accomplished; for example,
there is no certainty that 1,000,000 of decay bacteria derived from one
soil will accomplish exactly as much decomposition as the same number
of similar organisms from another soil. Otherwise stated, individual
cells differ in their physiological efficiency from other cells of the same
species.

QUALITATIVE REACTIONS. By modifying the composition of the
culture media different physiological groups may be favored in their
development. In this manner the silica jelly medium proposed by Wino-
gradski, or the gypsum plates proposed by Omelianski may be employed
for making numerical comparisons of nitro bacteria in different soils.
In like manner Beyerinck's mannit agar may be used for the numerical
comparison of Azotobacter, and other media could be adapted for the
quantitative-qualitative determination of urea, denitrifying, methane,
and still other physiological groups of microorganisms.

There is no doubt that the quantitative-qualitative method just out-
lined may be made to yield valuable information. Yet it, too, possesses
defects already noted in connection with the more strictly quantitative
method. Apart from the vast amount of work involved in the prepa-
ration of a large number of media and in the counting of colonies on
many plates, this method fails to indicate differences in physiological
efficiency. Furthermore, the colonies of the specific organisms sought
are almost invariably accompanied by those of foreign species not always
easily distinguished. With these limitations properly recognized and
with further improvement in the make-up of special media the method
may be made useful in supplementing data secured by other methods.

TRANSFORMATION REACTIONS. Instead of counting soil bacteria in
accordance with colonies produced in general or special media, soil
investigators have attempted to measure the bacteriological functions of
soils by comparing more or less definite quantities of the latter under
known conditions. This method was employed by Wollny and others
in studying the factors that affect the formation of carbon dioxide in soils.
It was also used by Schloesing and Miintz and their followers in similar
studies on nitrate formation. A method somewhat similar in principle
but different in its application was proposed by Remy in 1902. He

244

MICROBIOLOGY OF SOIL.

suggested the use of special media for the quantitative estimation of
different physiological reactions; thus, making a i per cent solution of
peptone and inoculating with equivalent quantities of soil, he caused the
decomposition of the peptone and the formation of ammonia, and secured
comparisons of the ammonifying power of different soils. In a similar
manner he used special solutions for comparing quantitatively the trans-
formation accomplished by nitrifying, denitrifying or nitrogen-fixing
bacteria.

Remy's method has been extensively tested by Lohnis, Ehrenberg,
Lipman and others. It has been shown to possess a serious defect in
that it deals with conditions unlike those occurring in the soil itself. For
this reason more recent investigations have been carried on in weighed
portions of soil rather than in culture solutions inoculated with 10 per
cent of soil as is done in Remy's method.

RATE or OXIDATION OF CARBON. The rate of decomposition of
humus or of other organic matter in the soil may be measured, as was
done by Wollny, by determining the amount of carbon dioxide evolved
in weighed quantities of material kept under definite conditions. The
influence of temperature, moisture, aeration, organic matter, antiseptics,
etc., has been determined in this manner. The same method may be
used in studying decay, and factors influencing decay, in soils in situ
that is, in the field.

More recently Russell and his associates have modified the method
in that they have determined the rate of oxidation of carbon not by
measuring the carbon dioxide evolved, but by estimating the amount of
oxygen absorbed. In either case decay is measured from the carbon
standpoint. The method based on this principle should find wide
application in future soil fertility investigations.

RATE OF OXIDATION OF NITROGEN. Another method or series of
methods for studying decomposition processes in the soil may be based
on the determination of nitrogen compounds formed in the breaking
down of proteins. Two of the derivatives of protein, namely, ammonia
and nitrate, have been used successfully to gauge the decomposition of
organic matter in the soil. The recent results secured by Lipman and
his associates demonstrate that ammonia formation from dried blood in
weighed quantities of soil may serve as a very accurate measure of decay
from the nitrogen standpoint. Corresponding determination of nitrates
may similarly be employed in tracing protein cleavage and trans-

MICROORGANISMS AS A FACTOR IN SOIL FERTILITY. 245

formation as influenced by the numerous factors of season, soil and
cultivation.

ADDITION OF NITROGEN. At least one other bacteriological factor
in soils should be mentioned here as deserving attention in a systematic
study of soil fertility from the nitrogen standpoint. It is known that
Azo-bacteria are widely distributed in arable soils, and that they are
more prominent in some regions than they are in others. The student
of soil fertility finds it desirable, therefore, to study azotofication in
different soils, and employs (for this purpose) mannit solutions like
those proposed by Beyerinck, sand cultures supplied with sugar solutions
like those proposed by Fischer, or weighed quantities of soil mixed with
sugar as suggested by Koch.

The methods referred to above make possible thus the study of
ammonincation, nitrification and azotofication under controlled conditions
and permit, thereby, the measure of bacteriological factors in soil fertility
from the nitrogen standpoint.

REACTIONS CONCERNING CALCIUM, MAGNESIUM, SULPHUR AND
PHOSPHORUS. In addition to the purely chemical methods available for
the study of these constituents, microbiological methods have also been
suggested. In some of his still unpublished experiments with A zotobacter
Lipman employed solutions of mannit in distilled water, provided with
small quantities of sterile soil which were to supply the organisms with
the essential mineral constituents. In this manner interesting data were
secured on the availability of phosphorus compounds in different soils;
similarly, Christensen has suggested the use of Azotobacter for determining
the lime requirements of soils, and Butkevich has experimented with
cultures of Aspcrgillus niger in determining the availability of the mineral
constituents.

CHAPTER II.
THE DECOMPOSITION OF ORGANIC MATTER IN THE SOIL.

CARBOHYDRATES

ORIGIN. The sugars, starches, vegetable gums and allied pectine
substances, as well as the cellulose, contained in roots and other crop
residues add large quantities of carbohydrates to the soil. The crop
residues are augmented still further by green manures and animal
manures whenever these are used. A good growth of timothy, for example,
may increase the content of organic matter in the surface soil by five
hundred or a thousand pounds per acre, and three-quarters of this
consists of carbohydrates. In the same manner, a green manure crop,
or an application of barnyard manure may add to the land as much as
fifteen hundred pounds, or even more, of carbohydrates per acre. These
carbohydrates contain a large proportion of cellulose.

THE DECOMPOSITION OF CELLULOSE. Pure cellulose (C 6 H 10 O 5 ) X
is a rather inert substance, as exemplified by the resistance of cotton and
flax fiber to decomposition processes. It is well known, nevertheless,
that even cellulose is in the end decomposed and resolved into simple
compounds. Plant roots, leaves and stems, as well as the trunks of
fallen trees, gradually distintegrate and vanish. Under favorable con-
ditions this may proceed rapidly, as is indicated by the processes in septic
tanks, or in manure heaps on the one hand, and in open sandy soils on
the other. The disappearance of cellulose may be accomplished by (a)
anaerobic organisms, (b) by aerobic organisms, (c) by denitrifying
bacteria, and (d) by molds.

THE PRODUCTION or METHANE AND HYDROGEN. The decomposition
of pure cellulose and the formation of methane and hydrogen mixed with
other gases was first noted by Popov in 1875. Some years later Tappeiner
and also Hoppe-Seyler confirmed Popov's observations that nearly pure
cellulose in the form of Swedish filter-paper, or cotton fiber may be fer-
mented by bacteria with the evolution of methane, carbon dioxide and
occasionally also of hydrogen. These investigators ascribed the decom-

246

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 247

position of cellulose to an organism found by Trdcul in decomposing
vegetable materials, and named by him Amylobacter in 1865, because of
the blue color assumed by it when stained with iodine.

Subsequent investigations by Omelianski begun in 1894 and continued
through a period of years demonstrated the presence of specific anaerobic
organisms in decomposing cellulose. He described two distinct species
of long, slender bacilli, assuming the clostridium form when in the spore
stage. Morphologically the organisms can hardly be distinguished, but
physiologically they show important differences in that one causes the
fermentation of cellulose with the production of gases consisting of carbon
dioxide and methane, while the gases produced by the other consist of
carbon dioxide and hydrogen; hence the one is designated by Omelianski
as the methane bacillus and the other the hydrogen bacillus. These
organisms do not stain blue with iodine, and do not belong, therefore, to
the butyric bacilli designated as Amylobacter by earlier investigators.
Omelianski's investigations make it appear that the butyric organisms
are not capable of fermenting cellulose proper.

In culture solutions containing mineral salts and nitrogen in the form
of ammonium compounds the decomposition of filter-paper and other
forms of cellulose proceeds with considerable rapidity. Calcium carbon-
ate must be added to neutralize the acids formed, otherwise the fermen-
tation soon comes to a standstill. In one of Omelianski's experiments
begun in October, 1895, and ended in November, 1896, 3.3471 g. of
cellulose was decomposed by a nearly pure culture of hydrogen bacilli.
The products consisted of 2.2402 g. fatty acids, 0.9722 g. carbon
dioxide and 0.0138 g. of hydrogen, a total of 3.2262 g. which nearly
accounts for all of the cellulose destroyed' The fatty acids were made
up of butyric and acetic acids with a slight proportion of some higher
homologue, probably valerianic acid.

In a similar experiment with an apparently pure culture of the methane
bacillus, begun in December, 1900, and ended in April, 1901, fermentation
began after an incubation period of about one month, and the entire
volume of gas gradually evolved was 552.2 c.c. This mixture consisted
of 190.8 c.c. methane and 361.4 c.c. carbon dioxide. The products formed
from the 2.0065 g- cellulose consumed included 1.0223 g- fatty acids,
0.8678 g. carbon dioxide and 0.1372 g. of methane, or a total of
2.0273 g. The slight difference in weight in favor of the fermen-
tation products falls within the limit of error. These experiments show

248 MICROBIOLOGY OF SOIL.

that about one half of the fermentation products is gaseous and that
the other half consists of acetic and butyric acids.

THE OXIDATION OF METHANE, HYDROGEN AND CARBON MON-
OXIDE. Aside from cellulose, methane may also be produced from
various other carbohydrates, organic acids and proteins. Large amounts
of methane are thus contributed to the atmosphere by swamps, manure
heaps and low-lying meadows. In a purely chemical way methane may
also be set free from volcanoes and mineral springs. The constant
additions of methane, ethane, hydrogen and carbon monoxide represent
a considerable amount of potential energy. It is important to know,
therefore, whether these materials are at all utilized.

That methane may be utilized by bacteria as a source of energy was
first shown by Sohngen in 1905. He isolated an organism named by
him B. methanicus that showed itself capable of growing in inorganic
solutions confined over an atmosphere of methane, oxygen and nitrogen.
The methane gradually disappeared and there were formed consider-
able quantities of organic matter. The ability to oxidize methane
has been claimed for a number of other organisms by Sohngen and
others.

Early observations on the ability of moist soil to cause the oxidation
of hydrogen are credited to de Saussure (1838). Many years later (1892)
Immendorff called attention to the same fact. It was not, however, until
1905 that the oxidation of hydrogen was shown to be a specific biological
process. In that year papers by Sohngen and Kaserer reported experi-
ments wherein inorganic solutions confined under an atmosphere of hydro-
gen, oxygen and carbon dioxide and inoculated with very small quantities
of soil developed a bacterial membrane at the surface. The hydrogen
was oxidized and organic matter produced at the expense of the energy
set free. The observations just noted have been confirmed by other
investigators, by means of mixtures and single species of soil bacteria.
Finally it should be added here that B. oligocarbophilus previously isolated
by Beyerinck and van Delden is able, according to Kaserer, to oxidize
also carbon monoxide.

THE CLEAVAGE AND FERMENTATION OF SUGARS, STARCHES AND
GUMS. -Sugars are a very acceptable source of food and energy for soil
bacteria. A culture solution containing suitable mineral salts and sugar
ferments readily when inoculated with a small amount of fresh soil.
When no combined nitrogen is added, Azotobacter, or B. (Clostridiuni)

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 249

pasteurianus (or both), may come to the fore. The cleavage products
then include alcohols, organic acids and carbon dioxide. With B.
(Clostridium) pasteurianus butyric acid is one of the prominent cleavage
products. When combined nitrogen is also added to the culture
solution other organisms will develop prominently, notably members
of the subtilis group, butyric bacteria, aerogenes, etc. In the soil
itself the addition of sugar leads to a very marked increase in num-
ber and, if acid production is favored, molds may subsequently become
prominent. In general it may be said that butyric, propionic, acetic,
formic and lactic acid, and ethyl, propyl, butyl and iso-butyl alcohol are
common cleavage products.

In the case of starch, pectins and pentosans, similar conditions hold
good. Diastatic enzymes seem to be produced by various bacteria, as
well, as molds and streptothrices. Members of the subtilis group and B.
fluorescens seem to be able to transform starch into sugar without difficulty.
It needs hardly be added here that the vast quantities of organic acids
and of carbon dioxide thus formed must play an important role in the
breaking down of the mineral constituents in the soil.

FATS AND WAXES.

ORIGIN AND DECOMPOSITION. Plant substances contain varying
proportions of fats and waxy materials. In the dry matter of grasses and
cereal straw crude fat is usually present to the extent of 1.5 to 2.0 per cent.
In hay made from clover and other legumes the proportion of crude fat
is rather more than 2 per cent. In cereal grains it may range up to
4 or 5 per cent while in soy beans the content of crude fat is 19 per cent,
in germ oil meal 22 per cent and flax seed meal 34 per cent.

Under the influence of enzymes produced by molds, yeasts and bac-
teria the fatty acids occurring as glycerides are decomposed into glycerin
and fatty acids. The extent of fat decomposition, brought about largely
by molds in the opinion of some, is shown by numerous experiments
with peanut cake, olive press cake, cottonseed meal, almond oil, corn
meal, etc. In a number of these experiments Aspergillus niger seemed
to be particularly efficient in decomposing fats. Analogous decompo-
sition processes may occur in the soil as proved by the experiments of
Rubner.

250 MICROBIOLOGY OF SOIL.

ORGANIC ACIDS.

SOURCE. The cleavage products of proteins include large quantities
of amino-acids. The latter are still further transformed and yield a
variety of fatty acids. The carbohydrates being present in larger quan-
tities than the proteins are still more important as a source of organic
acids. Finally, the fats, gums, and higher alcohols contribute additional
quantities of the latter. Among the more simple acids, acetic, propionic,
butyric, oxalic, succinic and lactic are common. The extent of acid pro-
duction was already indicated in connection with cellulose decomposition
by the methane and hydrogen bacilli. Apart from these organisms,
organic acids are formed by nearly every important species of soil bacteria;
moreover, the tissues of dead plants and animals are not the sole source
of organic acids in the soil. According to Stoklasa conditions may occa-
sionally occur in the latter, especially when atmospheric oxygen is
excluded, that favor the excretion by plant roots of appreciable quantities
of acetic acid.

TRANSFORMATION AND ACCUMULATION. Salts of organic acids are
suitable as food for a wide range of soil bacteria. Azotobacter will
readily make use of acetates, propionates and butyrates. A number of
denitrifying bacteria will grow vigorously with citrates as the only source
of organic nutrients. The fermentation of lactates by butyric bac-
teria has been known for a long time. The decomposition of malates,
succinates, tartrates and valerates may be accomplished by various
species, and even simple compounds like formates may yield food and
energy to certain soil bacteria, among them B. methylicus studied by
Loew and his associates. It is evident, therefore, that organic acids are
not liable to accumulate in well ventilated soils. Molds, as well as bacteria,
destroy them rapidly, and carbonates, carbon dioxide and water are the
final products of the decomposition of non-nitrogenous organic matter.

Notwithstanding the ready decomposition of the more simple organic
acids in the soil, it is well known that arable soils are frequently acid.
This acidity is largely due to the so-called "humic acids," organic com-
pounds whose composition is not well understood. They are composed,
to some extent, of rather complex organic acids or of their acid salts.
Continued cultivation seems to favor the accumulation of these acid
compounds, partly on account of the diminished supply of lime and of
other basic materials in older soils. When these soils are limed the

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL.

251

humic acids and acid humates are changed into neutral compounds and
are then subject to more rapid decomposition by microorganisms. Ac-
cording to the investigations of Blair the average acid soil in Florida
requires 1,500 pounds of lime (CaO) per acre to neutralize the acidity to
a depth of 84 mm. (9 in.). This means an acidity equivalent to more
than one ton of hydrochloric acid per acre. In peat and muck soils the
acidity is equivalent to many times this amount of hydrochloric acid.

PROTEIN BODIES.

AMOUNT AND QUALITY. The protein content of farm crops that
leave residues in the soil is variable, but in all cases quite considerab e.
Dried corn stalks contain 5 per cent of protein, timothy hay 6 per cent,
red clover hay 12 per cent or more, alfalfa hay 15 or 16 per cent. Even
wheat and rye straw may contain as much as 3 per cent of protein.
Cotton seed meal and other oil cakes, -tankage, ground fish, hair and
wool waste and dried blood (all used more or less extensively as sources
of nitrogen to crops) are made up in a large measure of protein compounds.

Being derived from plant residues, from microorganic, insect and
animal remains, and from fertilizers and manures applied, the nitrogen
in the soil humus exists, for the most part, in the form of protein com-
pounds. Hilgard reports the following humus and nitrogen content,
as based on the analyses of a large number of samples of humid, semi-
arid and arid soils:

(Humus)
per cent

(Nitrogen in
humus)
per cent

(Nitrogen in
soil)
per cent

Arid uplands

O. 01

I ? 21

O 12 C

Sub-irrigated arid soils

i .06

* ^o
8 ?8

Wl X O^

Oooo

Humid soils from humid and arid
regions (California)

2 . A.Z

? 20

O T 3 C

Humid soils from other states

7 OI

o *y

1 78

u - MD
o 20 z

o / u

^* ^yo

Taking the weight of an acre-foot of dry soil at 2,000,000 kg.
(4,000,000 pounds) and multiplying the nitrogen by 6.25 (the factor
usually employed for converting nitrogen into protein) we find the protein
content of these soils to range from about 11,339 kg. (25,000 pounds)

252 MICROBIOLOGY OF SOIL.

per acre to nearly three times as much. Similarly, the Illinois Ex-
periment Station reports quantities of nitrogen equivalent to 3,175 to
4,989 kg. (7,000 to 11,000 pounds) per acre to a depth of 101.6 cm.
(40 in.) in gray silt loams, of the lower Illinoisan glaciation. In the
brown silt loams the amount of nitrogen to the same depth is usually
more than 4,535 kg. (10,000 pounds) per acre; occasionally it is more
than 9,071 kg. (20,000 pounds) per acre. In one instance a black clay
loam of the late Wisconsin glaciation is reported to have about 13,154
kg. (29,000 pounds) of nitrogen per acre, to a depth of 101.6 cm. (40 in.).
This would be equivalent to more than 81,646. kg. (180,000 pounds)
of protein; of course, not all of the nitrogen in the soil exists in the form
of protein, some of it occurring as amino-compounds, and a small por-
tion as ammonia and nitrates. Nevertheless, by far the greatest part
of it occurs as protein compounds.

The protein compounds of the soil humus must be considered from the
standpoint of quality as well as from the standpoint of quantity. It is
well known that fresh plant residues are attacked more readily by micro-
organisms than older plant substances. For this reason soils frequently
supplied with fresh organic material supply greater amounts of available
food to crops than similar soils whose organic matter consists, largely
of older residues.

CARBON-NITROGEN RATIO. The decomposition of organic matter is
readily influenced by the relative content of nitrogenous and non-ni-
trogenous compounds. Substances of animal origin yield relatively and
absolutely more available nitrogen in a given length of time than sub-
stances of plant origin. The difference noted is due largely to the greater
proportion of protein in the animal materials; in other words, to the
narrower carbon-nitrogen ratio. On this basis Hilgard attempts to
explain the adequacy of the small proportion of humus in arid and semi-
arid soils. Because of the narrower carbon-nitrogen ratio the humus
compounds in these soils are decomposed with greater rapidity and yield
a sufficient amount of ammonia and nitrate to supply the needs of the
crop.

But when plant substances alone are considered the statement just
made requires qualification. It is true that cotton-seed meal or linseed
meal, having a narrower carbon-nitrogen ratio, will decay more readily
than corn-meal or wheat flour. It is also true that any given plant sub-
stance, as it undergoes decay, will lose in proportion more carbon than

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 253

nitrogen. Older humus has, therefore, a narrower carbon-nitrogen
ratio than humus of recent origin. It is likewise more resistant to decay
than new humus. Hence under any given climatic conditions and in
any given soil type, the carbon-nitrogen ratio may give important indi-
cations as to the availability of the humus nitrogen. Lawes and Gilbert,
as quoted by Hall, found the following carbon-nitrogen ratio in the
organic matter of different soils:

Cereal Roots and stubble 43

Leguminous stubble 23

Dung 1 8

Very old grass land 13 7

Manitoba prairie soils 13

Pasture recently laid down 11.7

Arable soil 10. i

Clay subsoil 6

Hall concludes, therefore, that humus with a wide carbon-nitrogen
ratio is more valuable than humus with a narrow carbon-nitrogen ratio,
since it will be attacked more easily by the soil bacteria.

THE TRANSFORMATION OF NITROGEN COMPOUNDS.

AMMONIFICATION. Experimental Study. By ammonification is meant
the production of ammonia by bacteria out of protein substances or
their cleavage products. That ammonia production in the soil is a
biological process was first demonstrated by Miintz and Coudon in 1893.
These investigators showed that no ammpnia is formed in sterile soils.
They also showed that ammonia may be produced out of nitrogenous
organic matter by molds as well as by bacteria. Marchal not only
confirmed these observations, but proved that various microogranisms
differ markedly in their ability to produce ammonia. Of the several
species of bacteria tested by him, B. mycoides (one of the common
soil bacteria) proved itself particularly efficient in the breaking down of
nitrogenous materials and the prodution of ammonia.

Since the publication of these experiments a large number of investi-
gators, both in Europe and America, have studied ammonia production
in culture solutions as well as in the soil itself. It has been shown that
under favorable conditions the breaking down of protein compounds and
the formation of ammonia may be very rapid; for instance, in some ex-
periments carried out by Lipman and his associates the following

254

MICROBIOLOGY OF SOIL.

proportions of nitrogen were transformed into ammonia in the course of

six days:

Dried blood 16. 74 per cent.

Concentrated tankage 56.66 per cent.

Ground fish 47 . 16 per cent.

Cottonseed meal 4- 95 P er cent -

Bone meal 16. 65 per cent.

Cow manure, solid and liquid excreta 32.60 per cent.

Cow manure, solid excreta ... 5-39 P er cent -

The experiments were carried out in equal quantities of soil and with
equivalent quantities of nitrogen in the different substances. It will be
observed that more than 56 per cent of the nitrogen in the concentrated
tankage was transformed into ammonia, whereas under the same condi-
tions cotton-seed meal yielded less than 5 per cent.

Mechanism of Ammonia Production. The relatively large protein
molecules are readily broken into larger or smaller fragments. This may
be accomplished by purely chemical means, as, for instance, by boiling
with acids or alkalies, or by biological activities. Among the first
cleavage products albumoses and peptones are quite prominent. These
in turn undergo further cleavage and the various amino-acids and their
derivatives, as well as ammonia, make their appearance. In so far as the
different species of bacteria are concerned, ammonia production seems
to depend, to a marked extent, on the ability to secrete proteolytic
enzymes. With the aid of such enzymes the proteins are more readily
hydrolyzed and further changed into amino- and hydroxy acids and
ammonia and carbon dioxide.

Influence of Soil and Climatic Conditions. Ammonia production in the
soil is affected by (a) its mechanical and chemical composition; by (b)
the amount and distribution of rainfall; by (c) the prevailing temperatures;
by (d) fertilizer treatment; and by (e) methods of tillage and cropping.
The mechanical composition of the soil influences the proportion of
aerobic and anaerobic species, while the chemical composition, particularly
that of the humus, influences the rate of multiplication and the character
of the chemical transformation accomplished. It is well known, for
example, that additions of fresh organic matter intensify the rate of de-
composition of the soil humus, and, likewise, ammonia production as
was already demonstrated by Breal. In a more general way it was proved
by Lipman and [his associates that, with a constant bacterial factor,

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL.

255

ammonia production in soils varies with the chemical and mechanical
composition of the latter. In some of these experiments 100 g.
portions of different soils were each mixed with 5 g. of dried blood,
sterilized in the autoclave, cooled and inoculated with equal quantities
of infusion from fresh soil. The following amounts of ammonia nitro-
gen were produced in six days:

Soil

Ammonia nitrogen found

A

T. i . 62 mg.

B

68. 20 mg.

c

117.06 mg.

D

107. 16 mg.

E

1^6.47 ing.

With all other factors constant, chemical and mechanical differences
in the soil used were responsible for striking variations in ammonia
production, as indicated by the figures given above.

The influence of temperature and moisture conditions is fully as
important as that of the chemical and mechanical composition of the soil.
The following data secured by Lipman may be cited in this connection
as showing the effect of moisture:

One-hundred-gram quantities of air-dry soil were each mixed with
3 g. of dried blood and varying amounts of water added. The ammonia
formed was distilled off and determined at the end of eight days.
The amounts of ammonia nitrogen found were as follows:

Water added

Ammonia nitrogen found

o c.c

4. IT, me.

I C.C

4. 13 mg.

^ c.c. .

=5.40 me.

<; c c

10.64 mg.

7 c.c

26. T.7 mg.

10 c.c . .

40. S7 mg.

12 C C

70.71 mg.

i e c.c

o^.oo mg.

256 MICROBIOLOGY OF SOIL.

It appears, therefore, that ammonia production in soils rises or falls
as the rainfall or irrigation is increased or decreased, or as the soil water
is more or less thoroughly conserved by proper methods of tillage. In the
same way, seasons of high temperature favor ammonification while
seasons of low temperatures discourage it. This point is well illustrated
by the observations of Marchal that at o to 5 only traces of ammonia
were formed in his culture solutions; that at 20 ammonia production
was quite marked, and that at 30 the maximum was reached. Moreover,
apart from the seasonal variations in any one locality, there is a wide
range in ammonia production, as we pass from the torrid to the temperate
and from the latter to the frigid zones.

Species and Numbers. Ammonia production is a function common
to most soil bacteria. Already in the earlier experiments of Marchal, 17
out of the 31 species tested were found capable of producing ammonia.
Prominent among these ammonifiers were B. mycoides, B. (Proteus) vul-
garis, B. mesentericus vulgattis, B. janthinus, and B. subtilis. Of a con-
siderable number of soil bacteria tested by Chester all but one were ob-
served to produce ammonia. In Gage 's experiments with sewage bacteria,
17 out of 20 species tested proved to be ammonifiers. Similarly, a num-
ber of species tested by the writer, among them B. coli, B. cholerae suis,
B. (Proteus) vulgaris, B. subtilis, B. tnegaterium, etc., all produced ammonia
in meat infusions. A mass of additional data, accumulated by different
investigators, furnish further proof that ammonia production is a common
function of soil bacteria.

The more prominent ammonifiers, including members of the B. subtilis
group and certain Streptothrices, are numerically important in all arable
soils. Their numbers are affected, however, by the amount and compo-
sition of the soil humus. It has been found, for instance, that additions
of straw and of strawy manure increase markedly the numbers of B.
subtilis and of other members of the group. An increase in the numbers
of certain ammonifiers is caused also by additions of lime or of green
manure. For example, in experiments carried out by Lipman and
his associates portions of fertile soil inoculated with B. mycoides were
found to contain, a month later, 2,000,000 of bacteria per g. of soil.
In similar soil portions that had also received additions of grass the num-
ber was twice as great.

Relative Efficiency of Different Species. In Marchal 's experiments
already referred to, the species employed showed marked differences in their

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 257

ability to produce ammonia out of egg albumen. The following propor-
tions of the protein nitrogen were converted into ammonia in twenty days:

B. mycoides ............ 46 per cent. B. suUilis ................ 23 per cent.

B. (Proteus') -vulgar is ... 36 per cent. B. janthinus ............... 23 per cent.

B. mesentericus vulgatus. 29 per cent. B. fluorescens putidus ...... 22 per cent.

Sarcina lutea .......... 27 per cent. B. fluorescens liquefaciens . . . 16 per cent.

Furthermore, apart from the variations from species to species, differ-
ences have been observed by Marchal and many other investigators
between one strain and another of any single species isolated from the same
or different soils. It must be remembered, therefore, that in the study
of ammonincation in soils and culture solutions, due consideration should
be given to differences in physiological efficiency as they are manifested by
strains and species of microorganisms.

Apart from the ammonifying bacteria already mentioned there is a
group of organisms studied by Miiller, Pasteur, van Tieghem, Leube,
Miquel, Beyerinck and others. These are the so-called urea bacteria,
capable of intensive transformation of urea and allied compounds into
ammonium carbonate, by means of the enzyme urease.

NH

2 H 2 O=(NH 4 ) 2 CO 3

I
NH 2

Morphologically these organisms include spherical and rod forms,
spore-bearing and non-spore bearing species. Most of the urea bacteria
are particularly prominent in the transformation of animal manures.

NITRIFICATION. Experimental Study. The term nitrification refers
to the oxidation either of ammonia or of nitrites to nitrates. In a broader
sense nitrification may be defined as the production of nitrates from
decomposing organic matter. Saltpeter or niter, the terms formerly
applied to potassium nitrate, possessed, for a long time, a peculiar interest
because of its relation to gunpowder. Whether it be true or not that gun-
powder was known to the Chinese before the beginning of the present era,
there is no doubt that for several centuries it played an important part
in the political and economic history of Europe. The large quantities of
gunpowder consumed in the almost incessant wars created a steady
demand for saltpeter that was not readily met by the saltpeter refiners of
India, Hungary and Poland. European nations, particularly France,

258 MICROBIOLOGY OF SOIL.

were therefore thrown on their own resources and were forced to develop
the domestic production of saltpeter. The industry came under govern-
ment control and experts were appointed to study the so-called saltpeter
plantations and the conditions affecting the appearance and increase of
nitrates in compost heaps and in the soil. Much knowledge was thus
gained about nitrification even though it was not suspected that living
organisms were concerned in the process.

With the rapid development of chemistry in the latter half of the
eighteenth century a nearer approach was made to the understanding of
the true character of nitrification. The observations of Cavendish in
1784 that potassium nitrate is formed when electric sparks are passed
through air confined over a solution of potassium hydrate formed the
starting-point for various theories that attempted to account for nitrate
formation on the basis of purely chemical reactions. The formation of
nitric acid and of its salts was thus assumed to be due to electric dis-
charges in the atmosphere, to combustion processes in nature, or to the
oxidation of organic matter and of calcium, magnesium iron and man-
ganese compounds in the soil. Much credence was given to the latter
explanation because of the almost universal occurrence of nitrates in
arable soils.

The first indication that nitrate production in the soil and in decaying
organic matter is due to biological activities was first given by Pasteur
in 1862. A few years later Miiller expressed his belief in the biological
origin of nitrates and nitrites in sewage and drinking water. It was
not, however, until 1877 that the true character of nitrification was made
clear. In that year Schloesing and Miintz demonstrated that dilute
solutions of ammonia could be changed into nitrate by being passed
slowly through long tubes filled with soil. The amounts of nitrate
nitrogen found in the leachings corresponded almost exactly to the
amount of ammonia nitrogen used up. When the soil in the tubes was
first sterilized by heating or by means of chloroform and other germi-
cides, the ammonia passed through unchanged. But when soils ster-
ilized by heat or chloroform were reinfected with small quantities of
fresh soils nitrification again proceeded in a normal manner.

The biological nature of nitrification having been thus established
numerous investigators tried to isolate the specific organisms in pure
culture. A large amount of work in this direction was done by Schloesing
and Miintz, Celli and Marino-Zuco, Munro, Warington, the Franklands

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 259

and many others. A large number of bacteria, yeasts and molds were
tested with negative results. Warington, who gathered a great mass
of valuable information about nitrification, almost succeeded in securing
pure cultures of nitrifying bacteria. Finally, Winogradski showed in
1890 not only that nitrification is caused by specific bacteria, but explained
also why the others failed in securing pure cultures. He proved that
these organisms do not develop colonies on the ordinary gelatin and
other organic media, a fact whose recognition was largely responsible
for his successful solution of the problem. The medium subsequently
employed by him consisted of silicate jelly properly supplied with inorganic
nutrient salts. After him other investigators proved that agar, deprived
of its soluble organic matter, gypsum and sandstone disks, filter-paper
pads, etc., could be used effectively as solid media.

Nitrous and Nitric Bacteria. Winogradski's investigations led to the
conclusion, foreshadowed by the earlier work of the Franklands and
Warington, that the oxidation of ammonia proceeds in two stages, viz.,

(i) 2 NH 3 +3O 2 =2HNO 2 +2H 2 O
( 2 )2HNO 2 +O 2 =2HN0 3

The organisms oxidizing ammonia to nitrites, and designated as
nitrous or nitrite bacteria, were called by Winogradski Nitrosomonas
and Nitrosococcus. The former include species or varieties isolated
from soils in Europe, Asia and Africa, and the latter those isolated from
soils in America and Australia. The organisms oxidizing nitrites to
nitrates and known as nitric or nitrate bacteria, were included by Wino-
gradski in the genus Nitrobacter.

Apart from these bacteria there is an organism, according to Kaserer,
that can oxidize ammonia directly to nitrate. He named it B. nitrator.
The reaction is illustrated by the following equation:

NH 3 +H 2 CO 3 +O 2 =HNO 3 +H 2 O+CH 2 O-4i Cal.
CH 2 O+O 2 =H 2 CO 3 -f-i32 Cal.

Enough energy for the completion of the reaction is obtained by the
oxidation of the formaldehyde (CH 2 O). Beyond the preliminary
announcement of Kaserer's there are no experimental data to prove the
existence of this organism, even though other evidence of an indirect
nature may be construed to lend support to his theory. But whether it be
proved or not that ammonia may be oxidized to nitrate by a single species,

260 MICROBIOLOGY OF SOIL.

it is evident that the number of species concerned in nitrate production is
relatively small.

Relation to Environment. The conditions that affect nitrate formation
in soils may be classified under the following heads: (a) supply of
oxygen; (b) range of prevailing temperatures; (c) amount and distribution
of moisture; (d) quantity of lime and of other basic materials; (e) quantity
of soluble mineral salts; (f) character and amount of organic matter;
(g) presence of toxic substances; (h) physiological efficiency of the nitrify-
ing bacteria.

The rapid disappearance of organic matter from sandy soils is due in
large measure to their better aeration. On the other hand, the decom-
position of vegetable and animal substances in heavy, ill-ventilated soils
is materially retarded by the limited supply and very gradual renewal of
oxygen. An intimate relation exists here between air and water in that
the latter replaces the former to a more marked extent in heavy than in
light soils. The influence of both aeration and the range of moisture is
illustrated by an experiment of Lipman's in which equal quantities of soil
were kept in large boxes under different moisture conditions. At the
end of a year the following quantities of nitrate nitrogen were found:

Moisture \ .

> 6. 52 per cent 14.75 P er cent 18.62 per cent 22.05 percent 22. 12 per cent

Nitrate

nitrogen \ 697 mg. 823 mg. 720 mg. Trace Trace

found

In examining the figures recorded above, we find that moisture was the
controlling factor in the development of the nitrifying bacteria, when the
proportion of water in the soil was 6.52 per cent. As the amount of
water increased to 14.75 P er cent there was a marked increase in the
amount of nitrate produced. Beyond that, however, the further in-
crease in the amount of water began to limit the supply of oxygen, and
the production of nitrate nitrogen with 18.62 per cent of water in the
soil was somewhat decreased. A still further addition of water up to
22.05 P er cen t l e d> practically, to saturation, and the encouragement of
reduction rather than oxidation processes. Hence, no nitrate was al-
lowed to accumulate in the soil. The data in question thus help to
explain why care was taken, on saltpeter plantations, to keep the
compost heaps moist, yet not too wet.

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 261

The influence of temperature on nitrate formation has been observed
by many investigators. Already Schloesing and Miintz recorded that
at 5 nitrification is quite feeble, at 12 marked and at 37 at its best.
Other investigators have obtained substantially the same results, except
that the optimum has been found to be considerably lower, often between
25 and 30. Under field conditions nitrification seems to take place at
relatively low temperatures, as is indicated by the rapid oxidation of
ammonium salts in the Rothamsted experiments in England; and the rapid
decay and nitrification of clover and of other legume residues in the
experiments at the New Jersey Experiment Station. These facts have,
therefore, an important bearing on the nitrogen feeding of crops in tropical,
subtropical and temperate zones.

The influence of lime and of other basic substances including the
carbonates of magnesium, potassium and sodium, and of the oxides of
iron is of far-reaching importance in all nitrification processes. It is
well known that applications of magnesian and non-magnesian lime,
marl or wood ashes promote nitrification in the soil and in compost
heaps, a fact that was well recognized by the ancient niter refiners. The
favorable action of lime is readily explained by its ability to neutralise
organic and mineral acids and to render, thereby, the soil reaction
favorable for the rapid growth of ammonifying, as well as of nitrifying
bacteria. Furthermore, the reserve of basic material serves to neutralize
the acid formed by the bacteria and prevents thus the accumulation of an
undue amount of acidity.

The role of certain mineral salts in promoting nitrification is quite
significant. Small amounts of sodium chloride have been found to favor
nitrification in the experiments of Pichard and also those of Lipman.
The former showed also that sulphates not only promote nitrification,
but that different sulphates display marked variations in this respect.
In the same manner nitrate formation was shown to be favorably affected
by phosphates in bone meal, Thomas slag, and acid phosphates. Gener-
ally speaking, therefore, nitrifying bacteria are stimulated in their de-
velopment by a proper supply of available mineral nutrients.

The exact relation of organic matter in the soil to the activities of
nitrifying bacteria is but beginning to be properly understood. Earlier
observations made it manifest that heavy applications of animal manures,
or of green manure may not only retard nitrification, but may actually
cause the disappearance of a part or of all of the nitrate in the soil. Sub-

262 MICROBIOLOGY OF SOIL.

sequent experiments by Winogradski and by Winogradski and Ome-
lianski showed that in pure cultures the presence of even slight amounts
of soluble organic matter may depress or even suppress the development
of the nitrifying bacteria. It was, therefore, concluded by these authors
that relatively small amounts of soluble organic matter may inhibit
nitrification. These conclusions, based on the study of liquid cultures
only, were given a very broad application by many writers on agricultural
topics. More recent experiments make it certain, however, that in the
soil itself small amounts of soluble organic matter, e.g., dextrose, are not
only harmless, but may really stimulate nitrification. It was shown,
likewise, that humus and extracts of humus may, under suitable condi-
tions, stimulate nitrification to a very striking extent.

Certain substances in the soil may exert a toxic effect on nitrifying
bacteria. Ferrous sulphate, sulphites and sulphides may thus act in-
juriously, as may also calcium chloride and excessive concentrations of
sodium carbonate, sodium bicarbonate, sodium chloride, magnesium
sulphate, etc. Injury by ferrous compounds, as well as by organic acids,
is not uncommon in low-lying fields and bogs; while injury from excessive
concentration of soluble salts may occur in the so-called alkali lands.

Finally nitrification in the soil should be considered from the stand-
point of the organisms themselves. There is no doubt that continued
growth under extremely favorable conditions leads to the development in
the soil of nitrifying bacteria, possessing a very marked physiological effici-
ency. On the other hand, in ill-aerated, sour soils the environment would
depress the physiological efficiency of the nitrifying bacteria. Differences
are thus undoubtedly established under actual field conditions, as is made
probable by the variable behavior of soils from different sources when
used as inoculating material in recently reclaimed or peat swamp lands.

Accumulation and Disappearance of Nitrates. As shown above, the
rate of formation of nitrates in the soil is dependent upon moisture,
temperature and aeration, as well as on the presence of organic matter
and basic substances. On the other hand, the accumulation of nitrates
depends, under any given conditions, largely on the character of the
growing crop. Observations on the rain gauges at Rothamsted showed
an average annual loss 14 kg. (31.4 pounds) of nitric nitrogen per acre
in the drainage water from uncropped soil. In one of King's experi-
ments, land that had been fallowed contained 137 kg. (303.24 pounds)
of nitric nitrogen per acre, to a depth of four feet. Adjoining cropped

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 263

land contained only 26 kg. (57.56 pounds) of nitric nitrogen per acre
to the same depth. Stewart and Greaves found in limestone soil in
Utah 64 kg. (142 pounds) of nitric nitrogen per acre, under corn;
98 pounds under potatoes, and only 12 kg. (27 pounds) under alfalfa.
Under the same conditions fallow land contained 74 kg. (165 pounds)
of nitric nitrogen per acre. The smaller amount of nitric nitrogen found
under alfalfa bears out the observations already made by a number of
other investigators that the accumulation of nitrates under legumes is
smaller than it is under non-legumes. While several explanations have
been offered to account for this fact, it is generally agreed that legumes
assimilate nitrate nitrogen more rapidly than non-legumes. Unusual
circumstances may favor, at times, the accumulation of quantities of
nitrate large enough to destroy all vegetation. It is reported, for
instance, by Headden that he has found in limited areas in Colorado as
much as 9,0718.5 kg. (100 tons) of nitrate per acre foot of soil.

The amount of nitrate nitrogen in the soil is influenced by the growing
crop not alone because of the nitrogen absorbed by the latter, but because
of the moisture relations as affected by growing plants. It is quite
apparent that a large crop dries out the soil more rapidly than a small
crop. When the soil moisture is sufficiently depleted, nitrification stops
and the further accumulation of nitrates becomes impossible, while their
disappearance is hastened by the constant demands of the crop. The
disappearance of soil nitrates is, likewise, hastened by the leaching action
of rain and by certain species of bacteria that transform them into other
nitrogen compounds.

DENITRIFICATION. Experimental Study. Denitrification may be de-
fined as the reduction of nitrates by bacteria, involving the evolution of
nitrogen gas or of nitrogen oxides. In a more general way, denitrification
has been defined as the partial or complete reduction of nitrates by bacteria.
The term direct denitrification has been suggested for complete reduction,
and indirect for the partial reduction to nitrites or ammonia. The term
denitrification should not be employed to designate losses of nitrogen gas
due to the oxidation of ammonia, or to the disappearance of nitrates
following their conversion into proteins by microorganisms.

The reduction of nitrates in the presence of fermenting organic matter
was noted by Kuhlmann as early as 1846. The same fact was recorded
many years later by Froehde and by Angus Smith. In 1868 Schoenbein
expressed the belief that nitrates may be reduced to nitrites by fungi.

264 MICROBIOLOGY OF SOIL.

For more than a decade after that, data were rapidly accumulating in
support of Schoenbein's contention, until in 1882 Gayon and Dupetit
made it certain that nitrate reduction with the evolution of nitrogen gas
may be caused by a "ferment." Finally, in 1886, the same investigators
described two organisms, B. denilrificans a., and B. denilrificans /?.,
capable of completely reducing nitrates. Subsequently the studies of
Giltay and Aberson, Burri and Stutzer, Severin, van Iterson, Jensen,
Beyerinck and of many others not only greatly increased the number of
known denitrifying bacteria, but added much to our knowledge con-
cerning the development and activities of these organisms. It has been
shown that a very large number of species can reduce nitrates to nitrites
and ammonia; moreover, a considerable number of organisms are already
known that can cause the complete destruction of nitrates with the evolu-
tion of nitrogen gas or of nitrogefi oxides. The following reactions illus-
trate diagrammatically the complete or partial reduction of nitrates.

2 HNO 3 =2HNO 2 +O 2

HN*O S +H 2 O=NH S +2O,

4 HNO 2 = 2H 2 O+ 2N 2 + 3<D 2

In the soil, manure or other culture media the denitrifying bacteria
which are, for the most part, aerobic develop also under anaerobic con-
ditions and transfer the oxygen of nitrates and nitrites to carbon com-
pounds. This is illustrated by the equations suggested by van Iterson:

5C + 4KNO 3 +2H 2 O = 4KH CO 3 +2N 2 +CO 2
3C+4KNO 2 +H 2 O= 2KH CO 3 + K 2 CO 3 + 2N 2

When nitrates are reduced to nitrites in the presence of amino-
compounds,~or even of ammonium compounds, elementary nitrogen may
escape as shown by the following reactions:

C 2 H 5 NH 2 + HNO 2 = C 2 H 5 OH+ N 2 + H 2 O
NH 4 C1+ KNO 2 = KC1+ 2H 2 O + N 2

An organism has been described by van Iterson that can decompose
nitrates in the presence of cellulose:

5C 6 H 10 O 5 + 24KNO 3 - 24KHCO 3 + 6CO 2 + i2N 2 + i3H 2 O

Still more interesting is Thlobacillus denitrificans described by
Beyerinck as capable of reducing nitrates in inorganic media. The
nitrate oxygen is used to oxidize elementary sulphur:

6 KNO 3 +5S+2CaCO 3 =3 K 2 SO 4 +2CaSO 4 +2CO 2 +3N 2

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 265

Relation to Environment. Nitrate reduction is favored by insufficient
aeration, as well as by an abundance of readily decomposable organic
matter. In fine grained, compact soils nitrate formation and nitrate
reduction may alternate, depending upon the more or less complete
replacement of soil air by water. Similarly, in soils receiving excessive
amounts of animal manure denitrifying bacteria may cause the reduction
of nitrates. In green-house soils excessive moisture, as well as excessive
amounts of organic matter, may be present and may prevent the accumu-
lation of nitrates. It has also been shown by Niklevski that, contrary to
opinions previously held, denitrification may occur in manure heaps. In
the better aerated surface portion of manure heaps conditions are favor-
able for the oxidation of ammonia to nitrites and nitrates. The nitrous
acid may combine with ammonia to form ammonium nitrite, the latter
decomposing, spontaneously, into water and nitrogen gas. It is very
likely, also, that the nitrites and nitrates are reduced by the denitrifying
bacteria in manure. On the other hand, in manure kept moist under the
feet of cattle nitrite and nitrate formation is prevented and losses by
denitrification are not likely to occur.

The economic significance of denitrification was overestimated at one
time, on account, largely, of the assertion of Wagner in 1895 that in all
soils receiving applications of horse manure, the nitrates in the soil itself
as well as those added in commercial fertilizers are almost certain to be
destroyed by denitrification. Subsequent experiments by many investi-
gators demonstrated that under field conditions, denitrification is a factor
of slight moment; however, in the green-house, in the manure heap (under
certain conditions) and in market gardening where manure is used at the
rate of 45,359 kg. to 54,431 kg. (50 to 60 tons) per acre, the danger
of denitrification is real.

ANALYTICAL AND SYNTHETICAL REACTIONS.

AMOUNT OF BACTERIAL SUBSTANCE IN THE SOIL. Various decom-
position processes in the organic matter of the soil may be designated as
analytical in that protein, carbohydrates and fats are split into more
simple compounds. At the same time, the microorganisms concerned
in the decomposition processes multiply very rapidly and fashion the
complex compounds of their cell-substance out of the simple cleavage
products in their medium. In other words, analytical and synthetical
reactions proceed hand in hand in the soil.

266 MICROBIOLOGY OF SOIL.

While it is not definitely known how large a proportion of the soil
humus consists of the dead and living cells of microorganisms there
is a mass of indirect evidence to show that these cells form a very con-
siderable proportion of the total quantity of organic substances in the
soil. For instance, it has been demonstrated that a large proportion of
the dry matter of solid animal faeces may consist of bacterial cells. At
various times and by different investigators the proportion of bacterial
substance has been estimated at from 5 to 20 per cent or more of the
total dry weight of fasces. A heavy application of barnyard manure
may introduce, therefore, several hundred pounds of bacterial cells per
acre of soil. Moreover, because of the extensive changes in the soil
humus itself, as is evidenced by the rapid formation of nitrates, large
masses of bacterial substance are constantly being formed and disin-
tegrated.

AVAILABILITY OF BACTERIAL MATTER. Substances of microorganic
origin are decomposed more or less rapidly, according to their composition.
The extent of transformation under favorable conditions is indicated by
an experiment performed by Beyerinck and van Delden, in which 50
per cent of the nitrogen in Azotobacter cells was transformed into nitrate
in seven weeks. On the other hand, the humus of peat and muck soils,
or that of worn-out soils, may contain microorganic residues of so inert
a character as to yield but little available nitrogen to crops.

TRANSFORMATION OF PEPTONE, AMMONIA AND NITRATE NITROGEN.
The cleavage of protein compounds into peptones, amino-acids and
ammonia, and the oxidation of the latter into nitrites and nitrates, may
be properly included among analytical reactions. It should not be
forgotten, however, that in the accompanying synthetical reactions the
compounds just mentioned may be transformed back into complex
proteins. It happens, thus, that large quantities of the available nitrogen
compounds may be withdrawn from circulation by microorganisms that
use these as building material. Under extreme conditions microorgan-
isms may become serious competitors of higher plants for available
nitrogen food.

Manure stored in heaps not infrequently deteriorates in quality,
even when losses by leaching are excluded This deterioration is largely
due to the change of the water-soluble ammonia and amino-compounds
into insoluble protein substances. While the extent of the change into
protein compounds is variable it may range from less than a tenth of the

DECOMPOSITION OF ORGANIC MATTER IN THE SOIL. 267

water soluble material to more than three-quarters or four-fifths of it.
Also in the soil the same processes take place, but not as intensively. A
large number of species of molds and bacteria have been isolated and
tested as to their ability to transform ammonia, amino- and nitrate
nitrogen into protein compounds. Among the more recent investi-
gations in this field those of Lemmermann and his associates report the
change in three weeks of 5 to 6 per cent of the nitrate added to the soil
into protein. In the presence of barnyard manure the proportion trans-
formed was increased to 15 per cent. In the case of ammonium com-
pounds the transformation may be even more far-reaching, amounting,
at times, to more than 25 to 30 per cent of the material originally present.
Generally speaking, molds will assimilate ammonia nitrogen more readily
while bacteria and algae will assimilate nitrate nitrogen by preference.
However, the preference of molds for ammonia nitrogen is often more
apparent than real, because of the rapid formation of acid residues in
culture media rich in certain ammonium compounds. Similarly, some
species of bacteria will assimilate ammonia nitrogen in preference to
nitrate nitrogen.

CHAPTER III.
FIXATION OF ATMOSPHERIC NITROGEN.

THE SOURCE OF NITROGEN IN SOILS.

EARLY THEORIES. When chemistry had made sufficient progress
to allow the analysis of soils and plants it was recognized that nitrogen is
always present in both. It was also recognized that the soil nitrogen is
almost wholly confined to the surface portion and is evidently of atmos-
pheric origin, since the unweathered, underlying rock is devoid of this
constituent. The vast accumulations of nitrogen, known to exist in all
arable soils, were ascribed, therefore, to the residues of many generations
of plants; and the assumption seemed to be justified that the atmosphere,
79 per cent of whose bulk consists of nitrogen gas, is the direct source of
this element to plants. It was not long, however, before plant physi-
ologists demonstrated experimentally that nitrogen gas as such could
not directly serve as food for plants. There thus arose one of the most
interesting and, for a long time, one of the most puzzling problems in
agricultural research. Among the earlier investigators de Saussure
believed, at the beginning of the nineteenth century, that nitrogen is
taken up from the soil in combined form. Liebig in 1840 advanced his
well-known "mineral theory" according to which plants secured their
nitrogen from the air, in the form of ammonia. He assumed, thus, that
plants cannot use elementary nitrogen, and that the supply of atmospheric
nitrogen in the form of ammonia was great enough to meet the needs of
growing vegetation. The latter view was not accepted by Lawes and
Gilbert of the Rothamsted Station in England. By a series of elaborate
and carefully controlled experiments they demonstrated in 1858 that
nitrogen in the elementary form cannot be used by plants. They further
demonstrated that the amount of combined nitrogen brought down in
the form of ammonia, nitrites and nitrates, by atmospheric precipitation
was but slight when compared with the quantities annually removed by
crops. Hence the problem as to the source and maintenance of com-
bined nitrogen in the soil seemed to be more puzzling than ever.

268

FIXATION OF ATMOSPHERIC NITROGEN. 269

CHEMICAL AND BIOLOGICAL RELATIONS. The second and third
quarters of the nineteenth century saw the birth of a number of theories
dealing with this problem. It was suggested that nitrogen compounds
may be formed in the soil by the oxidation of nitrogen to nitric acid. Com-
pounds of iron, manganese and lime were supposed in some way to make
such oxidation changes possible. It was likewise suggested that nascent
hydrogen may be generated in the decomposition of organic matter in the
soil, and reacting with elementary nitrogen, may give rise to ammonia.
The various hypotheses were not supported by experimental proof;
moreover, the situation was complicated by the knowledge, based on
empirical observations, that crops of the legume family seemed to be
more or less independent of the supply of combined nitrogen in the soil.
Indeed, clovers and other legumes had, apparently, the ability to increase
the content of combined nitrogen in the soil as was indicated by the ex-
periments of Boussingault and of Lawes and Gilbert. Finally, the
mystery was solved by the investigations of Berthelot and of Hellriegel
and Wilfarth who furnished the proof that elementary nitrogen may be
utilized by plants when certain biological relations are met. These
relations involve the presence and activities of microorganisms that by
themselves, or in conjunction with higher plants, make available to grow-
ing vegetation the great store of atmospheric nitrogen.

NON-SYMBIOTIC FIXATION OF NITROGEN.

HISTORICAL. Non-symbiotic nitrogen fixation, or Azofication, has
already been defined as the production of nitrogen compounds out of
atmospheric nitrogen by bacteria independently of higher plants. The
part played by bacteria in this process was not recognized until 1885,
when Berthelot published some of his data on the accumulation of com-
bined nitrogen in uncropped soils. His results seemed to explain a
number of scattered observations, made since the middle of the century,
on the apparent increase of the nitrogen content of cultivated soils.

While Berthelot's experiments proved that the nitrogen gains occurred
only in unsterilized soils and were, therefore, due to microorganisms,
it remained for Winogradski to demonstrate, in 1893, that the formation
of nitrogen compounds by certain types of bacteria may be accomplished
in culture media nearly or quite devoid of combined nitrogen. Soon
after that he succeeded in isolating his organisms in pure culture, and

270 MICROBIOLOGY OF SOIL.

described them as anaerobic bacilli allied to those of the butyric
group. In 1901 our knowledge of Azo-bacteria was enriched by Bey-
erinck's discovery of a group of large, obligate aerobic bacteria that he
designated as Azotobacter. Since that date it has been found that the abil-
ity to fix atmospheric nitrogen is possessed also by certain molds and by
various species of bacteria. However, this ability is not only extremely vari-
able, but is also very feeble as compared with that of the members of the
two groups described by Winogradski and Beyerinck. These two groups
may, therefore, be designated as including the nitrogen fixing bacteria
par excellence.

ANAEROBIC SPECIES. The species isolated by Winogradski was named
by him B. (Clostridium) pasteurianus (Fig. 66). It was found to grow

FIG. 66. B. (Clostridium) pasteurianus, a non-symbiotic nitrogen-fixing organism.

(After Winogradski from Lipman.)

readily under anaerobic conditions in culture solutions containing dextrose
and the necessary mineral salts, but no combined nitrogen. The products
of growth included protein, butyric and acetic acids, carbon dioxide and
hydrogen. In the presence of other bacteria B. (Clostridiwii) -pasteur-
ianus was found to develop also under aerobic conditions. Subsequently
studies by Winogradski and other investigators showed that.5. (Clostridium)
pasteurianus, and varieties of this species are very widely distributed in
cultivated soils. More recently Bredeman made a thorough and extended
study of anaerobic Azo-bacteria and demonstrated their almost invariable
presence in a large number of soil samples from Europe, Asia and America.
In his opinion they correspond more or less closely to B. amylobacter
described many years before by van Tieghem.

AEROBIC SPECIES. A more or less pronounced power to fix atmos-
pheric nitrogen is apparently possessed by a considerable number of
aerobic species. Lipman has demonstrated the fixation of small amounts

FIXATION OF ATMOSPHERIC NITROGEN. 271

of nitrogen by Ps. pyocyanea and Lohnis secured similar results with
Bad. pneumonia, B. lactis viscosum, B. radiobacter and B. prodigiosus.
Gottheil has detected fixation by B. ruminatus and B. simplex; Pill ai has
described a nitrogen fixing aerobic bacillus, B. malabarensis; Westermann
studied a similar organism that he named B. danicus; while Beyerinck
and van Delden observed, some years earlier, that certain strains of
B. mesentericus could fix relatively large amounts of nitrogen. Similarly
Ps. radicicola has been found to possess a slight, but nevertheless an
appreciable power to fix elementary nitrogen in culture solutions or in
the soil.

FIG. 67. Azutobacter vinelandi, a non-symbiotic nitrogen-fixing organism. (After

Lipman.)

But while nitrogen fixation among aerobic soil bacteria is not as un-
common as was at one time supposed, this function is so feeble and so
variable in most instances, as to be of negative, or, at best, of doubtful
economic significance. On the other hand, the aerobic Azotobacter, first
described by Beyerinck in 1901, may be regarded not only as possessing
a very pronounced ability to fix atmospheric nitrogen, but as playing a
role of some moment in maintaining the supply of combined nitrogen in
the soil.

To the two species of Azotobacter, A . chroococcum and A . agilis described
by Beyerinck and van Delden, Lipman added A. vinelandii, (Fig. 67) A.
beyerincki and A. woodsfownii, and Lohnis and Westermann, A. vitreum.

272 MICROBIOLOGY OF SOIL.

Of these species A. chroococcum and A. beyerincki are most common and
are widely distributed in cultivated soils of Europe and America, and prob-
ably also of the other continents. They are absent in acid soils deficient in
humus, and most common in limestone regions and in irrigated soils rich
in mineral salts. Their food requirements are covered by solutions con-
taining potassium phosphate, magnesium sulphate, calcium chloride
and ferric sulphate, and some organic nutrient, such as dextrose, sac-
charose, xylose, mannit, acetate, propionate, butyrate, malate, ethyl
alcohol, etc. An alkaline or neutral reaction and the presence of salts
of iron are essential for the vigorous development of Azotobacter, while
humates have been shown by Krzemieniewski to exert a stimulating
influence on the growth of these organisms, even though not acting directly
as a source of food and energy. As shown by Lipman and others, Azoto-
bacter may gain an increased power of fixing atmospheric nitrogen in the
presence of other organisms. It is resistant to drying, notwithstanding
the fact that it produces no spores, and has been successfully isolated
from soil samples that had been kept in a dry state for several years.
For some reason it may be detected in the soil most readily in the fall and
winter months.

ENERGY RELATIONS. In the fixation of nitrogen by bacteria the
necessary energy for the process is furnished by the carbohydrates,
organic acids, alcohols or other organic nutrients employed in the culture
media. Since any given quantity of organic nutrient possesses a definite
amount of potential energy the fixation of nitrogen is necessarily limited
by the supply of such potential energy. This limitation was already
recognized by Winogradski in his experiments with B. (Clostridium]
pasteuriamis. For every gram of dextrose used up there was produced,
on the average, 2 to 3 mg. of combined nitrogen. In the experiments of
Bredeman with B. amylobacter , and of Pringsheim with "Clostridhtm
americanum" the amounts fixed were, at times, considerably larger. On
the whole, however, it has been proved by a number of investigators that
A zotobacter can fix much larger quantities of nitrogen than the anaerobic
bacilli. The extended investigations of Lipman showed that A. vine-
landii has the ability to fix more nitrogen per unit of organic nutrient
consumed than either A. chroococcum or A. beyerincki. Under favorable
conditions A. mnelandii may at times fix 15 or even 20 mg. of nitrogen
per gram of mannit used up. Krzemieniewski found in experiments
with A. chroococcum that additions of humates to the culture solutions

FIXATION OF ATMOSPHERIC NITROGEN. 273

increased the nitrogen fixed from a maximum of 2.4 mg. to a maximum
of 14.9 mg.

The practical bearing of the foregoing data lies in the fact that the
fixation of nitrogen in cultivated soils is limited, among other things, by
the energy available, that is, by the quantity of readily decomposable
organic residues. An indication as to the extent of these is given by the
amount of humus present; nevertheless, this must remain an indication
merely, for most of the humus is too inert to serve as a source of energy
to Azotobacter. From the data at present available different investigators
have estimated the quantity of nitrogen fixed by Azotobacter at 6.8 kg. to
1 8 kg. (15 to 40 pounds) per acre, per annum. Assuming favorable
conditions for fixation, so that one pound of nitrogen could be fixed for
every one hundred pounds of carbohydrate consumed, it would still take
an equivalent of 680 kg. to 1,814 kg. (1500 to 4000 pounds) of sugar
to produce this quantity of combined nitrogen. It may be noted in
this connection that Azotobacter have been demonstrated to live in sym-
biosis with algae, obtaining thereby the necessary energy for their activi-
ties. This may explain, perhaps, the remarkable facts observed by
Headden in Colorado, relating to the accumulation of such enormous
quantities of nitrate in the soil, as to destroy all vegetation. In some
instances the nitrates were found to be present to the extent of 90718 kg.
(100 tons), or more (per acre), to a depth of a few inches. If the accumu-
lation of combined nitrogen was due to Azotobacter, as is claimed by
Headden, and the bacterial residues oxidized by nitrifying bacteria to
nitrates, it is difficult to account for the source of the 1,000 or 2,000 tons of
carbohydrates necessarily used up in the process of fixation, unless
it could be proved that the energy was furnished by algae.

SYMBIOTIC FIXATION.

HISTORICAL. Empirical observations extending well back into
ancient agriculture have led to the recognition of the soil-enriching qual-
ities of certain crops of the legume family. Columella mentions the fact
that many Roman farmers regarded beans as possessing these qualities,
but does not accept this belief for himself. On the other hand, he points
out that luzerne (alfalfa) , lupins and vetches improve the land and act as
manure. He points out, also, that it was the practice of Roman farmers
to plow under lupines in order to enrich the soil. In the centuries follow-
18

274 MICROBIOLOGY OF SOIL.

ing the fall of Rome the use of legumes for soil improvement persisted to
some extent in Italy, France and other countries; yet the practice was
not followed consistently and the fertility of European soils was declining
for lack of available nitrogen, and, to a large extent, also of phosphoric
acid. The more general introduction of clover into Germany and
England in the eighteenth century helped to restore the fertility of many
farms, and led, ultimately, to the recognition of the peculiar place held
by legumes in the maintenance of soil fertility. But while practical
farmers knew of the soil-enriching power of legumes, and while they
retained their belief in it even when it seemed contrary to scientific
authority, they did not know the secret of this power. It remained for
Hellriegel and Wilfarth to demonstrate in 1886, and more fully in 1888,
that this power, already hinted at by the investigations of others, is the
resultant of the combined activities of the plants and of bacteria that
enter their roots, and produce there the well known nodules or tubercles.
They showed in no uncertain manner that legumes can improve the soil
only in so far as they add nitrogen to it with the aid of the bacteria in the
tubercles; in other words, legumes were shown to enter into a symbiotic
relationship with certain bacteria and to acquire, thereby, the ability to
fix atmospheric nitrogen.

The presence of tubercles on the roots of leguminous plants was first
recorded by Malpighi in 1687. He regarded them as root galls. The
botanists who studied them in the first half of the nineteenth century clas-
sified them as modifications of normal roots or as pathological processes.
In 1866 the Russian botanist Woronin found that the tubercles were filled
with minute bodies resembling bacteria and concluded that they were
pathological outgrowths. Some years later Frank not only showed that
tubercles are almost invariably present on the roots of legumes, but
that their formation may be prevented by sterilizing the soil. Frank
was thus in possession of facts that might have revealed to him the true
nature of the root-tubercles. However, he later modified his belief in
the origin of tubercles as due to outside infection, and accepted the
interpretation of 'his pupil Brunchhorst who claimed that the bac-
teria-like bodies in the tubercles were merely reserve food materials.
Because of their resemblance to bacteria Brunchhorst named them
bacteroids.

The studies of Marshall Ward, published in 1887, proved not merely
that tubercle formation is due to outside infection, but that such infection

FIXATION OF ATMOSPHERIC NITROGEN. 275

may be brought about at will by placing the roots of young plants in con-
tact with pieces of old tubercles. Hellriegel in his preliminary communi-
cation of 1886 also showed that outside infection is necessary for the
production of tubercles, and called attention to the true function of the
latter as laboratories wherein nitrogen compounds are manufactured
out of elementary nitrogen. The true worth of HellriegePs investigations
was brought out more clearly in another paper that he published jointly
with Wilfarth in 1888. The authors showed that in sterilized soils
legumes behaved precisely like non-legumes, and died ultimately of
nitrogen hunger when not provided with nitrates or other suitable nitrogen

FIG. 68. Ps. radicicola. i, From Melilotus alba; 2 and 3, from Medicago saliva; 4,
from Vicia v illosa. (After Harrison and Barlow from Lipman.)

compounds. On the other hand, when the sterilized soil was later
infected with a few drops of leachings from fresh soil that had supported
a normal growth of legumes, the starving plants recovered and grew
vigorously. Under the same conditions non-legumes did not recover.
The recovery of the starving legumes was found to be coincident with
the formation of tubercles.

Hellriegel and Wilfarth's studies were soon confirmed by the inves-
tigations of others. Wigand showed in 1887 that the tubercles contained
within them true bacteria. In the following year Beyerinck reported the
successful isolation of these bacteria on artificial media, and named the
organism B. radicicola (Fig. 68). Prazmowski also isolated pure cultures
of B. radicicola, and followed the entrance of the organisms into the root

276

MICROBIOLOGY OF SOIL.

hairs of young plants, their passage through the cell-walls, and their
transformation into bacteroids. These facts were all confirmed by
other investigators, and it was further shown by Schloesing and Laurent
that properly inoculated legumes not only can grow in soils devoid of
combined nitrogen, but that when growing in such soils in a confined
atmosphere they decrease the quantity of nitrogen gas surrounding them
by transforming it into nitrogen compounds. It was, therefore, made
clear by these investigations, and by others not mentioned here for lack
of space, that the belief of practical farmers in the soil enriching qualities
of legumes was amply justified. It was shown, further, that the later
experiments of Boussingault, as well as those of Lawes, Gilbert and
Pugh failed to solve the problem because these investigators treated

FIG. 69.- Sections through root tubercles, i, Cell from tubercle of Pisum sativum,
showing bacterial filament; 2 and 3, cells with bacterial filaments from tubercle of
Trifolium pannonicum. (After Stefan from Lipman.)

their soil so as to prevent the survival and subsequent entrance of B.
radicicola, and deprived the leguminous plants of the ability to utilize
atmospheric nitrogen.

MODES OF ENTRANCE AND DEVELOPMENT. Tubercle bacteria con-
sisting of small motile rods usually enter the legumes by way of the root-
hairs. For this reason young tubercles, with but few exceptions, are
found on young roots. The organisms multiply at the point of infection
and penetrate into adjacent plant-tissue by means of a hypha-like hollow
thread or tube that seems to consist of a gelatinous material (Fig. 69).
The tubes branch out as they pass from cell to cell and carry the invading
organisms with them. The bacteria which may be readily detected
within the tubes and cells are the involution forms of Ps. radicicola and
assume various irregular shapes. They are designated as bacteroids.

FIXATION OF ATMOSPHERIC NITROGEN. 277

Stefan has suggested that bacteroids may be produced within the tubes
and, possibly, from the buds or swellings that appear on the tubes. While
still young, the bacteroids are capable of dividing, but as they grow they
swell up -and finally degenerate.

RESISTANCE, IMMUNITY AND PHYSIOLOGICAL EFFICIENCY. The
invasion of legumes by Ps. radicicola and the acquisition by the plant,
thanks to this invasion, of the power to fix elementary nitrogen are cited
as a case of symbiosis. However, some writers would regard the pres-
ence of Ps. radicicola in legume roots as a case of parasitism. According
to them symbiosis presupposes the living together of two organisms with
resulting benefit to both. In the present instance, however, conditions
may arise when the host plant is injured, rather than benefited; and
similarly, conditions may arise when the invading bacteria are suppressed
by the plants. Making due allowance for the objections raised we still
find, nevertheless, that in the broad relation of the two groups of organ-
isms there is an apparent benefit to both plants and bacteria. The
former gain an adequate supply of nitrogen and the latter a supply of
carbohydrates and of mineral salts.

A more detailed study of this relation shows that the plants resist
the entrance of bacteria. When an abundance of available nitrogen
compounds is supplied tubercle formation may be largely or wholly
suppressed. In that case the plants secure their nitrogen from the soil
and are not only independent of the bacteria, but are strong enough to
resist their entrance. It is further claimed by Hiltner that tubercle
bacteria differ in their virulence, and that the more virulent the organ-
isms, the more readily will they penetrate the root tissue. Moreover,
he believes that when a plant is invaded by organsms of any degree of
virulence, the host plant becomes immune to a large extent and can keep
out all but the most virulent bacteria. The use of the term virulence, in
this connection, has been objected to, since it is borrowed from animal
pathology and is likely to be misleading. It is better to employ the term
physiological efficiency as implying not only a more pronounced ability
to enter the plant roots, but also to fix atmospheric nitrogen. It is con-
ceivable that strains of Ps. radicicola may be developed that would grow
rapidly and yet possess but a feeble nitrogen-fixing power. In other
words, they would possess a high vegetative power and a low physiological
efficiency.

MECHANISM OF FIXATION. It is generally believed that the fixation

278 MICROBIOLOGY OF SOIL.

of nitrogen is accomplished by the bacteria within the tubercles. The
claim, at one time, advanced by Stoklasa, that the fixation is accomplished
by the plants themselves with the aid of enzymes produced by the bacteria
in their roots, has been disproved. It is known that the period of active
nitrogen assimilation by the plants coincides with the appearance of the
bacteroids in the tubercles, and it is supposed that the microorganisms
fashion nitrogen compounds out of atmospheric nitrogen by using the
carbohydrates and organic acids in the plant juices as a source of energy.
The plants then seem to utilize the soluble nitrogen compounds that pass
out of the bacterial cells. It is further supposed that bacteroid formation
is an attempt on the part of the microorganisms to adjust themselves to
the drain caused by the activities of the host plant.

VARIATIONS AND SPECIALIZATION. Apparent differences in bacteria
from different legumes were noted by Hellriegel. Some of his experi-
ments indicated that bacteria from clovers could not produce tubercles
on lupines and serradella. Analogous differences were found by
Nobbe and his associates, nevertheless they were finally led to conclude
that the root invasion of legumes is caused by a single species. However,
continued association with any particular legume accomplished in the
end a certain modification, or specialization, as it were, of the micro-
organisms, and they were then no longer able to invade the roots of other
legume genera. Latterly, Hiltner and Stormer have been led to modify
this view and have arranged the tubercle bacteria in two groups, possess-
ing, according to them, well defined morphological and physiological
differences. One of these groups is included under the species "Rhizobium
radicicola" and the other under "Rhizobium beyerinckii." The former
comprises the organisms from lupines, serradella and soy beans while
the latter comprises all of the others.

RELATION TO ENVIRONMENT. Nitrogen fixation by leguminous
vegetation is readily influenced by soil conditions, particularly the supply
of lime and of other basic substances; the supply of organic matter and the
aeration of the soil. As to the first of these it is well known that all legumes,
with the exception of lupines and serradella, are stimulated in their
growth by generous applications of lime. The top dressing of lawns
with lime, marl or wood ashes encourages the appearance of white clover;
an adequate supply of lime makes possible the successful growing of
alfalfa in almost any soil, while the leguminous vegetation of limestone
soils is proverbially vigorous. The favorable influence of lime is due to the

FIXATION OF ATMOSPHERIC NITROGEN.

279

'direct action on the plants as well as on the bacteria in the soil. Similarly,
the tubercle bacteria are favorably affected in their survival and multi-
plication by an abundant supply of organic matter. On the other hand,
acid soils or those deficient in humus and inadequately aerated are but ill
suited to the activities of Ps. radicicola.

_ - o -These two pea plants were grown in clean quartz sand to which had been
added small quantities of all the necessary elements of plant food except nitrogen. The
conditions were exactly identical except that plant A was without root nodules (see
Fig. 71) and plant B had numerous nodules well developed (see Fig. 72). (Mich.
Exp. Station.)

280

MICROBIOLOGY OF SOIL.

SOIL INOCULATION.*

By soil inoculation is now understood the adoption of some artificial
method for supplying suitable quantities of nitrogen-fixing organisms to
soils deficient in these types. The first attempts at soil inoculation were
made in 1886 by Hellriegel and Wilfarth during the course of their
studies on the cause of nitrogen accumulation by legumes. They
found that when leguminous plants were grown in sterile sand, nodules

FIG. 71. Roots of Plant A without nodules. (Figs. 70).

were formed on the roots only after the addition of a small portion of
aqueous extract of fertile soil, or an extract of crushed nodules, or in
some cases (lupines and seradella) by soil itself from a field on which
these crops had been grown. The first successful artificial production
of nodules by the aid of pure cultures was made in' 1889 by Prazmowski
in the course of studies on the method of entrance of the organism to the
root hairs of the host plant.

*Prepared by S. F. Edwards.

FIXATION OF ATMOSPHERIC NITROGEN. 281

The first inoculation experiments in a large way were those made in
1887 at the Moor Soil Experiment Station, Bremen, Germany, where earth
taken from fields that had borne luxuriant crops of various legumes was
scattered over reclaimed heath or swamp soils upon which legumes had
not previously grown, with the result that in every instance the yield on the
inoculated portions of land was greater than on the uninoculated plots.
After such favorable results, it was but a natural step to try the effect

FIG. 72. Roots of plant B with nodules (.Fig. 70).

of similar applications of soil rich in the nodule-forming bacteria to
ordinary cultivated soils of varying character. While results in some cases
were eminently satisfactory in others there was no increase in the vigor or
amount of the crop as a result of the inoculation.

METHODS OF SOIL INOCULATION. From these early experimental
results there evolved two general methods of inoculation, namely, the
application of soil from an already inoculated field, and the application
of pure cultures of the nodule-forming bacteria to the seed before sowing.

282 MICROBIOLOGY OF SOIL.

Inoculation with Legume-Earth. The use of soil as inoculating mate-
rial was tried by various experiment stations of the United States, with
results not varying widely from those secured in the pioneer experimental
work at Bremen. It was found in general that the commonly grown
crops, such as the common clovers, peas and beans, made little or no
increase as a result of inoculation with old legume-soil. With new crops,
however, such as alfalfa and soy beans when they were first introduced,
it was found impossible in many places to secure a successful stand until
the fields on which these crops were to be grown had received a top-dress-
ing of soil from land that had already grown the crop in question; and it
became a common practice to inoculate soil in this manner before seeding
with these new crops. It was early observed, however, that this method
of soil transfer for inoculation purposes was not an unmixed benefit.
Aside from the expense and difficulty of handling and transportation of
soil, fungus and bacterial diseases, not only of legumes but of other crops,
as well as the seeds of noxious weeds, were transmitted from one field to
another and even from one section of country' to another. It was to avoid
this difficulty that the preparation of pure cultures was introduced.

Inoculation with Pure Cultures. Nitragin. The first pure culture
method was launched in 1896 by Nobbe and Hiltner, German investigators
who prepared cultures of the legume bacteria on nutrient gelatin and
arranged with a firm of manufacturing chemists to place them on the
market under the trade name of Nitragin.

Nitragin met with an enthusiastic reception on the part of experi-
menters and farmers. The results of its use, however, were so
varying and uncertain that the product fell into disrepute and its manu-
facture was discontinued. Improvements have been made by Hiltner
and his associates, and a new Nitragin which has been placed on the
market within the past four years has given results much more positive
and uniform than were obtained with the old preparation.

Dried Cultures. -In the United States the matter of pure cultures
was first taken up by the Department of Agriculture about 1902.
Cultures of the nodule-forming bacteria were cultivated in nitrogen-
free culture media, dried on cotton and distributed to farmers with a
small package of salts from which a culture solution was to be made
by the farmer and applied to the seed. This method gave poor results,
chiefly because the bacteria could not withstand the drying on cotton.
Afterwards the cultures were sent in a liquid condition with somewhat

FIXATION OF ATMOSPHERIC NITROGEN. 283

more satisfactory results. The dry cotton cultures were exploited for a
time by a commercial firm under the name of Nitro-culture, and some-
what similar cultures were placed on the market in England under the
name of Nitro-bacterine. Cultures of both kinds, however, have been
shown to be valueless, both by microbiological and by planting tests.

Cultures on A gar. The most promising results thus far in the use of
pure cultures appear to have been secured at the Ontario Agricultural
College, where Harrison and Barlow originated the method of growing the
bacteria upon a nitrogen-free medium. By this method the farmer has
simply to apply the bacteria to the seed just before sowing. These cul-
tures used upon all of the common legumes, sown in all kinds of soil,
have given favorable results in about 65 per cent of trials. Similar agar
cultures are now prepared by commercial firms which have adopted the
method of Harrison and Barlow, and also by some of the U. S. Agricul-
tural Experiment Stations.

Alinit. Alinit was the trade name applied to cultures placed on the
market in 1897, of B. ellenbachensis, isolated by Caron, owner of the
Ellenbach estate in Germany, and claimed by him to be one of the non-
symbiotic nitrogen-fixing bacteria. Alinit was examined and tested by
numerous investigators and the organism was found to be incapable of
nitrogen-fixation.

Azotobacter Cultures. A great deal of experimental work is being
conducted at the present time in the use of artificial cultures of the azo-
tobacter species. The results thus far, however, are contradictory, and
much more work needs to be done to prove the efficiency of such cultures.

The lack of complete success thus far with artificial cultures for soil
inoculation does not afford sufficient ground for condemnation of the
method. Failure may often be attributed to unsuitable soil conditions
rather than any inherent failing in the culture used, and no method of
inoculation will compensate for poor physical or chemical condition of the
soil itself. The principle of using artificial cultures to be applied with
the seed is sound, and if the cultures contain large numbers of living
bacteria, there is little reason why they should not prove of benefit when
used under soil conditions that would seem to warrant their need.

CHAPTER IV.

CHANGES IN INORGANIC CONSTITUENTS.
WEATHERING PROCESS.

ORIGIN AND FORMATION OF SOIL. Rock surfaces exposed to the
action of rain, sunshine and frost lose their fresh appearance, become
pitted and uneven, and gradually crumble into larger and smaller frag-
ments. In the course of time the layer of disintegrated material be-
comes deeper and its constituent particles smaller thanks to the uninter-
rupted process of subdivision. Finally, lichens, algae and bacteria make
their appearance, the organic debris accumulates, and higher plants begin
to find a suitable environment for their development. The rock has
changed into soil.

INFLUENCE OF BIOLOGICAL FACTORS. Soil-formation is not entirely
a mechanical or chemical process. Even before the layer of weathered
rock acquires any appreciable depth microscopical and macroscopical
forms of life gain a foot-hold on the uneven surface. With the aid of
sunlight they build organic compounds and make use of combined or
elementary nitrogen of the atmosphere. Their life activities result in the
production of carbon dioxide and of varying organic and inorganic acids
which in their turn react with the constituents of the rock particles. In
this manner the biological activities become of utmost moment in the
transformation and migration of mineral substances in nature. They
assume an important role in the circulation of calcium and magnesium,
with the accompanying phenomena that find most striking expression
in the formation of caves and canyons in limestone strata. They assume
a no less important role in the circulation of sulphur ; in the accumulation
and removal of available potash compounds in the soil, as well as in the
transformation of phosphorus and its migration from inorganic to organic
compounds.

LIME AND MAGNESIA.

REMOVAL AND REGENERATION OF CARBONATES. Lime and mag-
nesia are present in soils in different combinations. They may occur as

284

CHANGES IN INORGANIC CONSTITUENTS. 285

silicates, carbonates, phosphates, humates, sulphates, etc. In humid
climates the carbonates are being continually removed from weathered
rock material, as is plainly shown by the composition of drainage
waters. The losses become much greater in cultivated soils thanks
to the humus and the microorganisms present in them. The absolute
amounts lost from year to year will depend on the proportion of lime and
magnesia in the soil, the mechanical composition of the latter, its content
of humus and the methods of tillage and fertilization. According to Hall
the soils of the experiment fields at Rothamsted, containing about 3
per cent of calcium carbonate, are losing lime at the rate of 362 kg.
to 453 kg. ( 800 to 1,000 pounds) per acre annually. In certain
sections of Scotland where liming has been practised for a long time the
farmers estimate the loss of lime from the land at 6 bushels per
acre, annually; that is, approximately at the rate of 226 kg. to 272
kg. (500 to 600 pounds). In New Jersey, New York, Pennsylvania and
other eastern states farmers who use lime more or less regularly apply one
ton of it at the beginning of each five year rotation. This would provide
for an annual loss of 181 kg. (400 pounds) per acre. The loss oi
lime and magnesia is increased under intensive methods of agriculture.
When animal manures and green manures are employed, microbial
activities are stimulated, the production of carbon dioxide is encouraged
and the loss of the soluble calcium bicarbonate made greater. The re-
moval of lime is hastened even to a more striking extent when ammonium
salts are applied to the land. The resulting nitrification and loss of lime
are illustrated by the following equation:

(NH 4 ) 2 SO 4 + 2 CaC0 3 + 4O 2 = Ca(NO 3 ) 2 + CaSO 4 + 4 H 2 O+ 2 CO 2

As was already indicated, the loss of calcium and magnesium carbonate
from the soil is effected largely through the activities of bacteria and of
other microorganisms. At the same time microorganic life is responsible
for the restoration of varying amounts of carbonates. It has been dem-
onstrated that, in the weathering of the complex silicates, carbonates and
silicic acid may be formed in considerable quantities. In the presence
of decaying organic matter and the consequent evolution of carbon diox-
ide the formation of carbonates from silicates may be extensive enough to
balance the losses. Similarly, calcium carbonate may be formed in the
soil from humates and from the calcium salts of simpler organic acids.
They may be formed, also, through the activities of denitrifying and other

286 MICROBIOLOGY OF SOIL.

reducing bacteria from the corresponding nitrates and sulphates. As
pointed out by Nadson ammonium carbonate produced in the decom-
position of protein compounds may react with calcium sulphate as follows:

(NH 4 ) 2 CO 3 + CaSO 4 = CaCO 3 + (NH 4 ) 2 SO 4

Moreover, calcium sulphate may be reduced to sulphide and may react
with carbon dioxide as follows:

CaS + CO 2 + H 2 O= CaCO 3 + H 2 S

Magnesium would be subject to similar reactions and Nadson has observed
the formation of a mixture of calcium and magnesium carbonates
(corresponding to dolorite in composition) in media inoculated with a pure
culture of B. (Proteus) vulgaris.

LIME AS A BASE. The carbon dioxide generated in vast amounts in
the life processes of most soil bacteria, the nitrous and nitric acids formed
by the nitro-bacteria, the sulphuric acids produced in the oxidation of
hydrogen sulphide and of sulphur by the so-called sulphur bacteria,
and the great variety of organic acids formed in the decomposition of
carbohydrates, fats and proteins all react with basic substances in the soil.
Of these basic substances calcium carbonate is by far the most prominent.
Combining with the different acids it maintains a favorable reaction for
microorganic life in the soil.

The calcium salts thus formed are more or less soluble. In this man-
ner enormous amounts of lime are annually carried to the ocean as bi-
carbonates, and to an appreciable extent also as nitrate and sulphate
Thus soil bacteria help to furnish shell fish and other forms of marine life,
the material necessary for the building of their skeletons. In the course
of ages the latter become a portion of the solid land and as coral reefs,
chalk cliffs and marl beds offer to microorganisms a new opportunity to
start calcium carbonate on its migrations.

EFFECT OF CALCIUM AND MAGNESIUM COMPOUNDS ON BACTERIAL
ACTIVITIES. Being basic in character calcium and magnesium car-
bonates are of great service in maintaining a suitable reaction in the soil.
But somewhat apart from this service calcium and magnesium compounds
seem to be particularly important for the growth of certain organisms.
It has already been observed by Winogradski and Omelianski that magne-
sium carbonate is especially useful in facilitating the isolation and culture
of nitrate bacteria. Heinze and others have noted the favorable action
of calcium carbonate on the growth of Azotobacter, while the beneficial

CHANGES IN INORGANIC CONSTITUENTS. 287

influence of calcium carbonate and sulphate on the development of Ps.
radicicola has been repeatedly observed by different investigators.

PHOSPHORUS.

AVAILABILITY OF PHOSPHATES. Phosphorus exists in the soil largely
in the form of phosphates of calcium, magnesium, iron and aluminum.
A small portion of it occurs in organic combination in lecithin, phytin
and other compounds. The soil phosphates possess a very slight degree
of solubility and often fail to become available rapidly enough to meet the
demands of the growing crop. Fortunately the presence of carbon dioxide
generated from decaying organic matter hastens the solution of the inert
phosphates, thus:

Ca 3 (PO 4 ) 2 + 2CO 2 + 2H 2 O= Ca 2 H 2 (PO 4 ) 2 + Ca(HCO 3 ) 2

For this reason a maximum supply of available phosphates may be
secured by plants in the presence of readily decomposable organic matter.

Apart from carbon dioxide as a means for making available inert
phosphates, bacteria produce organic and inorganic acids that are of
direct service. The influence of nitrous, nitric and sulphuric acids, all
of them products of bacterial activity, is undoubtedly of some importance.
The influence of lactic, acetic and butyric acids, as well as of the more com-
plex humic acids, must be of considerable moment. For instance, in the
decomposition of bone meal by B. mycoides, Stoklasa found that 23 per
cent of the phosphoric acid had become soluble, whereas in similar un-
inoculated portions of bone meal only 3 per cent of soluble phosphoric
acid was found. The significance of organic acids produced by micro-
organisms is brought out even more strongly in the loss of phosphates
from acid soils.

In so far as the organic phosphorus compounds are concerned bac-
terial activities are important in that the processes of decay restore the
phosphorus to circulation. Hence, it will be seen that microorganisms
are directly concerned in the migration of phosphorus from the soil to the
plant and from the plant back to the soil.

RELATION OF PHOSPHORUS TO DECAY AND NITROGEN-FIXATION.
Just as bacteria influence the transformation of phosphorus compounds
in the soil, so phosphorus itself affects the growth and activities of bacteria.
As one of the essential constituents of living cells it reacts on the growth
of microorganisms and influences species relationships. There are un-

288 MICROBIOLOGY OF SOIL.

doubtedly species whose phosphorus requirement is greater than that of
other species. Indeed, conditions may arise that favor the rapid assimi-
lation of soluble phosphates by bacteria. In that case the microorganisms
would act as competitors to the higher plants. Among the species
favorably affected by an abundant supply of phosphates Azotobacter is
quite prominent. Hence nitrogen-fixation is in a measure dependent
upon a proper supply of phosphorus compounds.

SULPHUR.

SULPHUR COMPOUNDS IN THE SOIL. Sulphur occurs in the soil in
the form of sulphates and in that of organic compounds. In ill-
aerated soils the reduction products of sulphates; viz., sulphites, sulphides
and even elementary sulphur may be present in small amounts as a transi-
tion stage. According to Berthelot and Andre the protein compounds
of the soil humus are quantitatively more important than the sulphates.
However, this is not true of arid and semi-arid soils in which sulphates
represent a larger store of combined sulphur than is contained in organic
substances.

SULPHUR BACTERIA. In the decomposition of protein compounds
with a limited supply of air, hydrogen sulphide and mercaptans are evolved.
The quantities of hydrogen sulphide produced may be large enough to
become perceptible to the sense of smell, as happens in the putrefaction
of eggs. At the bottom of seas, rivers, lakes and ponds (in canals, ditches,
swamps, etc.) as well as in finer-grained soils the production of hydrogen
sulphide goes on almost uninterruptedly owing to the activities of a great
variety of bacteria. The hydrogen sulphide thus generated serves as a
source of energy to a group of organisms known as sulphur bacteria.
The oxidation of the hydrogen sulphide by these bacteria may be ex-
pressed by the following equations:

2H 2 S+O 2 =2H 2 O + S 2

S 2 +2O 2 =2SO 2

The sulphur dioxide produced is further changed into sulphuric acid
in the presence of oxygen and water. In its turn the acid reacts with some
base, usually calcium carbonate, resulting in the formation of calcium
sulphate. Thus:

SO 2 +O + H 2 O = H 2 SO 4
H 2 SO 4 + CaCO 3 = CaSO 4 + H 2 O+ CO 2

CHANGES IN INORGANIC CONSTITUENTS. 289

We owe much of our knowledge concerning the sulphur bacteria to
Winogradski. This investigator showed that in places where hydrogen
sulphide is generated in considerable quantities sulphur bacteria grow
vigorously and accumulate granules of sulphur within their cells. When
the cells containing sulphur granules are removed to suitable media, in
which no hydrogen sulphide is present, the sulphur seems to be gradually
oxidized and disappears and the bacteria finally die of starvation. Thanks
to the sulphur bacteria, the higher plants are enabled to utilize again the
sulphur once locked up in plant and animal tissues, and liberated thence
by decay bacteria. The circulation of sulphur is thus made possible
and the cycle is completed when the sulphates are again used by plants to
build protein compounds. It may also be noted in this connection that
" Thiobacillus denitrificans," described by Beyerinck, may also oxidize
elementary sulphur. In this case, however, the oxygen is derived from
nitrates instead of the atmosphere. Thus:

SULPHATE REDUCTION. The fact that sulphates may be reduced to
sulphides in the presence of organic matter has been known for many
years. In compost heaps, and at the bottom of seas, lakes and rivers,
the reduction of calcium sulphate is of common occurrence. Similarly,
ferrous sulphate may be reduced in water-logged soils and in swamps
and may give rise to deposits of bog iron. But while sulphate reduction
is of common occurrence in certain localities, it has been shown by Bey-
erinck and also by van Delden, that the reduction can be accomplished
in artificial media by specific microorganisms. Two species isolated by
these investigators have been named Sp. desidpliuricans and Msp.
(estuarii. When grown under anaerobic conditions in culture media sup-
plied with combined nitrogen and organic nutrients these organisms were
found capable of reducing sulphates. The oxygen withdrawn from the sul-
phates was used for the oxidation of organic matter in a manner analogous
to that in nitrate reduction where the oxygen is derived from the nitrates.
Apart from the two microspiras that cause the specific reactions just
noted, there are many common soil bacteria that may be responsible for
sulphate reduction in a less direct manner. Nadson has observed that
when the supply of oxygen is limited calcium sulphate may be reduced
to sulphide by B. my co ides and by B. (Proteus') vulgar is. The calcium
19

2QO MICROBIOLOGY OF SOIL.

sulphide according to him may react with carbon dioxide and water,
giving rise to the formation of hydrogen sulphide. Thus:

CaS+CO 2 +H 2 O=CaCO 3 +H 2 S

The hydrogen sulphide derived from sulphates or from proteins
becomes a source of energy to the sulphur bacteria as already noted in
the preceding pages.

POTASSIUM.

THE TRANSFORMATION OF POTASSIUM COMPOUNDS IN THE SOIL.
Potassium occurs in the soil largely in the form of silicate minerals.
Smaller amounts occur as nitrate, carbonate and in organic compounds.
The portion present as silicates is often very large in clay loam soils,
amounting not infrequently to 22,679 kg. to 34.019 kg. (50,000 to
75,000 pounds) per acre foot. Unfortunately for the farmer, the grow-
ing crops fail, in many cases, to secure sufficient quantities of available
potash for their rapid development, notwithstanding these enormous stores
of potassium compounds. However, when sufficient quantities of readily
fermentable organic matter are present and the generation of carbon
dioxide is rapid the silicates weather sufficiently fast to meet the demands
of maximum harvests. The part played by carbon dioxide in the trans-
formation of inert potash compounds may be illustrated by the following
reaction:

A1 2 O 3 K 2 O 6SiO 2 +CO 2 +2H 2 O = Al 2 O 3 2SiO 2 2H 2 O+K 2 CO 3 +4SiO 2

Under actual conditions it is the aim of the farmer to stimulate
bacterial activities (and, therefore, the production of carbon dioxide) in
his land by the use of animal manures or green manures and of commercial
fertilizers. Apart from the influence of carbon dioxide available potash
compounds may likewise be formed on account of nitric, sulphuric,
acetic, lactic, butyric and other acids produced by different soil bacteria.

OTHER MINERAL CONSTITUENTS.

IRON. The investigations of Ehrenberg, Winogradski, Molisch,
Adler, Ellis and others have accumulated a mass of data relating to the
so-called iron bacteria. These organisms belong to the class of higher
bacteria and have a marked ability to precipitate iron oxide out of solutions
of iron salts. Winogradski believed that the reaction is a physiological

CHANGES IN INORGANIC CONSTITUENTS. 29 1

one in that the microorganisms oxidize ferrous to ferric compounds, and
utilize for their growth the energy thus made available. The investi-
gations of Molisch, Adler and Ellis show, however, that the iron bacteria
can exist very well without iron compounds; and that the precipitation
of iron oxide is due to mechanical rather than chemical influences. But
whether physiological or mechanical the influence of these microorganisms
is felt in the formation of bog iron, and in the filling up of iron pipes;
in the latter instance much annoyance is occasionally experienced by
those in charge of municipal water supplies.

Compounds of iron are of considerable significance in the life pro-
cesses of many bacterial species. For instance, it was shown by the Lipman
and after him by Koch, that Azotobader will not develop in culture
media devoid of iron compounds. In field practice small applications of
ferrous sulphate often seem to exert a favorable effect on crop growth,
and there is reason to suspect that soil-microbial activities are of some
moment in bringing about the results noted.

ALUMINUM, MANGANESE, COPPER. Weathering processes and the
relation of carbon dioxide to these processes have already been discussed
in connection with calcium and potassium compounds. To a great
extent aluminum is affected by these reactions, for in the decomposition
of feldspar kaolinite is one of the important products formed. Hence,
bacteria become a factor of considerable importance in the formation of
hydrated silicates of aluminum, at least, in the presence of organic matter.
Moreover, it is recognized in the ceramic industries that after it is dug
clay must undergo ripening in order to be suitable for certain purposes.
The ripening process involves the activities of bacteria. Unfortunately
very little is known about the reactions that occur in the ripening of clay.

As to manganese and copper there is scarcely any experimental evi-
dence available as to the part played by their compounds in the soil,
particularly in so far as they affect microorganic life. To some extent,
it is known that where Bordeaux mixture has been employed for spraying
potatoes, cranberries, fruit trees, etc., plant growth is subsequently
stimulated to a striking extent. In view of the very slight quantities of
copper that are actually added to the soil by these sprays, it is possible
that the effects noted are caused by stimulated or changed microbial
activities. This view finds some support in the influence exerted by
copper sulphate on the growth of algse in lakes, ponds, and shallow
treams.

DIVISION IV.
MICROBIOLOGY OF MILK AND MILK PRODUCTS.

CHAPTER I.*
THE RELATION OF MICROORGANISMS TO MILK.

IMPORTANCE OF MILK AS A FOOD.

Fresh normal milk is one of the most important of human foods.
It has a pleasant taste and aroma and is generally liked as a food or drink;
but unless properly cared for will not long remain in its normal condition.
No article of human diet is more susceptible to undesirable changes, due
to the delicate nature of the milk itself and to the conditions naturally
surrounding its production and handling. The injurious changes which
commonly occur in milk are of two kinds.

ABSORBED TAINTS AND ODORS.

Milk is very quickly affected by odors of any sort. The foreign
odor may be absorbed before the milk leaves the udder if the cow has
eaten strong feeds, such as cabbage, onions, etc., or it may be absorbed
after the milk is drawn from the cow. If milk is exposed to any
strong odor, such as silage or foul air, resulting from lack of venti-
lation in the stable at milking time, these odors will be taken
up by the milk with surprising rapidity. If placed in an ice chest
with fresh strawberries or pineapple, or foods like cabbage or turnips,
the milk will very quickly absorb the odor of these foods. The absorp-
tion of any foreign odor gives to milk a decidedly disagreeable taste.
This is true even when the odor which is absorbed is pleasant in itself
as is the case of strawberries or pineapples. When the "off" flavors are

* Prepared by W. A. Stocking with the exception of the paragraphs treating the acid-
forming bacteria, prepared by E. G. Hastings.

292

THE RELATION OF MICROORGANISMS TO MILK. 293

due to absorption they are strongest at the outset and become less pro-
nounced as the milk becomes older, especially if it is subjected to some
method of aeration.

CHANGES DUE TO MICROORGANISMS.

While absorption of foreign odors is not uncommon, perhaps most
of the undesirable flavors, found in milk when it reaches the consumer,
are caused not by absorption but by the growth of microorganisms in
the milk. In this class the changes are slight at first and increase with
the age of the milk. Changes of this sort include the common phe-
nomena of souring and curdling, the so-called sweet curdling, ropy or
slimy milk, bitter flavors, gassy milk and a large variety of changes
usually known as barny or cowy odors and flavors. If milk could
be kept free from microorganisms it might be kept for some time
without showing perceptible changes in appearance or taste. No
other food product will undergo fermentation changes as rapidly as milk
because it is an ideal culture medium for the growth of most kinds of
microorganisms, especially bacteria and yeasts. Not only does milk
contain the needed food elements but, being in liquid form, they are
easily available for the use of the microorganisms. The proteins and
milk sugar are most easily attacked and it is the breaking down of these
which causes most of the changes in the milk.

MICROBIAL CONTENT or MILK.

When we recognize the extreme ease with which milk undergoes
bacterial changes we are not surprised to find that ordinary milk, when
delivered to the consumer, contains relatively large numbers of bacteria.
The amount of care exercised in the production and handling is a most
important factor in determining the bacterial infection of milk. On this
basis milk may be roughly divided into three classes.

COMMON MILK. Age is one of the chief factors in determining the
germ content of milk. We are, therefore, not surprised to find the milk
in large cities having a much higher germ content than in smaller cities
and towns. The normal germ content of ordinary milk as it is found in
the cities may be shown by the following tables.

294

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

Bacteria in Boston Milk.*

Average taken from 2,394 Samples.
From June to September.

Per cent

Below 100,000 bacteria per c.c 42 . o

Between 100,000 and 500,000 per c.c 29. 75

Between 500,000 and 1,000,000 per c.c 9-75

Between 1,000,000 and 5,000,000 per c.c 12.75

Above 5,000,000 per c.c 5.0

Uncountable plates o. 75

Bacterial Counts of Chicago (Raw} Milk.^

Date

Number of
samples

Average
count

Lowest
count

Highest
count

January, 1910

64

|
1,067,000

27,000

< =;oo ooo

April, 1910

A3

?, 048,000

14,000

I ^O OOO OOO

Tulv IQIO .

183

12 ?48 OOO

8 ooo

IOO OOO OOO

Bacteria in Milk of Connecticut Cities. J

Bacterial count Number of samples

Under 50,000 1,707

50,000-100,000 130

100,000-500,000 459

500,000-1,000,000 98

Over 1,000,000 73

These figures give the results of 2,467 samples collected in seventy-five different
towns in the State from October i, 1908 to October i, 1909.

Goler gives the average bacterial count for 1,057 samples of market milk collected
in Rochester during the year 1909 as 446,099 per c.c. Of these samples 1.79 per cent
were above 5,000,000 and 38.4 per cent below 100,000.

In Montclair, N. J., the average bacterial count for the year 1909 from samples
representing fifty-seven dairies was 53,000 per c.c.

In Ithaca, N. Y., 148 samples were taken for the year beginning April i, 1909, and
ending March 31, 1910. The average bacterial count of these samples was 221,000.

* Data given by Hill and Slack.
t Data given by Tonney.
j Data given by Conn.

THE RELATION OF MICROORGANISMS TO MILK. 295

The immense numbers of bacteria found in milk in the large cities
are usually the result of the rapid growth of the Bad. lactis acidi group
resulting from the age of the milk and the temperature at which it has
been kept. Such milk may also contain large numbers of those sapro-
phytic organisms which occur in the atmosphere and about the stables and
milk-house. The number of this group depends largely upon the sani-
tary conditions of production and the initial contamination. In ordinary
milk organisms of the Bact. lactis acidi type will constitute a very large
percentage of those present when the milk reaches the city even before
it shows any perceptible signs of souring. During the past few years
great progress has been made in the production of clean milk, and at
present quite an important part of the general milk supply of our cities
has a very much lower germ content than it had a few years ago.

SPECIAL MILKS. In this class may be considered those milks known
as Selected, Inspected, or Guaranteed. As commonly used these terms
mean milk which has been produced and handled with considerably
more care than ordinary market milk but not with the extreme care
required for certified milk. Guaranteed milk is produced by herds which
have been shown by the tuberculin test to be free from tuberculosis.
Considerable care is exercised in all the operations of handling the milk.
The result is that these milks usually have a much lower germ content
than the ordinary milk supply of the same city. Sometimes the germ
content of such milk compares favorably with that of certified milk.
These milks may contain various types of normal milk organisms but
they should not contain any tubercle bacteria.

CERTIFIED MILK. Certified milk means milk which has been produced
according to the regulations of and under the supervision of a medical
milk commission. The stables and cows are kept extremely clean. No
dust is allowed in the stable at milking time. The cow's flanks and udder
are washed just before milking, the milkers wear white suits and wash
their hands before milking each cow. Small-top pails are used and the
milk is cooled as soon as drawn from the cow. The extreme care ex-
ercised in the production and handling of this milk has a very marked
effect on the number of bacteria found in it. The following counts are
typical of certified milk.

296

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

Bacterial Counts of Certified Milk in Different Cities.*
Boston, Oct. i, 1909 to Sept. 30, 1910.

Dairy number

Number samples

Average bacteria

i

i?

5,794

2

13

4,176

3

30

6,825

4

12

1-475

5

7

2,294

New York City, Oct., 1909 to Sept., igio.t

Farm number

Average count

2

11,132
10,516

3

8,504

4

5
6

2,863
11,246

7
8

9

23,705
5,370
15,062

10

459

Chicago.

Dairy Number

Number counts

Average number
bacteria

i

5i

5, 612

2

60

4,078

3

43

6,502

4

J 7

2,553

Brooklyn.

Moak gives the average of 321 counts for certified milk delivered in Brooklyn
during the first six months of 1910 as 4,095 bacteria per c.c. The best average from any
one farm was 561 bacteria per c.c.

* Data given by Arms,
t Data given by Park.
} Data by Heinemann.

THE RELATION OF MICROORGANISMS TO MILK.
SOURCES OF MICROORGANISMS IN MILK.

297

The sources from which bacteria get into the milk have been the sub-
ject of much investigation during the past few years, until now the chief
sources of contamination are pretty well understood. These sources
may be grouped in a general way under the following heads:

FIG. 73. Vertical section of one quarter of udder showing teat, milk cistern, and larger
milk ducts. (After Ward and Hopkins.)

INTERIOR OF THE Cow's UDDER. Healthy Udders. Milk as it is
secreted by the normal udder of a healthy cow is comparatively free from
bacteria. It is very difficult, however, to obtain milk from the udder which

298 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

does not contain bacteria in greater or less numbers. This is due to the
fact that immediately after secretion the milk becomes contaminated by
bacteria which exist in the interior of the udder. Early investigators,
notably de Freudenreich and Grotenfelt, believed that milk while in the
udder was entirely free from microorganisms. Later investigations, how-
ever, by Moore, Ward, Bolley, Hall and others, have shown that the healthy
udder normally contains bacteria in appreciable numbers. It has been
found that bacteria are present even in the upper portions of the udder in
the small milk passages leading from the secreting cells. These organ-
isms, which normally exist in the milk passages of the udder, gain en-
trance through the orifice in the end of the teat where they find suitable
conditions for growth and, once inside, work up through the milk cistern
to the larger milk ducts and finally though all parts of the udder (Fig.
73). The number of bacteria found in the udder varies widely in differ-
ent cows as may be seen by the following figures:

Bacterial Content of Entire Milk of Different Cows.

Cow No. i, 850 bacteria per c.c.

Cow No. 2, 750 bacteria per c.c.

Cow No. 3, 25 bacteria per c.c.

Cow No. 4, 112 bacteria per c.c.

Cow No. 5, 70 bacteria per c.c.

Cow No. 6, 1850 bacteria per c.c.

If portions of milk are taken at different intervals during the process
of milking in such a way that all external contamination is prevented, it
will be found that the first few streams or "fore-milk" contain many
more organisms than the milk drawn later. After the first ten or twelve
streams the number of organisms will decrease quite rapidly, normally
becoming less and less until the final strippings, when there is usually a
marked increase. This condition indicates that the larger number of
organisms exist in the milk cistern and larger milk ducts in the lower
part of the udder and are therefore removed during the early part of the
milking. The increase at the end of the milking is probably due to the
greater manipulation, resulting in dislodging some of the organisms
which have adhered to the walls of the milk passages.

Not only does the number of organisms in different cows vary, but
there is a marked difference in the different quarters of the same udder,
as shown by the following figures:

THE RELATION OF MICROORGANISMS TO MILK.
Bacteria in Different Quarters of Cow's Udder.

299

Right

Right

Left

Left

fore

hind

fore

hind

quarter

quarter

quarter

quarter

Cow No. i

18

IT.

<:<;

n

Cow No 2

40

2^1

CO

7O

Cow No. 3

7AO

60

GO

T.2

The number of organisms normally found in the udder is much smaller
than would be expected when we consider the fact that ideal conditions
of food and temperature are provided there for bacterial growth. The
relatively small number of organisms is perhaps due to some germicidal
action existing in the udder. Attempts to increase the germ content in
the udder by injecting cultures of different species of saprophytic bacteria
have failed to produce a continued increase, the injected organisms usually
decreasing very rapidly in numbers until they disappear at the end of a
few days. From the standpoint of ordinary market milk, the number of
bacteria found in the healthy udder is so small that it is of little com-
mercial importance. In dairies where a very small germ content is
desired, however, this source of infection must be taken into account and
in certain cases individual cows, which normally have a high bacteria
content in the udder, can be discarded to advantage.

It is evident that many species do not find the conditions in the udder
suitable for their growth, since investigations have shown that compara-
tively few species exist for any length of time in the healthy udder. Cer-
tain types of cocci are the predominating forms with occasional cultures
of other species. The Bad. lactis acidi type does not thrive in the udder.
The types of organisms commonly found there do not seem to develop
rapidly in the milk when it is held at low temperatures and fail to
produce any appreciable changes in it.

Diseased Udders. If, however, the cow is suffering from disease
in the udder, the bacterial condition may be quite different from that
described above. In this case, the milk may be filled with the specific
bacteria before it leaves the udder. In cases of inflammatory trouble or
tuberculosis in the udder the milk may contain very large numbers of or-
ganisms, frequently many millions per c.c. at the time the milk is drawn.

300

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

EXTERIOR or Cow's BODY. The nature of the cow's coat and the
condition under which she is normally kept favor the accumulation of dust
and bacteria upon her body. Unless special care is taken to keep the
cow's body free from dust, the organisms which fall into the milk from
this source at milking-time will constitute one of the most important

**

FIG. 74. Colonies developing in agar plate held for ten seconds in position of milk pail
after udder was brushed gently with the hand. (Original.)

sources of contamination. The importance of this source of contami-
nation may be recognized when we see what large numbers of micro-
organisms may be carried by small particles of dust or an individual
cow hair.

The importance of this source of contamination depends very largely
upon the conditions under which the cows are kept and the care exercised

THE RELATION OF MICROORGANISMS TO MILK.

301

in cleaning just previous to milking. In many of the certified milk dairies
this source of contamination is reduced to a minimum and has little
effect upon the milk.

ATMOSPHERE OP STABLE AND MILK HOUSE. Next to the cow's
body, the atmosphere of the stable is often the most important factor in
determining the bacterial content of fresh milk. In sanitary dairies this

FIG. 75. Colonies developing from cow-hairs planted in agar plate. (Original.)

factor is fully recognized and every effort is made to prevent the presence
of dust in the atmosphere at the time of milking. The atmosphere is
sometimes sprayed either with the hose or with steam in order to settle
every particle of dust at milking time. In stables where the importance
of this factor is not recognized and dust is allowed to exist in the atmos-
phere at milking time, the number of bacteria in the milk will be materi-
ally increased.

302

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

THE MILKER. Not infrequently the milker himself is an important
source of contamination. If his clothing and hands are dirty or if he
brushes against the cow, the dust thus dislodged may carry into the
milk large numbers of microorganisms. This is shown in the diffe ence
in the germ-content of milk drawn by two men milking in the same barn
under identical conditions.

FIG. 76. Colonies developed from a bit of dust found in cow stable. Agar plate culture.

(Original.)

Difference in Number of Bacteria in Milk Drawn by Men in Same Stable.

Number of milkings

Number of bacteria per c.c.

Milker No i

10

2 4. CO

Milker No. 2

10

17 IOO

THE RELATION OF MICROORGANISMS TO MILK. 303

THE UTENSILS. If properly cared for, the dairy utensils should not
add to the germ content of the milk. Not infrequently, however, they
are faulty in construction. In open seams and other places the milk
may accumulate and not be thoroughly washed out. Usually when
utensils of this sort are used, the methods for washing and sterilizing
are not sufficient and bacteria multiply in large numbers in the cracks
and crevices and contaminate each new lot of milk put into them. Some-
times utensils which are properly constructed may contaminate the milk
because they have not been properly cleansed and sterilized. The use
of steam is the most efficient means of sterilizing all dairy utensils, but
boiling water may give very satisfactory results if used at actual boiling
temperature. If not used at the boiling temperature some of the more
resistant organisms will not be killed and will be left to inoculate the fresh
milk. The ropy milk organism, B. lactis viscosus, often remains in the
utensils from day to day in this way.

WATER SUPPLY. Sometimes the water used for washing the dairy
utensils is a serious source of contamination. Serious epidemics of dis-
ease have been traced to this source where the utensils were washed with
water contaminated by typhoid or other disease organisms and were not
sufficiently sterilized to kill those remaining in the utensils. Such dairy
troubles as ropy milk and gassy milk may be caused by the water used for
washing puposes.

METHODS OF PREVENTING CONTAMINATION OF MILK.

INDIVIDUAL Cows. Normally the number of microorganisms found
in the udder is not sufficient to be a serious source of contamination for
market milk. There are, however, certain cows which have a much
higher germ content than others, and where a very low count is desired
in the milk it may sometimes be advisable to eliminate such cows from
the herd.

CARE OF THE Cow's BODY. In order to reduce to the minimum the
contamination from the cow's body she should be kept as clean as possible.
Dust should not be allowed to accumulate in her coat. It is well to keep the
hair of the flank and udder clipped in order to prevent the accumulation
of dust and also to facilitate the process of cleaning. The use of a damp
cloth for wiping the flank and udder at milking time is a very efficient
means of reducing this source of contamination. The beneficial effect
of this method may be seen in the following table:

34

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

Effect of Wiping Udder and Flank "with a Damp Cloth as Shown by Bacterial Counts of

Milk.

Number of experiments

Date

Treatment Bacteria per c.c.

I

Not wiped

2,780

I

Apr. 13

Wiped

e;^o

2

Not wiped

I.3IO

2

Apr. 15

Wiped

7IO

!. .

Not wiped

800

3

Apr. ID <

Wiped

7CA

4- .

\

Not wiped

I.I7O

4- .

May 28 <

Wiped

qoo

Even when considerable care is taken to clean the surface of the
cow's body, there will still be some organisms which may fall into the pail
at milking time. This number can be very materially lessened by re-
ducing as far as possible the area through which dust can fall into the
milk pail. This can be accomplished by the use of a milking pail with a
small top.

Value of Small Top Pail in Reducing Germ Content of Milk.

Experiment

Kind of pail

Bacteria per c.c. of milk

No i

( Open

JS.S 00

No 2

\ Small top
/ Open

7,750
3,7oo

No T.

bmall top
| Open

I,IOO

30,000

^ Small top

4,700

AVOID DUST IN THE ATMOSPHERE. Many of the necessary operations
of the cow stable stir up large quantities of dust and fill the air with
microorganisms. It is astonishing to see how many bacteria can adhere
to a small piece of hay or may be found in a gram of any of our common
dairy feeds. When these materials are fed dry just previous to milking

THE RELATION OF MICROORGANISMS TO MILK.

35

time, the atmosphere of the stable will be completely filled with organisms
which may settle into the milk while it is exposed during the process of
milking. The effect of this source of contamination may be seen by the
following experiments:

FIG. 77. Some different styles of small top milking pails which
are practical and efficient. (Original.)

Bacterial Content of Milk as Affected by Feeding Dry Hay and Grain.

Experiment

Date

Nature of sample

Number bacteria per c.c.

No. i...

May 4 I

Before feeding

35

No. 2

May 17 \

After feeding
Before feeding

i,45
2,900

No. T....

May 18 <

After feeding
Before feeding

4,400
4,100

After feeding

7,200

DAIRY UTENSILS. All utensils which are to be used in connection
with milk should be so constructed that there are no cracks or crevices
in which the milk can accumulate and from which it is not easily washed
out. A milk pail with an open seam may be the cause of serious trouble
in the dairy. The dairy utensils should be simple in construction, and
so made that they can be thoroughly cleansed with ease and made of
such material that they can be thoroughly sterilized either with water
which is actually boiling or in steam.

20

306 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

THE MILKER. No food material requires greater care and cleanliness
on the part of those handling it than does milk. All persons having to do
with the handling of this delicate food product should constantly keep
in mind that clean hands and clothing and extreme cleanliness in every
operation is very necessary if milk of good quality is to be obtained.

GROUPS OR TYPES OF MICROORGANISMS FOUND IN MILK
AND THEIR SOURCES.

In studying the types of bacteria which are found in milk, it is con-
venient to arrange them in groups based upon their action on the milk and
their effect upon persons consuming it. There are certain types of organ-
isms which are very troublesome to the milk dealer but which are not
injurious to the consumer. Other species which may be of little or no
significance from their action upon the milk are of greatest significance
from the standpoint of the consumer since most of the disease organisms
which may be carried by milk have no appreciable action upon it. Still
other forms are of but little importance to either the dealer or the con-
sumer and others are troublesome to both.

GENERAL SIGNIFICANCE OF ACID-FORMING BACTERIA. Of all the
bacteria that find their way into milk, those that are able to ferment the
milk sugar, producing from it different kinds and amounts cf acids, find
more favorable conditions for growth at ordinary temperatures, 15 to 45,
than do those belonging to other groups. Because of their greater rapid-
ity of growth and because of the inhibiting effect of their by-products upon
the other groups of bacteria, the acid types tend to predominate in milk
and the specific change which they produce, the souring, is of such
common occurrence that it is often looked upon as something inherent
in milk.

GROUPS OF ACID-FORMING BACTERIA.* The acid-forming bacteria
that are constantly present in milk represent many kinds which differ in
morphology, in cultural characteristics, and in their products of fermen-
tation. They may be divided into four groups that vary greatly as far as
their importance in the handling of milk is concerned. If milk is pro-
duced under clean conditions and is kept at temperatures ranging from
15 to 35, the acid fermentation will be almost wholly due to a group of
bacteria closely allied to one of the pathogenic forms, Strept. pyogenes
(Rosenbach). To representatives of this group, which is of the greatest

* Prepared by E. G. Hastings.

THE RELATION OF MICROORGANISMS TO MILK. 307

importance in all phases of dairying, have been given various names by
different investigators. The most important organism of this group is one
to which the name Bad. lactis acidi is applied. The group undoubtedly
includes a large number of organisms, all of which produce, however, a
similar change in milk.

Second in importance is a group of organisms, of which the best known
representatives are B. coll comnmnis and Bact. lactis aerogenes. A large
number of organisms of this group have been described and named.
The most important characteristics of the representatives mentioned will,
however, suffice to characterize the group. A third group is represented
by Bact. bulgaricum and the rod-shaped organisms that have been studied
especially by de Freudenreich. A fourth group includes many acid-forming
cocci, some of which exhibit proteolytic properties while others do not.
Organisms of the third and fourth groups exert little or no effect in the
normal acid fermentation of milk, although they are constantly present in
varying numbers, as can be demonstrated by appropriate means, and un-
doubtedly are of importance in certain phases of dairy manufacturing.

In any sample of milk the relative number of bacteria belonging to
each of the first two groups is dependent upon the conditions surrounding
production, especially with reference to cleanliness. The bacteria belong-
ing to the first group come largely from the milk utensils and are also
found in the dust of the barn and on the coat of the animal. The source of
the second group is largely the fecal matter that gains entrance to the milk,
although they are also found in the upper layers of the soil and enter the
milk from the coat of the animal. The cleaner the conditions of pro-
duction, the smaller will be the number of these two groups of organisms
found in milk.

The manufacture of the leading type of butter and of all kinds of cheese
is dependent on the action of microorganisms, hence dairy manufacturing
should be classed as a true fermentation industry. In all such industries
one of the factors determining the quality of the product is the type of
microorganism employed to produce the desired fermentation, and the
importance of insuring the presence of desirable organisms, and the ex-
clusion of harmful kinds is well recognized.

The most important properties of organisms employed in the fermen-
tation industries are the physiological rather than the cultural or mor-
phological, since the quality of the product is dependent on the by-
products of the fermentation. Hence in characterizing, the groups of

308 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

acid-forming bacteria, the biochemistry of each group will be emphasized
rather than the cultural and morphological characteristics of the mem-
bers of the group.

Characteristics of the Bact. Lactis Acidi Group.* The organisms of
this group are widely distributed in nature, as is shown by the constancy
with which milk undergoes the characteristic fermentation produced by
the members of the group.

The cells are oval in form, about o.6/* to i/j. in length, and 0.5/4 in diame-
ter. The shorter cells appear nearly spherical, which, together with the fact
that chains of cells often occur, has led some to classify them among the
cocci and Kruse has applied the name Strept. lacticus to a member of the
group. In milk the cells are usually in twos, the outer ends of the two
cells being pointed. None of the group is motile; spores are not formed
and capsules are often noted. The members of the group are Gram-
positive.

The optimum temperature for growth lies between 30 and 35, the
minimum growth temperature ranging from 10 to 12, while the maxi-
mum is 42. They are to be classed as facultative anaerobes. The growth
on all culture media is marked by its meagerness; in the absence of a fer-
mentable carbohydrate, no growth usually occurs; peptone favors the
growth even in milk. In the case of freshly isolated cultures, the growth
is almost invisible, on slopes of sugar agar appearing as small discrete
colonies. On sugar agar plates the colonies are small, often surrounded
by a hazy zone, and always occur below the surface of the medium.
In lactose-agar stab cultures growth occurs along the entire line of inoc-
ulation, but there is no surface growth. No liquefaction of gelatin occurs.
In bouillon the medium is uniformly turbid or it remains clear with a slight
sediment. On potato, growth is slight or is absent. Milk is usually
curdled within twenty-four hours at the optimum temperature by mem-
bers of the group, although some fail to curdle the milk, since the maxi-
mum amount of acid produced is not sufficient to cause this phenomenon.
Still others cause curdling in the presence of small amounts of acids, in
which case a rennet-like enzyme may be present. No gas is produced in
the fermentation of lactose, hence the curd formed in milk is perfectly
homogeneous; it shows but little tendency to shrink and to express whey.
In litmus milk the color is discharged from the entire mass of medium
before curdling occurs, due to the reduction of the litmus to the colorless

* Prepared by E. G. Hastings.

THE RELATION OF MICROORGANISMS TO MILK.

39

leuco-compound. Through the action of the oxygen of the air the litmus
is slowly reoxidized and the pink layer, which immediately after curdling is
but a few millimeters in depth, is slowly extended until the entire mass of
curd has a uniform pink color. Saccharose, dextrose, maltose, and
mannit are fermented.

The maximum amount of acid produced by organisms that are most
typical of the group is determined by the composition of the medium.
It is often said that the organisms causing the normal souring of milk
represent a group that can grow in a strongly acid medium. This is true
as far as acid salts are concerned, but free acid totally inhibits growth. In
a culture medium, which contains no substance that can combine with the
acid formed and thus remove it from the sphere of action, no growth or
but very slight growth occurs. In sugar bouillon and in milk, the amount
of acid formed is determined by the content of substances in these liquids
that can combine with the acid. In milk such compounds are the casein
and some of the ash constituents, especially the phosphates. In normal
milk, the maximum acidity attained ranges from 0.9 to 1.25 per cent cal-
culated as lactic acid. If the content of neutralizing compounds per unit
volume is varied by concentration, dilution, or by the addition of such
substances as calcium phosphate, the maximum amount of acid produced
by typical cultures will be changed. In sugar bouillon the maximum
acidity produced rarely exceeds 0.25 per cent.

The fermentation of lactose is usually expressed as follows:

Ci 2 H 22 Oii + H 2 O = 4C 3 H 6 O 3

Thus 342 parts of lactose should yield 360 parts of lactic acid. The
theoretical yield of lactic acid is never obtained, for the action of the organ-
ism on the carbohydrate is much more complex than is represented by the
equation given. In the following table are given data obtained by a num-
ber of investigators:

Sugar content of

Sugar fermented

Lactic acid calcu-

Lactic acid found

milk

lated

Per cent of theo-

Per cent

Per cent

Per cent

retical

4-54

0.60

0.632

89-56

4.96

0.56

0.590

98.13

4-94

0.65

0.684

97.89

310 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

These data signify that other compounds than lactic acid are formed in
the fermentation of lactose by these acid-forming bacteria. Acetic acid
(CH 3 .COOH); formic acid (H.COOH); propionic acid (C 2 H 5 .COOH);
traces of alcohols, aldehydes and esters have been found. The lactic acid
formed is the dextro modification. It is believed that the fermentation is
due to an enzyme, lactacidase, one of the intracellular enzymes that can
be demonstrated only with difficulty.

Milk fermented by members of this group has a mild acid taste, an
agreeable odor, and the curd can be so finely divided by agitation as to
produce almost as perfect an emulsion as in raw milk. The organisms
are to be classed as desirable from the stand-point of the dairy manu-
facturer and the fermentation produced by them may be called a true
lactic fermentation.

Characteristics of the B. Coli-aero genes Group* This group includes
a considerable variety of organisms, which differ in morphology, in cul-
tural characteristics and undoubtedly in the character and amounts of
their by-products. They are more distinctly bacilli than the members of
the preceding group; are mo tile or non-motile; none produces spores and
they are usually negative to Gram's stain. The optimum growth
temperature, 35 to 40, is somewhat higher than for the preceding group, .
the vegetation range being 15 to 45. They are to be classed as facul-
tative anaerobes.

The conditions for development are less narrow than for the Bact. lactis
acidi group, growth occurring on all the ordinary culture media and in the
absence of carbohydrates. Indol and hydrogen sulphide are often formed
and nitrates are reduced. The growth is usually profuse, the colonies
large and surface growth occurring in stab cultures. Gelatin is not usually
liquefied.

Lactose, dextrose and saccharose are fermented, with the production
of varying amounts of gas in which have been found carbon dioxide;
hydrogen, methane, and free nitrogen. The maximum amount of acid
produced in any culture medium is quite similar to that formed by the
members of the previous group. The relative proportions between the
non-volatile and volatile acids are far different, lactic acid comprising less
than 30 per cent of the total acid formed, while volatile acids as acetic and
formic make up the remainder. Traces of succinic acid (C 2 H 4 (COOH 2 ))
and alcohol have also been found. The lactic acid is of the laevo-form.

* Prepared by E. G. Hastings.

THE RELATION OF MICROORGANISMS TO MILK. 311

Milk is usually curdled, although some members of the group do not
produce enough acid to cause curdling. Depending on the amount of
gas formed, the curd may be almost perfectly homogeneous or it may be
very spongy. In all cases the curd shrinks to a greater or less extent and
thus becomes so firm that it is difficult or impossible to emulsify it again.
The odor of the fermented milk is offensive and the taste disagreeable and
sharp. The organisms of this group are to be classed as undesirable and
the fermentation produced by them cannot correctly be called a lactic
fermentation.

The representatives of these two great groups of acid-forming bacteria
are to be found in every sample of market milk in varying proportions.
Both find in milk favorable conditions for growth, and the normal souring
is produced conjointly by them, each producing its own specific products,
the relative amount of which is largely dependent on the number of each
group that were originally introduced into the milk. The value of milk
for butter and cheese is determined by the relative amount of the prod-
ucts of the desirable and the undesirable acid-forming bacteria.

The difference in taste and odor between milk fermented by pure
cultures of Bad. lactis acidi, and that which has soured spontaneously
emphasizes the difference in the products of the fermentations pro-
duced by the two groups of acid-forming bacteria.

Characteristics of theBact. Bulgaricum Group* The organisms of this
group are to be classed as true lactic bacteria, since they produce almost
exclusively lactic acid from the sugar fermented and only small quantities
of other acids as formic, acetic, and propionic. They vary widely in
form and size; but are usually large rods, 2 // to 3 /* long and 0.5 p. to 0.75 //
wide. There is a tendency to form long threads. They are Gram-
positive and with methylene blue often show distinct granules in the cells ;
with Neisser's stain the appearance of some cultures is similar to that of
the diphtheria bacteria. They are non-motile and do not form spores;
capsules are seldom noted. The optimum growth temperature is from
40 to 50 and the minimum is asserted to be 25, although for many
members of the group it must be much lower.

The growth on all ordinary culture media is meager or is absent; the
colonies are often microscopic in size and show radiating threads. Free
acids do not inhibit development and the term acidophilous has been applied
to the group. They grow slowly in milk, even at the optimum tempera-
ture, and curdling may not occur for several days; the curd is homogeneous

* Prepared by E. G. Hastings.

312 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

and in litmus milk reduction occurs. The maximum amount of acid
varies from 1.25 to 4.0 per cent. Some members of the group produce
dextro-, others laevo-acid, and it is claimed that racemic acid is formed
in some cases. The curd can be easily broken by agitation, and through
the solvent action of the acid is partially dissolved. The organisms do not
liquefy gelatin, but the casein of milk is partially changed into soluble de-
composition products, as was first shown by de Freudenreich, and later
confirmed by Hastings.

It has been supposed by many that this group was confined to and
cha acteristic of certain of the fermented milks, especially those of eastern
Europe and western Asia, such as Yogurt and Matzoon. The work of
de Freudenreich has demonstrated their presence in Switzerland, and
more recently, Hastings has shown that similar organisms are constantly
present in milk and other dairy products in this country. They can be
demonstrated by placing a sample of milk in a corked bottle, and incu-
bating at 37. The acidity of the milk increases rapidly at first, due
to the growth of the members of the two previous groups. These ordinary
acid-forming organisms are soon inhibited by the appearance of free acid,
but the acidity of the milk nevertheless continues to increase slowly, and
with this continued increase a change in flora is noted, the short, plump
bacilli ceasing to predominate and long slender rods constantly increasing
in numbers. The source of this group is undoubtedly the alimentary tract
of the animal.

Characteristics of the Coccus Group* This group is well represented
by the bacteria which form the characteristic flora of the udder. They
vary greatly in size and in other properties. They retain Gram's stain;
many are chromogenic, the color ranging from a white to a deep orange.
They grow slowly on all ordinary culture media, but the growth is not
necessarily meager. Generally they are aerobic, although many grow
under anaerobic conditions. Gelatin may be liquefied or not. Milk may
or may not be curdled, the curd often resembling that formed by rennet-
like enzymes. They produce no lactic acid, but only acetic, propionic,
butyric and caproic acids, and hence cannot be classed as lactic bacteria

BACTERIA HAVING No APPRECIABLE EFFECT ON MILK. This group
is made up of many different forms. They produce no changes which
can be detected either by the eye or the taste. They do not develop very
rapidly in milk, and some species gradually disappear while others in-

* Prepared by E. G. Hastings.

THE RELATION OF MICROORGANISMS TO MILK. 313

crease in numbers. Many of the organisms in this group are chromogenic,
orange and lemon yellows being among the more common forms. They
are mostly cocci and do not liquefy gelatin. From the standpoint of the
commercial milkman these organisms are of little significance and this is
probably also true from the standpoint of the consumer.

THE CASEIN-DIGESTING OR PEPTONIZING BACTERIA. These organ-
isms digest the casein either with or without coagulation. Many of them
coagulate the casein with an alkaline reaction. They liquefy gelatin.
Most of the organisms of this group are rods of various shapes and sizes,
some of them being the largest rods found in milk. Some are motile and
some non-motile. Some representatives of this group produce little or no
odor, but many of the species develop very strong putrefactive odors.
Barny or cowy odors or other off-flavors sometimes found in milk and dairy
products are caused by the action of this type of organisms. They are
associated with filth and their presence in milk indicates unsanitary con-
ditions of production or handling.

PATHOGENIC ORGANISMS. This group of organisms produces no
changes in the milk which would indicate their presence. Some of them
do not even develop in milk, as is the case with the Bad. tuberculosis
Others, as the diphtheria bacteria and typhoid fever bacilli, may grow in
milk with great rapidity. This group also contains certain species which
produce diarrhoeal disorders, especially in infants and young children.
Some of them are probably organisms which are also included in the
peptonizing group of organisms. The specific pathogenic organisms,
possibly with the exception of Bad. tuberculosis, get into milk, either
directly or indirectly, from human patients suffering with the particular
disease.

FACTORS INFLUENCING THE DEVELOPMENT OF MICROORGANISMS IN MILK.

The number of microorganisms found in fresh milk shows its bacterial
condition at that time, but it gives little idea of the organisms which may
be found in the same milk at later periods. There are many factors to be
considered if we wish to study the development of the various types
which get into ordinary milk. These factors may be considered briefly
under the following heads:

INITIAL CONTAMINATION. Fresh milk varies widely in the number of
organisms which it contains as a result of the conditions under which it

3*4

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

has been produced. There are differences not only in the numbers of
organisms but also in the species which may be found in different samples
of fresh milk. Both of these factors are important in the later changes
which may take place. The effect of numerical initial contamination
may be seen in the following tables where milk starting out with different
numbers of organisms was kept under similar conditions until coagulation.
Plate cultures made from these three samples show the relative develop-
ment of the number of organisms.

Effect of Initial Contamination on Development of Bacteria and Keeping Quality of Milk.
Milk Having Moderately High Initial Contamination.

Bacteria per c.c. in fresh milk

Bacteria 12 hours

Bacteria 36 hours

Hours to curdling

187,000

432,000

633,500,000

45

Milk Having Moderate Initial Contamination.

Bacteria per c.c. in fresh milk Bacteria 12 hours Bacteria 36 hours Hours to curdling

3,000

14,000

I 49, 6 5; 00

99

Milk Having Small Initial Contamination.

Bacteria per c.c. in fresh milk

Bacteria 12 hours

Bacteria 36 hours

Hours to curdling

3 2 5

i,7 12 .

10,125,000

121

These samples were all kept at a constant temperature of 21
and the difference in the curdling time can therefore be fairly attributed
to the difference in the initial contamination of the three samples.
All three of the samples showed a normal development of the lactic

THE RELATION OF MICROORGANISMS TO MILK.

315

organisms, which constituted over 99 per cent of the total organisms
present at the time of curdling. While this may be considered as showing
the normal effect of the original contamination upon the milk, it is well
to bear in mind the fact that there are many apparent exceptions due to
some particular type of organism predominating and interfering with the
normal development of the lactic types.

STRAINING. The straining of milk is one of the most common opera-
tions in connection with its handling and is considered by most dairymen
as one of the most essential from the standpoint of the quality of the milk.
If milk is strained through cheese cloth or wire gauze much of the insoluble
dirt can be removed. This has led to the general belief that straining
improves the sanitary and keeping qualities of the milk.

The effect of straining on removal of insoluble dirt is shown by the
following results of tests:

Dirt Removed by Passing Milk through Two Thicknesses of Fine Cloth.
(Weight of insoluble dirt given in milligrams per liter of milk.)

Experiment

Before straining After straining Per cent removed

No. i

8. a*

4- ?o

47-5

No 2 .

C. CC

4.<K

10.8

No.?

C. I C

2. (K

42.7

No. 4.

2.4X

O. 2O

oi. 8

No. <;--

s.o?

3 . IO

38.6

It may be noticed that even after straining the milk contained
appreciable quantities of insoluble dirt which had passed through the
strainer cloth. The difference in per cent of dirt removed in different
samples is due to the nature of the dirt itself. The coarser the dirt the
greater the proportion that will be removed by straining.

It is not true, however, that the keeping quality is necessarily improved
by the simple process of straining. It depends largely upon the condition
of the milk and the nature of the strainer. Not infrequently passing milk
through a strainer not only fails to improve its keeping quality but ac-
tually injures it. This has been shown by a number of investigators.
The effect of straining upon the germ content may be seen in the following

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

figures where the milk was passed through a strainer composed of three
thicknesses of fine cheese cloth supported by wire gauze.

Effect of Straining upon Bacterial Content of Milk.

Experiment

Before straining
Bacteria per c.c.

After straining
Bacteria per c.c.

No. i

3,600

3,600

No 2 . . ...

7,400

6 ooo

No. 3

12,800

10 =;oo

No. 4

8 800

II 37^

No. 5

8,800

2 7OO

The effect of straining upon the keeping quality is shown in the fol-
lowing experiments where the milk was strained through the same form
of strainer mentioned above and the samples kept at constant temperature
of 21 until coagulation.

Effect of Straining upon Keeping Quality of Milk.

Not strained
Hours to coagulation

Strained
Hours to coagulation

Experiment No. i

42

42

Experiment No. 2

C7

cc

Experiment No 3

2 e

-if

Experiment No 4

SQ

oj

CA

Experiment No 5

co

O'r
CQ

j^

It will be seen that in no case was the keeping quality of these samples
increased by the straining process while in some cases it was materially
injured.

Cotton filters are more efficient than cheese cloth and in some cases the
keeping quality of the milk may be improved by this process.

AERATION. This is the process of exposing the milk to the atmosphere
by allowing it to run over the surface of the aerator in a very fine film

THE RELATION OF MICROORGANISMS TO MILK. 317

If milk has been produced under such conditions that it has absorbed
foreign odors, this process may be of value in getting rid of the absorbed
odors, but from the bacterial standpoint the process of aerating is not
desirable, since it gives one more opportunity for the milk to become
contaminated with organisms from the atmosphere and from the aerator
itself. It is possible to aerate milk under such conditions that the germ
content will not be increased, but if aeration takes place in the cow stable
or other place where the atmosphere contains dust the number of organ-
isms will be greater after aeration than before, the amount of increase being
proportional to the sanitary conditions under which the aeration is done.
It is even possible that the milk may absorb foreign odors during the
process of aeration and be of poorer quality than it was before. It is
thought by many that the process of aeration is necessary in order to get
rid of the so-called animal odors commonly found in milk. These odors
are, however, not normal to the milk but are absorbed from the foul air
in the stables or other sources. This is shown by the fact that some of the
very finest quality of certified milk is bottled while still containing the
animal heat with the least possible exposure to the air, tightly sealed
at once and plunged into ice water. Such milk contains no suggestion of
animal odor. Aeration may be of value in removing undesirable odors
from milk which is not produced under good sanitary conditions, if done
in an atmosphere free from all dust and odors, but it is not necessary for
milk of good quality. The common belief that aeration is valuable is
probably due to the fact that most aerators are coolers as well, and the
beneficial results are due to the cooling and not the aeration.

CENTRIFUGAL SEPARATION. It is a common practice in some dairies
to pass the milk through a centrifugal separator to remove any dirt which
it may contain. This operation is effective for the removal of much of
the insoluble dirt which may be in the milk, but it is of very doubtful
value from the standpoint of the bacterial content and the keeping quality
of the milk. In spite of the fact that the separator slime is very rich in
bacteria, the milk and cream as they come from the separator will nor-
mally show larger bacterial counts in agar and gelatin plates than will the
milk before it enters the separator. The usual effect upon the germ con-
tent of passing milk through a separator may be seen in the following
experiments:

318

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

Influence of Passing Milk through a Centrifugal Separator upon the Germ Content of the

Milk and Cream.

Bacteria in
whole milk

Bacteria in
skim milk

Bacteria in cream

Sample No. i

30,000

60,000

7"?, ooo

Sample No. 2

44,000

76,000

700,000

Sample No 3

t;6 ooo

7^,OOO

820,000

Sample No 4

200 ooo

"^6,000

730,000

\J\J i

There is usually a marked increase in the number of bacteria which
will develop in plate cultures both from the skim milk and the cream in
excess of the number found in the whole milk. This does not mean that
there is an actual increase in individual bacteria in these samples due to
the action of the separator. What it does mean is that the small clusters or
groups of organisms, as they exist in the whole milk are thrown apart by
the force of the separator and therefore develop individual colonies in the
plate cultures. The effect of this breaking up of the clusters of bacteria
in the milk is also injurious to its keeping qualities. This is probably
due to the fact that the multiplication of the organisms is greater where
they exist in the milk as individuals than it would be had they remained
in their original clusters. So marked is this injurious effect upon the
keeping quality of the milk that in some cases the commercial milk plants
have been obliged to give up the practice of clarifying milk by means of
the centrifugal separator.

TEMPERATURE. The temperature at which milk is kept is one of the
most important factors determining the development of its microbial
content. Every one at all familiar with milk knows that it spoils very
quickly if allowed to stand at warm temperatures. If, however, the milk
is held at temperatures of 10 or lower, the keeping quality of the milk is
greatly increased. Most of the ordinary species of organisms which gain
entrance to milk do not grow rapidly at temperatures of 10 or lower.
There are, however, certain species which will grow with considerable
rapidity at temperatures below 10, especially some of the spore-bearing
non-acid forms. If the temperature of the milk is allowed to rise above
10, the growth of the common species increases rapidly. The influence
of temperature upon the development of bacteria may be seen in the fol-

THE RELATION OF MICROORGANISMS TO MILK.

3*9

lowing experiment where a given lot of milk was thoroughly mixed and
divided into six portions, which were then held at the temperatures
indicated for twelve hours, at the end of which time they were plated for
the total germ content.

Effect of Different Temperatures upon the Development of Bacteria in Milk.

Temperature maintained
for 12 hours

Bacteria per c.c. at end
of 12 hours

Hours to curdling
at 21

4.5

4,OOO

75

7

9,OOO

75

10

l8,OOO

7 2

12.5

38,000

49

15-5

453.00

43

21

8,800,000

52

26-5

55,300,000

28

The fresh milk showed a count of 5,000 per c.c. and curdled in fifty-
two hours at a temperature of 21. The curdling time of these samples
was determined by placing them at a constant temperature of 21 at the
close of the twelve-hour period and holding them at this temperature
until coagulation took place. The difference in time of curdling there-
fore is due to the maintenance of the special temperature for 12
hours only and not for the entire period up to the time of curdling.

PASTEURIZATION. The term pasteurization is used to designate the
process of heating milk to a temperature sufficient to destroy a portion of
the bacteria and then cooling it to a temperature which will prevent the
rapid development of the organisms that are left. The temperatures
commonly used for this purpose vary from 60 to 85. The
length of time the milk is exposed to the high temperature may also
vary from a few seconds to thirty minutes, depending upon the method
employed. The two chief purposes for the pasteurization of milk are to
increase its keeping quality and to destroy any pathogenic organisms which
the milk may contain. The purpose for which the pasteurization is done
will determine the method used. In commercial pasteurization, where
the chief purpose is to destroy the lactic organisms and thus improve

320 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

the keeping quality of the milk, the method used is that known as the
continuous or instantaneous method, where the milk is subjected to a
high temperature for a few seconds only and then cooled. In this method
of pasteurization varying degrees of efficiency are obtained, depending
upon a number of factors, chiefly the bacterial condition of the milk
to be pasteurized, the degree of heat and the length of the exposure and
the temperature to which the milk is cooled. With all these factors to be
considered it is not surprising that the germ content in the pasteurized
milk varies widely. In the instantaneous method of pasteurization, the
lactic organisms are killed first, while any spores which are present
are allowed to pass through the machine and remain in the milk. These
spores may result in the rapid development of certain putrefactive types-
of organisms in the pasteurized milk, until its bacterial condition at the
end of a few hours may be worse than before pasteurization. If, however,
the milk was of reasonably good quality, the process carefully done and
the milk properly cooled, the bacterial quality of the milk may be materi-
ally improved as a result of the pasteurization. This method of pasteur-
ization cannot be depended upon to kill all of the disease organisms which
may be in the milk.

Where the chief purpose of pasteurization is to render the milk free
from disease-producing organisms, the so-called holding method is
employed. This consists in raising the temperature of the milk to about
60 to 63 and holding it at this temperature for a period of twenty to
thirty minutes. If this method is properly done, all of the organisms
except certain spore forms should be killed and the milk at the end of
the pasteurizing process contain but few organisms compared with its
original germ content.

It is believed by many that heating milk to a high temperature hastens
the growth of any organisms which may remain in it or afterward gain
entrance. Others maintain that this is not true. The weight of evidence,
however, seems to indicate that it is a fact. This being the case it is
evident that extreme care should be taken with pasteurized milk to pre-
vent subsequent contamination and also the development of any organ-
isms which may not have been killed by the pasteurizing process.

THE USE or CHEMICALS. The addition of certain chemicals to milk
will retard the growth of bacteria. The chemicals most commonly used
for this purpose are borax and formalin. While the keeping quality
of milk may be materially increased by the use of such chemicals, their

THE RELATION OF MICROORGANISMS TO MILK. 321

use has been opposed by health authorities and is contrary to the Pure
Food Laws. If milk is handled with any degree of care, there should be
no need for the use of chemical preservatives. They are simply a means
of counteracting the unsanitary conditions of the production and handling.
The same results can be obtained by cleanliness in the production of the
milk and the use of low temperatures for preventing the contamination
and subsequent growth of the bacteria in the milk. The developments
in the production of clean milk of the past few years have illustrated very
clearly that the use of chemical preservatives is not necessary.

NORMAL DEVELOPMENT OF MICROORGANISMS IN MILK.

The flora of any particular sample of fresh milk is determined by the
conditions under which it is produced. In stables where extreme clean-
liness is practised the flora may be practically limited to those species
which occur in the udder of the cows, but under ordinary conditions there
will be in addition to the normal udder types such others as may occur
in the dust and atmosphere of the stables. Market milk, therefore,
when first obtained from the cow ordinarily contains a mixed flora, the
different types present depending upon the sanitary conditions under
which the milk is produced.

The future development of this initial flora is largely dependent upon
the temperature at which the milk is kept. If the milk is held at tempera-
tures between 10 and 21 there will result what may be considered as the
normal development of milk fermentations. These changes may be
divided for convenience into four periods or stages.

FIRST STAGE. GERMICIDAL PERIOD. It has been shown by a number
of investigators that instead of an increase in the numbers of bacteria in
fresh milk there is normally a decrease in the number during the first few
hours after its production. The rapidity of this decrease and the length
of time over which it extends seem to be determined largely by the tem-
perature at which the milk is kept. The higher the temperature the more
rapidly the number of organisms decreases and the more quickly the end
of the germicidal period is reached. If the temperatures are kept fairly
low the rate of decrease is much slower but the decline will extend over a
considerably longer period. This is shown by the following examples
given by Hunziker.

21

322 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

Table Showing the Germicidal Action in Cows' Milk.

Name

Temp.* After After After

After

After

After

After After

L/OW

of

of

3

6

9

12

IS

24

32

48

COW

warm

milk hours hours hours hours hours hours

hours hours

f

40

i, 080

1,220

1,040

I.O20

1,120

1,360

1,040

400

May

1,212 \

55

1,260

I,4OO I.5OO

1,460

1,360

i, 080

3,500; 17,740

I

70

1,000

1,340

1, 860

3,460

3.460

64,000

800,000

f

40

4,400

4,260 i 3,62O

3,7oo

3,900

4,000

3,900

3,840

Ida

5,120 \

55

3,900

3,460 2,980

2,800

2 , 9 2 3,26o

3,220 3,240

I

70

3,560

2.I2O

1, 880

i, 880

1,240 4,960

58,400

[

40

1,170

1,070

1,120

870

1,120

990

1, 060 1, 080

Julia

1,345 \

55 1,080

990 980

1,400

1, 080 1, 080

3,110 68,800

(

70

1,000

I.OOO I.20O

5,6oo

17,720

1,600,000

*Fahrenheit.

The exact reason for this decline is at present not well understood.
Some investigators believe that milk possesses a certain germicidal action
or property which results in the destruction of a portion of the organisms
found in the milk at the outset.

The work of other investigators seems to show that the so-called
germicidal action is felt by certain species and not by others as is indicated
by the following sample.

Age of milk

Total
bacteria

Acid
bacteria

Per cent acid
bacteria

Liquefying
bacteria

Fresh

12, =^0

1.2 W

10

200

3 hours

I2.2CJO

2,000

16

200

6 hours

10,6^0

2.2CJO

23

800

o hours

c6,ooo

2O,2 SO

36

^o

1 2 hours

114,2^0

68,4OO

60

I.OOO

This would seem to indicate that the decrease in number is due not so
much to a definite germicidal property possessed by the milk as to the
gradual dying out of certain species which for some reason do not find the
milk a suitable environment for development, while other types, finding
the milk suitable to their needs, develop uniformly from the start.

THE RELATION OF MICROORGANISMS TO MILK. 323

SECOND STAGE. PERIOD FROM END or GERMICIDAL ACTION TO
TIME OF CURDLING. The period following immediately after the ger-
micidal action is characterized by the rapid development of the lactic
organisms. Under normal conditions this group develops much more
rapidly than any other type. Not only do they increase rapidly in
actual numbers but their percentage also rises rapidly. There may
be a continual increase in numbers in the other species, but their growth
is much less rapid than that of the Bad. lactis acidi type. As this period
advances certain of the miscellaneous types may cease to grow entirely.
During this time the gas-producing acid organisms of the B. coll and
Bad. ladis aerogenes type may develop more or less rapidly, but if the milk
is held at temperatures not much above 20, the Bad. ladis acidi type will
develop much more rapidly, so that by the time the milk becomes sour
and curdles, this type will constitute 99 per cent approximately of the total
number in the milk. From the standpoint of the milk consumer milk
ceases to be of value when the end of this period is reached, but there are
further developments which are of importance in certain lines of dairy
manufactures, notably cheese making.

THIRD STAGE. PERIOD FROM TIME OF CURDLING UNTIL ACIDITY
is NEUTRALIZED. At the time milk curdles it contains enormous num-
bers of the lactic bacteria. The number usually runs into the millions
and may be even higher than one thousand million per c.c. By the
time the coagulation takes place the acidity of the milk is so high that
the growth of the lactic organisms is checked and from this time on
their number decreases with more or less rapidity.

During the period following the curdling certain other types of
organisms which have existed in the milk during the earlier stages now
begin to grow. The organisms especially important in this stage are
Oidium ladis, certain species of molds, and yeasts. These organisms
are able to grow in a highly acid medium, and as a result of their devel-
opment the acid is decreased until the milk finally presents a neutral
or alkaline condition resulting from the decomposition of the proteins
in the milk.

FOURTH STAGE. FINAL DECOMPOSITION CHANGES. The reduction
of the acidity affords favorable conditions for the growth of certain types
of organisms which have remained in the milk during the earlier stages
but have been practically dormant. In this fourth stage the conditions
are suitable for the growth of the liquefying and peptonizing bacteria

3 2 4

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

and they now grow rapidly, causing the decomposition of the casein.
The changes resulting from this type of organisms are of special signifi-
cance in cheese making and are discussed more fully in another chapter.

ABNORMAL FERMENTATIONS IN MILK.

GASSY FERMENTATION. It frequently happens that instead of the
normally rapid development of the Bact. lactis acidi type of organisms
in the milk, other acid producers develop rapidly, with the production
of more or less gas. The organisms most prominent in this type of
fermentation are the B. coll communis and the Bact. lactis aerogenes types.
This group of organisms contains a number of varieties, some of which
produce little or no gas while others develop large amounts. Their
action in milk is usually accompanied by disagreeable odors and flavors.
They grow readily in the presence of air and therefore develop abundant
colonies on the surface of plate cultures. This distinguishes the members

of this group quite clearly from those of the
true lactic group which grow chiefly below the
surface of the medium. The members of this
group do not form spores, but certain varieties
are quite resistant to heat and will oft-times sur-
vive pasteurizing temperatures which completely
destroy the Bact. lactis acidi group. They grow
most rapidly at high temperatures between 20
and 37.

SWEET CURDLING FERMENTATION. This
phenomenon is caused by a variety of organisms
which cause the milk to coagulate without the
production of acid. The coagulation is brought
about by a rennet-like enzyme produced by this
type of bacteria. The resulting milk is either
neutral or alkaline in reaction. Usually the

coagulation of the milk is followed by the digestion of the casein as a
result of another enzyme which is also produced by these bacteria. The
coagulation caused by these organisms is slower than in the case of the
acid formers and the curd is usually soft and mushy as compared with
the curd formed in the normal acid fermentation. The members of this
group get into the milk from and along with dust and dirt associated with

FIG. 78. Ropy cream
lifted with a fork. (After
Ward.)

THE RELATION OF MICROORGANISMS TO MILK.

3 2 5

unsanitary conditions. Some of the species produce spores and are not
killed by the ordinary methods of pasteurization. This fact accounts
for the occurrence of sweet curdling of pasteurized milk. This group
of organisms is unable to develop rapidly in the presence of the lactic
bacteria and for this reason we do not commonly get the sweet curdling
of raw milk. The presence of these organisms is evidence of unsanitary
conditions. Frequently they develop very disagreeable flavors in the
milk.

ROPY OR SLIMY FERMENTATION. One of the most common milk
infections causing trouble to the milk dealer is that which causes a ropy

%"'"" ',*'
OV^

-.^i..k*f.-;i-7,

^

FIG 79. Bacillus lactis viscosus from a milk culture.
(After Ward )

or slimy fermentation of milk. This is sometimes spoken of as stringy milk
(Fig. 78). Several species of organisms are capable of producing this con-
dition. These organisms grow most freely in the presence of an abundant
supply of oxygen and for this reason the cream usually becomes slimy
before any changes are apparent in the underlying layers of milk. B.
lactis viscosus is perhaps the most common species in this group. The
slimy condition in the milk is supposed to be the result of a very viscid
capsule surrounding these organisms (Fig. 79). Representatives of this
group are quite resistant to heat and frequently pass uninjured through
the methods of cleansing and scalding used under ordinary dairy condi-

326 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

tions. Because of this, dairy utensils once infected become a constant
source of infection. This trouble can be effectively stopped by a
thorough scalding of all utensils coming in contact with the milk.

BITTER FERMENTATION. Bitter flavors in milk may be the
result of bacterial changes after the milk has been drawn, or due to
certain strong feeds which the cows have consumed. If the cows
are allowed to eat certain kinds of vegetation, such as "rag weed"
and certain other plants, they may impart a bitter taste to the milk,
in which case the abnormal flavor will be apparent when the milk
is fresh and usually becomes less pronounced as the milk becomes
older, because of the volatile nature of the substances causing the
bitterness. Most of the cases of bitter milk and cream, however, are
due to the growth of certain types of bacteria in which case the bit-
terness increases in intensity with the age of the milk. Some of the
species capable of producing bitter milk grow at quite low temperatures,
which accounts for the fact that the most trouble with bitter flavors is
found in milk and cream which has been held at low temperatures for
some time.

ALCOHOLIC FERMENTATION. The bacteria as a group are not able to
act on the milk sugar and produce alcohol, but it sometimes happens that
yeasts get into the milk in sufficient numbers to ferment the milk sugar,
producing appreciable amounts of alcohol. To the milk handler this
trouble is not usually serious but the action of the yeasts is frequently
of considerable importance in the cheese industry.

OTHER FERMENTATIONS. It frequently happens that a considerable
variety of disagreeable flavors and odors develop in milk. These may
be due to the direct absorption of odors from the foul stable atmosphere
or strong smelling feeds, such as silage; or they may be, and no doubt
frequently are, the result of the growth of certain types of bacteria which
have entered the milk from dirty surroundings. The growth of some
of these organisms is frequently the cause of the so-called cowy and
stable odors and flavors.

COMMERCIAL SIGNIFICANCE OF MICROORGANISMS IN MILK.

RELATION OF DIRT CONTAMINATION TO GERM CONTENT. To the
commercial milkman bacteria are of importance only as they influence
the length of time the milk will keep in a salable condition. The consumers

THE RELATION OF MICROORGANISMS TO MILK.

3 2 7

do not want milk that is sour or has unpleasant flavors and odors. In
order to sell his milk, therefore, the milkman must avoid the presence
of these undesirable conditions, and in proportion as he recognizes the rela-
tion between germ life and the quality of his product, will he pay attention
to the presence and development of microorganisms in his milk. In
like manner, the presence or absence of dirt contamination is important
from the commercial standpoint since it bears a relation to the bacterial
count, and, therefore, affects the keeping properties of the milk. Under
normal conditions there is a fairly direct relation between the amount of
visible or insoluble dirt and the number of bacteria found in any given
lot of fresh milk. This relation may be shown by the following samples
taken from four different milk producers:

Producer

Number of
samples

Average mg.
dry dirt per
liter

Average num-
ber bacteria per
c.c.

Average hours
to time of curd-
ling

A

c

ei . f

1 1 c; ooo

17^

B

16

<;8 8

273 600

78

c

21

70 o

428 6OO

/ u
7C

D

17

71 Q

QAQ AOO

/ J

68

This relation does not always hold for the reason that a gram of one
kind of dirt may contain infinitely more organisms than an equal amount
of some other kind. The difference in the solubility of various forms
of dirt always causes apparent discrepancies in this normal relation. In
the majority of cases, however, the relation shown in the above examples
will hold reasonably true in the case of fresh milk. There is also a general
relation between the number of bacteria in fresh milk and the length
of time it will keep before souring and curdling. In this case the relation
is in inverse ratio, the smaller the initial contamination, the longer the
keeping time, and vice versa. This relation is also shown in the table
given above. There are many irregularities, however, in this relation
because of differences in the flora of fresh milk. It may frequently
happen that a sample of milk containing a relatively high number of
organisms will not sour as quickly as another sample with a smaller
original germ content. The associative action of the different species

328 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

of organisms is an important factor here. In making comparisons of
this sort, it is, of course, necessary that the different samples be held at
the same temperatures.

MILK AS A CARRIER or DISEASE-PRODUCING ORGANISMS.

It is not the purpose of this chapter to discuss in detail the diseases
which may be carried by milk, but a chapter on bacteriology of milk
would be incomplete without a brief discussion of this important subject.

From the standpoint of their relation to the health of the consumer
the microorganisms in milk may be divided into three groups.

THOSE MICROORGANISMS WHICH ARE BENEFICIAL AND DETRIMENTAL
TO HEALTH. Acid Forms. The preservative properties of sour milk
have been known since very ancient times. Its use as a preservative
for meat, eggs and other perishable food products demonstrates the value
of sour milk as a means of preventing decomposition. It has also been
known for a long time that sour milk has a certain therapeutic value
because of the action of the lactic bacteria in preventing harmful
fermentations in the digestive tract. More recently the work of Metch-
nikoff has shown the usefulness of sour milk both for the treatment and
prevention of intestinal disorders by preventing the development of the
putrefactive bacteria in the digestive tract. In view of the value of sour
milk for preventing certain forms of disease and its inhibiting action on
certain undesirable organisms the Bact. lactis acidi type of bacteria must be
regarded as beneficial organisms, and from the standpoint of the health of
the consumer their presence in the milk is to be welcomed rather than
discouraged. As the value of sour milk drinks becomes better known
the importance of this group of milk bacteria will be more fully recognized

Neutral Forms. In ordinary milk there is a large class of bacteria
which, so far as known, have no appreciable effect either upon the com-
position of the milk or the health of the persons consuming it. This group
includes a number of species, many of them being coccus forms,
some of them appearing in plate cultures as chromogenic colonies.
They grow more or less freely in milk, depending upon the conditions,
but they are usually held in check by the acid-forming bacteria and do
not constitute a very important part of the flora of normal milk. They
are, therefore, of little significance from the practical standpoint except
as they indicate the conditions under which the milk has been produced
and handled (p. 312).

THE RELATION OF MICROORGANISMS TO MILK. 329

Injurious Organisms. The diseases which may be carried by milk
are of two classes.

Epidemic Diseases. The human diseases most commonly carried
by milk are typhoid fever, diphtheria and scarlet fever and occasionally
other diseases such as cholera and foot-and-mouth disease. The first
three are by far the most important of milk-borne diseases. The
outbreaks of typhoid fever which are traceable to milk occur most fre-
quently. There is a large accumulation of data showing the occurrence
of epidemics caused by infected milk. An epidemic caused by the milk
supply has certain characteristics which distinguish it from epidemics
resulting from other causes. A considerable number of cases of the
particular disease will appear almost simultaneously and will be dis-
tributed along some particular milk route. Usually the epidemic stops
as suddenly as it began except for a few secondary cases contracted from
those first taken. The source of the disease organisms is a human
patient suffering from the disease. The infection of the milk may be
direct, as when a sick person handles the milk, or it may be indirect as
when a person caring for a patient also works about the milk. In other
cases it may be caused by contamination of the water used in washing the
utensils or by cows wading in water of infected streams and getting the
organisms on their body whence they fall into the milk pail at milk-
ing time. Unfortunately the specific organisms of these diseases grow
readily in milk and a small infection is all that is necessary to render the
milk dangerous by the time it reaches the consumer. The return of
milk bottles from the sick room sometimes is the means of infecting the
milk supply.

Non-epidemic Diseases. There is another class of diseases which
may be carried by milk which are not characterized by a sudden
outbreak, and for this reason are not so readily recognized as being
associated with the milk supply. One of these diseases, namely tuber-
culosis, is caused by a specific, well-known organism, Bact. tuberculosis,
which may get into the milk from the udder of a tuberculous cow or by
the organisms which have been given off from the digestive tract of the
animal becoming scattered about the stable and finally getting into the
milk with particles of dust and filth. In some cases the milk may become
infected by persons having the disease being permitted to handle the milk.
Fortunately for mankind Bact. tuberculosis does not multiply in milk.

Regarding the danger of contracting tuberculosis from the use of

330

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

milk there is at present much difference of opinion, but the consensus
of opinion at the present time seems to be that there may not be very

MmxccpoX K\X\v SU\\Xo\vs
SYmv Chart

600
80
60
AO
20
500
80
60
40
20
400
80
60
40
20

300
80
60
40
20
200
80
60
10

20
IQ.

Jon

Feb-

Mar.

Apr

May

Jun

July

Aug.

5ep

OCT.

Nov.

3ec.

600

40
2.0

\

\

\

500
80
60
40
20
400
80
60
40
->

\

\

1

\

\

\

\

\

\

1

Pv

\

300
80

\

/

5

\

\

/

\

60
40
20

s,

/

i

\

^

\

1

S

\

/

'\

<J

i

N

b

200
80

o

-y

\

\

\

/

\

60

S^

J

\

p

<

40

V

20

|00

FIG. 80.

great danger for healthy adults, but that a considerable percentage of
the cases of tuberculosis of children may be traced to infection from the
milk supply.

THE RELATION OF MICROORGANISMS TO MILK. 33!

There is another class of disorders not so well defined as the above
but which are nevertheless of great importance from the standpoint of
public health, especially of young children and also to some extent of
adults. This group includes such disorders as infantile diarrhoea, summer
complaint, cholera infantum and other disorders of the digestive tract.
The organisms producing these troubles doubtless belong to the group
of putrefactive bacteria which come from filth. Some of the gas pro-
ducers and some of the peptonizers are probably responsible for these
troubles. Shiga isolated from a large number of cases of infant diarrhoea
a bacterium which he named Bad. dysenteric, but in general the specific
organisms responsible for these intestinal troubles are not well known.
Their importance, however, is shown by the relation of the germ content
of milk to infant mortality (See Fig. 80).

BACTERIOLOGICAL ANALYSIS OF MILK.

The development of our knowledge of the relation of bacteria to the
wholesomeness of foods has led to a study of the bacterial content of milk
as a means of determining its wholesomeness. The methods used for this
purpose have followed quite closely those of the water bacteriologists.

PLATING METHOD. The early workers in milk bacteriology attempted to deter-
mine only the number of organisms in the milk. This was done by plating in nutrient
agar. From the results of this analysis they attempted to judge the sanitary quality of
the milk. By this method the number of colonies developing in the plates is assumed to
represent the germ content of the milk. It should be borne in mind that such counts
are only approximate and always less than the actual number of organisms in the given
sample of milk. Even if the best methods are employed it is not possible to determine
the exact number of bacteria in any lot of milk.

THE DIRECT MICROSCOPIC METHOD. The plating method is expensive because of
the large amount of time and materials needed. It is not possible for one person to
handle a large number of samples at one time. In routine work in the city laboratories
this labor has been a serious drawback to this method. In order to decrease the
labor and give greater possibilities to the work Stewart devised a method by which the
bacterial condition of milk can be studied by direct microscopic examination. His
purpose was to determine only the species present, but later Slack developed the method
so that it Is now used in some cities for determining the approximate numbers and at
the same time the general species present in a given sample of milk.

LEUCOCYTES. The microscopic examination of milk sediment revealed the fact
that freqently a sample would be found which showed the presence of leucocytes in
greater or less numbers. The presence of these cells was regarded as important because

332 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

it was assumed that they showed the presence of inflammation and pus formation in the
udders of the cows producing the milk.

Several methods have been used for determining the leucocyte content of milk.
"The Smear Sediment" and "Blood Counter" are methods which more strictly belong
to laboratory practices and will not be considered in this place.

BACTERIOLOGICAL MILK STANDARDS.

Thtyelation of the bacterial content of milk to its wholesomeness has
led to the adoption of certain standards by the boards of health in our
cities. These standards recognize the fact that the germ content of milk
in the large cities is greater than in the smaller ones because of the greater
distance from which it is shipped and its age on arrival in the city. New
York City in 1900 adopted a maximum limit of 1,000,000 per c.c. Later
Boston established a limit of 500,000. Chicago's limits are 1,000,000
from May to September, inclusive, and 500,000 from October to April,
inclusive. Rochester's standard is 100,000. Other cities have similar
standards.

Stokes' standard for the number of leucocytes permissible in normal
milk was 5 per field of the 1/12 objective in his smeared sediment prepara-
tion. Bergey found so many samples running above this number that
he made the limit 10 cells per field and felt that no milk containing more
than this number should be used for food. Later Slack raised the limit
to 50 cells per field. The reason for changing the standard was due partly
to the larger numbers found as a result of improved methods but more
especially to the discovery that milk from apparently healthy cows nor-
mally contains leucocytes in excess of the first standards set.

It is held by some that a numerical standard is of little value since the
actual number of organisms present in a given lot of milk may not be a
correct measure of its wholesomeness. For this reason some cities pay
little attention to the numbers of bacteria present but base their stand-
ards wholly on the species, and the quality of the milk is judged on the
presence and numbers of streptococci, B. coli, leucocytes, sediment.
Milk is passed or condemned on the basis of any one or combination of
these conditions.

In recent years there has been a tendency to combine these two
standards using the total germ content as a measure of the care the milk
has had and the presence or absence of certain groups or species as an
indication of the occurrence of pathological conditions in the cows pro-
ducing the milk. The practice in most city laboratories now is to make

THE RELATION OF MICROORGANISMS TO MILK. 333

use of both the numbers and the species present in determining the
quality of the milk supply.

VALUE OF BACTERIOLOGICAL MILK STANDARDS AND ANALYSES.

Regarding the value of bacteriological standards for milk there is still
some difference of opinion among milk bacteriologists. The germ con-
tent of any lot of milk is largely dependent upon three factors: the
number of organisms getting into the fresh milk; the temperature at
which it is kept; the age of the milk when analysis is made.

The high bacterial count in any lot of milk may be the result of any
one of these conditions or a combination of them. A high count means
that there has been carelessness either in the production, resulting in high
initial contamination, or in the subsequent handling permitting a rapid
multiplication of the organisms, or that the milk is old.

On the other hand milk with a low germ content can be obtained only
where the original contamination is small and it has been held at low
temperatures. A low count, therefore, means care both in the production
and later handling of the milk.

While the germ content may be regarded as a general index to the
care the milk has received, it may not at all indicate its wholesomeness.
A high count may be the result of the rapid growth of the lactic bacteria,
in which case the milk may be perfectly safe and wholesome. On the
other hand the count may be quite small but contain pathogenic species.
The bacteria count is valuable as showing the sanitary conditions of
production and handling, but much care should be used in the interpreta-
tion of such results. In some ways a direct microscopic examination of
the milk sediment is much more satisfactory. The skilled analyst can
recognize certain types which may indicate the sanitary quality of the
milk. With sufficient experience one can recognize streptococci, other
groups and leucocytes. The presence and abundance of one or more
of these groups may indicate the nature of the original contamination
and the existence of diseases in the udders of cows. If rightly interpreted
the information thus obtained is of much value. The weakness of this
method lies in the fact that it is not always possible to recognize the above
types of organisms. In a smear preparation it is not possible to differenti-
ate between pathogenic and non-pathogenic streptococci or between
B. coli and certain other types. The presence of unusual numbers of

334 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

streptococci and pus cells may indicate the existence of disease in the
cows and when this condition is found in the city milk it may be possible
to trace it back to the farm and locate the diseased cow and prevent her
milk from being used for human consumption.

The tendency at present is to combine the quantitative and qualitative
analyses and the results thus obtained in the hands of the careful worker
are of much practical value in controlling the quality of a city's milk
supply.

CHAPTER II.*

THE RELATION OF MICROORGANISMS TO BUTTER.

Butter is the fat of milk that has been largely freed from the other
constituents of milk by the processes of creaming and churning. If
milk is allowed to stand, the fat, which is in the form of minute globules,
accumulates in the upper layers of the milk because its specific gravity
is much lower than that of milk serum. In modern practice the fat is
concentrated in a portion of the milk by passing the milk through a cream
separator. In the rapidly revolving bowl of the separator the centrif-
ugal force exerted is many times greater than that of gravity and the fat
is rapidly and efficiently removed. The cream, which is obtained by
these methods, contains varying amounts of fat which is further concen-
trated, by subjecting it to agitation in the churning process. The globules
of fat cohere to form larger and larger masses until the entire amount of
fat is brought into a single mass, the butter.

TYPES OF BUTTER.

SWEET-CREAM BUTTER. If little or no increase in the acidity of the
milk or cream develops, previous to churning, the butter will have certain
marked characteristics and is called sweet-cream butter. It is especially
characterized by its low flavor, since it has only the flavor of the fat of
milk which is not marked. This is usually known as the primary flavor
of butter. Sweet-cream butter is also marked by the rapidity with which
it undergoes decomposition changes, especially when it is made from
raw cream.

SOUR-CREAM BUTTER. If the cream is allowed to undergo the acid
fermentation, the butter will differ markedly both in degree and kind of
flavor from that prepared from sweet cream, and as a rule its keeping
qualities are much better than those of sweet-cream butter. This type
of butter is made throughout northern Europe, England and her colonies,

* Prepared by E. G. Hastings.

335

336 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

and in America. It may be said to be the standard butter of the world
since it is the type made in all the great dairy countries. Sweet-cream
butter is made especially in southern Europe, and in limited amounts in
other countries.

The intensity and kind of flavor of butter is thus dependent on the
acid fermentation of the milk or cream. It is not believed that the fat
undergoes any changes during the acid fermentation of the milk which
could produce the flavor of sour-cream butter, but rather that the increase
in flavor is due to the absorption by the butter fat of certain of the com-
pounds formed in the acid fermentation. It is not essential that the fat
be present during the acid fermentation in order to impart flavor to the
butter. If sweet cream is mixed with sour milk and churned at once,
the flavoring compounds are absorbed by the fat from the fermented
milk, and the butter will have much the same flavor, both as to intensity
and kind, as though the fat had been present during the fermentation.
The churning of a mixture of sweet cream and sour milk is used commer-
cially and is identical with the methods employed by the manufacturers
of oleomargarine and renovated butter to impart flavor to the flavorless
fats they employ. It is impossible to recognize these substitutes for butter
by their flavor since it is identical with and derived from the same source
as the flavor of butter.

In the past many ideas have been expressed as to the source of the
flavor of butter; some have asserted that it is due, in part, to the decom-
position of the proteins of milk by proteolytic bacteria. Both practical
experience and experimental work have demonstrated the connection
between the acid fermentation of milk and the flavor of butter, and it
is certain that what is now considered the finest type of butter can be
made from cream in which only acid-forming bacteria (see Chapter I)
have grown.

FLAVOR or BUTTER.

CONTROL OF BUTTER FLAVOR. The commercial value of any sam-
ple of butter is largely determined by its flavor. If it is lacking in flavor
and aroma, or if it has a poor flavor, it brings a low price. The impor-
tance of being able to control the flavor, both as to degree and kind, in the
manufacture of butter has increased greatly in recent years, because of
the introduction of the creamery system, which has largely supplanted
the making of butter on the farm. The financial success of any creamery

THE RELATION OF MICROORGANISMS TO BUTTER 337

is largely dependent upon the ability of the butter maker to control the
flavor of the product, so that it shall be uniform from day to day. It is
asserted that one of the factors in the remarkable invasion of Denmark
into the butter markets of the world is the uniformity of the Danish
butter, not only from a single creamery, but from all the creameries of
the country. To the Danes we owe the most improved methods for the
control of the flavor of butter.

The other points, texture, color and salt, which the judge of butter
takes into consideration, can be easily controlled, since they are due to
mechanical operations. The flavor, on the other hand, is due to the by-
products which are formed by microorganisms in the fermentation of the
milk and cream, and which are absorbed and held by the fat. If any of
the products formed possess a disagreeable taste or an offensive odor, the
flavor and aroma of the butter will be impaired. It is thus evident that
the control of the flavor of butter is dependent on the control of the acid
forming bacteria that ferment the milk and cream. This is the problem
of the modern butter maker and the modern methods seek to give him
this control, to enable him to eliminate the undesirable bacteria, Bact. coli
aerogenes, the second group,* and to insure the predominance of the desir-
able bacteria, Bact. lactis acidi. This general statement is not to be inter-
preted as meaning that all bacteria that injure the flavor of butter are
to be included in the group mentioned, for many other types of bacteria,
when present in milk in large numbers, may injure the flavor of the butter
prepared from it.

The acid fermentation of the cream is most frequently called the
ripening of cream and sour-cream butter is frequently called ripened-
cream butter. The ripening of the cream not only increases the flavor
of the product, but it enhances its keeping quality. The ripening of the
cream also aids in the mechanical process of churning, the sour cream
churning more easily and with less loss of fat in the butter milk.

KINDS AND NUMBERS or BACTERIA IN CREAM. The number and
kinds of bacteria found in cream are dependent upon the number and
kind in the milk from which the cream is obtained. The cream will,
however, contain a greater number of bacteria per unit volume than the
milk, since the immense number of fat globules passing through the milk
serum carry mechanically a considerable proportion of the bacteria of
the milk into the cream. This phenomenon is to be noted in gravity

* See Chap. I, Div. IV, in which the groups of bacteria are considered.
22

338 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

creaming, but to a much greater extent in the removal of the cream by
use of the separator.

SPONTANEOUS RIPENING OF CREAM. By this expression is meant
the fermentation of the cream by those acid-forming bacteria that have,
from one source and another, gained entrance to it, but which have not
been intentionally added. Under these conditions the butter maker can
exert but little control over the fermentation. A very considerable part
of the butter made from such cream has an excellent flavor, because at
the temperature at which cream is usually kept, Bact. lactis acidi and
related organisms are the primary factors concerned in its fermentation
and their by-products produce desirable flavors in butter. It is often
asserted that the highest type of butter can be made only from spon-
taneously ripened cream.

As the cream from many farms was assembled at a creamery for the
manufacture of butter, it became evident that some means of controlling
the type of fermentation in the cream was needed. If the milk had been
produced under clean conditions, and had been received at the creamery
before the acid fermentation had gone on to any extent, and if the cream
was then kept at temperatures most favorable for the lactic bacteria, the
product was likely to be of good quality, but such ideal conditions did
not always obtain. Cream containing a large proportion of harmful
bacteria, or in an advanced state of fermentation, or possessing an unde-
sirable flavor was often received, and the butter maker could not control
the quality of the product under such conditions.

USE OF CULTURES IN BUTTER MAKING. As the science of microbiology
progressed and the role of microorganisms in all kinds of fermentation
became known, it was evident that the control of the causal organism is
an important factor in determining the quality of any product of the
fermentation industries. In the manufacture of butter, the first step in
this direction was the addition of some fermented milk, cream, or of
buttermilk, to the cream to be ripened. In this manner the number of
acid-forming organisms in the cream was greatly increased, and the fer-
mentation went on more rapidly and in more definite directions than
without such additions, as the bacteria added were largely of the desirable
group, Bact. lactis acidi. The addition of fermented milk to accelerate
the souring of cream antedates by many hundred years the science of
bacteriology.

The next logical step in the development of the process was the use of

THE RELATION OF MICROORGANISMS TO BUTTER. 339

the same types of bacteria from day to day. Cultures of these were obtained
by allowing a quantity of milk to sour, and if it had the desired flavor, a
small amount of it was added to another quantity of milk that had been
heated, in order to destroy the acid-forming bacteria it contained. By
the daily preparation of some heated milk, and the inoculation of it with
the soured milk previously prepared, the butter maker could use the same
types of bacteria for an indefinite time for addition to the cream.

It had been found by Hansen that, in order to control the flavor of
beer, pure cultures of yeasts must be used for the fermentation of the
wort. The success of this method in the brewing industry led to the
introduction of pure lactic cultures for the fermentation of cream. The
use of such cultures was suggested independently by Storch, a Danish
bacteriologist and by Weigmann, the director of the dairy experiment
station at Kiel in Germany, in 1890. Many cultures were isolated and
tested as to their effect on the flavor of butter. Those found to be de-
sirable could be maintained in the laboratory, and could be furnished to
butter makers to be used and propagated in a manner similar to the
method employed with the impure and less constant home-made starters.
The pure cultures of lactic bacteria are widely used at present in the
butter-producing countries of the world and their use is being constantly
extended, as butter makers come to recognize the importance of con-
trolling the ripening of cream.

It was found that the butter made from cream ripened by pure lactic
cultures did not possess as high a flavor as did the finest butter made
from naturally ripened cream. This led to the search for organisms
that could be used alone, or together, with the lactic bacteria, and which
should give the high flavor desired. Such cultures were found, but
their use did not prove practical, either because they did not maintain
their properties on continued cultivation, or because of their effect on
the keeping quality of the product. The difference in flavor in the case
of butter made from naturally ripened cream and that from cream ripened
by pure lactic cultures is undoubtedly due to the products of the B. coli-
aero genes group.

The acid in spontaneously soured milk is very evident to the taste
when the acidity is 0.6 per cent and above; the volatile acids formed by
the members of the colon-aerogenes and coccus groups impart a sharp,
pungent taste. In milk of like acidity fermented by pure cultures of
Bact. lactis acidi, the acid is scarcely evident to the taste and there is no

340 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

sharpness, due to the absence of volatile acids. This same difference
appears in the butter made from the two kinds of milk.

The low flavor of the butter made from cream ripened by pure cultures
was one of the factors that prevented the rapid introduction of the cultures
in this country. The demands of the butter market have changed and
the mild flavored butter, which is now considered to be the finest, can
be made by the use of pure cultures in the fermentation of pure sweet
cream.

COMMERCIAL CULTURES. In this country the preparation and dis-
tribution of cultures for the ripening of cream is largely in the hands of
commercial firms; hence, the term "commercial culture" is applied to
them. The different pure cultures are propagated in the laboratory
of the maker; they are sent out either as liquid cultures, a small mass of
milk or bouillon inoculated with the organism, or in a dry form, the
latter being prepared by mixing a culture of the organism with an inert
substance, such as milk sugar, milk powder, or starch, and drying at a
low temperature. In a liquid the organisms are exposed to the effects
of their own by-products, and the vitality of the culture is rapidly lost.
Such cultures must be used when fresh in order to give good results, and
they cannot be kept in stock by the manufacturer or dealer. The resist-
ance of Bad. lactis acidi to desiccation is great; it thus lends itself to the
preparation of the dry cultures, in which the organisms remain in a dor-
mant condition and retain their vitality for long periods.

Most of the cultures now sold are pure, as this term is used in bac-
teriology, still others contain non-acid-forming organisms, intentionally
added or introduced accidentally during the process of preparation. If
the lactic bacteria are present in such cultures in large numbers, the
impurities are usually of small practical significance. In the past so-
called "duplex" cultures have been sold which were supposed to contain
an acid-forming organism and a second organism that was to enhance
the flavor of the product. Such cultures are no longer sold.

For the propagation in the creamery the contents of the container
purchased are added to a small mass of milk that has been heated to
destroy all non-spore-forming bacteria and other microorganisms; the
milk, after being inoculated, is incubated at favorable temperatures and
when curdled can be used for the inoculation of a second and larger
quantity. The process of inoculating a quantity of milk is carried out
daily. It is impossible for the butter maker to propagate the culture so

THE RELATION OF MICROORGANISMS TO BUTTER. 341

as to maintain the original purity, but with care in the heating of the milk
the sterilization of all utensils and the maintaining of proper temperatures,
the contamination that occurs will not injure the culture for practical
work. The cultures propagated under such conditions gradually deteri-
orate and recourse must be had sooner or later to a fresh culture. The
contamination that is of the greatest practical significance is undoubtedly
that with other acid-forming bacteria rather than with the forms that
remain in the milk after heating.

Many of the cultures gradually lose their fermentative properties,
and do not form acid rapidly and in sufficient amounts to insure ex-
haustive churning and to produce the desired degree of flavor in the product.
Cultures frequently become slimy or ropy on propagation. This is not
necessarily due to contamination with specific slime-forming organisms
but rather to a change in the lactic organism itself. Such an abnormality
usually persists for only a short period and the conditions that govern its
appearance and disappearance are not known. It is asserted by practical
butter makers that the development of too high an acidity in the cultures
as they are propagated in the creameries permanently impairs the value
of the culture.

The cultures are propagated in skim milk. Where this is not avail-
able, unsweetened, condensed milk has been employed. Efforts have
been made to grow the bacteria in some other kind of medium than milk,
but without success. The starter is said to be ripe or in the best condition
for use soon after curdling, or when the acidity is 0.5 to 0.7 per cent, as at
this time it contains the maximum number of living cells. The practical
man thus uses the curdling as an indication of the ripeness of the starter.
The curdled milk should show no free whey, and the curd should be
easily broken up to form a creamy mass that can be uniformly incor-
porated with the cream. The temperature of incubation and the amount
of initial inoculation determine the rapidity with which the acid fermen-
tation will progress, the maker seeking to regulate these so that the culture
shall be ripe at the desired time each day.

USE or PURE CULTURES IN RAW CREAM. The cream as it reaches
the creamery contains a greater or less number of acid-forming bacteria
that ultimately will cause it to ripen and the flavor of the butter will be
due to the by-products of the mixture of bacteria. If, through the addi-
tion of a pure culture, the relative number of organisms that are known
to be favorable is greatly increased, the flavor of the product should be

342 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

improved. This has been found to be true in practice and it is now
believed that pure cultures are of value not only in the ripening of sweet
cream, but that the addition of a relatively large amount of starter to
cream that is already fermented will enhance the value of the butter.

USE or PURE CULTURES IN PASTEURIZED CREAM. It is evident that
the maker has but imperfect control over the fermentative processes when
raw cream is treated with a pure culture. To insure more perfect con-
trol the destruction of the contained bacteria and the subsequent inocula-
tion of the cream with a pure culture is indicated. The introduction
of the process of pasteurization of cream for butter making was due to
Storch. In Denmark this method is used almost exclusively. It has
been introduced into the other dairy countries of the world and is con-
stantly spreading. Pasteurization combined with the use of the pure
culture represents the highest type of modern butter making, and where
the raw product can be obtained in a fresh condition the butter maker
has perfect control over the bacteria that cause the ripening; hence can
control the flavor of the butter, both qualitatively and quantitatively.

The intensity of flavor of butter is dependent upon the amount of
acid that is developed in the cream or on the ratio between the amount of
fat and the by-products of the acid fermentation. If these by-products
are small in amount, as in cream having a low acidity, the flavor of the
butter will be low. If the acidity is allowed to reach the maximum,
the flavor will be much higher. Thus the maker can control the intensity
of flavor of butter as accurately as he can the kind o.J flavor. With rich
cream, the acidity that can be developed is small and the ratio between
the fat and the products of fermentation is low; thus, the flavor of butter
made from very heavy cream is certain to be low.

PURE CULTURES IN OLEOMARGARINE AND RENOVATED BUTTER.
It was previously mentioned that the manufacturer of butter substi-
tutes employs the same methods to impart butter flavor to his pro-
ducts as does the butter maker. The oleomargarine manufacturers
employ pure cultures of lactic bacteria for the fermenting of milk that
is mixed with the fats they employ. The same practice is followed
by the manufacturer of renovated butter. Many of the creameries
of the western states receive cream that is shipped long distances,
and is collected from the farms but once or twice a week. It is
thus in an advanced state of fermentation when it reaches the cream-
ery. In order to prepare from this grade of cream, which often has a

THE RELATION OF MICROORGANISMS TO BUTTER. 343

most undesirable flavor, a merchantable product, various means are
employed to remove the flavoring substances and to replace them with
desirable flavors from the pure cultures. The acidity may be reduced by
the addition of lime so that the cream can be pasteurized; the cream may
be aerated by passing air through it, or it may be mixed with water and
reseparated. After such treatment it is mixed with a large proportion
of milk fermented by a pure culture and churned. The resulting pro-
duct is constantly sold as the highest grade of creamery butter.

ABNORMAL FLAVORS OF BUTTER. The abnormal flavors of butter
are traceable to the partial replacement of the desirable acid-forming
bacteria with other types of microorganisms. Many samples of butter
having abnormal flavors have been examined, and the organisms believed
to be the cause isolated and studied but it cannot be said that any
particular group of microorganisms can be associated with any of the
abnormal flavors met. It is asserted that "oily" butter, i. e., that having
the taste of machine oil, is caused by bacteria and by microorganisms
that decompose the fat, as Oidium lactis, yeasts, and liquefying bacteria.
Organisms of the B. coli group that produce a turnip-like flavor in butter
have been described by Weigmann. The flavors of putrid butter, fishy
butter and also many other abnormal flavors have been ascribed to bacteria.

The abnormal flavors may be due to the presence in the milk of certain
aromatic principles contained in the feed and excreted in the milk.
Cabbage, turnips, and others of the cruciferae impart their characteristic
taste to the milk and butter.

DECOMPOSITION PROCESSES IN BUTTER.

Butter is a finished product at the time it is removed from the modern
churn and all subsequent changes are likely to cause more or less dete-
rioration. The specific causes of these changes are not well known but
it is very evident from a study of the conditions that favor or retard the
appearance of the flavors, characterizing these changes, that biological
factors are concerned. Sweet-cream butter has very poor keeping
qualities. As the proportion of acid-forming bacteria in butter is increased,
either by the fermentation of the cream, by the addition of pure cultures,
and through the use of the latter in connection with pasteurization, the
keeping qualities are enhanced. Of the butter made from ripened cream,
that prepared from cream, handled in a clean manner, and thoroughly

344 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

pasteurized and ripened with a pure culture of Bact. lactis acidi, has
the best keeping qualities. It is asserted from very limited data that
if fresh, sweet, clean cream is pasteurized, the butter will have better
keeping qualities than when made from the same cream pasteurized
and ripened with a pure culture. If this is true, it is evidence that not
only the bacteria other than Bact. lactis acidi are harmful, but that
this organism, that has been considered without influence on the keep-
ing quality, must be classed as one of the factors in the decomposition of
butter.

It has been shown that the bacterial content of the water used for
the washing of the butter has an influence on the keeping quality. If
the water is of surface origin and contains the bacteria peculiar to these
types of waters, its influence may be marked and some method of treat-
ment must be followed. Filtering or heating the water has been resorted
to, the latter with marked success. A pure water will contain so few
bacteria that they will not exert any noticeable influence on the keeping
quality of the butter.

Storage temperature also has a marked influence on the deterioration
changes in butter. Modern butter-storage rooms are kept below
o F.; the butter is quite unchanged on removal from storage, but
deteriorates much more rapidly than would have been true at the same
temperature before storage. Another factor that is of influence in the
keeping of butter is the amount of salt used. In salted butter, the con-
tained water is a concentrated brine; in such a medium most forms of
bacteria are unable to grow. Small packages deteriorate more rapidly
than large ones, because the proportion of the mass of butter exposed to
the air is relatively greater. Exposure to light is also claimed to exert
a harmful influence. Antiseptic substances such as borax and boric acid
have a marked effect on the deterioration changes. The New Zealand
and Australian butter exported to the English markets is treated with
preservatives.

A large amount of experimental work has been done in order to
determine the effect of specific organisms on the keeping quality of butter.
The results obtained have not been definite and it is not certain that the
organisms employed are constantly concerned in the deterioration
changes. It is very probable that both bacteria and molds exert an
influence. The chemical changes that take place in the spoiling of butter
are no better known than are the causal factors. It has been asserted.

THE RELATION O'F MICROORGANISMS TO BUTTER. 345

that there is a decomposition of the glycerides with a resulting increase in
free acids. It has been shown that this does not always occur; that a
butter may be in an advanced state of decomposition and its content in
volatile acids not be higher than when fresh. Two types of changes are
usually distinguished, rancidity and the appearance of a tallow-like odor.
The latter may be due to purely chemical factors, while the former is
quite certainly biological.

Moldy butter is a frequent trouble encountered by the butter maker.
Butter itself is not a good substratum for mold growth, but the parch-
ment paper in which the butter is wrapped and with which the containers
are lined, is an excellent substratum. Air and moisture favor mold
growth; hence if the papers and containers are contaminated with mold
spores, the butter is likely to reach the market in an objectionable con-
dition. Butter tubs are scalded, steamed, soaked in brine, or treated
with a dilute solution of formaldehyde, in order to destroy the mold
spores present. The most "efficient treatment is to coat the inside of the
container with paraffin. This prevents trouble from the container but not
from the paper, which, if suspected as the source of trouble, may be treated
by heating in water.

PATHOGENIC BACTERIA IN BUTTER.

If the milk contains pathogenic bacteria, they are certain to pass into
the cream and be incorporated in the butter. It is not believed that
butter is an important agent in the distribution of the organisms of
tuberculosis and typhoid fever, although both are able to exist in salted
butter for over two months. Foot-and mouth-disease may be caused in
humans by the use of butter made from the milk of infected animals.

CHAPTER III.*
THE RELATION OF MICROORGANISMS TO CHEESE.

GENERAL.

Cheese consists of the fat and casein of milk, together with the insol-
uble salts; however, along with these constituents are carried some of the
moisture of milk, in which are dissolved small quantities of sugar, albu-
min, and salts. The amount of moisture and soluble constituents found
in cheese is determined by the amount of whey incorporated in the curd.

In the process of making cheese, it is necessary to curdle the milk,
thus enabling the separation of the cheese constituents from the bulk of
whey and soluble constituents of the milk. Two methods are employed
to accomplish this purpose, and, as a result, two types of cheeses are
produced.

TYPES OF CHEESE.

These types may be designated as "Acid-curd Cheeses" and "Rennet-
curd Cheeses."

ACID-CURD CHEESES. The curdling maybe accomplished by allowing
the milk. to undergo acid fermentation, either spontaneously through the
action of the normal flora of the milk, or through the addition of pure
lactic cultures. This method furnishes the so-called acid-curd cheeses,
which are ready for use as soon as the whey has been removed by draining
and the curd salted. Acid-curd cheeses are not commercially important in
that they are made for local consumption and are to be classed as a form
of sour milk. They owe their flavor to the products of the acid fermen-
tation, especially lactic acid. The moisture content is high, which,
together with the acid reaction, favors the growth of molds and yeasts.
These biological agents may soon spoil the cheese.

RENNET-CURD CHEESES. All of the important varieties of cheeses
are made by the use of rennet for the curdling of the milk. Over four
hundred kinds are known, but only twelve to fifteen are of great com-

* Prepared by E. G. Hastings.

346

RELATION OF MICROORGANISMS TO CHEESE. 347

mercial importance. With few exceptions, they are made from cow's
milk. From the same raw material milk, rennet, and salt therefore,
a wide variety of products, differing in texture, taste and odor, is obtained.
This fact indicates the importance of biological factors in the changes
the curd undergoes during the ripening process.

The rennet-curd cheeses may be divided into: i, hard cheeses, 2, soft
cheeses; the initial difference is largely in the amount of whey left in the
curd during the making of the cheese and they meet in types which are
classified with difficulty.

The rennet-curd cheeses of the cheddar group are at first tough and
rubber-like in texture, due to their curd, which is not easily digested,
is soluble in water only in small part, and, further, is devoid of flavor
and aroma. The curd must pass through a complex series of chemical
and physical changes, altering its texture, solubility and digestibility, and
giving to it flavor and aroma by which the different kinds of rennet-curd
cheeses are especially to be differentiated. In the hard cheeses the factors
concerned in these changes act in a uniform manner throughout the entire
mass of the cheese, making it possible to manufacture such cheeses in any
desired size. In the case of the soft cheeses, the ripening changes are
largely controlled by agents existing only on the surface, the products
of such agents by means of diffusion gradually affecting the entire mass.
In order that this may take place within a reasonable time, it is essential
that these cheeses be made in small sizes. Then, too, the soft texture
of such cheeses makes it impossible to handle them commercially in

large sizes.

CONDITIONS AFFECTING THE MAKING OF CHEESE.

QUALITY OF MILK. In the curdling of milk by rennet the solid bodies
present in the milk are retained in the curd, thus the fat globules are held,
as are also the bacteria. The latter continue to grow as they would have
done in the milk except that growth must take place in the form of colonies
as in the solid culture media of the bacteriologist. The bacteria, however,
produce the same fermentation in the curd as they would have done in
the uncurdled milk.

The butter maker can control, through pasteurization and the use of
pure lactic cultures, the fermentation of the cream. The pasteurization
may be so efficient as to destroy all non-spore-forming bacteria since the

348 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

quality of the product will not be impaired by the use of temperatures
approximating the boiling point. The cheese maker is much more
dependent on the original quality of the milk, since effective pasteuri-
zation so changes the milk as to effect profoundly the expulsion of the
whey from the curd and the development of flavor during the ripening

, _^

FIG. 81. -The type of curd obtained from milk in which the acid-forming flora consists
largely of organisms of the Bacl.coli aerogenes group. Many gas holes and few irregu-
lar-shaped, angular, mechanical holes due to imperfect "matting." (Original.)

process. If harmful forms of microorganisms are present in the milk,
they will pass into the cheese and there produce their harmful effects.
Through the addition of pure lactic cultures to the milk the proportion of
desirable bacteria can be increased and a partial control of the fermen-
tation thus secured.

RELATION OF MICROORGANISMS TO CHEESE.

349

TESTS FOR THE QUALITY OF MILK. -Methods by which the cheese
maker can determine, in a rough manner, the kinds of bacteria present
have been devised. The bacteria most dreaded and most frequently
present are those of the B. coli-aero genes group.

The method most frequently used for their detection consists in
incubating a sample of the milk to be tested at temperatures ranging

FIG 82 The type of curd obtained from milk in which the acid-forming flora
consists almost wholly of Bact. lactis acidi. No gas holes and no marked mechan-
ical holes as the curd has "matted" almost perfectly. (Original.)

from 35 to 40 for a few hours and noting the type of curd that is formed.
Milk suitable for cheese making should show the solid curd character-
istic of the Bact. lactis acidi group, while gassy curds or soft and partially
digested curds are indicative of bacteria that are likely to be harmful
in the cheese.

An improvement over the fermentation test of foreign origin has

350 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

been devised by Babcock and Russell and is known as the Wisconsin
Curd Test. It has for its basis the same principle as the simple fermen-
tation test; however, a modification is introduced; the milk is curdled by
the addition of rennet and the curd is cut and drained to free it from the
whey as completely as possible. The jars employed in testing are kept
at temperatures favorable for the kinds of bacteria sought; those for the
colon group, requiring a higher optimum temperature than the lactic
bacteria, should have a temperature of incubation from 35 to 40, those
for the lactic group from 30 to 35. The great advantage of the Wisconsin
Curd Test is its greater delicacy, since the bacteria are concentrated in a
small volume, and thus their presence is more evident than would be the
case in the larger mass of curd obtained when no rennet is added. The
curd can also be removed from the jar, cut, tasted, and its texture deter-
mined, all of which aid in judging the quality of the milk. The curd
should have a clean acid odor and taste; it should be free from sliminess
on the surface, and possess a uniform texture. Such a curd can be
obtained only in the presence of a considerable number of lactic bacteria.
Very clean, fresh milk is likely to give an undesirable result, since milk
always contains microorganisms which will grow rapidly at the high tem-
perature in the absence of the acid-forming bacteria and which will
usually produce undesirable flavors in the curd. This fact should be kept
in mind in the testing of market milk.

RIPENING OF MILK.* It has been shown that the growth of acid-
forming bacteria in milk is not followed by a parallel increase in the acidity
of the milk. Indeed milk may contain hundreds of thousands of acid-
forming bacteria per c.c. and yet the acidity be no greater than when the
milk was drawn from the cow. Ultimately the acidity begins to increase,
and for some time increase in bacteria and acidity run parallel. The
period during which bacterial proliferation is taking place but without
corresponding increase in acidity is known as the "period of incubation. "f
Its length is determined by the temperature at which the milk is kept and
by the amount of initial seeding with acid-forming bacteria.

* In order to illustrate the role of microorganisms in the making and ripening of cheeses, a
somewhat detailed summary of the present knowledge concerning their action in Cheddar cheese
will be given. Many of the factors concerned in the ripening of this kind of cheeses also function
in the ripening of other rennet cheeses. In their description only such additional factors need be
considered as are not active in Cheddar cheese.

j" This should not be construed to mean that no acid is produced during this "so-called"
period of incubation, but that such acid does not respond to the usual tests and may be found in
combination with or neutralized by the constituents of the milk, such as casein.

RELATION OF MICROORGANISMS TO CHEESE. 351

In order to insure proper rennet action the maker of Cheddar cheese
desires the milk to have an acidity of about 0.2 per cent. He thus wishes
milk that has passed through the period of incubation and in which the
acidity has begun to increase. If the milk shows the desired acidity when
it reaches the factory, the making process is immediately begun. If the
milk is too sweet, the period of incubation is shortened by warming the
milk to temperatures most favorable for the lactic bacteria, 30 to 32, and
by the addition of pure lactic cultures, which are identical in nature and
in method of propagation with those used in butter making. The devel-
opment of a slight acidity is known as the "ripening" of milk.

CURDLING OF MILK. Under the influence of a favorable temperature
and the slight acidity, the milk is quickly changed by the rennin* to a
firm, jelly-like mass that is cut, with appropriate knives, into small cubes.
The curd encloses 95 to 99 per cent of the bacteria in the milk. The same
factors that favor the curdling of the milk favor the shrinking of the curd
and the expulsion of the whey from the cubes. The development of acid
within the curd is rapid, due to the concentration of large numbers of
bacteria in a small volume and to the favorable environment. During the
six to eight hours that elapse between the curdling of the milk and the pres-
sing of the curd, the increase of acidity is over o. i per cent per hour. The
following table gives the acidity of milk and the whey expressed from
the curd at various stages in the making of a typical Cheddar cheese.

Acidity of milk before adding rennet 0.20-0.21 per cent.

Acidity of whey immediately after cutting curd. o. 14-0. 145 per cent.

Acidity of whey when removed from the curd. o. 16 o. 18 per cent.

Acidity of whey when curd is packed o. 24-0. 30 per cent.

Acidity of whey when curd is milled o. 65-0. 75 per cent.

Acidity of whey when curd is salted o. 90-1 . 10 per cent.

MANIPULATION OF THE CuRD.f The curd particles at first show little
tendency to cohere; but, as the acidity increases, the nature of the curd
changes, and, when the whey is removed, the pieces of curd soon cohere
and ultimately form a single mass in which the original cubes of curd
cannot be detected. The fusion of the curd particles is known as "mat-
ting" and is an important step in the Cheddar process. The lack of acid

* The rennet used in cheese-making is obtained by extracting the abomasum the true diges-
tive stomach of the calf with a solution of sodium chloride. The extract contains two enzymes
a clotting or curdling enzyme, rennin, and a proteolytic enzyme, pepsin.

f Cheddar cheese.

352 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

formation within the curd prevents matting while the curd is in the vat,
and may even render difficult the fusion of the particles under pressure.
The nature of the change which the curd undergoes at this stage in the
manufacture is not well understood, but probably is due to a com-
bination between the paracasein and the lactic acid, the resulting
compounds differing from the paracasein in physical properties and in
solubilities.

RIPENING OF CHEESE. Cheese in ripening undergoes profound
physical and chemical changes under the influence of a number of factors,
which for purposes of discussion may be divided into two groups:
those by which the content of soluble nitrogen in the cheese is increased
and the digestibility enhanced; and those which cause the formation of
flavoring substances. During the ripening of the cheese the maker
can do little toward the control of the factors which ultimately determine
its commercial value. As in butter, the flavor is the most important
characteristic of the ripened cheese and the most difficult to control.

Theories of Cheese Ripening. Many theories have been advanced to
explain the changes that occur during the ripening process. Duclaux,
a French microbiologist, studied the bacterial flora of Cantal cheese by
aid of the crude methods available before the introduction of the gelatin-
plate method. By the use of the dilution method, using bouillon as the
nutrient medium, he isolated a number of kinds of spore-forming bacteria.
The organisms formed two enzymes, one a curdling enzyme related to
rennin, the other a proteolytic enzyme to which was given the name
casease. A chemical study of the by-products of the organisms, when
growing in milk, revealed a number of compounds that had previously
been found in ripe cheese, such as leucin, tyrosin, and the ammonia salts
of acetic, valeric and carbonic acids. The cultures often possessed a
cheese like odor. These facts led Duclaux to believe this class of organ-
isms were responsible for the ripening of the hard cheese in question.
The generic name Tyrothrix was applied on account of the supposed
relation to cheese. This term is still found in current bacteriological
literature. The methods employed by Duclaux were such as favored
the growth of the liquefying, rather than the acid-forming bacteria.
To the latter more recent investigators have devoted attention.

The theory that the proteolytic bacteria function in the ripening of
hard cheese has been more recently emphasized by Adametz. It is
sufficient to say that the number of spore-forming proteolytic bacteria

RELATION OF MICROORGANISMS TO CHEESE. 353

in cheese is not sufficiently large, nor is their presence so constant that
any importance can be attached to them. Any agent to be considered
as a factor in the ripening process must be present in every cheese in
sufficient numbers to account for the change for which it is considered
responsible. Such agents should be capable of demonstration. It should
be remembered that, by following the rules laid down by the practical
maker, a normal cheese can invariably be made, hence the factors of
importance in the ripening must be constantly present in the milk or
rennet. It is doubtful whether the liquefying bacteria will satisfy this
requirement. It has been shown by de Freudenreich that such organisms,
even when added to milk in large numbers, exert no influence on the
ripening of hard cheese, since the conditions within the cheese are not
such that growth can occur.

De Freudenreich, a Swiss microbiologist, by the aid of modern methods,
demonstrated the constant presence of certain classes of acid-forming
bacteria in Swiss cheese, and to them ascribed an important role in the
ripening of this hard cheese. He was led to this conclusion by their great
numbers in the fresh cheese, and by the fact that cheese made from milk
drawn under aseptic conditions, which thus contains no lactic bacteria,
do not ripen; through the discovery, also, that certain of the lactic bacteria
predominating in Swiss cheese, those of the Bact. bulgaricum group, exert
a solvent effect on the casein of milk, although they are devoid of action
on gelatin.

Babcock and Russell demonstrated the presence of an inherent proteo-
lytic enzyme in milk, to which the term galactase was applied. This
enzyme can be demonstrated by preserving a sample of fresh milk with
chloroform or other mild antiseptic. At 37 curdling occurs in three to
four weeks; the content of soluble nitrogen in the milk is slowly augmented.
The presence of this proteolytic enzyme, together with the fact that a
normal cheese cannot be made from milk in which this enzyme has been
destroyed by heat, led these investigators to consider this inherent enzyme
of milk an important factor in cheese ripening.

Present Knowledge of Causal Factors* The role of certain factors in
the ripening of Cheddar cheese has been established beyond doubt by the
chemical and bacteriological investigations of recent years. It is certain
that acid-forming bacteria are essential factors in the ripening of this
kind of hard cheese, and probably all kinds of rennet cheeses.

* Cheddar cheese.
23

354 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

As has been shown the growth of acid-forming bacteria is rapid
during the making of Cheddar cheese. The growth continues during
the pressing and subsequent thereto; the maximum number of lactic
bacteria is found when the cheese is one to five days old. As many as
1,500,000,000 per gram of the moist cheese have been demonstrated.

Causes of Proteolysis. The proteolytic action of rennet extract on
the paracasein of cheese was demonstrated by Babcock and Russell, and
by Jensen. This property is due to the fact that rennet extract also con-

A B

FIG. 83. Proteolytic action of rennet extract in the absence and in the presence of

acid-forming bacteria. A, sterile milk agar; a strip of filter paper treated with rennet

was allowed to remain on the medium for i hour at 37. No digestion of the casein.

B, milk agar inoculated with Bad. lactis acidi; incubated for 24 hours at 37, then

reated as A. True digestion of the casein is indicated by the clearing. (Original.)

tains the enzyme pepsin, which for its action outside the body requires con-
ditions similar to those which obtain in the stomach; in other words, the
presence of sufficient acid to activate it. The hydrochloric acid secreted
by the walls of the stomach acts as the activating agent in the body.
The acidity resulting from the fermentation of the sugar in the curd
is sufficient to activate the pepsin. Under its influence the paracasein
is partially converted into soluble decomposition products such as albu-
moses and peptones. In the absence of acid-forming bacteria no acid is
formed; consequently the pepsin does not become active and no proteo-
lytic effect is produced. Under these conditions the curd remains tough
and elastic and the solubility is not increased. It is thus evident that

RELATION OF MICROORGANISMS TO CHEESE. 355

acid-forming bacteria are essential factors in cheese ripening. The
pepsin of the rennet extract and the galactase suffice to account for the
initial proteolysis of the paracasein. Since neither of these enzymes
forms ammonia, which is always found in ripe cheeses, the origin of this
simple decomposition product of protein is still unexplained. It may
owe its origin to microorganisms not yet discovered.

Prevention of Putrefaction. The various stages in the decomposition
of milk have been outlined in a previous chapter. Briefly they are as
follows: The first evident change is the curdling due to the acid-forming
bacteria. Succeeding this, the acid, semi-solid mass or curd is a favorable
substratum for the characteristic mold of milk, Oldium lactis, which soon
forms a white, velvet-like layer over the surface of the milk. Like other
molds, this form can use acids as a source of energy. The acid is then
oxidized to carbon dioxide and water, and thus the reaction of the milk
is slowly changed until a point is reached which allows the putrefactive
bacteria, that have remained dormant during the period of unfavorable
environment, to develop. The curd is accordingly peptonized and
putrefaction occurs. If the acid reaction is maintained through the
prevention of mold growth, the milk will be preserved from the attacks
of putrefactive organisms and will remain unchanged for an unlimited
time.

The second r61e of the acid-forming bacteria in cheese is to protect
it against the putrefactive organisms that are constantly present in milk
and hence in cheese. The acid reaction of the cheese, due to the per-
sistence of lactic acid, or to the formation of volatile acids after the initial
fermentation, is sufficient to prevent the growth of the putrefactive bac-
teria within the cheese. If the cheese is made from milk which contains
no acid-forming bacteria and few putrefactive ones, or if the sugar is
removed from the curd by washing it with water, the cheese will not
ripen since there is no acid to activate the pepsin; the curd will remain in
much the same condition as when it was removed from the press. Cheese
made from milk containing no acid-forming bacteria but many putre-
factive bacteria is likely to undergo putrefaction, since the latter class
of organisms finds conditions for growth in the absence of an acid re-
action. Such a condition is rarely noted in a hard cheese under normal
conditions, but may be produced experimentally. The biological acid
may be replaced by other acids added to the curd in appropriate amounts,
since these will activate the pepsin and protect the cheese against the

35 6

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

attacks of putrefactive bacteria; but it is not certain that the cheese will
develop a normal flavor when lactic acid is replaced by mineral acids.

Flavor Production in Cheese. The factors that have been discussed
are undoubtedly the most important ones concerned in the proteolysis of
the curd, and are thus the factors concerned in the changes of texture,
solubility and digestibility. The flavor, which develops during the
ripening process, has been regarded as due to the proteolysis of the para-
casein. A thoroughly ripened cheese contains a large amount of ammonia
and related compounds. It was thus natural to consider the flavor due
to these simple products of protein degradation. More recently it has
been discovered that the intensity of flavor does not necessarily cor-
respond to the content of the cheese in these products; indeed a cheese
may have a high content of nitrogen as ammonia and yet be low in
flavor.

The Wisconsin Experiment Station has found that the volatile fatty
acids of Cheddar cheese increase as the ripening progresses. In the
following table are given the data obtained from the detailed study of a
normal Cheddar cheese.

Acids in 100 Grams of Dry Matter.

c.c. of N/io alkali neutralized.

3 days

42 days

3 months 51/2 months

10 months.

Lactic acid

84.09

n-59
0.41

o-73
o.oo

90.28

29.44
2.15
2.17
0.36

124.00
24.25
3-42

3-5
0.96

103.70
25-86
1.07
4.82
1.25

74.10
12.64
2.63

5-45
2.23

Acetic acid

Propionic acid

Butyric acid

Caproic acid

It will be noted that the content of the higher volatile acids, those
especially marked in odor, continually increases. It is possible to separate
other volatile compounds found in cheese from the volatile fatty acids by dis-
tilling with steam, neutralizing the distillate with an alkali and redistilling;
the second distillate will contain the alcohols and esters present in the
cheese. Such a distillate prepared from Cheddar cheese is found to possess

RELATION OF MICROORGANISMS TO CHEESE. 357

the characteristic aroma of the cheese in question. The esters it contains
are largely those of ethyl alcohol. The acid-forming bacteria, as stated
previously, produce varying amounts of volatile acids and slight amounts
of alcohols and esters. It is likely that the larger part of the volatile
compounds found in the ripening cheese is formed in fermentations which
take place subsequent to the initial fermentation of the lactose. The
flavor of Cheddar cheese, therefore, owes its origin very probably to the
fermentation of the lactose, and to the further change which the products
of the initial fermentation undergo under the influence of factors yet
unknown. The death and disintegration of the lactic bacteria may
liberate intracellular enzymes, or possibly, the period of initial bacterial
activity is followed by a second in which unknown forms are concerned,
the result of whose action is the production of flavoring substances.

By appropriate means the constant presence of other types of bacteria
than the lactic can be demonstrated, but little is known concerning their
number and still less of their probable action in cheeses because of the
lack of knowledge of the compounds on which they may act or those they
may form. That some biological factor is concerned in the production
of flavor in Cheddar cheese is indicated by the fact that if changes are
made in the methods of manufacture, changes in flavor are likely to result.
If the salt is omitted, the typical flavor does not appear. This can be
explained only by the action of the salt on certain types of bacteria, which,
in its absence, are able to grow and produce compounds that are not
found in a normal cheeses. Apparently the methods of manufacture
establish a certain equilibrium in the bacterial life which results in the
production of definite substances in amounts varying within certain
limits. If any condition is varied too widely, a deviation in the microbial
balance is produced and the products formed in the cheeses are changed
in kind or in amounts, either of which may result in a change of flavor.

ABNORMAL CHEESES.

The development of a normal texture and flavor in Cheddar cheese is
largely dependent on the presence of definite types of bacteria. If these
are replaced, wholly or in part, by other kinds, the product is likely to
suffer in texture, flavor or both. As has been emphasized previously,
the bacterial content of the milk is of the greatest importance in cheese,
since the organisms in the milk pass into the cheese and there produce

358 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

the same products as they would have done in the uncurdled milk. All
abnormalities of the cheese so far as they are occasioned by bacteria are
due to the abnormal flora of the milk. To the raw material the maker
must direct his attention if a fine product is to be prepared.

GASSY CHEESE. The most frequent trouble encountered and the
one of greatest economic importance is the fermentation caused by organ-
isms belonging largely to the B-. coli- aero genes group. It has been seen
that these produce in milk gases, such as carbon dioxide and hydrogen,
and offensive smelling and tasting compounds. In cheese similar com-
pounds are formed by these organisms; the gas causes the more or less
abundant formation of holes which give to the cheese judge an indication
of what may be expected with reference to flavor. All milk contains some
of the gas-forming organisms, but it is only when they are numerous that
marked injury is done.

Gassy cheese may also be due to the presence of lactose-fermenting
yeasts which are usually found in milk in such small numbers that they
cannot compete with the lactic bacteria in the fermentation of the sugar
in the cheese. At times the number may be increased to such an extent
that the major part of the sugar is fermented by them, alcohol and carbon
dioxide being produced. An outbreak of gassy Swiss cheese was found
by Russell and Hastings to be due to such yeasts that had gained entrance
to the milk from the whey-barrels because of careless washing of the
milk cans. The cheese makers of the country are realizing the impor-
tance of the contamination of the milk from the transportation of whey
and milk in the same can. The most practical means of preventing
trouble from this practice is to heat the whey to 68 as it passes from the
cheese vat to the storage tank. This temperature destroys the harmful
microorganisms, and if the storage tank is kept in a sanitary condition
the whey is sweet when returned to the farm in the milk can. It has
been demonstrated that such a treatment of the whey results in a marked
improvement in the quality of the product.

MISCELLANEOUS ABNORMALITIES OF CHEESE. Bitter cheese is
produced by bacteria that form a bitter principle. An outbreak of
bitter cheese investigated by Hastings was found to be due to the replace-
ment of the normal acid-forming flora by a lactic organism which pro-
duced such an intense bitterness as to mask the acid taste in the milk
and cheese.

RELATION OF MICROORGANISMS TO CHEESE. 359

Colored cheese is produced by chromogenic bacteria. In case the
colonies are not numerous and the pigment formed is not soluble in any
of the constituents of the cheese, the color will appear as colored specks,
such as the rusty spot investigated by Connel and Harding, which is due to
red forms of B. rudensis. If the colonies are very numerous, or if the
pigment is soluble, the curd may be uniformly colored.

Putrid cheese is caused by the absence of sufficient acidity to hold
the putrefactive bacteria in check. This trouble is rare in cheddar
cheese, since such cheese is made from ripened milk. Fruity flavors are
asserted to be due to yeasts which form fruit esters.

Moldy Cheese. In the moist air of the curing-room the cheese forms
an excellent substratum for the growth of common molds whose pigmented
spores discolor the surface of the cheese and thus impair its value because
of the appearance rather than by any effect in the flavor. Cheddar
cheese is protected effectively from molds by dipping the cheese, when
two or three days old, in melted paraffin which excludes the air from the
spores on the surface of the cheese.

SPECIFIC KINDS OF CHEESE.

There are cheeses made in this, and especially in foreign countries,
which are of great commercial importance. Only a few can be men-
tioned. It has been found possible to manufacture a few so-called
"foreign cheeses" in this country; however, with some "foreign cheeses"
the manufacture has been successful only in such localities where such
types originally developed, and where the climate and other conditions
are favorable to a normal ripening.

CHEDDAR CHEESE. Cheddar cheese, treated in much detail in the
foregoing considerations because it is the most important American
cheese, is made in England and her colonies and in the United States.
It appears in many varieties and by the American consumer is often called
American cheese in distinction from the foreign cheese. This distinction
is not wholly applicable at the present time.

EMMENTHALER CHEESE. Swiss or Emmenlhaler cheese originated in
Switzerland, but is now made in various other countries. A large amount
is made in Wisconsin, Ohio and New York (Fig. 84). It is charac
terized by its sweetish flavor and by the so-called "eyes," which are
holes formed by gas, produced in a fermentation that occurs subsequent
to the fermentation of the lactose. The number of eyes is not large and

360

MICROBIOLOGY OF MILK AND MILK PRODUCTS.

they are evenly distributed throughout the mass of the cheese except
near, the surface.

The cheese is made from as fresh milk as it is possible to secure.
The rennet used is prepared by placing a piece of the dried rennet in
whey and incubating the same for twenty-four to thirty-six hours at 30.
This is employed in place of the commercial extract used by the Cheddar
maker. It serves not only to curdle the milk, but adds to it a large
number of acid-forming bacteria that have grown in the rennet solution
during the period of incubation. The number is not, however, sufficient
to cause any development of acid during the making process which
differs from the preparation of Cheddar cheese in the method of firming

* *

FIG. 84. Typical development of "eyes" in Swiss cheese. (Original.)

the curd. This is accomplished by heating the curd to 52 to 60, and by
cutting it into pieces scarcely larger than grains of wheat. The salt is
applied to the exterior of the cheese by immersion in brine for one to four
days and by sprinkling salt on the surface.

The fermentation of the lactose proceeds rapidly during the pressing
and subsequent thereto, so that within a few days the sugar has disap-
peared. The lack of the development of acid during the making probably
results in a somewhat different relation between the acid and protein
from that existing in a Cheddar cheese, which, together with the absence
of salt gives a somewhat different environment, thus making possible
the development of a different flora. There is no ground for believing
that the agents concerned in the proteolytic changes are other than those
that function in Cheddar cheese. The flavor must, however, be due to
other factors; this is indicated by the fact that if the milk is ripened as in
the Cheddar process, or if salt is added to the curd the flavor will approxi-

RELATION OF MICROORGANISMS TO C1IEESR. 361

mate the Cheddar flavor. The formation of the eyes is inhibited by salt,
as is indicated by their relative scarcity in the outer layers of the cheese.
Jensen has shown that the eyes are due to the fermentation of lactates
with the formation of propionic and acetic acids, and carbon dioxide.
The causal organism is found in the milk and the whey rennet. It is
believed that lactic bacteria of the Bad. bulgaricum group are important
factors in the ripening of Swiss cheese. They are present in large numbers
in the rennet and cheese. Mixed cultures of an organism of this group
and a mycoderma are used with success for the inoculation of the whey in
which the rennet is to be soaked. The exact role of this form of lactic
organism is not known; de Freudenreich considered them to be concerned
in the proteolysis of the paracasein, since he had found that the content
of sterile milk in soluble nitrogen increased when inoculated with the
organism. It is probable that the formation of eyes and the flavoring
compounds are due, in part at least, to the same factors.

In the other kinds of cheeses to be described, the role of the acid-forming
bacteria is similar, if not identical, to their role in Cheddar cheese, i.e.,
in activating the pepsin of the rennet and in preventing the growth of
putrefactive bacteria. The factors concerned in flavor development are
different.

ROQUEFORT CHEESE. This cheese, which is prepared almost ex-
clusively in the Department of Aveyron in southern France, is made
from sheep's milk. Its most striking characteristic is the marbled or
mottled appearance of the interior, due to the growth of a mold, Penicillium
roqueforti, Thorn. The curd is inoculated with the mold, when it is
placed in the press, by sprinkling the curd with bread crumbs on which
the mold has grown. The growth and sporulation of the mold in the
interior of the cheese are favored by piercing it with small needles, thus
admitting air. The characteristic flavor is due, partially at least, to the
mold.

This cheese is cured in caves having a temperature below 15. The
fermentative processes are apparently closely dependent on the moisture
and temperature conditions of the curing room. This emphasizes the
importance of biological factors in the ripening process.

GORGONZOLA CHEESE, prepared in Italy from cow's milk, and STILTON
CHEESE, made in England are similar to Roquefort in appearance and
contain the same mold Penicillium roqiteforli.

CAMEMBERT CHEESE. The soft cheeses are best represented by this

362 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

important French cheese made from cow's milk by the addition of rennet.
The milk is ripened to an acidity of 0.20 to 0.25 per cent before the addi-
tion of the rennet. The curd, which thus contains many acid-forming
bacteria, is neither cut nor heated in order to retain the maximum amount
of whey. The curd is placed in small hoops and allowed to drain with-
out pressure. Salt is applied to the surface of the cheese.

The milk sugar is rapidly fermented and the resulting acidity is high,
for the cheese contains 60 to 70 per cent of moisture when fresh and
50 per cent when ready for consumption. The high moisture content
of the cheese and the humidity and temperature conditions of the curing
room favor the rapid development of microorganisms on the surface of
the cheese. Both molds and bacteria thrive under the influence of these
favorable conditions, changing the cheese to a soft, smooth and butter-
like mass, while a characteristic flavor is developed.

In three or four days the cheese becomes covered with the growth of
Oidium lactis; the characteristic mold of Camembert cheese, PenicUlmm
camemberti, appears later, within five to six days. These molds reduce
the acidity of the curd, and through the enzymes, which they produce
and which gradually diffuse into the cheese, proteolyze the curd very
completely. The appearance of the cheese when cut indicates the depth
to which the enzymes have penetrated; when the entire mass is acted
upon, the cheese is ready for use. The reduction of the acidity by the
molds exposes the cheese to the attacks of putrefactive bacteria and it
soon becomes unfit for use after it is completely ripened. A number of
different kinds of bacteria are found in the slimy surface layer, but their
role is not known.

The development of the characteristic flavor and aroma is dependent
on a certain relation between the various biological agents concerned
in the ripening. This balance is dependent on very narrow conditions
of temperature and humidity; slight changes in these environmental
conditions favor or retard the individual types in varying degrees. If the
equilibrium essential for the development of typical flavor is destroyed
this cheese fails to ripen properly and is of low value. The manufacture
of Camembert cheese is a delicate problem in the ecology of microorgan-
isms, and because of this fact the manufacture is attended with greater
difficulties than is the case with most types of hard cheese.

CHAPTER IV.*

RELATION OF MICROORGANISMS TO SOME SPECIAL

DAIRY PRODUCTS.

GENERAL.

There are a number of special dairy products which do not normally
come into a discussion of market milk, butter or cheese, but which are of
considerable importance. A book of this sort would not be complete
without a discussion of some of these products from the bacteriological
point of view. Most of these special products have been developed as
commercial enterprises and the processes of manufacture have been
zealously guarded as trade secrets. The result is that there is very little
available data on the manufacture of these products and very little
authoritative knowledge about their bacteriological condition. It is,
therefore, difficult to give a full discussion of the microbiology of these
products. A few of the more important ones will be discussed, however.

CONDENSED MILK.

There are at least three quite distinct kinds of condensed milk made
under conditions which result in an entirely different bacteriological
condition in the finished product. These different products must, there-
fore, be considered separately. Condensed milk means simply milk
from which a large part of the water has been removed, thus decreasing
its bulk, the purpose being to lessen the cost of transportation and to
increase the keeping quality of the product. Water is removed from
milk by some process of heating, either with or without vacuum, the
heating process being more or less equivalent to pasteurization.

SWEETENED CONDENSED MILK. This product is made by removing
a large part of the original water by means of heat and the addition
of cane sugar. It is then put up in sealed cans. It is not intended to be
sterile. The degree of heat to which it is subjected is not sufficient to

* Prepared by W. A. Stocking.

3 6 3

364 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

kill all of the microorganisms present and it is also subject to infection
after the condensing is completed. Cane sugar is added to the milk,
making the final product contain about 25 per cent of water, 35 per cent
milk solids and 40 per cent cane sugar. The low percentage of moisture
together with the added sugar tends to preserve this product against the
action of microorganisms. There may be some bacterial growth, the
rapidity depending upon the temperature at which the product is kept,
but it is usually slow and milk prepared in this way will keep for a con-
siderable time without undergoing marked bacterial changes. Some gas
producing bacteria exist in the milk and if cans containing the organisms
are allowed to remain at warm temperatures, they will develop in spite
of the large percentage of sugar, producing sufficient amounts of gas to
cause the ends of the cans to bulge out. Such cans are known com-
mercially as "swell-heads."

UNSWEETENED CONDENSED MILK. In this form of condensed milk
approximately the same amount of moisture is removed as in the sweet-
ened product but no sugar is added. The decreased amount of
moisture tends to prevent the rapid growth of bacteria, but this is not
enough to guarantee the keeping quality of the product. After the milk
is condensed it is put into the can, hermetically sealed, and then placed
in steam sterilizers and subjected to temperatures somewhat above the
boiling-point. In this way the milk is heated a sufficient length of time
to insure perfect sterilization of the contents of the cans. If this process
is properly done, the finished product contains no living microorganisms
and from the bacteriological standpoint the milk should keep indefinitely.

Sometimes the unsweetened product is sold in bulk in cans. In this
case it is subject to more or less contamination after heating and is not
sterile, but because of the small amount of moisture and the concen-
tration of the milk solids, the bacteria do not develop rapidly and if kept
at a cool temperature, the milk will keep several days without undergoing
appreciable biological fermentations.

CONCENTRATED MILK. There is now on the market a form of con-
densed milk prepared by a different process, which is commonly known
as concentrated milk. By this method the water in the milk is removed
by means of dry air. The milk is first heated and then air under pressure
is forced through it. By this process the milk is heated to a temperature
of 60 (140 F.), and this temperature maintained for two hours, during
which time air is forced through the milk causing violent agitation and

RELATION OF MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS. 365

the removal of the moisture. At the end of this time the milk is reduced
to one-fourth its original volume.* The result of this process is a pas-
teurized milk, with a marked reduction of the original germ content.
Investigations by Conn failed to show the presence of B. colt in milk
prepared by this process. The reduction in the bacterial content of the
milk is similar to that secured by other methods of pasteurization. No
additional sugar is added to this milk so the product is, therefore, a pas-
teurized milk containing a small amount of moisture. Because of the
small amount of moisture and the concentration of the milk sugar, the
bacteria which survive the heating process do not grow rapidly at low
temperatures. The following figures will serve to illustrate the effect of
this process upon the bacterial content of milk:

Number of bacteria per c.c. in original
milk

Number of bacteria per c.c. in finished
product

1,250,000
3,000,000
518,000
894,000
796,000
150,000

15,000
21,000
26,000

9,95

10,000

5,000

The rate at which the bacteiia develop in this milk is shown by the
following counts:

Number of bacteria per c.c.

Number of sample

2 days old

4 days old

6 days old

i

2

18,000
55,000

39,000
28,000

46,000
39,000

3
4

3,5o
4,400

11,000
5. 2 7o

10,000
4,630

The lack of moisture and concentration of milk sugar prevents the
rapid growth of these organisms so that bacterial changes do not take

* Data furnished by H. W. Conn.

366 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

place as rapidly as in ordinarily pasteurized milk retaining its normal
moisture.

POWDERED MILK. This product is produced by carrying the extrac-
tion of the water farther than in the case of the condensed milks. The
water is removed to a point where the milk solids can be reduced to a
powdered form. This product contains the original milk solids with a
very small percentage of moisture usually not more than 21/2 per cent.
There are several forms of powdered milk now on the market produced
by somewhat different methods. In some cases the moisture is removed
from the milk by its being exposed to a heated surface in a thin layer.
Sometimes the drying is done in vacuum. The resulting product is dry
and can be ground to the form of flour.

Another process is to remove the moisture by spraying the milk by
means of an atomizer into the top of a hot chamber, the moisture being
removed while the fine particles of milk are falling to the floor. By
this process the product accumulates on the floor as a very dry flour and
does not require any grinding. In the first process the heat is sufficient to
pasteurize the milk while in the latter process it is pasteurized before
being subjected to the drying process. The powdered milks do not claim
to be sterile but are preserved against subsequent action of microorganisms
because of the very low percentage of moisture which they contain. It
is probable that there is no appreciable increase in the number of bacteria
in milk flour and the product will keep for a long time without undergoing
bacterial fermentations.

CANNED BUTTER AND CHEESE.

Some effort has been made to put up butter and cheese in hermetically
sealed cans, the purpose being to increase the keeping qualities of the
products and influence the flavor by controlling the development of the
aerobic bacteria. Only a limited amount of bacteriological work has been
done on these canned products and the biological changes which take
place in them are not very well known.

SPECIAL MILK DRINKS MADE BY THE ACTION OF MICROORGANISMS.

From time immemorial fermented or sour milk has been used as an
article of food. We are told that Abraham* placed "curdled milk"

* Genesis 18:8. The Hebrew word "hemah" translated in the English authorized version
of the Bible " butter" means "curdled milk." Century Bible, Vol. Judges and Ruth, p. ja.

RELATION OF MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS. 367

before his guests and that Moses told the Israelites that curdled milk was
one of the blessings which Jehovah had given to his chosen people.*
History also tells us that the wandering tribes of Arabia used fermented
milk as a beverage. For centuries many of the tribes of eastern Europe
and western and middle Asia and parts of Africa have used sour milk
for food. Each of these regions appears to have had its own particular
milk beverage resulting from the particular bacterial flora of the region.

The sour milk products which are now on the market under a variety
of names have been derived from these original sour-milk drinks of an-
tiquity. Fermented milk beverages have become very popular during the
last few years among all the civilized peoples, partly because they make
a pleasant drink but more especially because of their supposed therapeutic
value, f

KUMYSS (KOUMISS, KUMISS, ETC.). Kumyss derives its name from the
Kumanes, a Russian tribe which lived along the river Kuma. This drink
was prepared from mare's milk by placing it in a leather bag and adding
a small amount of old kumyss as a starter. J In this country kumyss
is made from cow's milk. This product is now placed upon the market
by a number of companies who keep their methods, so far as possible,
from their rivals by maintaining strict secrecy in regard to the methods
of preparation. Dr. Piffard who has done special work on this product
states that kumyss is fermented by the action of yeasts and lactic bacteria.
This fermentation produces approximately i per cent of alcohol and
about 0.75 per cent of acid. Kumyss is strongly effervescent. The
lactic organisms used in the preparation of this material appear to be
a strain of the common Bact. lactis acidi. Whether or not the yeasts
are the common forms used by bakers cannot be stated with certainty.

Kumyss can be easily prepared in the household by the addition of
cane sugar and baker's yeast to fresh, warm milk which should be kept
at a temperature of about 38 (100 F.) until gas begins to form. It should
then be bottled and kept at a cool temperature. In one or two days
a slight amount of alcohol will be formed and a sufficient amount of carbon
dioxide to cause marked effervescence.

KEFIR (KEFYR, KEPHIR, KEFR, Etc.). Kefir was orginally made
and used by the inhabitants of the Caucasus Mountains. It was made

* Deut. 32:14.

f Metchnikoff's Prolongation of Life.

J Milch Zeitung, September, 1889.

New York Medical Journal, January 4, 1908.

368 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

from the milk of goats, sheep or cows and was fermented by the addi-
tion of "kefir grains" to the milk. The origin of these kefir grains is
unknown but the natives believe that they were the gift of Mahomet and
are carefully preserved by them.

Kefir was prepared by the natives by placing milk in a goat skin
bag and shaking it at intervals until it began to ferment. The kefir grains
were then removed, dried and preserved for future use. The fermented
kefir was also used as a starter for inoculating new lots. This beverage
is now commonly made by more scientific methods.* The principal points
to be observed in the preparation of kefir are cleanliness and proper temper-

FIG. 85. A large sized kepfir grain and the three species of bacteria of which it is
composed. (From Conn, after de Freudenreich.)

ature for fermentation and the regulation of the fermentation so that not
the acid but the alcoholic fermentation will prevail. | Good kefir should
be highly effervescent, should be free from lumps and contain about i per
cent of acid but show no marked tendency to whey off. According to
Kern, kefir is fermented by a mixed culture of yeasts and bacteria in
symbiosis. He found but one form of bacteria present in the cultures he
studied. De Freudenreich J made an extended study of the flora of kefir.
He prepared the kefir from the kefir grains and isolated the organisms
present, putting these organisms together in different combinations in
order to determine which were necessary for the proper fermentation of
the kefir. He found the kefir contained four different organisms: yeasts,
streptococci, micrococci, and bacilli. The yeasts and streptococci were

* Milch Zeitung, 1885, p. 209.

f F. Stohman, Milch und Molkerei Products, p. 1006 to 1013.

j Centr. fur Bakt. Abt. t, Vol. 3, 1897.

RELATION OF MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS. 369

plated in gelatin without difficulty but it was very difficult to grow the
other two organisms present on any artificial media. He concluded that
the yeasts present in kefir are not identical with the species commonly used
in making beer and named it Saccharomyces kefir. The streptococcus
curdled milk in less than forty-eight hours at a temperature of 37 but the
micrococcus did not curdle milk at all, although it produced a consider-
able amount of acid.

De Freudenreich changed the name of the bacillus from Dispora caucasica,
given it by Kern to B. caucasicus, because it did not produce spores as Kern sup-
posed. He also found that this organism would not grow at all on media without
sugar, very slightly on milk, serum, agar, and best of all in milk, in which it produces
both gas and acid without curdling the milk. This organism is 5 jj. or 6 fj. in length
by i j in width, is slightly motile and retains Gram's stain. It has a thermal death-
point of 55 for five minutes.

The preparation of good kefir seems to depend upon the combined
action of the four types of organisms described. Kefir is sometimes
prepared without the use of the kefir grains* by placing milk in bottles
to which is added a small amount of compressed yeast and sucrose. The
bottles are then held at a temperature of 10 to 15 about fifteen hours
and shaken occasionally. Kefir prepared in this way gives an effervescent
mild flavored drink.

LEBEN. For centuries the Egyptians have used a fermented milk
drink known as leben or leben raib. This was prepared from the milk of
cows, buffaloes, and goats. In general it resembles the other fermented
milk drinks in the fact that the fermentation is produced by yeasts and a
variety of other microorganisms working together. At least one yeast
and three species of bacteria seem to be normal to this product. A
fermented milk drink very similar to leben is also used in Algeria. Just
the action of each microorganism concerned in the fermentation of this
product is not certain, but it is probable that all of the species are essential
for the production of the particular flavor and consistency of the fermented
product. It is claimed that the fermentation that takes place in the milk
renders it more digestible than raw milk. For this reason it is recom-
mended for the use of invalids and persons having weak digestion.

YAHOURTH OR MATZOON (YOGURT, YAHOURD, MADZOON, ETC.). A
fermented milk drink known by one of the above names has been used
by the Bulgarian tribes for a long time. It has recently been studied and

* Milch Zeitung, 1888, p. 393.
24

370 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

brought to public notice by the investigations and writings of Metchni-
koff,* who was struck by the longevity of the tribes using this product as
a part of their regular diet. As a result of his investigations, Metchni-
koff has advanced his theory regarding the antiseptic power of certain
strains of lactic bacteria in the digestive tract. His theory is that cer-
tain species or types of bacteria which are able to resist the action of
the stomach and can, therefore, pass through into the intestines have
the power of checking the growth of the putrefactive bacteria existing
there and thereby prevent the production and absorption of bacterial
toxins which cause autointoxication. As a result of his experiments,
Metchnikoff came to the conclusion that the acid organism (Bact. bulgari-
cum) found in yahourth was able to establish itself in the intestinal tract
and produce enough lactic acid to hold in check the putrefactive pro-
cesses which otherwise exist there.

Yahourth is made by the Bulgarians in skin bags in the same way
that the Russian tribes prepare kumyss. It is similar to the other fer-
mented drinks already described in the fact that it is produced by a mixed
flora of microorganisms. At least one yeast is present and two or more
species of bacilli capable of producing lactic acid in relatively large
amounts. These two organisms are known as Bact. bulgaricum and Bacillus
paralacticus. Herter states that Bact. bulgaricum is 4^ to 6/< in length
by ifj. in width and grows singly or in pairs and occasionally in
chains. It stains with ordinary aniline dyes and by Gram's method. It
grows with difficulty on ordinary laboratory media and is therefore hard
to obtain in pure cultures. These organisms produce a much higher
percentage of acid than the common Bact. lactis acidi and also grow at
a much higher temperature.

This makes it possible to secure it in practically pure cultures by
growing it in milk at a high temperature. Grown in pure cultures, the
Bact. bulgaricum will produce from i to 2 or more per cent of acidity.
It grows well at temperatures between 37 and 40 and even higher.
Recently a number of fermented milk drinks have been put upon the
market which have evidently been derived from the yahourth. These
are sold under such trade names as zoolak, vitalac, yogurt, fernienlactyl,
etc. The flora of these preparations appears to be practically the same
as that of the original yahourth.

All of the fermented milk drinks thus far discussed are similar in that

* El., Metchnikoff, Prolongation of Life.

RELATION OF MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS. 371

each contains a variety of microorganisms, made up of at least one species
of yeast with one or more species of bacteria, capable of producing greater
or less amounts of acid. In some, as in the case of kefir, the yeast fer-
mentation is allowed to predominate, while in others, like yahourth, the
action of the yeasts is held in check by the rapid development of the acid
by the Bad. bulgaricum. All of these drinks are commonly recommended
by physicians because of their beneficial effect upon the digestive tract.

ARTIFICIAL BUTTERMILK. Quite recently there has developed an
important industry in the manufacture of artificial buttermilk. This
is usually made by inoculating skim milk with a culture of lactic bacteria,
either our native Bad. lactis acidi, or one of the imported species, such
as Bad. bulgaricum. In making the artificial buttermilk, yeasts are not
commonly added. After the milk becomes coagulated, it is then churned
in order to give it a smooth, creamy consistency, after which it may be
bottled and kept for some time by holding at low temperatures. Some-
times a small percentage of whole milk is added at the time of churning
to make the finished product more closely resemble natural buttermilk.
In making artificial buttermilk, the skim milk is frequently pasteurized
in order to get rid of the miscellaneous flora which it contains. The
finished product, therefore, differs from ordinary buttermilk in the fact
that it contains nearly pure cultures of the lactic organisms while the
natural buttermilk will contain a more or less miscellaneous flora in
which the acid organisms predominate. It is possible to obtain a more
uniform product in the artificial buttermilk than in the natural product,
and this is perhaps responsible for the rapid development of this industry.

FROZEN MILK.

Some effort has been made to put upon the market milk which has
been frozen into cakes or bricks. This has been tried both in Europe
and in this country. Some difficulty has been met in satisfactorily freez-
ing the milk and holding it in a frozen condition. The process has proved
to be rather expensive and not very satisfactory. One difficulty with
this process seems to be that the quality of the frozen milk after it has
been melted is not as good as it was before it was frozen. From a bac-
teriological standpoint, this process is of some interest, but it is doubtful
whether it becomes of much importance commercially.

372 MICROBIOLOGY OF MILK AND MILK PRODUCTS.

ICE CREAM.

Ice cream is one of the important manufactured dairy products and
its use seems to be increasing steadily. Its bacterial flora varies with
the materials used in its manufacture and the conditions under which it
is made. It may be made from fresh cream which is only a few hours
old and under good sanitary conditions. On the other hand, it may be
made from cream which has been produced and handled under unsani-
tary conditions, kept in storage for a number of days and finally manu-
factured in surroundings not conducive to a low bacterial content. We
are not surprised, therefore, to find a very wide variation in the germ
content of ice cream, as it is placed upon the market.

An examination of 263 samples of ice cream collected in the city of
Washington* showed an average germ content of over 26,600,000 per c.c.
The lowest count obtained was 37,500 and the maximum was 365,000,000.
A similar study of commercial ice cream in Philadelphia! showed the
average bacterial content to be very high. The lowest count found was
50,000 per c.c., while the highest count was 150,200,000. In this work
it was found that the bacterial content of the ice cream was in quite
direct relation to the sanitary conditions of the establishment where the
ice cream was manufactured. When ice cream is manufactured in a
city from materials which have been shipped in from considerable dis-
tances and frequently held for several days in cold storage, it is not sur-
prising that the germ content of the manufactured product should be
high. In some establishments the cream is pasteurized before manu-
facturing, while in others it is used in its raw condition.

In normal cream held for sometime, the lactic bacteria should exist
in considerable numbers, but when cream is held at low temperatures
these organisms do not develop rapidly. Pennington found that cer-
tain species of streptococci developed quite rapidly in cream held at
refrigerator temperatures. Streptococci were found in fifty-five (80 per
cent) of the sixty-eight samples examined. It was found that at refrig-
erator temperatures the relative growth of these organisms was greater
than at higher temperatures, a fact which may account, in part at least,
for the frequency with which these organisms occur in ice cream.

Frequently ice cream is held for a considerable time in a frozen con-
dition before it is sold. It has generally been supposed that there is no

* Results of work done under the direction of Dr. G. W. Stiles,
f Work done under the direction of Dr. M. E. Pennington.

RELATION OF MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS. 373

bacterial growth in material which is held below the freezing temperature.
This, however, did not seem to be the case in samples examined by the
investigators already mentioned. They found in samples held about a
month that there was normally a decrease in the bacterial count and
also in the amount of gas production for a number of days, after which
there was frequently a marked increase in the bacterial counts. These
results would seem to indicate that even in the frozen condition there
may be some increase in the number of bacteria present. The number
of these experiments, however, is not sufficient to justify very general
conclusions. The work of Conn and Esten* in holding milk at i may
throw some light upon this question.

If the cream from which the ice cream is made has been produced and
handled under sanitary conditions, the bacterial content should consist
chiefly of organisms of the Bad. lactis acidi type, in which case the high
count in the ice cream might not be objectionable. If, on the other hand,
the cream has been held in cold storage for some time under conditions
which inhibit the growth of the lactic organisms and permit the develop-
ment of putrefactive types, bacterial poisons may be developed in the
cream, which will be highly objectionable. There seems to be little
doubt that this is the cause of the cases of ptomain poisoning, resulting
from the use of ice cream. It is known that certain types of bacteria,
especially those belonging to the so-called putrefactive group, are capable
of developing at very low temperatures and can, therefore, produce con-
siderable quantities of toxic products in the cream. Whether or not
these products are developed before the cream is manufactured or whether
they may develop in the frozen product cannot at present be stated. In
general it can be said that the total bacterial count does not indicate the
wholesomeness of the ice cream any more than does a similar count in
buttermilk or in thf commercial fermented-milk drinks. The kinds of
organisms present is a far more important question from the standpoint
of the wholesomeness of the ice cream.

* Annual Report, Storr's Experiment Station, 1901.

DIVISION V.
MICROBIOLOGY OF SPECIAL INDUSTRIES.

CHAPTER I.
DESICCATION, EVAPORATION, AND DRYING OF FOODS

FACTORS THAT BRING ABOUT CHANGES IN DRIED FOODS.

The factors that bring about changes in dried foods may be considered
under two general heads, chemical and microbial. Enzymes, although
the product of living cells, may represent the chemical changes, and the
activity of bacteria, yeasts and molds, the microbial changes. Enzymes
are normally present in food stuffs derived from animals or plants which
have not been subjected to heating. All living cells contain enzymes,
and these may remain active for a considerable time after the death of the
cell. Some of these enzymes attack carbohydrates, some fats, some
proteins, and some other compounds. Enzymes are responsible for
the stiffening of the muscles after death (rigor mortis), others later
break down the tissues and bring about a ripening of meat whereby it
becomes more tender. Autolytic enzymes may in some instances
produce rancidity in food products by a splitting of the fats. Bacterial
enzymes are known that duplicate the action of practically all those pro-
duced by higher animals and plants. Some of the changes produced are
desirable, others undesirable, particularly if action is allowed to continue
for too long a time. In foods dried for preservation, it is therefore im-
portant that sufficient heat be used to destroy the enzymes or that enough
water be removed to inhibit their activity. Ordinarily the activity of
enzymes will be inhibited by the removal of water sufficient to prevent
the growth of microorganisms. The action of enzymes is characterized as
reversible, that is, after a certain concentration of enzymic products has
been reached, the transformation ceases until a part of the accumulation
has been removed by diffusion or otherwise. Since many of these actions

* Prepared by R. E. Buchanan.

374

DESICCATION, EVAPORATION, AND DRYING OF FOODS. 375

are hydrolytic in nature, water for both diffusion and hydrolysis must be
present before the enzyme can act.

Bacteria are introduced in large numbers when food is handled and
probably constitute the most important factor in its destruction. If
moisture and temperature conditions are favorable, they bring about
undesirable changes. The amount of water present in foods may be
used as a basis for their classification into four groups: first, those in
which moisture is present in appreciable quantities in the interstices,
that is, those which seem wet. Under these conditions bacteria not only
multiply but spread rapidly through the medium by actual space growth
by diffusion currents, and by their own motion. Second, some foods
may contain sufficient moisture for the abundant growth of bacteria,
but not free water for diffusion and distribution. In these the spread of
infection must be largely by direct growth of the organism and will neces-
sarily be slower than in the preceding. Third, the substratum may be
so dry that little or no growth of organisms may take place, yet there is
sufficient moisture so that they remain viable. Fourth, the food may be
so dry that only those organisms that can withstand relatively complete
desiccation will survive. These groups cannot be differentiated entirely
upon the basis of the percentage of water present, for the character of
the food itself and of the material in solution are also important.

Yeasts usually require sugars for their best development and are
therefore commonly present in foods containing this substance. They
are of importance therefore in fewer foods than bacteria. In nature,
they are frequently found upon fruits, particularly those which contain
considerable quantities of sugar in the sap. They will be found also
upon the cut ends of twigs or grass culms where sugary sap has oozed
out. Colonies of considerable size may sometimes be seen upon corn
stubble during damp weather. They are commonly distributed by
flies and other insects which feed upon the sugary plant juices. They
are not motile, hence the spread of infection in any food must be by
direct growth.

Molds, like bacteria, are ubiquitous and under proper conditions will
destroy almost any food. They grow readily in solutions and on saturated
substrata, but ordinarily are prevented by the bacteria which find the
optimum condition for their growth in such conditions. For example,
it is commonly observed that wet silage rots when exposed to air sup-
ports a luxuriant growth of bacteria, while drier silage becomes moldy

376 MICROBIOLOGY OF SPECIAL INDUSTRIES.

Unlike bacteria, the molds extend through and over food when there is
no visible water film. The spores are much better adapted to air dis-
persal than are bacterial cells, and the hyphae penetrate more rapidly
than will the bacterial colony. In certain foods, therefore, as meals and
flours, molds are more destructive than are the bacteria. Usually they
will multiply with less moisture.

INHIBITION OF GROWTH OF MICROORGANISMS IN DRIED FOOD.

In a few cases, the development of microorganisms is prevented by the
absence of sufficient moisture in the medium to support growth. This
is not nearly so common as might appear at first thought. It occurs in
some foods as olive oil, starches, meals, cane sugar, etc., that have little
or no free water. Frequently the drying results in a concentration of
the solutes, beyond the point to which microorganisms can adapt them-
selves to the osmotic pressure. When it is remembered that a 50 per cent
solution of cane sugar is capable of exerting a pressure of nearly 226.796
kg. (500 pounds) per square inch, it will be realized that considerable
readjustment is necessary in the cell of a yeast plant that can grow in
such a medium. Drying also sometimes changes the former relationship
of cells and tissue constituents so that protective layers may be formed.
For example, in curing pork, the fat which is structurally isolated in
distinct cells for the most part becomes diffused throughout the outer
layers of the tissues and forms a water-free and water-proof exterior.
Foods are sometimes subjected during the process of drying to sufficient
heat to destroy the microorganisms contained. At other times they are
exposed to the germicidal action of the direct rays of the sun or to the
fumes of some disinfectant or bleaching agent as sulphur dioxid or smoke.

METHODS OF DRYING.

The reduction of the water in foods below the minimum required
for the growth of microorganisms is accomplished in a variety of ways.
Most commonly heat is employed, either the sun's ray or some artificial
source. In localities where the humidity of the air is low, as in
many of the irrigated fruit districts of the western United States, ex-
posure to the rays of the sun results in rapid drying. With other types
of foods and in more humid regions, artificial heat is used to reduce this
relative humidity. Some foods cannot be dried at high temperatures
because of their instability. In most cases such foods must be dried

DESICCATION, EVAPORATION, AND DRYING OF FOODS. 377

quickly for they are readily attacked by microorganisms. These are
usually dried at a low temperature and in a partial vacuum. Other foods
are dried without recourse to evaporation by the use of the hydraulic
press or by centrifugal action, the latter in the manufacture of cane sugar.
The water available for the growth of microorganisms may be reduced
by the addition of some crystalline substance such as sugar or salt. The
usefulness of the latter depends largely upon their ability to create a
concentration of solutes too great for the growth of bacteria. At the
same time a considerable proportion of the water from that part of the
food into which the solutes will not penetrate, is abstracted by osmosis.

Many food products do not require any additional drying, as they
naturally contain little moisture. Such are the grains and the products
manufactured from them, as flour. The drying in this instance has oc-
curred during the ripening process of the grain. When for any reason
this does not occur, the grain will mold. It has been found necessary in
many instances to kiln-dry corn. Grain, nuts, etc., are by their nature
adapted to keep under normal conditions for considerable periods.
Other foods require artificial drying. In these we have the intergrading
classes, which have been discussed above, those which contain a very
small percentage of water and those which have considerable water but
a high concentration of solutes. The absolute amount of water in the
food is by no means an index to the amount that is available for the
growth of microorganisms. Many foods are hygroscopic. Foods having
the same water content and percentage of solutes will behave very differ-
ently with reference to delivering up the water to any organism present.

The effect of the concentration of solutes by drying is perhaps the
most important factor in the preservation of food. These substances
dissolved in the water may be actually antiseptic when concentrated, as
the acids of the juices of certain fruits. More often the sugars reach a
concentration so great as to prevent growth by plasmolyzing the cell con-
tents of the organisms. For every organism there is a maximum con-
centration reached sooner or later, beyond which growth is impossible.

Dried foods may be divided into three groups, using the relative
abundance of carbohydrates, fats, and proteins as a basis for classification.

Carbohydrate foods are usually preserved by drying. Many, such as
grains and nuts and the flours and meats prepared from them, do not
require artificial heating. They are, however, somewhat hygroscopic
and in damp climates enough moisture is taken up to allow the growth of

378 MICROBIOLOGY OF SPECIAL INDUSTRIES.

injurious molds and bacteria. Moldy corn has long been regarded as the
probable cause of the disease, called pellagra, in man. Still other carbo-
hydrate food stuffs require more or less care in the drying or curing, such
as hay and fodder in general. This is usually dried by simple exposure
to the air and sun until most of the water has been evaporated. Fodder
that has become moldy through the presence of too much moisture is a pro-
lific cause of trouble in horses and less frequently in cattle. The many
deaths due to the so-called equine cerebrospinal meningitis are suspected
many times to be due to the consumption of moldy hay. In localities
where the air is too moist or it rains so frequently as to make it difficult to
dry hay, curing is effected by a process of self fermentation. The hay is
piled in a mass while still green and undergoes a process of heating. The
temperature rises usually to about 70. The causes of this rise are some-
what uncertain, but it is probably due to the combined action of enzymes
and microorganisms. Just how much of this keeping quality is due to the
heating, how much to the loss of water, and how much to the accumula-
tion of products of fermentation is uncertain. In other cases, the heated
hay is spread out and quickly dries sufficiently so that it may be stored.
A certain small percentage of the nutriment of the hay is necessarily lost
in the development of the heat energy.

Fruits are quite generally preserved by drying. In many instances,
as in peaches, apples, and berries, it is probable that enough moisture is
usually removed to prevent organisms from growing, but in many other
cases, as in the preparation of currants and raisins, the concentration of
the sugar and other solutes is the controlling factor. Frequently as much
as 30 per cent of the dried fruits is water. Fruit drying is often
accomplished by the heat of the sun's rays, in other cases artificial heat
or even hydraulic presses are used.

Many manufactured products, particularly baker's goods, as crackers,
biscuits, dried yeast cakes, etc., are preserved by the elimination of water.

Macaroni and vermicelli are prepared by forcing a thick paste of expecially prepared
flour and water through openings of different sizes. The product is then dried in the
air until it is brittle and may then be kept indefinitely.

Copra, one of the principal exports of certain of the islands of the Pacific and Indian
Oceans, is prepared by cutting the meat of the cocoanut into pieces and drying them in
the sun. From this copra, much of our desiccated and powdered cocoanut is prepared.

Syrups, molasses, jellies, jams, and many other carbohydrate foods
are preserved through the concentration of the solutes. Many of these

DESICCATION, EVAPORATION, AND DRYING OF FOODS. 379

are partially sterilized by the heat used in the process of manufacture,
but there is usually plenty of opportunity for subsequent infection. They
are more frequently attacked by molds and yeasts than by bacteria.
An exception may be noted in Streptococcus mesenterioides which sometimes
causes considerable trouble by a gelatinous fermentation in syrups from
which sugars are manufactured commercially.

Foods with considerable quantities of fat usually contain little water.
Cottonseed, olive and other vegetable oils, the plant and animal fats,
as lard, tallow, and butter, are quite resistant to change by bacteria
unless water is present. With these foods the water is necessary for the
growth of the organism and also for the activity of the lipolytic enzymes,
which might hydrolyze fats and aid in the development of rancidity.
Butter forms an exception to the rule that fat foods contain little water,
as it usually has from 12 to 16 per cent. Where it is necessary to keep
butter-fat for long periods or under unfavorable conditions, it is melted,
the water removed, and the clear fat preserved. Bacteria, enzymes,
and a few molds have been described that attack fats. In the process
of preparation or manufacture of many fat foods, sufficient heat is used
to sterilize the material and infection thereafter spreads to the interior
very slowly. The heat destroys the enzymes as well as the bacteria.

The third class of foods preserved by drying includes those that
contain a high percentage of protein, in large part flesh foods and flesh
derivatives.

Desiccation, however, is only one of the agencies acting to preserve
the flesh.

Jerked meat is sometimes prepared in localities with a hot dry climate. Lean
meat is cut into thin slices and exposed to the direct rays of the sun until dry. The
bactericidal action of the sunlight and the rapid extraction of moisture prevents micro-
organisms from producing undesirable changes during the curing process.

Dried beef is lean meat which usually has been treated with certain condiments or
smoked and salted and then dried.

Dried fish such as cod, mackerel, and herring, is prepared by the use of condiments,
salt, and smoke in addition to the drying.

Pemmican is prepared by drying lean meat, grinding it, and mixing it with sugar
and fat, dried fruits, spices, etc. It is highly nutritious, not unpalatable, and compact,
and will keep for a long period. It is frequently used as a concentrated form of food
by Arctic explorers, etc.

Beef extract is prepared by cooking minced beef and water in a receptacle under
a slight steam pressure. The digestion is continued for several hours. The liquid is
filtered off and concentrated in a partial vacuum to the desired consistency.

380 MICROBIOLOGY OF SPECIAL INDUSTRIES.

Gelatin is prepared by boiling bones and tendons, sometimes also horn and hide
scraps and concentrating the gelatin which dissolves from these.

Somatose, sarco-peptone and related so-called predigested protein foods are mixtures
of albumoses and peptones prepared by the artificial digestion and drying of proteins,
usually flesh. The product is marketed as a powder.

M ilk, either with or without its butter fat, is dried by being sprayed into a warm
compartment from which the air is partly exhausted. It dries immediately, in the form
of a very fine powder. This powder, if thoroughly dry, will keep well and is finding an
extensive use. The high sugar content of this powder is instrumental in preventing the
development of microorganisms (p. 366).

Eggs are dried in much the same manner as milk and the product is being used
extensively at the present time by bakers.

Meats are frequently preserved by a combination of drying and the
action of certain antiseptics. The salting of meat owes its effectiveness
in part to the abstraction of water. In most cases, the surface of the
meat and probably even the other portions are protected in large measure
by the diffusion of the fat and the saturation of tissues and by the forma-
tion of water-proof fat films. The autolytic enzymes are active in the
fresh meat and soon become inert upon the removal of water. The
organisms responsible for decay of preserved meats and flesh foods are
usually bacteria. Some of these break down the protein into simpler
chemical compounds, of which a few are poisonous.

CHAPTER II.*
HEAT IN THE PRESERVATION OF FOOD PRODUCTS.

HISTORICAL RESUME.

The principle involved in the preservation of food by heat may be
said to have had its origin in the experiments of Spallanzani, who in 1765
boiled meat extract in hermetically sealed flasks for an hour, after which
treatment no change occurred in the material. An application of this
principle was suggested as early as 1782 by the Swedish chemist, Scheele,
who advised the exposure of vinegar in bottles to the temperature of boil-
ing water in order to effect its preservation. Some years later the principle
was applied to the conservation of food by a French confectioner, Nicholas
Appert, who in 1811 published an exhaustive treatise on "The Art of
Preserving Animal and Vegetable Substances." His method was to
enclose the food in a glass jar which was then corked tightly, and placed
in boiling water, the length of time of heating varying with the article
treated.

In 1810 Peter Durand secured a patent from the English government
for the preservation of fruits, vegetables and fish in hermetically sealed
tin and glass cans. He did not claim to be the discoverer of the process,
but said it had been communicated to him by a "foreigner residing abroad."
Although the secret of the process was jealously guarded, the employees
of different establishments became familiar with its essentials, and in
this manner the industry found its way to America. One of the first
to introduce the process was Ezra Daggett, who, with his son-in-law
Thomas Kensett, in 1819 engaged in the manufacture of hermetically
sealed goods, the principal foods packed being salmon, lobsters, and
oysters. In 1820, William Underwood and Charles Mitchell, emigrant
employees from a canning factory in England, opened a factory in Boston
where they canned plums, quinces, cranberries and currants.

In the earliest days of canning, glass jars were used exclusively, but
were gradually abandoned as it was found that they could not readily

* Prepared by S. F. Edwards.

382 MICROBIOLOGY OF SPECIAL INDUSTRIES.

withstand the extremes of temperature, and were expensive, bulky, and
costly in transportation. In 1825, Thomas Kensett secured a patent
on the use of tin cans in preserving food, and in the same year began
using the process in his factory. The early manufacture of tin cans
was by hand and crude, the bodies being cut with shears and the side
seams made with a plumb joint (that is meeting but not overlapping),
and then soldered together. Heads were made to set into the body and
were soldered in place in a very crude manner. The making of 100 cans
was considered a good day's work for one man. Improvements were
gradually made, however, in their manufacture, until finally can making
became a distinct industry and now all the parts are made and put
together by mechanical devices.

In the original Appert process, the goods were cooked in open kettles,
the highest temperature obtainable by this method being the boiling
point. A little later common salt was used to aid in securing a higher
temperature, and this was followed later by the use of calcium chloride
which made possible a temperature of 115. In 1874, a closed kettle
was invented for superheating water with steam, and this was immediately
followed by another improved kettle in which dry steam was used, the
principle employed being that of the modern autoclav, by which method
any desired temperature may be obtained and modified to suit the re-
quirements of different classes of food.

ECONOMIC IMPORTANCE.

FROM STANDPOINT OF HEALTH AND DIETETICS. The value of a
variety of foods, especially fruits and vegetables, is recognized by dieti-
cians. Unfortunately, however, the season for fresh fruits and vege-
tables is comparatively short Moreover, many foods grown exclusively
in one section of country will not withstand shipping in a fresh condition
to other sections. In spite of improved methods of refrigeration, it is
not practicable to ship fresh sea foods to far inland towns, or to send
some perishable products of warm climates to cold countries. The
canning and preserving industry overcomes these difficulties by supplying
pure, clean, wholesome fruits, vegetables, meats, and fish to any region
the year round, and at prices comparatively low.

FROM STANDPOINT OF COMMERCE. In its commercial aspect, the
importance of the industry can scarcely be estimated. Canned products

HEAT IN THE PRESERVATION OF FOOD PRODUCTS. 383

make possible the carrying of larger stores of provisions by armies and
navies and expeditions for exploration than would otherwise be possible.
In fact, the stimulus which prompted the investigation of Appert was a
prize offered by the French Navy Department for a method of preserving
foods for provisioning ships more satisfactory than by pickling, drying,
or other methods in use up to that time.

Although the preserving industry was established in three great com-
mercial centers in the United States as early as 1825, it did not become
of much importance until the last quarter of a century. There were
many hindrances to the progress of the industry, such as the secrecy
observed in the process, skepticism of the public regarding the health-
fulness of canned foods, the general prejudice against them, and the high
cost of production. These obstacles have gradually been surmounted,
and at the present time the several branches of the industry have collec-
tively assumed large proportions.

An idea of the importance and magnitude of the industry in the
United States and Canada may be gained from statistics for 1909 com-
piled by the National Canners' Association, the California Fruit Grower,
and Dominion Canners Limited. In the United States the pack of
tomatoes was 10,984,000 cases; of corn, 5,787,000 cases, and of peas
5,028,000 cases. This does not include fruits, the pack of which in
California alone was 3,047,000 cases. Nor do these figures include the
great variety of other vegetables, fruits from other states than California,
meats or fish. In Canada the pack was estimated at approximately
3,000,000 cases. The average case holds two dozen cans, and sells at
an approximate average price of two dollars and forty cents. It is ap-
parent from these figures that the canning and preserving industry is one
of immense value, and that it constitutes a large factor in the feeding of
the race.

ALTERATION OF FOOD.

PHYSICAL CHANGES. Appearance. Some physical changes attend
the conservation of foods by heat, approaching more or less closely the
changes incident to the ordinary preparation of fresh foods for the table.
In the preserving of some vegetables, notably peas and asparagus, the
canner subjects them to a blanching process which consists in submitting
the vegetables to the action of hot water for a short time, the object being,
first, to remove the mucous substance from the outside and a part of the

384 MICROBIOLOGY OF SPECIAL INDUSTRIES.

green coloring matter so as to have a clear liquor in the can, and second,
to drive water into the vegetables so that all will be tender. There is a
consequent tendency toward a paleness in products which undergo the
blanching process. Sometimes an artificial color is produced by the
addition of small amounts of copper sulphate, but this practice has never
become general in the United States.

Mechanical Disintegration. In the case of very soft fruits or vege-
tables, the high temperature essential to sterilization causes a slight
amount of mechanical disintegration, which is not objectionable, however,
unless excessive, as there is little deterioration in appearance and none at
all in food value. In the case of meats, practically the only physical
change is the shrinkage during the parboiling previous to placing in cans.

CHEMICAL CHANGES. Appearance. The chemical changes in foods
preserved by heat may be considered under two heads: first, those in
which the appearance is modified, and second, those in which the food
itself is altered. Change of color often occurs and results from various
causes. In colored vegetables, such as peas, string beans, and asparagus,
a part at least of the loss of color is due to oxidation of the chlorophyl
as a result of which the vegetables assume an unsightly yellowish or
grayish green appearance. Goods may sometimes be discolored by the
formation in the container of metallic sulphides which arise through the
liberation of simple sulphur compounds, by decomposition of protein
portions of the product, the sulphur afterward combining with the tin.
Abnormal color is sometimes attributed to the presence of iron in the
water used in the process. Some fruits packed in glass gradually lose
their color by oxidation on exposure to the light.

Chemical Change. So far as chemical alteration of the food itself is
concerned, there is little change and none other than would occur in the
ordinary preparation of the food for immediate consumption. The
albumins are coagulated. The fats probably remain unchanged. Of
the carbohydrates, the chief action is on the sugars. The cane sugar is
wholly or partly inverted by the combined action of the heat and the fruit
or vegetable acids. The starch undergoes little, if any, cleavage, inas-
much as this change only occurs in the presence of acids, and in foods
with a high acid content the proportion of starch is relatively low. The
other amyloses probably undergo little if any change.

Palatability and Digestibility. It is often contended that canned
foods are less palatable than fresh foods of the same kind. This deterio-

HEAT IN THE PRESERVATION OF FOOD PRODUCTS. 385

ration of agreeableness to the taste is, however, more seeming than real,
and arises largely from prejudice of the consumer against food conserved
in tin cans, rather than from any actual change. When the preserving
is properly done, the product should be no less attractive to the eye, no
less pleasing to the palate, and of no less value from the standpoint of
digestibility than the same food when served in the fresh condition.

BIOLOGICAL CHANGES. Vital Disorganization. The entire industry
of conservation of foods by means of heat is based on a microbiological
process. It is a universally recognized fact that the ordinary spoilage of
food is a microbiological change, hence the individual desirous of
protecting food from spoilage must give consideration to the microbial
agents responsible for the change.

Normal Flora and Fauna. Aside from a few types of organisms
causing disease or specific poisonous conditions, we are unable to desig-
nate definite species as those causing canned goods to spoil. Considering
the great variety of foods preserved by heat, and the different conditions
under which they are grown and secured, it naturally follows that the
normal flora and fauna of food to be preserved in this manner would
embrace a wide variety of species, including some higher fungi molds,
yeasts, bacteria, and low animal forms. Generally speaking, the mi-
crobial flora of fruits consists mostly of molds and yeasts, although
bacterial forms may also be present. In the case of vegetables, and of
fruits coming in contact with the earth, more species of bacteria are apt
to be present, many of them spore formers able to withstand a high tem-
perature. Finally, in meats and fish the living forms may include not
only molds, yeasts, and bacteria, but animal forms as well, such as the
organisms of taeniasis (tapeworm) and trichinosis. In the preserving
industry, therefore, consideration must be given to all these forms, not
only from the standpoint of the successful preservation of the various
foods, but also from that of protecting the health of the consumer from
possible poisonous substances present or produced in the conserved food,
and from possible infection with pathogenic organisms present in the food
before it is packed.

PASTEURIZATION.

ECONOMIC CONSIDERATIONS. In the preservation of food by heat,
two processes are applicable, pasteurization and sterilization. In pasteur-
ization, the aim is not to effect the permanent preservation of foods or
25

386 MICROBIOLOGY OF SPECIAL INDUSTRIES.

drinks by destroying all life present, but rather to accomplish the specific
purpose of destroying certain species of organisms in the material to be
pasteurized, thus effecting a partial arrest of the natural fermentation
along certain chosen lines by removing from the field of activity those
organisms whose presence serves to inhibit or pervert the desired fer-
mentative result.

The principle of pasteurization may be said to have originated in the
early work of Spallanzani and Scheele, already mentioned, and was
employed by Appert in his later investigations. The operation as employed
by Appert does not, however, appear to have found general application
until Pasteur revived the method, and as a result of his activities in attempt-
ing to secure a general adoption of the practice to prevent the spoiling of
wine, the process was named from him.

SPECIFIC APPLICATION. Beer. Pasteurization is of economic im-
portance particularly in the dairy and the fermentation industries. In
brewing, "the process of pasteurization is in use with even the smallest
brewers in the United States, beer being pasteurized even for local con-
sumption." The beer is pasteurized in bottles by being submerged
for one half-hour in water heated up to 60 to 65. In European countries
pasteurization of beer in jugs, as well as in bottles, is practised. Experi-
ments have been made in pasteurizing beer in large metal containers,
steel, copper, aluminum and tin having been tried, but without complete
success as yet. The process destroys the flavor.

Fruit Juices. The essentials in the pasteurization of wine and fruit
juices are similar to those for beer. There is, however, no universal
rule of application. Details of the process must be arranged to suit the
character of the different liquids under treatment.

Cream and Milk. Pasteurization as employed in the dairy industry va-
ries in its method of application according to the purpose for which it is
used. In factory butter-making, it must be employed to secure the best
results. Milk as ordinarily received at creameries contains a widely vari-
ant microbial flora, many of the species exerting a greater or less influ-
ence in determining the flavor of the finished product. By pasteurization
of the cream, the butter-maker destroys most of the organisms present
and by the use of a culture starter of lactic bacteria, a necessary conco-
mitant of pasteurization, he is able to control definitely the fermentation
and is assured of a uniform quality of product from day to day throughout
a season. An added value of pasteurization is that thereby all pathogenic

HEAT IN THE PRESERVATION OF FOOD ^PRODUCTS. 387

organisms are destroyed, thus aiding in the prevention of such diseases
as might otherwise be conveyed through this product. In creameries,
the usual method of pasteurization is what is known as the continuous
process, in which the milk is subjected to a momentary heating at about
85, the flow through the pasteurizing apparatus being so regulated as
to bring all the milk up to the desired temperature, the heating being
immediately followed by rapid cooling, and subsequent addition of the
lactic starter.

In the pasteurization of milk for infant or invalid feeding, a lower
temperature is employed. A temperature sufficiently high to destroy
the organism of tuberculosis (the standard for pasteurization) by momen-
tary heating, imparts to the milk a cooked flavor, making it less palata-
ble; coagulates some of the protein constituents, rendering it less di-
gestible; and affects the enzymes present, thus "devitalizing" the milk.
The desired end may be reached, however, by employing a lower tempera-
ture for a longer period of time, and the method generally recommended
is to heat the milk to 60 to 65 for twenty minutes. This heating is
sufficient to render harmless any pathogenic organisms likely to be present
in milk, without the objectionable features attendant upon heating to a
higher degree (p. 319).

Condensed Milk. It is commonly stated that Gail Borden invented the
process for preparing condensed milk in 1856. Previous to this, how-
ever, milk had been condensed in France, Germany and England as
early as 1825 to 1835. While he cannot, therefore, be called the inventor
of condensed milk, to Borden belongs the credit of having first prepared
it by a rational process and in a practicable form.

In the manufacture of condensed milk, good fresh milk is evaporated
in a vacuum pan similar to those used in sugar factories, at a temperature
of 40 to 50, until the volume is reduced to one-third or one-quarter of
the original. The evaporation must be conducted with great care,
otherwise the lactose crystallizes out and this causes the product to feel
"sandy" on the tongue. Condensed milk is made either with or without
the addition of cane sugar. When used at all, it is added in the propor-
tion of about 10 to 12 per cent of the weight of the milk. When the
evaporation of the milk is finished, the yellowish white syrup is sealed
up in tins which hold about 450 g., and this quantity is equivalent to
about i 1/2 liters of normal milk. The addition of cane sugar acts as
a preservative, and although the finished product may contain some living

388 MICROBIOLOGY OF SPECIAL INDUSTRIES.

organisms, it is said to keep indefinitely if unopened, and will even keep
for a number of days after opening. Occasional losses do occur, however,
by spoilage of the finished product, either from the growth of occasional
types of bacteria tolerant of the high percentage of cane sugar, or from
yeasts (p. 363).

STERILIZATION.

ECONOMIC CONSIDERATIONS. For certain classes of food products,
pasteurization is widely applicable, and is of immense value from an
economic standpoint. Preservation by pasteurization is at best, however,
temporary. Spores of spore-forming bacteria are certain to be present
on many kinds of foods, and these, unharmed by pasteurizing tempera-
tures, develop vegetative cells, and spoilage occurs.

For permanent preservation, therefore, sterilization must be adopted
and it is upon the principle of sterilization, coupled with prevention of
future contamination by hermetically sealing the container, that the whole
canning and preserving industry is based. The method is applicable to
nearly every class of food, and with less alteration in the food than any
other method of conservation. The principle employed to-day is essen-
tially the same as that used by Appert 100 years ago. Although he
knew nothing of microorganisms or their relation to the spoilage of
food, Appert's experiments taught him that not only must the food to be
conserved be heated thoroughly, but that it must be so sealed as not to
allow air to enter the container. In the light of microbiological science,
it is clear that the success of the process depends, not upon keeping out
the air, but upon keeping out organisms which are carried in the air.

SPECIFIC APPLICATION. The process of preservation by sterilization
is not so extensively practised for fruit juices and fermented liquids as
that of pasteurization. If too high temperature is employed for fruit
juices, certain compounds of agreeable taste and aroma are destroyed,
with a consequent deterioration in the flavor of the product. Fruit juices
may, however, be sterilized by heating at a low temperature for several
successive days.

The method of Appert has its widest application in the conservation of fruits and
vegetables, meats and fish. Whatever modifications are made in the handling of the
different classes of foods the essentials are the same. The raw material after thorough
cleaning and removal of waste if any, is filled into the cans and submitted to the steriliz-
ing process, the degree of heat and time of processing varying with different products

HEAT IN THE PRESERVATION OF FOOD PRODUCTS. 389

From the character of the flora, fruits as a rule require a comparatively low temperature
for sterilization, while some vegetables and meats require a very high temperature
to destroy the bacterial spores sure to be present. Briefly, the methods employed in
canning some foods follow:

Meat. In the meat canning industry, lean meat is largely selected for two reasons.
Fat, well-finished carcasses bring a better price when offered for sale in the fresh condi-
tion; and in the second place, lean meat has a better appearance in the canned state than
fat meat. The selected meat is cut into pieces of approximately from i to 4
pounds in weight, according to the size of the tins in which it is to be preserved. The
pieces are cut as nearly as practicable the same size, not only for purposes of appearance
in the cans when opened, but also that the process of sterilization may be more uniformly
carried out. If the pieces were of different size, the smaller ones would become
thoroughly cooked and disintegrated before the larger ones were sterilized.

After the pieces have been selected and dressed they are parboiled before being
placed in the containers, the time ranging from eight to twenty minutes, according to
the size of the pieces of meat. The object of parboiling is to secure the shrinkage which
always takes place on heating. Meats put into tins in the fresh state and sterilized
shrink to about two-thirds of their original volume. When the meat is put directly into
boiling water, there is less loss of protein than when the meat is placed in cold water
and heated gradually. During parboiling, the meat loses about i per cent of the
protein content, about one-third of the total meat bases, and 50 per cent of the
mineral matter.

This shrinkage by parboiling tends to make a more concentrated article, thus
favoring transportation, and, pound for pound, the nutritive value is not lowered.
Practically, the nutritive value of a pound of properly canned beef is about one-third
greater than that of i pound of fresh beef of the same kind. After parboiling, the
meat is placed in tins, by hand or by machinery, and to each can is added a small
quantity of "soup liquor," the manner of preparation of which is not disclosed by the
packers. It may be regarded as a thin soup, the object of which is to fill up the spaces
between the meat, and to add condimental substance to render the meat more palatable.

After the cans are filled, they are closed and processed in suitable retorts by steam
under pressure, as previously described, the temperature ranging from 110 to 120.

A modification of the usual method consists in exhausting the cans in vacua, and
automatically sealing them in the exhausted state, thus removing all the air and other
gases, after which they are placed on an endless conveyor and dipped into an oil bath at
a temperature of 1 1 5, the speed of the conveyor being so regulated that the cans remain in
the bath a sufficient length of time to complete sterilization before they are carried out
at the opposite end. They are next carried automatically into a solution of carbonate of
soda, and finally into pure water, after which they are dried, painted with a shellac or
lacquer and labelled.

Fresh meats other than beef or pork are canned in a fresh state. Game and wild fowl,
as well as domesticated chickens, ducks, geese, turkeys, and pigeons are used, the general
process being as already described. Horse meat is used more or less commonly in some
European countries, but probably rarely in the United States.

Fish. The process of fish canning does not differ materially from that of other
meats. On account of its proneness to rapid decomposition, especial care must be

3QO MICROBIOLOGY OF SPECIAL INDUSTRIES.

observed that the fish are in a perfectly fresh state before canning, and that the steriliza-
tion be most thorough. Salmon is the principal fish for the preservation of which
dependence is placed on sterilization alone, most fish being preserved by other
methods.

Vegetables and Fruits. Corn. The young, tender ears of sweet corn are picked
from the stalk, preferably in the early morning, keeping the husks on, and are taken in
this condition to the factory. They are husked and the silks are removed and passed
through machines with sets of knives which cut the grains evenly from the cob, care
being observed not to cut the corn so closely as to cut off particles of the cob with the
corn. After the corn is cut off the cob, some canners add a "syrup" of water, salt, and
sugar, and cook the corn for a few minutes at a temperature of 80, after which it is filled
into cans, sealed and sterilized. Another method is to fill the uncooked corn directly
into the cans, fill them with "syrup," hermetically seal, and sterilize. The tempera-
ture employed varies somewhat, but usually lies between 110 and 120 for thirty
minutes for pints. Proportionately longer time is required for larger packages. Most
of the operations are carried on by machinery. The sterilization is sometimes done in
the ordinary canners' retort, or the cans may be placed on an endless conveyor, dipping
into water or brine of a proper temperature, the speed of the conveyor being so
regulated that the cans are sufficiently heated to sterilize them during the passage.

Peas. In the pea canning industry the vines are cut with a mower, loaded on to
racks like hay, and hauled to the vining machines. The viner is a machine consisting of
an outer and an inner cylinder revolving in opposite directions, the inner one bearing pad-
dles or beaters so arranged that as the vines pass through the machine the paddles break
open the pods. As the peas are thrown out they pass through perforations in the outer
cylinder, while the vines are discharged at the opposite end. The shelled peas are
next washed to remove all dirt and also the mucous substance from the surface, thus
insuring a clearer liquor in the can. They are next graded for quality. This is
accomplished by passing them through tanks containing salt solutions of different
densities. The first grade includes all peas which will float in a salt solution having a
specific gravity of 1.040. The second grade consists of all peas which will float in a solu-
tion of specific gravity 1.070, while the third grade consists of peas which will sink in
the latter solution. Grading for size is next done by passing the peas over sieves, or into
a revolving cylinder having four sections with perforations of different sizes. The small
peas are of the best quality, and the quality decreases as the size of the peas increases.
The peas are next blanched in hot water to remove the mucous covering, and to drive
water into the peas so that all will be tender. The time of blanching varies from one-
half to one minute to five or more minutes, large mature peas requiring longer for
the blanching than smaller ones. After blanching the peas are filled into the cans by
special machines, the cans are filled with "liquor" consisting of water, salt, and sugar,
sealed and sterilized. The time of processing varies considerably, the average being
115 for twenty-five to forty minutes for the ordinary sized can.

Fruits. The essentials in the canning of fruits do not differ from those for vegeta-
bles. Stone fruits may be canned either with or without the pits. In case of such fruits
as cherries, or other acid fruits, the tin container must be coated on the inside with some
substance which will protect the tin from the galvanic action of the juice, which when
it occurs causes a deterioration in color of the fruit. The time and temperature of

HEAT IN THE PRESERVATION OF FOOD PRODUCTS. 391

processing of fruits is usually less than that required for vegetables, for the reason
that in the presence of the fruit acids the organisms are more easily destroyed than in
foods in which acids are not present.

CONTROLLING FACTORS IN SUCCESSFUL CANNING.

CLEANLINESS. Too much emphasis could hardly be placed upon the
importance of cleanliness throughout the whole preserving process, and
especially in the preparation of the product for preserving. Vegetables
and fruits that have come in contact with the soil are pretty certain to
harbor many spores of bacteria, and if as many of these are removed as
is possible by thorough preliminary cleansing, sterilization may be affected
with greater ease and surety. The necessity of cleanliness on the part
of factory employees is needful only of mention, not only from the aesthetic
standpoint, but also from that of good health.

THE SOUNDNESS OF RAW MATERIAL. The necessity of sound and
wholesome raw material is fully as great as that of cleanliness of handling.
Foods are never so good as when they are fresh, providing they are sound.
It makes no difference how long nor by what method they may be cooked,
the quality cannot be bettered; and if food is unsound when it is put into
containers for canning, it will never be wholesome for food; and this fact
is equally true whether the unsoundness is the result of diseased condi-
tions of meats, fruits or other products, or whether it is due to ordinary
decay.

WATER SUPPLY. Another essential for the success of the canner is
an ample supply of pure water, chemically and bacteriologically. Water
containing iron or sulphur compounds cannot be used for syruping or
brining operations, nor is a water containing any percentage of iodine or
bromine permissible. It is a well-known bacteriological fact that out-
breaks of spoilage have occurred in canneries which could be traced to
organisms getting into the goods from the water supply.

RECEPTACLE. The commercial canner recognizes two essentials for
suitable containers for his goods, and these are equally important for the
home canner. First, they must be tight, both to prevent the escape of
the contained product and the entrance of contaminating material.
Second, they must be of a material which will withstand erosion or corro-
sion for a reasonable length of time without giving up any notable quan-
tity of foreign material to the food product with which they may be in
contact. Glass is most satisfactory from this consideration, but for

3Q2 MICROBIOLOGY OF SPECIAL INDUSTRIES.

reasons previously stated it is impracticable for use on a commercial
scale. The difficulty from erosion in tins is partly overcome by coating
the interior of the can with an enamel or lacquer which is not affected by
the heat of sterilization, nor by chemical action of the contained material
within a reasonable length of time.

DEGREE OF HEAT REQUIRED. The actual sterilization of food
products after placing in the containers is termed by the commercial
canner processing, and he appreciates fully that upon the care with which
the processing is done depends the success of the entire pack. The
degree of heat necessary to accomplish sterilization varies considerably
with different products.

One factor lies in the chemical composition of the fruits or vegetables
to be sterilized. It is, for example, well known that peas and asparagus
are rendered germ-free with much greater difficulty than beans, not-
withstanding the fact that the heating of the can contents of the former
is accomplished much more easily than that of the latter. The higher
acid content of the beans facilitates the sterilization, and this same prin-
ciple holds true in a broad way for all products of an acid character.
The canner and the housewife have long known that tomatoes were
easy to preserve as compared with other vegetables. The canner also
finds a variation from season to season. In some seasons the acid content
of fruit and vegetable products will be higher than in others, and conse-
quently a lower processing temperature will suffice for sterilization.

In sterilization under pressure, as in the canners' retort, it is important
that the steam forced into the autoclaves should completely displace all
the air, for otherwise at a certain pressure the corresponding temperature
will not be obtained. Large cans require a longer time for thorough
heating than small cans; closely packed cans are heated with greater
difficulty than loosely packed ones; the inner temperature is frequently
lower than that of the outer parts of the can.

In addition to these factors, the canner must consider the possible
presence or absence of bacterial spores, which may gain entrance to his
factory, and necessitate a higher temperature than that usually employed
to accomplish the desired result.

STORAGE. Canned goods should be stored in a cool temperature.
With some foods, there is a slow physico-chemical change occurring even
in the absence of microorganisms by which the texture is loosened with
consequent deterioration in value. These changes proceed very slowly,

HEAT IN THE PRESERVATION OF FOOD PRODUCTS. 393

being hardly noticeable in two or three years, and the change will be even
more gradual at a low temperature.

SPOLIATION OF PASTEURIZED AND STERILIZED GOODS.

CHEMICAL. Aside from the change just mentioned, the chief chemical
action is that of the fruit acids on the tin containers, with consequent
formation of poisonous salts of lead or tin. The use of enamelled cans,
however, effectually prevents electrolysis from the action of the fruit acids
on the tin with impairment of color of the product, and also prevents
formation of lead or tin salts.

A kind of spoilage which sometimes occurs in goods packed with the
seeds or with the pits, and which causes a bulging of the ends of the cans,
designated by the canner as "springers" or "nippers," is caused by
insufficient heat being applied during the processing to destroy the germ
of the seed.

MICROBIOLOGICAL changes occur when the goods have not been
processed at a temperature sufficiently high to destroy all microorganisms
which may have been present in the uncooked product. In some in-
stances, the organisms present decompose the contents of the can, with the
formation of gas, causing bulging of the cans, sometimes to the point of
bursting at the seams. Such cans are designated at the factory as " swells"
in distinction from the springers mentioned above. In other instances
the bacteria in the imperfectly sterilized cans produce an acid fermenta-
tion, with consequent souring of the contents. Such cans are termed
"sours" by the canner.

Spoilage may occasion great monetary loss, as sometimes a whole
season's pack may be lost.

DETECTION or SPOILED GOODS. In cases of spoilage accompanied
with gas production, detection of the spoiled cans is usually easy, from
the swelled appearance of the can. On account of the exhaustion of air
from the cans during processing, the ends of sound cans should be slightly
concave. If, then, the ends of the cans are convex, it indicates some
abnormal condition of the contents, and such cans should be rejected.
In the case of sours, detection is not so easy. The can may appear
normal, and there may be no change in the contents apparent to the eye
on opening the can. Taste, however, reveals a more or less pronounced
disagreeable acid flavor.

394 MICROBIOLOGY OF SPECIAL INDUSTRIES.

Canned meats, fish, or crustaceans are likewise liable to spoilage if
sterilization has been imperfectly carried out. In these goods the change
may or may not be accompanied by gas production, but detection is
usually comparatively easy because of the objectionable odors and flavors
present. The degradation products from the breaking down of these
foods sometimes possess poisonous properties.

DISPOSAL OF FACTORY REFUSE.

The disposal of the factory refuse has at times become a serious problem to the
commercial canner. Of late years, however, methods have been devised for utilizing
much of the material that formerly was allowed to accumulate about the factory in
fermenting heaps to the extent of sometimes becoming a nuisance to the neighborhood.

At pea canneries several methods of utilising the vines are in use. They may
be converted into silage, either by putting into silos or stacking in large stacks. In some
sections the pea vines are cured for hay. They are also valuable as a soiling crop, and
as a fertilizer.

Corn husks and cobs are also utilized for silage. Experiments were made by the
United States Department of Agriculture on the feasibility of using the refuse from the
canning of corn for the production of alcohol. It was found that, on account of the
expensive machinery and other apparatus required in the manufacture, a small factory
could not profitably utilize the corn waste for alcohol. It was shown, however, that
where several factories were located within a short radius, by shipping their wastes to a
central plant, they might be utilized to advantage.

In the canning of tomatoes, many factories manufacture the cores, mutilated and
inferior tomatoes into pulp which is stored in barrels, to be made up later into the cheaper
grades of catsup. The skins are usually hauled out for fertilizer, although they " may
be used as a filler for cayenne pepper, and for ' cattle spice'." The tomato seeds are
sometimes graded for size, the larger ones being used for seed, and the smaller for the
manufacture of a yellow dye stuff.

Apples cores, "chops," peelings, etc., are usually either used for vinegar making,
or are made up into apple jelly. From one factory visited by the writer, the apple
cores and peelings were dried, baled, and shipped to Europe "to be made up into
champagne."

Peach pits are sometimes sold to nurserymen for seed. Sometimes the pits are
cracked and the meats removed and used for almond meats. In many factories no use
is made of the peach stones.

In the classes of food in which the amount of waste is not large, the refuse is
either hauled away to a dumping ground near the factory or is taken away by farmers
for its manurial value.

CHAPTER III.*
THE PRESERVATION OF FOOD BY COLD.

INTRODUCTION.

In recent times cold storage has become of very great importance
in the preservation of perishable food stuffs, and foods preserved by cold
usually command a higher market price than those preserved by othei
methods. This is probably due primarily to the fact that the general
appearance of refrigerated food resembles that of the perfectly fresh
article, in many instances very closely. Moreover, in many instances
cold storage, for a reasonable length of time, preserves not only the
appearance and the nutritive value, but also the chemical composition,
and even the delicate flavors of the original articles, so important in deter-
mining market value. The great economic importance of this industry
is at once apparent, for it aims to preserve unchanged the over-abundance
of one locality for transportation to another, and the over-production of
one season of the year for subsequent use.

THE EFFECTS OF REFRIGERATION UPON FOOD STUFFS IN GENERAL.

The decomposition of foods depends upon the activity of their own
intrinsic enzymes to some extent, but more especially upon the activity
of foreign microorganisms bacteria, yeasts and molds. Cold acts as a
preservative, not by destroying these microbes, but by retarding or in-
hibiting their activity. In general, cold not only retards the growth of
the microorganisms but delays' their death also, tending to preserve
them as well as the food stuff unchanged.

In discussing the refrigeration of foods we may consider three periods
of treatment, (i) the removal of the heat or chilling of the food, (2) the
prolonged storage at low temperature, (3) the subsequent warming of
the food before sale or consumption.

CHANGES DURING CHILLING. The period of cooling is a relatively

* Prepared by W. J. MacNeal.

395

396 MICROBIOLOGY OF SPECIAL INDUSTRIES.

short one, varying from a few hours to a few days in length. The chief
physical change is the intentional removal of heat by conduction and
convection, but there is usually also some loss of water by evaporation.
If cooled to a sufficient degree the water content of the food may crys-
tallize, altering to a considerable extent the physical structure of the
food substance (frozen food). Most foods are either actually living
when chilling begins, or they are only recently dead and various chemical
changes due to intrinsic enzymes continue at a diminishing rate as the
heat is removed. Decomposition changes due to microbes may also be
in progress and continue during the process of chilling. At this time the
microbes living in the cold-storage chamber gain access to the newly
arrived food stuff, others are added in the process of handling. The
extent to which these will grow and multiply depends upon their ability
to flourish under the storage conditions. In general the bacteria which
flourish at ordinary temperatures, producing the familiar decomposition
of the particular food, are greatly retarded in their activities and other
kinds outstrip them under the new conditions. The changes taking
place during chilling are very important in some special instances, and
often a very definite procedure must be followed to obtain a satis-
factory result.

CHANGES DURING STORAGE. This is often a relatively long period
and causes acting very slowly may ultimately produce marked alteration.
There is ordinarily some loss of water by evaporation, as well as the
evaporation or diffusion of other volatile constituents, some of them at
times important factors in the flavor of the food. Other volatile sub-
stances may be absorbed from the air of the storage room. The chemical
changes of the chilling period continue at a greatly diminished rate, or
may be entirely inhibited if the food is frozen. The behavior of the
microbic content of the food is the most important factor to be considered
during this period. Besides those already present, various other micro-
organisms, bacteria, yeasts or molds, may gain access to the food from
time to time, either from the circulating air or by contact with other
things. The fate of the implanted microbes will depend upon their
nature and adaptation to the conditions existing in the stored food.
Many of them perish, but many also survive the entire period of storage,
and some may actively multiply. There can no longer be any doubt
that some bacteria can grow at the temperature of zero, and many kinds
multiply at a fraction of a degree above that point. In order definitely

THE PRESERVATION OF FOOD BY COLD. 397

to inhibit microbic activity, the food must be frozen. When it is not
frozen, bacteria continue to multiply slowly at the lowest temperature of
storage, and small variations in the temperature and in the humidity
of the atmosphere serve to accelerate their activity. Such variations
also accelerate diffusion currents in the food substance and so tend to
distribute the microorganisms and their products. The extent of the
resulting chemical changes in the food will depend upon these factors and
upon the nature of the food, the temperature and the length of the period
of storage.

CHANGES AFTER STORAGE. This is a relatively short period, but in
many instances a very important one as regards change in the food stuff.
If warmed too rapidly, vigorous currents may be set up in the food mass
by the great difference in temperature between the outer portion and
the interior, serving to distribute microorganisms and their products.
In the case of frozen foods rapid warming fails to restore the original
physical structure. Dry cold food stuffs are likely to condense moisture
from the warmer atmosphere unless it is particularly dry, and this con-
densed water becomes another cause of diffusion currents. In frozen
foods the water, in melting, may fail to reenter the food structure, and
exude and drip away, carrying a portion of the soluble constituents with
it. At this time still more microbes are likely to be added to the food,
and, together with those already present, they multiply with increasing
rapidity as the temperature rises. As they may be already pretty well
distributed throughout the mass of the food, the resulting chemical decom-
position is the more rapid. It is well recognized that, in keeping quali-
ties, foods removed from cold storage are much inferior to the corre-
sponding fresh foods.

REFRIGERATION OF CERTAIN FOODS.

MEAT, FISH AND POULTRY. Meat, in this sense the flesh of mammals,
is preserved by cold in two ways, by storage above the freezing-point
(chilled meat) and by storage at 10 to 4 (frozen meat). Fish and
poultry are usually frozen for storage, the former often in the undrawn
condition, the latter sometimes so.

Mammals killed for chilled or for frozen meat are slaughtered and
carefully dressed. For chilled meat the temperature is reduced by storage
in a cold air chamber to about + 2 in 48 hours, and the meat is stored at a

398 MICROBIOLOGY OF SPECIAL INDUSTRIES.

temperature between + 1 and +2. Under these conditions the enzymes
of the dead flesh continue to act and bacterial decomposition proceeds
slowly, bringing about a process of ripening which, up to a certain point,
improves the market value of the flesh by making it more tender and giv-
ing to it a more desirable flavor. The extent to which the slow bacterial
decomposition may proceed before the flavor becomes disagreeable varies
with different tastes, but in general the beginning of proteolytic change
which follows after the almost complete fermentation of the muscle sugar
may be said to mark the desirable limit. This point is reached in from
a week to three months, depending upon the condition of the animal, skill
and care in slaughter and dressing, especially the extent of bacterial
contamination at this time, and the accurate control of the storage con-
ditions. In the production of frozen meat the carcasses are rapidly
chilled in an air chamber at 20, where the meat re mains until frozen solid.
It is then kept at a temperature below 4. Freezing produces a marked
change in the finer physical structure of the meat, as the water crystallizes,
eaving the protein material with which it was formerly intimately mixed
in a shrunken and shriveled state between the crystals. Enzymic and
bacterial activities are practically if not absolutely suspended under these
conditions, and, save for slight surface evaporation, such meat remains
unchanged for long periods. The subsequent thawing presents certain
difficulties and requires particular care. If warmed very slowly the
melting water crystals are imbibed by the protein material and the original
structure of the flesh almost completely restored. The warmer air must
be dry and must be kept in motion to avoid condensation of moisture on
the exterior of the thawing meat. Bacterial activity is likely to gain con-
siderable headway during this process and the penetration of the microbes
into the flesh is favored by the diffusion currents. The more prolonged
the warming process the greater the opportunity for bacterial decompo-
sition. Ordinarily, in order to avoid this, the thawing is carried out
rapidly and the finer structure of the meat is not restored. It is softer,
darker, and more moist than fresh or chilled meat, and usually sells at a
lower market price.

Fish and poultry are usually frozen for storage. As these foods are
especially subject to rapid objectionable decomposition changes they are
rapidly chilled by immersion in ice water or packing in ice immediately
after death, and are frozen as quickly as possible. During storage in the
frozen condition microbic activity is suspended, but in the subsequent

THE PRESERVATION OF FOOD BY COLD. 399

thawing the same physical and biological changes occur as in frozen meat.
When fish and poultry are stored in the undrawn condition there is an
abundant supply of bacteria at hand in the intestinal contents ready to
multiply energetically during the chilling and thawing stages. Practically,
the presence of the entrails has been found objectionable in the case of
frozen poultry because of the ease and rapidity with which poisonous
decomposition changes take place in this kind of food. It is desirable
that skill and care in killing and dressing should be employed in the
preparation of frozen poultry, and that the chilling period should be
shortened as much as possible. The tendency of such food rapidly to
undergo decomposition changes after thawing should be clearly recognized.
Its sale as fresh or as chilled food is a fraud upon the purchaser.

The nature and source of the bacteria which produce poisonous
changes in poultry are not definitely known, but there is some evidence
indicating that they belong to the para-colon group and that they are
derived from the intestinal contents of the fowls.

EGGS. The cold storage of eggs is an industry which has attained
large proportions in recent years. A very constant storage temperature
between +0.5 and + 1 is essential for the best results. The humidity of
the atmosphere is also of very great importance, as a dry air causes
extensive evaporation from the egg and a too moist air favors the develop-
ment of microorganisms on the exterior of the shell and the absorption of
their products and even their penetration into the egg. A constant
humidity of 70 per cent saturation has been found to be the best. Storage
at this temperature and humidity greatly retards the growth of micro-
organisms and definitely inhibits the ordinary putrefaction of eggs. The
activity of the intrinsic enzymes of the egg are not necessarily inhibited
by this temperature, nor is the growth of all microorganisms prevented.
Unquestionably there is a marked difference between the ordinary cold-
storage egg and the strictly fresh egg, but to what extent this deterioration
may be due to errors in storage such as inaccurate control of temperature
and humidity, use of odoriferous crates for packing, decomposition
changes previous to storage, too rapid chilling of the eggs, or too rapid
warming of them after removal from storage, and to what extent it is
inherent in the most perfect cold-storage procedure, is still somewhat
uncertain. Doubtless a certain amount of deterioration, especially the
loss of the peculiar flavor of the fresh egg, is unavoidable in any method
of prolonged storage. The discrimination in price in favor of new-laid

400 MICROBIOLOGY OF SPECIAL INDUSTRIES.

eggs in the market is an indication of difference in actual value, and the
sale of cold-storage eggs for new-laid or strictly fresh eggs is generally
recognized as a fraud by the purchaser and doubtless will in time be so
recognized by law. The cold-storage egg is nevertheless a very valuable
food and the economic importance of saving the over-abundant supply
produced during the spring for use during the colder season of the year
makes this industry a great benefaction to the public. Suitable regula-
tion may be expected to remove its objectionable features.

MILK AND BUTTER. Milk as ordinarily sold at retail is not subject
to sufficient seasonal change in market price to make its prolonged storage
advisable. But milk is so rapidly changed by bacterial activity at ordinary
temperatures that efficient dairy methods necessarily include prompt
cooling of the milk after it is drawn from the animal, and the maintenance
of a low temperature until it is delivered to the consumer. At the low
temperature bacteria slowly multiply, unless the milk is actually frozen,
but at a temperature slightly above the freezing-point very clean milk
may be kept in perfect condition for a week, and it may be kept sweet
for several weeks. Refrigeration of milk cannot compensate for un-
healthy animals producing it, nor for careless and uncleanly methods of
handling. The cold does not destroy the microbes in the milk but only
retards their multiplication and chemical activity. In practice, espe-
cially in the transportation of milk into large cities it is frequently most
economical to freeze the milk and trust to insulation and the latent cold
in the ice to maintain a low temperature during transportation. Such
milk should arrive at its destination in a partly frozen condition.

The cold storage of butter is essential even when it is kept for only
short periods, and the seasonal variation in price is sufficient to warrant
its storage from summer to winter. The keeping qualities of butter
depend upon many factors,* and the most efficient cold storage cannot
compensate for previous deficiencies. In refrigerated butter there is a
gradual diminution in the total number of living bacteria, with possibly
a multiplication of a few particular kinds. There is a slow increase in
acidity. In frozen butter the bacterial content and the chemical com-
position remain practically unchanged.

FRUITS AND VEGETABLES. These foods are for the most part adapted
for preservation for short periods at ordinary temperatures, and cold
storage at a temperature slightly above zero is very effective in diminishing

* See chapter on the microbiology of butter.

THE PRESERVATION OF FOOD BY COLD. 4OI

the rate of change in them. The humidity of the storage chamber should
be kept constant at about 60 per cent saturation in order to diminish
evaporation as far as possible without favoring the development of molds.
These foods generally remain alive during storage and the changes due
to intrinsic enzymes are often important. Some fruits need to undergo
further ripening in storage before they are ready for consumption and
this change may be accelerated or delayed by changing the temperature
of the storage chamber. The development of bacteria and molds with
consequent rotting is best delayed by maintaining dry clean fruits and
vegetables in an atmosphere of very constant humidity and very constant
temperature slightly above the freezing-point.

LEGAL CONTROL OF THE COLD-STORAGE INDUSTRY.

At present there is a rather widespread prejudice against cold-storage
food products, and in some respects this is not without justification.
Cold storage preserves so well the external appearance of fresh foods that
deception in the sale of them to the consumer is too frequently practised.
This is extremely unfortunate for all parties concerned in such transac-
tions. The proper branding of all cold-storage foods, clearly indicating
their character and the length of time held in storage, would ultimately
benefit the producer, the consumer and also the cold-storage industry.
Where cold storage is efficient such a practice would proclaim its efficiency.
Where it is inefficient the cold-storage industry can ill afford to allow the
consumer to be deceived concerning the food he is purchasing. Laws
compelling the p - oper labeling of su:h foods and prohibiting their sale
except when branded as such would quickly remove unjust prejudice
against cold storage, and would place this industry upon a secure founda-
tion, greatly increasing the possibilities of its service to the food producers
and consumers, and at the same time promoting the legitimate interests
of the cold-storage industry.

CHAPTER IV.*
THE PRESERVATION OF FOOD BY CHEMICALS.

The addition of preservative substances to foods is a very ancient
practice, and as no extensive equipment is required it is one of the cheap-
est ways of preserving food, especially on a small scale. The resulting
alteration of the food in appearance and composition is greater than when
it is preserved by cold storage, for the preservative substance added be-
comes a more or less permanent constituent of the food, but the changes
are not necessarily undesirable. The addition of chemical preservatives
is often practised in conjunction with desiccation or cold, or sometimes
even in canned or bottled foods sterilized by heat. All the substances
employed as preservatives owe whatever efficiency they may possess to
their ability to restrict the activity of microorganisms, that is, their anti-
septic properties.

THE EFFECTS OF PRESERVATIVES UPON FOODS IN GENERAL.

In only a few instances are chemical preservatives added to foods to
be sold as fresh foods, and these practices are generally regarded with
disfavor. Their most important use is in the prepared foods, the preserv-
ative being incorporated with the food during the process of preparation
for storage.

THE PROCESS OF CURING. The procedures employed necessarily
vary with different foods. Physical alterations in the food, such as changes
in form, texture and water content are usually involved, as well as the
solution of the preservative in the juices of the food. Chemical changes
due to the intrinsic enzymes of the food, to the various accessory pro-
cedures such as drying, cooking or soaking in pickling solution may
produce marked alteration. In some cases the preservative reacts
chemically with some constituent of the food. During the curing
process microbic activity may be more or less prominent at various
times, playing its part in the chemical changes. Bacteria, yeasts and

* Prepared by W. J. Mac Neal.

402

THE PRESERVATION OF FOOD BY CHEMICALS. 403

molds are likely to be introduced into the food by the various manipula-
tions and some of these may find conditions favorable for their prolifera-
tion. In some instances the activity of certain kinds of microbes appears
to be essential to the proper curing and subsequent adequate preservation
of the food, the preservative, the constituents of the food and the micro-
organisms mutually reacting to bring about the desired result. It is
worth noting that the added chemical preservative is never sufficiently
potent to destroy with certainty pathogenic microbes which may be
present in the food.

THE PERIOD OF STORAGE. Unless the food has been sterilized and
stored in sealed containers, slow changes in water content, in consistency
and in physical appearance usually take place during storage. The
added preservative may continue to react with the food substance and
its decomposition products. During this period there is relatively little
intimate manipulation of the food and therefore little opportunity for the
penetration of new microbes. Some of those already present may con-
tinue their activities at a diminished rate, producing slow chemical
changes often of a desirable nature rather than otherwise. Accessory
conditions, such as desiccation, cold storage, or sterilization and sealing,
may greatly retard or check altogether microbic activity.

THE AFTER-STORAGE CHANGES. The immediate preparation of
preserved food for consumption is frequently important. The preserva-
tive may be largely removed mechanically, or extracted with water.
During cooking peculiar chemical reactions may occur, and cooking is
also important in the destruction of microorganisms remaining alive in
the food up to that time.

THE CHEMICAL PRESERVATION OF CERTAIN FOODS.

MEATS AND FISH. The preservation of meat and of fish by salting
depends upon the increase of osmotic tension in the food, a physical
change sufficient to prevent or greatly delay the growth of microorganisms.
Sodium chloride (NaCl) probably owes its preservative value solely to
this physical effect. In practice its action is often supplemented by the
addition of a small amount of saltpeter (KNO 3 ), and sometimes also
cane sugar (C 12 H 22 O n ). The fluids of the flesh are in part removed
by this treatment, carrying away a part of the soluble constituents. The
fluids which remain contain the added preservative substance in solution,

404 MICROBIOLOGY OF SPECIAL INDUSTRIES.

and the whole mass of food substance is permeated by them. Potassium
nitrate (saltpeter) reacts with the flesh, being reduced in part to nitrite.
This enters into a combination with the coloring matter of meat, which
upon cooking produces the characteristic red color of saltpeter-cured
meat.

The various manipulations during the process of pickling or dry
curing serve to introduce numerous microorganisms. Many of these
may flourish in the pickling fluids, but in a sufficient concentration of
salt and at a sufficiently low temperature, decomposition ordinarily does
not progress so as to become objectionable, and proteolytic decomposition
(putrefaction) is effectually prevented. This protection of the protein
depends to some extent upon the acidity of the medium, which in turn
is due largely to the bacterial decomposition of the muscle sugar. The
powerful putrefactive bacteria (B. cedematis group, B. putrificus) flourish
only in an alkaline medium. On the other hand too high a degree of
acidity becomes in itself objectionable on account of the sour or rancid
taste, and it is therefore important that the acid-producing bacteria should
be held in check somewhat. In practice, saltpeter has proved of value
for this particular purpose, and its action apparently depends upon the
antiseptic effect of minute quantities of nitric acid (HNO 3 ) and nitrous
acid (HNO 2 ) set free from the salt by the excess of organic acids produced
by the bacteria. The curing of meats by pickling solutions is often supple-
mented by desiccation and impregnation with the antiseptic substances
of wood smoke.

The dry-salting of codfish is an example of preservation by increasing the osmotic
tension. The fish is cleaned and beheaded, split longitudinally, and the vertebral
column removed. It is then carefully washed, and all visible blood is removed. The
pieces are next covered with dry salt and packed in open casks. The salt rapidly
extracts water from the flesh and a strong brine results. After a few days the casks are
emptied out, and the pieces of fish, now smaller because of the loss of water, are again
thoroughly washed and again packed in dry salt so that the adjacent pieces of fish are
completely separated by an intervening layer of solid salt. The contents of the cask
is subjected to high pressure to remove air, and the cask is finally closed.

The curing of hams is an example of preservation by increased osmotic tension
combined with the addition of chemical preservatives. After slaughter and chilling
the hams are injected with a solution containing 25 per cent common salt, 15 percent
granulated sugar, and 12 per cent saltpeter, and are then stored at a low temperature,
preferably between o and +4, in a brine containing about 20 per cent common salt, 5
per cent sugar, and i per cent saltpeter. The brine is renewed once or twice at intervals
of a week or ten days. After about a month the hams are washed in warm water, dried

THE PRESERVATION OF FOOD BY CHEMICALS. 405

and hung in wood smoke for several days. They are then stored in a cool place.
The proportions of the various constituents of the pickling solutions are subject to
rather wide variation, and in general it may be said that the higher the temperature of
the storage room, the more concentrated must be the pickling solutions to insure
satisfactory preservation.

BUTTER. Butter is usually salted with sodium chloride to impart
the desired taste, and this salt also acts to some extent as a preservative
by increasing the osmotic tension of the moisture remaining in the butter.
Antiseptics such as boric acid, saltpeter, salicylic acid and formaldehyde
have been employed in the preservation of butter, the first-mentioned
appearing to be the most satisfactory. One half of i per cent of boric
acid incorporated with high-grade butter previous to storage greatly
delays rancid change. Fresh milk and cream are also sometimes treated
with antiseptics such as formaldehyde, but the use of any chemical pre-
servative whatever in these dairy products is unnecessary and generally
disapproved.

PREPARED VEGETABLE AND FRUIT FOODS. These foods are some-
times preserved by vinegar, sugar or alcohol, the presence of which is of
course very evident to the consumer. Other substances less readily
detected, such as sulphurous acid and sulphites, boric acid, salicylic acid,
benzoic acid and sodium benzoate, and formaldehyde, are also employed
in foods which must be kept some time after exposure to the air. These
substances are incorporated with the food before it is packed, and serve
to prevent the activity of microorganisms which gain access to it.

THE NUTRITIVE VALUE OF PRESERVED FOODS.

The nutritive value of a food depends upon the amount of utilizable
food principles it contains. The food-principle content can be readily
measured by chemical analysis, and in general there is no important dif-
ference between a preserved food and the corresponding fresh food in
this respect. The utilization of the food principles, however, depends
upon a number of factors and may be greatly influenced by individual
peculiarities of the consumer. One important factor in the utilization
of a food, and probably the most important factor in determining its
market value, is palatability. In general, preserved foods are pleasant
to the taste when eaten at intervals, but upon prolonged daily ingestion,
the appetite for them fails and they may even become distasteful. It
would therefore appear to be erroneous to regard preserved foods as in

406 MICROBIOLOGY OF SPECIAL INDUSTRIES.

every respect as valuable from the standpoint of nutrition as the corre-
sponding fresh foods. The difference is not dependent upon a change in
the food-principle content, but must be sought rather in slightly altered
composition of the food and the specific effects of newly formed substances,
and especially in the possible effects of the continued ingestion of the con-
tained chemical preservatives upon the consumer.

THE EFFECTS OF FOOD PRESERVATIVES.

The essential characters of a food preservative include antiseptic
action to prevent decomposition of the food, and absence of evident
poisonous or deleterious influence upon the consumer. It follows there-
fore that the effects of food preservatives upon the consumer, if they exist
at all, are at any rate not easily recognized, and on account of the economic
importance of the questions here involved this field of scientific research
has been energetically cultivated by investigators with different viewpoints,
and the results of investigation have been discussed with some heat. The
modern pure-food laws have required more specific knowledge upon these
points for their proper administration, and have stimulated extensive
investigations, but it is still too early to regard the facts as finally and
definitely ascertained.

SUBSTANCES WHICH PRESERVE by THEIR PHYSICAL ACTION. The
preservative effects of sodium chloride seem to depend entirely upon the
high osmotic tension of strong salt solutions, and the same may be said of
cane sugar. When diluted so as to be eaten with relish, these substances
are themselves properly classed as foods, without deleterious effects upon
ordinary individuals.

SUBSTANCES WHICH PRESERVE BY THEIR CHEMICAL ACTION. These
preservatives inhibit the activity of microorganisms in a different way,
not by withdrawing water from the microbic cell, but by entering into
chemical combination with the living substance in such a way as to hinder
its activity, or by entering into chemical reactions with the food to produce
new substances capable of attacking the microbic protoplasm in this way.
The ideal chemical food preservative would be one which, without altering
the food substance, would exhibit this poisonous property toward living
protoplasm until the food was ready for consumption, and then would
suddenly and permanently lose this property. None of the ordinary food
preservatives approaches this ideal very closely.

THE PRESERVATION OF FOOD BY CHEMICALS. 407

INORGANIC FOOD PRESERVATIVES. Boric acid and borax are weak
antiseptics, practically a saturated solution of boric acid being necessary
to inhibit ordinary bacterial growth. When employed as a dry powder
on the surface of meats, boric acid prevents the growth of mold, and most
of it is removed from the food before consumption. When incorporated
with butter it is eaten, and 0.5 to i.o g. may be taken daily in this
way alone. The effect of such amounts of boric acid upon the consumer
is still a disputed question. Wiley,* after a recent extensive investigation,
concluded that small doses of either boric acid or borax continuously
administered for a long period create disturbances of health.

Nitric acid and nitrous acid and their salts are food preservatives of
some theoretical interest because it is well known that some bacteria
readily decompose fairly strong solutions of nitrates, and also oxidize or
reduce nitrites. Apparently, however, this is true only in neutral or
alkaline solutions, and in the presence of free acid the activity of these
microbes is quickly inhibited. The preservative effect of nitrates and
nitrites is best ascribed to the liberation of minute quantities of free nitric
and nitrous acids from these salts, and these substances are without value
as preservatives in foods which are alkaline in reaction. The effects of
the ingestion of nitrate or foods preserved with nitrate upon the consumer
has been investigated by Wiley, who concluded that the deleterious effects
are slight and less clearly detected than in the case of the other preserva-
tives. Minute but variable amounts of nitrites occur in foods preserved
with nitrates, but whether these amounts are sufficient to produce the
specific nitrite effect upon the blood circulation of. the consumer has not
yet been definitely ascertained.

Sulphurous acid and the sulphites are rather extensively used in
chopped meat (Hamburg steak) and in cider and wines. The addition
of sulphite to chopped meat serves a three-fold purpose, retarding bac-
terial decomposition, producing a red color on the exposed surface, and
removing odors of decomposition. It thus not only delays decomposition,
but also to a certain extent conceals the decomposition which has already
occurred. The ingestion of moderate quantities of sulphites in food
has at times been followed by acute gastric derangement in man, and
prolonged feeding of meat containing sulphites has been followed by
inflammatory changes in the kidneys of experimental animals.

Fluorides have been used to a slight extent in beverages, but acute

*U. S. Dept* AgT., Bureau of Chemistry, Bull. No. 64, Part I.

4O8 MICROBIOLOGY OF SPECIAL INDUSTRIES.

gastric derangement and depression of the heart are caused by rather
small quantities, and probably on this account the salts of hydrofluoric
acid have not come into very general use as food preservatives.

ORGANIC FOOD PRESERVATIVES. Formic acid (H-COOH) and
acetic acid (CH 3 'COOH) are produced by microbic activity and their
preservative action appears to depend more upon the degree of acidity
than upon the character of the acid radical. Both these acids appear to
be utilized as food in the body of the consumer.

Benzoic acid and benzoates are rather extensively employed in pre-
pared vegetable food products, such as jams and catsups. The antiseptic
effect seems to be due wholly to free benzoic acid, even where it is added
in the form of the salt, but the action is not due merely to the acidity
(i. e. the hydrogen ion). Benzoic acid is not utilized as a food in the
body, but is excreted by the kidneys in the form of hippuric acid. It has
been said to produce irritant effects upon the stomach and the kidneys,
and to arrest the action of digestive enzymes in dilute solutions, but the
Referee Board* of the United States Department of Agriculture, after
extensive investigations, concluded that small doses of sodium benzoate
mixed with food are not injurious to health, and do not impair the quality
or nutritive value of the food.

Salicylic acid and the salicylates have been used for much the same
purposes as benzoic acid, and there does not appear to be much dif-
ference between the two acids, either in their efficiency as preservatives
or in their possible deleterious effects upon the consumer. Salicylic acid
is more expensive. After extensive investigation Wileyf has concluded
that the addition of salicylic acid and salicylates to foods is reprehen-
sible in every respect, this conclusion corresponding to the results of
similar work by the same investigator! upon benzoic acid.

Formaldehyde is very efficient as an antiseptic, delaying microbial
decomposition when added to foods in very small quantity. Its use for
this purpose is generally condemned, partly because of its hardening or
"fixing" effect upon the protein constituents of the food, tending to make
them more indigestible. Its use in milk and milk products, though still
practised to some extent, has been prohibited by law in some states.

Alcohol (CH 3 -CH 2 OH), in sufficient concentration, is an excellent

* U. S. Dept. Agr. Report No. 88, May, 1909.

t U. S. Dept. Agr., Bureau of Chemistry, Bull. No. 84, Part II, 1906.
j U. S. Dept. Agr., Bureau of Chemistry, Bull. No. 84, Part IV, 1908..

THE PRESERVATION OF FOOD BY CHEMICALS. 409

preservative, but its presence in foods is readily detected, and it gives rise
to characteristic effects upon the consumer. Furthermore, such foods are
subject to special taxation as alcoholic products. Its use as a food pre-
servative is therefore limited.

Wood smoke has been employed for centuries in the curing of meats.
Its antiseptic properties probably depend for the most part upon creosote
and pyroligneous acid, constituents of wood smoke which are antiseptic
and also undoubtedly poisonous in sufficient doses. Smoking is a time-
honored custom, however, and the amount of these substances actually
consumed with the smoked meat is doubtless exceedingly minute.

SUBSTANCES ADDED TO FOODS TO IMPROVE THE APPARENT QUALITY.
Several chemical substances are employed in various foods to improve
the appearance, or to simulate the taste of a higher-grade product. In
some cases the presence of these agents is known to the consumer, and
desired by him; in other instances they are employed to deceive the pur-
chaser. Butter coloring is quite generally used to produce the color of
June butter the year around; nitrates bring about a pleasing red color in
cooked pickled meats; copper sulphate is used to give a more brilliant
green color to prepared vegetable foods; sulphites restore the red color of
freshly cut meat to meat far from fresh; saccharine devoid of food value
gives a taste resembling sugar to a variety of preparations at a great sav-
ing in cost to the manufacturer; carbonates of the alkalies or alkaline
earths added to milk, neutralize the acids resulting from bacterial decom-
position and so keep the milk sweet; inorganic acids added to weak
vinegar increase its acidity. Some of these practices are so universal and
so well known that they are no longer criticized, others, such as the use
of chalk in milk, are generally disapproved.

LEGAL CONTROL OF THE PRESERVATION OF FOODS BY CHEMICALS.

The desirability of legal regulation of the use of chemical food pre-
servatives is now generally recognized, but there is still considerable di-
versity of opinion concerning what this regulation should be. Few, in-
deed maintain that a substance exerting antiseptic action upon microorgan-
isms outside the body is wholly without influence, after ingestion, upon
the enzymes and bacteria of the normal digestive tract, even if we dis-
regard the possible effects of the substance after absorption. It seems
necessary to grant the existence of some effect, even though it be so slight

410 MICROBIOLOGY OF SPECIAL INDUSTRIES.

as to have escaped detection. Over against the possible injury to the con-
sumer must be placed the economic saving through the use of the preser-
vative, often involving a considerable amount of money. In the absence
of accurate and trustworthy knowledge concerning the actual influence of
preservatives in the human body it would seem wise to prohibit all decep-
tion in regard to their presence. The principle advocated by Pasteur
(1891) would still seem to be best, that is, to allow the use of preservatives,
which are not known to be dangerous, upon the condition that their
presence and the exact amounts be definitely and clearly stated on an
appropriate label for the benefit of the purchaser and the ultimate con-
sumer. Such regulation would not only protect the consumer against
deception and fraud but would go far toward removing unjust prejudice
against preservatives, for even now there is little or no objection to those
preservative substances whose presence can be detected and the amount
roughly measured by the senses, such as salt, sugar, spices, vinegar and
wood smoke.

CHAPTER V.*
MICROBIAL FOOD POISONING.

GENERAL CONSIDERATIONS.

Illness following the ingestion of food and more or less definitely
ascribable to the food has been long recognized. The Mosaic regulations
in regard to foods forbidden to the Jews are evidently designed in part
to avoid the occurrence of food poisoning. In recent times recognized
instances of food poisoning have been sufficiently frequent to make the
subject one of considerable practical importance, but there are undoubt-
edly many instances of actual food poisoning in which the causal relation
of the food remains unrecognized or even unsuspected.

Food poisoning is usually suspected at once upon the occurrence of
sudden acute illness in a number of people at the same time, after they
have partaken in common of some particular food or foods. The causal
relation is especially evident when, as sometimes happens, a large number
of people are affected in the same way immediately after eating together
at a banquet, not having been associated with each other either before or
after the meal. When a smaller number of individuals is involved, the
connection with food may be more obscure. For this reason most of the
well authenticated instances of food poisoning are instances in which
many persons have been affected at the same time. Acute food poisonings
involving only a few persons probably occur very frequently in the home,
but they receive little public notice unless fatal, and are often dismissed as
mere "errors in diet," or as "indigestion." A careful study of these cases
is likely to be made only where there is suspicion of criminal poisoning,
or some other practical end to be served by the investigation. Chronic
forms of food poisoning are for obvious reasons very difficult to recognize
with certainty, and some of the forms of disease now regarded as due to
chronic food poisoning may eventually prove to be due to other causes.
On the other hand chronic food poisoning may really be more important
than is recognized at present. The subject is still in a very doubtful
state.

* Prepared by W. J. Mac Neal.

411

412 MICROBIOLOGY OF SPECIAL INDUSTRIES.

To establish by laboratory investigation the poisonous character of
foods requires toxicological training and experienced judgment, a dis-
cussion of which would lead beyond the scope of the present chapter. For
a general review of this field of work and references to further information
the articles cited at the end of this chapter should be consulted.

Several different classes of food poisonings may be recognized accord-
ing to the source of the poisonous substance.

The substance of plants or animals may be naturally poisonous to
man as a result of the physiological activity of their own living substance.
Poison of this kind may be constantly present throughout the tissues,
or it may be confined to certain parts, or it may occur only at particular
times or seasons. Some instances of poisoning with fish and with mush-
rooms belong to this class, and possibly also some of the instances of
poisoning with potatoes of high solanin content.

Plants and animals may feed upon substances not poisonous to
themselves, and these substances may remain a constituent part of their
bodies to poison man when consumed by him. Some poisonings with
freshly killed game are considered to be of this nature.

Any food may contain foreign poison added to it by design or by
accident, such for example as the salts of the various poisonous metals.
The amount of tin or lead passing into solution in canned or tinned foods
may conceivably be sufficient to cause poisoning, but there is no reliable
evidence that it has ever occurred.

Animals may be infected with pathogenic bacteria or with other
parasites capable of infecting man, and the use of food products from such
animals may cause disease. Tuberculosis, trichinosis, and tapeworm
may be acquired in this way.

Any food may serve as the passive carrier of infectious agents, such
as B. typhosus, and some foods may even favor the multiplication of patho-
genic bacteria gaining access to them.

A food may undergo chemical changes due to microorganisms in-
capable of infecting man, resulting in the production of poisonous sub-
stances in the food, Undoubtedly the great majority of instances of food
poisonings belong in this class. The bacteria causing these changes have
been designated as pathogenic saprophytes.

The last three classes comprise the microbial food poisonings, and these
are the kinds of food poisoning with which we are at present more par-
ticularly concerned.

MICROBIAL FOOD POISONING. 413

INFECTIONS OF FOOD-PRODUCING ANIMALS TRANSMISSIBLE TO MAN.

Animals dead of infectious diseases or slaughtered in the last stages of
disease are not ordinarily used for food, nor is the milk of such animals
ordinarily considered wholesome. This custom is certainly an ancient
one, and is doubtless founded upon observation of unfavorable results
following the consumption of such food. Exact knowledge of the nature
of the diseases transmitted in this way is a more modern development, and
this more exact knowledge is now being applied to some extent through
food-inspection regulations to prevent the transmission of such diseases.

Tuberculosis of cattle has been shown by Smith to be due to a germ
somewhat different from that causing the ordinary human tuberculosis,
and this discovery has called into question the necessity of avoiding
the use of food products from tuberculous animals. After a considerable
amount of controversy it may now be regarded as definitely established
that the bovine type of tubercle bacterium is capable of infecting man, and
that a very considerable proportion of cases of tuberculosis in children are
due to this type of organism, the infection probably arising through the
use of milk from tuberculous animals. Anthrax, glanders, actinomycosis,
and acute enteritis of animals are also transmissible to man. Food
products from animals afflicted with these diseases should not be used
until they have been passed upon by competent authority. Further in-
formation concerning them will be found in the sections dealing with these
particular diseases.

In this connection it may be mentioned that some of the animal para-
sites, especially trichinae and various sorts of tapeworms, gain access to
the human body with the food. Thorough cooking usually serves to kill
these parasites, as well as the pathogenic bacteria, but ordinary cooking
should not be too implicitly relied upon to accomplish this result.

HUMAN INFECTIONS TRANSMITTED IN FOOD.

Food may serve as the passive carrier of the germs of any human in-
fectious disease capable of indirect transmission upon dead material.
In some foods, especially milk, these infectious agents may actually
multiply. Typhoid fever, diphtheria, and scarlet fever appear to be
rather frequently disseminated through the agency of food, and para-
typhoid fever seems to be commonly transmitted in this way. Especial
precautions are advisable to prevent persons afflicted with dangerously
communicable diseases and those who are chronic germ-carriers from

414 MICROBIOLOGY OF SPECIAL INDUSTRIES.

engaging or continuing in occupations concerned with the immediate
preparation of food for consumption, particularly such occupations as
market-dairying, cooking, and serving food. Numerous serious epi-
demics have been traced to such sources in recent years.

FOOD POISONING DUE TO THE GROWTH OF SAPROPHYTIC BACTERIA TN

THE FOOD.

Most food poisonings are due to food derived from perfectly healthy
and wholesome animals or plants, which has subsequently undergone
some bacterial decomposition giving rise to poisonous products. Our
knowledge of the specific causes of the poisonous changes is, however, very
incomplete, and on account of the difficult nature of investigation in this
field, some of the conclusions reached by careful men are still open to
question. The bacteria which have been most frequently identified with
various epidemics of food poisoning are the following: B. enteritidis in meat
poisoning; B. botulinus in meat and in sausage poisoning; B. paratyphosus
in poisoning with meat, chicken, shell-fish, and vegetables; B. coli in cheese
poisoning and in milk poisoning; B. vulgaris in meat and in vegetable food
poisonings. Doubtless other microorganisms, as yet unrecognized, play
an important part in many food poisonings, and there is reason to believe
that some of these important unknown forms are anaerobic bacteria.

POISONOUS MEAT AND SAUSAGE. The flesh of a healthy animal is
ordinarily free from bacteria at the time of slaughter, and bacterial changes
must begin at the surfaces of the pieces of meat and gradually extend in-
ward . In diseased animals, b acteria more frequently circulate in the blood,
and the flesh may be contaminated throughout when the animal dies of the
disease or when it is slaughtered, not only with the specific germs of the
disease but also with bacteria derived from the intestinal tract of the ani-
mal. It is a matter of observation that the flesh of diseased animals is
more liable to undergo early putrefactive and poisonous changes than that
derived from healthy animals. Hashed meat is, of course, much more
prone to bacterial decomposition, because in it the bacteria have become
well distributed throughout the mass, and ideal conditions are provided
for the development of anaerobic as well as aerobic bacteria. Minced
chicken and chicken pie appear to be very frequent sources of acute
poisoning in the United States, and epidemics of sausage poisoning have
repeatedly occurred, especially in Germany. The bacteria found to be
concerned in these instances have been B. enteritidis, B. paratyphosus,

MICROBIAL FOOD POISONING. 415

B. coli, and B. botulinus. These organisms, or at least some varieties of
them, produce powerful poisons which are not destroyed by boiling,
and therefore may remain in the food after thorough cooking. Moreover,
meat rendered poisonous by these bacteria may show no evidence of putre-
faction. B. (Protens) vulgaris has also been found in some samples of
poisonous meat, and this finding is usually associated with definite evi-
dence of putrefaction.

The symptoms of meat poisoning are usually those of acute gastro-
enteritis, vomiting, cramps, and diarrhcea. The patients often recover
very quickly, but occasionally the illness is rapidly fatal, or it may merge
into a subacute form resembling or identical with paratyphoid fever. In
those instances of poisoning due to the presence of B. botulinus the symp-
toms are of a different kind, consisting almost solely of nervous disturb-
ances, secretory and motor paralyses, without fever, resembling in many
respects poisoning with atropin. In this form of meat poisoning the
death rate is relatively high, occurring in about 40 per cent of the cases.

FISH POISONING is of two general kinds, that due to poisons natural
to the fish, and that due to poisons formed by bacterial activity in the
flesh of the fish. Blan chard has applied the Spanish name " Siguatera" to
the first kind and the term "Botulism" to the second. In the Japanese
fish of the genus Tetrodon the roe is poisonous, giving rise to severe
gastro-intestinal irritation and convulsions. The remainder of the fish
is not poisonous. In some other fishes the sexual glands are poisonous
during the spawning season; others are provided with special poison
glands connected with protective spines or barbs. These are examples
of poisons natural to fish. Bacterial poisons are likely to be formed in
any kind of fish, given the suitable conditions, and thus give rise to the
kind of fish poisoning designated as botulism. Cases of this kind have
resulted from eating (spoiled) canned salmon and sardines. Poisoning may
also result from eating diseased fish, the effects being due to poisons
elaborated by the infecting bacteria in the body of the fish before consump-
tion. This appears to be a rather common form of fish poisoning in
Russia. B. paratyphosus has been isolated from some poisonous fish, and
certain toxicogenic anaerobes have been found in others.

POISONING WITH SHELL-FISH is so well recognized that this forrn of
food is not customarily used at all during the warmer part of the year,
May to August inclusive, the months without an r in their names. Shell-
fish may serve as carriers of human infectious diseases, such as typhoid

416 MICROBIOLOGY OF SPECIAL INDUSTRIES.

fever; they may be poisonous on account of actual disease or through
serious contamination due to living in dirty water; or they may be poison-
ous because of decomposition which has taken place after removal from
the water. According to the symptoms produced, there appear to be at
least three distinct varieties of shell-fish poisoning, one a purely gastro-
intestinal disorder, the second an involvement of the nervous system with
itching skin eruption and convulsions, and a third type resembling very
closely alcoholic intoxication. The exact nature of the microbic agents
concerned in these different types of poisoning is unknown. It is pretty
well established, however, that the poisonous character of shell-fish is
due either to their living for some time in dirty water, or to their too long
preservation, especially at high temperature, after removal from the water.

MILK, ICE-CREAM AND CHEESE sometimes give rise to poisoning, and
although these instances are small in number in comparison with the
enormous amount of milk and milk products consumed, yet in the aggre-
gate they are numerous. That many human infections may be trans-
mitted by milk has already been pointed out. In the summer, milk is
undoubtedly a great factor in the infant morbidity and mortality, and
this poisonous action is largely due to bacterial changes in the milk.
Extraordinary precautions are therefore essential in the production and
care of milk to be used as food for children, particularly during the warmer
season of the year. Severe poisoning of adults with milk, ice-cream, or
cheese, is relatively less frequent. Cases which have been studied have
been traced to the development of B. coli or B. paratyphosus in these foods.
There is some evidence that other bacteria, probably strict anaerobes, are
also sometimes concerned. Strict cleanliness, proper refrigeration, and
pasteurization of milk of uncertain character, may usually be relied upon
to prevent milk poisoning. Ice-cream should be made only from whole-
some materials and with due regard to cleanliness in making it. The
causes of serious cheese poisoning are not definitely known, but such
poisoning may be avoided, to a large extent at least, by using only stand-
ard varieties of cheese of the proper odor and flavor.

VEGETABLE FOOD POISONING, in an acute form, has followed the use
of sprouting and partly decomposed potatoes, and also various canned
vegetables, particularly those of high protein content, such as beans.
The large majority and possibly all of these cases are due to decomposition
changes in the foods, B. botulinus and B. proteus appearing to be the mi-
crobes most frequently concerned.

MICROBIAL FOOD POISONING. 417

There are also certain definite, more or less chronic diseases which
have been attributed to the use of certain grains as foods. Ergotism,
characterized by cachexia, gangrene, and convulsions, is caused by eating
the fungus, Claviceps purpurea, which grows as a parasite upon rye. The
grain of this parasite has a considerable commercial (medicinal) value suffi-
cient to pay for its separation from rye where it occurs, so there is little
economic excuse for food poisoning from this cause. Beriberi or kakke
is a chronic nervous disorder which occurs especially among the Japanese,
at one time ascribed to the use of fish and later to the use of rice as food.
The rice hypothesis has considerable observational and experimental
evidence to support it, but can hardly be regarded as definitely established.
Pellagera is a cachexia, characterized by a definite sort of skin eruption,
which has been ascribed to the use of maize (Indian corn) as food. This
disease is discussed in a separate section.

THE CHEMICAL NATURE or FOOD POISONS.

The poisonous substances in foods are for the most part of the same
nature as the poisons of the pathogenic bacteria. The simplest in structure
of these poisons belong to the alkaloidal substances, substituted ammonia
and ammonium compounds, called ptoma'ins (p. 115). Several of these
have been prepared in a pure state, for example, mytilotoxin (C 6 H 15 NO 2 )
from poisonous shell-fish, and neurin (C 2 H 3 -N(CH 3 ) 3 'OH) from putre-
fied horse, beef, and human flesh. Although ptomai'ns undoubtedly occur
at times in poisonous foods, they are not now considered of so much im-
portance in food poisoning as formerly, for in the majority of samples of
poisonous food the search for ptomams has been in vain. The poisonous
effects are believed rather to be due for the most part to much more com-
plex bodies resulting from the earliest analytic changes in the food
protein, or else to bodies built up by actual synthesis by the bacteria.
Such substances are classed with the toxic proteins and the true toxins.
Their chemical composition and structure are not definitely known.

REFERENCES.

Dieudonne, A., translation by Bolduan, C. F., Bacterial food poisoning. E. B.
Treat and Co., New York, 1909.

Nervy, F. G., Food poisons, Osier's Modern Medicine, Vol. I. Lea Bros., and Co.,
Philadelphia, 1907.

Thresh and Porter, Preservatives in food and food examination. J. and A.
Churchill, London, 1906.

Vaughan and Novy, Cellular toxins. Lea Bros., and Co., Philadelphia, 1902.
27

CHAPTER VI.*

THE MICROBIOLOGY OF ALCOHOL PRODUCTS.

WINE.

Wine may be defined shortly as the product of the alcoholic fermenta-
tion of grapes and the usual cellar treatment.

The classifications of wines are numerous and the varieties innumerable. They
may be separated, however, into a few main groups, depending on chemical composition
and methods of manufacture. Dry wines are those in which practically all the sugar
has been removed by fermentation; sweet wines, those in which enough sugar remains
or is added to be noticeable to the taste; fortified wines, those that have received an
addition of distilled wine spirits; and sparkling wines, those highly charged with carbon
dioxide, produced by supplementary fermentation in the bottle. Each of these
groups includes white wines made from the expressed juice of the grape, and red wines
made from both the juice and skins of red grapes.

GRAPE JUICE AND WINE AS CULTURE MEDIA.
Grape juice, known technically as must, is a sugary, acid, organic so-
lution very favorable to the growth of yeasts and of many other fungi,
but unfavorable to most bacteria. Wine is of a similar composition but
contains alcohol instead of sugar and is therefore less favorable to the
growth of most microorganisms. Both liquids are of highly complex
composition. Their character as culture media is indicated by the fol-
lowing table:

Composition of Must and Dry Wine.

Must.

Wine.

Specific gravity

Fermentable Sugar .
Alcohol by volume. . .
Acidity (as Tartaric) .
Nitrogenous matters
(soluble)

Tannin

Dry extract

Ash.

i. 0600 to 1. 1090
12. o to 25.0 per cent
none

.5 to i. 25 per cent

.2

Traces

to . 4 per cent

.20 to .70 per cent

. 9850 to i . oooo

o to . 5 per cent
8.0 to 15.0 per cent
.25 to i. o per cent

variable but small
traces to .30 per cent
.15 to .40 per cen
.13 to . 50 per cent

* Prepared by F. T. Bioletti.

4l8

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 419

Fortified wines (sweet wines are usually fortified) contain enough
alcohol to make them practically antiseptic to all microorganisms.

THE MICROORGANISMS FOUND ON GRAPES.

On the surfaces of grapes, as they are brought to the cellar, may be
found any of the bacteria and fungi usually carried by the air and by in-
sects. Many of these are incapable of growing in grape must, and are,
therefore, without effect on the wine.

MOLDS. The spores of the common saprophytic molds, Penicillium,
Dematium, Aspergilhts, Mucor, are always present in abundance, and they
find in must excellent conditions for development. Botrytis cinerea, a
facultative parasite of the leaves and fruit of the vine, is also nearly con-
stantly present in larger or smaller quantities. All these molds are harm-
ful to the grapes and the wine. Some of them, such as Penicillium, may
give a disagreeable, moldy taste to the wine, sufficient to spoil its com-
mercial value. Others, such as some Mucor s and Aspergilli may injure
the wine but slightly except by destroying the sugar and diminishing the
alcohol. Dematium pullulans may produce a slimy condition in weak
white musts and most of them may injure the brightness and flavor to
some extent.

On sound ripe grapes these molds occur in comparatively small num-
bers and being in the spore or dormant condition they are unable to develop
sufficiently to injure the wine under the conditions of proper wine making.
On grapes which are injured by diseases, rain or insects, they may be
present in sufficient quantities to spoil the grapes before they are gathered.
On sound grapes which are gathered and handled carelessly, they may
develop sufficiently before fermentation to injure or spoil the wine.

An exception to the generally harmful effect of these molds is Botrytis
cinerea (Sderotinia fuckeliana) which under certain circumstances may
have a beneficial action. When the conditions of temperature and mois-
ture are favorable, this mold will attack the skin of the grape, facilitating
evaporation of water from the pulp. This results in a concentration of
the juice. The mycelium then penetrates the pulp, consuming both sugar
and acid, principally the latter. The net result is an increase in the per-
centage of sugar and a decrease in that of acid. This, where grapes ripen
with difficulty, is an advantage, as no moldy flavor is produced. Two
harmful effects, however, follow: the growth of mold results in the

420 MICROBIOLOGY OF SPECIAL INDUSTRIES.

destruction of a certain amount of material, and a consequent loss of quan-
tity, which is, in certain circumstances, more than counterbalanced by an
increase in quality (wines of the Rhine, Sauternes) ; again, an oxydase
is produced which tends to destroy the color, brightness and flavor of the
wine. This can be counteracted by the judicious use of sulphurous acid.

YEASTS. The true yeasts occur much less abundantly on grapes than
the molds. Until the grapes are ripe they are practically absent, as first
shown by Pasteur. Later, they gradually increase in number and on very
ripe grapes often become abundant. In all cases and at all seasons,
however, their numbers are much inferior to those of the molds and pseudo-
yeasts. The cause of this seems to be that in the vineyard the common
molds find conditions favorable to their development at nearly all seasons
of the year, but yeasts only during the vintage season.

Investigations of Hansen, Wortmann and others show that yeasts
exist in the soil of the vineyard at all times, but hi very varying amounts.
For a month or two following the vintage, a particle of soil added to a
nutritive solution contains so much yeast that it acts like a leaven. For
the next few months, the amount of yeast present decreases until a little
before the vintage, when the soil must be carefully examined to find any
yeast at all. As soon as the grapes are ripe, however, any rupture of the
skin of the fruit will offer a favorable nidus for the development and in-
crease of any yeast cells which reach it. Where these first cells come from
has not been determined, but as there are still a few yeast cells in the soil,
they may be brought by the wind, or bees and wasps may carry them from
other fruits or from their hives and nests.

The increase of the amount of yeast present on the ripe grapes is often
very rapid and seems to have (according to Wortmann) a direct relation to
the abundance of wasps. These insects, passing from vine to vine, crawl-
ing over the bunches to feed on the juice of ruptured berries, soon inocu-
late all exposed juice and pulp. New yeast cultures are thus produced,
and the resulting yeast cells quickly disseminated over the skins and other
surfaces visited.

The more unsound or broken grapes present, and the more honey -dew
or dust adhering to the skin, the larger the amount of yeast will be. The
same is true, however, also of molds and other organisms.

In the older wine-making districts, much of the yeast present on the
grapes will consist of the true wine yeast, S. ellipsoideus. The race or
variety of this yeast will differ, however, in different districts. Usually

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 421

several varieties will be found in each district. The idea prevalent at one
time, that each variety of grapes has its own variety of yeast seems to have
been disproved, though there seems to be some basis for the idea that
grapes differing very much in composition, varying in acidity and tannin
contents, may vary also in the kind of yeast present. Several varieties
of S. ellipsoideus may occur on the same grapes. In new grape-growing
districts, where wine has never been made, 5. ellipsoideus may be com-
pletely absent.

Besides the true wine yeast, other yeasts usually occur. The com-
monest forms are cylindrical cells grouped as S. pasteurianus. These
forms are particularly abundant in the newer districts where they may take
a notable part in the fermentation. Their presence in large numbers is
always undesirable and results in inferior wine. Many other yeasts may
occur occasionally and are all more or less harmful. Some have been
noted as producing sliminess in the wine. Many of these yeasts produce
little or no alcohol and will grow only in the presence of oxygen.

Pseudo-yeasts. Yeast-like organisms producing no endospores always
occur on grapes. Their annual life-cycle and distribution are similar
to those of the true yeasts, but some of them are much more abundant
than the latter. They live at the expense of the food materials of the must
and when allowed to develop cause cloudiness and various defects in the
wine.

The most important and abundant is the apiculate yeast, S. apiculatus .
According to Lindner this is a true yeast, producing endospores. The
cells of this organism are much smaller than those of S. ellipsoideus and
very distinct in form. In pure culture these cells show various forms,
ranging from ellipsoidal to pear-shaped (apiculate at one end) and lemon-
shaped (apiculate at both ends). These forms represent simply stages of
development. The apiculations are the first stage in the formation of
daughter cells, the ellipsoidal cells, the newly separated daughter cells,
which later produce apiculations and new cells in turn.

Many varieties of this yeast occur as in the case of S. ellipsoideus.
They are widely distributed in nature, occurring on most fruits, and are
particularly abundant on acid fruits such as grapes. Apiculate yeast ap-
pears on the partially ripe grapes before the true wine yeast and even on
ripe grapes is more abundant than the latter. The rate of multi-
plication of this yeast is very rapid under favoring conditions and much
exceeds that of wine yeast. The first part of the fermentation, especially

422 MICROBIOLOGY OF SPECIAL INDUSTRIES.

at the beginning of the vintage and with acid grapes, is, therefore, often
almost entirely the work of the apiculate yeast.

The amount of alcohol produced by this yeast is about 4 per cent,
varying with the variety from 2 to 6 per cent. When the fermentation
has produced this amount of alcohol the activity of the yeast slackens and
finally stops, allowing the more resistant ellipsoideus to multiply and
finish the destruction of the sugar. The growth of 5. apiculatus,
however, has a deterring effect on that of the true wine yeast so that
where much of the former has been present during the first stages of
fermentation the latter often fails to eliminate all the sugar during the
last stages.

Wines in which the apiculate yeast has had a large part in the fer-
mentation are apt to retain some unfermented sugar and are very liable
to the attacks of disease-producing organisms. Their taste and color
are defective, often suggestive of cider, and they are difficult to clarify.
This yeast attacks the fixed acids of the must, the amount of which is
therefore diminished in the wine, while on the other hand the volatile
acids are increased.

Many other yeast-like organisms may occur on grapes, but, under
ordinary conditions, fail to develop sufficiently in competition with apicu-
latus to have any appreciable effect on the wine. Most of them are small
round cells, classed usually as Torulce. They destroy the sugar but pro-
duce little or no alcohol.

A group of similar forms, known collectively as Mycoderma vini,
occurs constantly on the grapes. These, being strongly aerobic, do not
develop in the fermenting vat, but under favoring conditions may be
harmful to the fermented wine.

BACTERIA of many kinds occur on grapes as on all surfaces exposed
to the air. Most of these are unable to develop in solutions as acid as
grape juice or wine. Of the acid-resisting kinds, a number may cause
serious defects and even completely destroy the wine. These, the
"disease-producing bacteria" of wine, are mostly anaerobic and can
develop only after the grapes are crushed and the oxygen of the must
exhausted by other organisms. Practically all grape must contains
some of these bacteria, which, unless the work of the wine maker is
properly done, will seriously interfere with the work of the yeast, thus
causing injury to the wine. The only bacteria which may injure the
grapes before^crushing are the aerobic, acetic bacteria, which may develop

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 423

on injured or carelessly handled grapes sufficiently to interfere with fer-
mentation and seriously impair the quality of the wine.

THE MICROORGANISMS FOUND IN WINE.

Wine microorganisms may be conveniently divided into two groups:
those which grow only in the presence of notable supplies of free
oxygen, and those which require or grow better in the absence of free
oxygen.

AEROBIC ORGANISMS. Mycoderma. If a normal wine, especially
one strong in alcohol, is left with its surface exposed to the air, it will
usually, in a few days, be covered with a whitish film, thin and smooth
at first but gradually becoming thicker and finally rough and plicate.
This is what is known to wine-makers as "wine flowers." This film con-
sists of yeast-like cells, somewhat longer and more cylindrical than S. el-
lipsoideus, reproducing by budding and forming large aggregations.

Pure cultures show that there are many varieties of this organism
differing in the color and texture of the film, in the cloudiness of the
liquid and in the character of the deposit. They are called collectively
Mycoderma vini, though one form which has been found to produce
endospores has been called S. anomalous.

These organisms are strongly aerobic and can develop only on the
surface in full contact with the air. They are a serious enemy to the
wine, rendering it insipid and cloudy. They attack the extract, fixed
acids, and alcohol, producing at first volatile acids and finally causing
complete combustion of the organic matters to carbon dioxide and
water, destroying the wine completely.

Acetic Bacteria. The film formed on wines exposed to the air,
especially on those of low alcoholic content, will often differ from that
due to Mycoderma vini. It will be thinner, smoother and consist of
bacteria. These are the vinegar bacteria described on page 448. They
grow not only on the wine at the expense of the alcohol, but on crushed
grapes and must at the expense of the sugar, producing acetic acid in
both cases.

Acetic acid in small amounts is produced by the yeast and is a normal
constituent of wine. Unless in excess its effect is not injurious. There
may be present from .09 g. in 100 g. in light white wine to .14 g. in a
heavy red wine without deterioration of quality. In sweet wines, even
a somewhat larger amount may be present without causing injury.

424

MICROBIOLOGY OF SPECIAL INDUSTRIES.

Much larger amounts are injurious in two ways. When the acetic
acid is perceptible to the taste, the wine is spoiled. When an abnormal
amount of acetic acid is produced before or during fermentation it
stops or interferes with the work of the yeast. In such cases, the wine
"sticks," that is, fails to eliminate all its sugar and becomes especially
liable to the attacks of other bacteria.

Wines high in alcohol are less liable to acetic fermentation than weaker
wines. Sound wines containing over 14 per cent by volume of alcohol
are almost immune, but such wines may be spoiled during fermentation
by the growth of acetic bacteria on the exposed floating "cap" of pomace
or on the crushed grapes, especially at high temperatures.

ANAEROBIC ORGANISMS (Facultative and Obligate). Some of the
worst, most frequent, and most difficult to treat of the diseases and
defects of wine are due to organisms which develop only in the absence
of oxygen. These organisms are all bacteria and appear to include a
large number of forms, though, owing to difficulties of isolation and
culture, the diferent forms have not been well studied or described.

\

^ ^

*>* "S:^~.

'^v -

J C3 i

V,v

r:'^ : :

^-/

FIG. 86. Bacteria of slimy wine. A, B, C, pure cultures of various forms; D, muci-
laginous sheath of slime bacteria. (After Kayser and Manceau.)

Slime-forming Bacteria. -Musts and wines become slimy rarely
through the action of Dematium pullulans (Wortmann) and wild yeast
(Meisner) in the presence of oxygen but more frequently through the
action of bacteria. In most cases only young wines after fermentation
and when contained in closed casks or bottles exhibit this defect. A
slimy wine has an oily appearance, pours without splashing and in
extreme cases, becomes cloudy and will hang from a glass rod in strings.

In such wines, the microscope reveals large numbers of almost
spherical or more or less elongated bacteria in long chains. Other ob-

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 425

servers have noticed a diplococcus and a sarcina. Kayser and Manceau
have recently investigated the subject very thoroughly and described a
number of forms which are mostly short rods of from i/.< to 2/j. by .y/<
to i.2/i. One large form, 3/z to^X i.6fj. to i.-j/n, was also noted. They
all form chains, usually of considerable length. They all produce an
abundant slimy sheath and stain easily with carbol-fuchsin and other
aniline dyes and are Gram-positive (Fig. 86).

These bacteria attack the sugar but neither the glycerin nor the alcohol
and produce mannit, carbon dioxide, lactic and acetic acids and ethyl
alcohol. The disease is usually not serious and disappears under the
ordinary cellar treatment. Alcohol above thirteen per cent, free tartaric
acid, tannin and sulphurous acid in small amounts prevent their
growth.

Propionic and Lactic Bacteria. The most serious and perhaps
the commonest disease of wines is characterized by persistent cloudiness,
disagreeable odors and flavors, increase of volatile acid and injury to or
complete destruction of the color. Wines affected are characterized
commonly as mousey, lactic or turned wines (Pousse and Tourne of the
French) .

The disease is due to bacteria. Enormous numbers are readily revealed
by the microscope in badly affected wines. There seem to be several or
many closely related forms, all short rod-shaped, isolated in the first stages
of the disease, but later forming chains or filaments of various lengths.
The most noticeable change caused in the composition of the wine is
the decrease of fixed and increase of volatile acidity. The tartaric acid
and tartrates are destroyed, and carbonic, acetic, lactic, propionic and
other acids formed.

Light wines of low acidity are most subject to this disease which
may be prevented by measures which increase the acidity and alcohol,
by rapid and complete defecation and attenuation of the wine with
the proper use of sulphurous acid, and finally by timely filtration and
pasteurization. Wines noticeably affected can be used only for distilling
those badly affected are valueless.

Mannitic Bacteria. Very sweet grapes of low acidity in hot climates
are subject during fermentation to a similar trouble characterized by
increase of volatile acidity and a persistent cloudiness and a vapid sweet-
sour taste. The disease is commonly confused with the preceding but
is caused by bacteria of different forms. The form described by Gayon

426

MICROBIOLOGY OF SPECIAL INDUSTRIES.

is a very fine short rod which does not unite in filaments. It attacks
the sugar, especially the levulose, producing volatile acids and mannit.
The latter may reach over 2 per cent and the former 5 per cent,
giving a sweet-sour wine which is completely spoiled.

The bacteria grow abundantly only at high temperatures approaching
40 and can be controlled by cool fermentation, increase of acidity and
proper use of sulphurous acid.

Butyric Bacteria. In the cooler climates, wines, especially old red
wines in bottles, often become bitter. This trouble is due to comparatively
large rod-shaped bacteria, first described by Pasteur. The cells re-
main united in angular filaments, short at first, but becoming longer
and finally thicker with age by incrustations of coloring matter.

s I

\

/\ \

V

1 \w

\

A

}

r

\

/
i>

FIG. 87. Bacteria of wine diseases. A, bacteria of "turned wine," young wine
(After Bioletti); B, bacteria of "turned wine," old wine (After Bioletti}; C, mannitic
bacteria (After Maze and Pacottet); D, bacteria of "bitter wine" (After Pacottet).

The tannin, coloring matter, and glycerin of the wine are attacked,
acetic and butyric acids being formed. In small amounts the bacteria
do little or no harm, in larger amounts they may spoil the wine. Means
which increase the alcohol, tannin and acidity diminish the liability to
the disease. Prompt attenuation and clarification and in extreme cases
pasteurization will cure wines not too badly affected.

All the above anaerobic bacteria of wine diseases probably exist in

most wines. Which develop most, or whether any develop sufficiently

to injure the wine depends on conditions, chiefly the composition of the

must and the temperature at which the wine is fermented or stored.

Most diseased wines show a mixed infection of several forms.

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 427

CONTROL OF THE MICROORGANISMS.

Given grapes of suitable composition, the quality of the wine depends
on the work of microorganisms. The art of the wine maker consists
almost entirely in the control of these microorganisms. His success in
facilitating the work of the useful form (true wine yeast) and in preventing
or hindering the work of injurious forms determines the quality of his
product.

BEFORE FERMENTATION. On the skins of sound ripe grapes as they
hang in the vineyard the microorganisms are comparatively few and in
an inactive condition. With the utilization of intelligent methods
they cannot injure the wine. On broken or injured grapes, the number
is greater and the forms more active. If many such grapes occur they
should not be mixed with the sound grapes if the best wine is to be made.

Care should be taken to avoid unnecessary bruising of the fruit if it cannot be
worked immediately. Molds, wild yeasts and acetic bacteria multiply rapidly on
grapes wet with juice.

The sooner the grapes can be crushed and placed in the fermenting vat or
pressed the easier it is to obtain a sound fermentation.

Cleanliness is essential. Grapes, which are gathered in moldy, vinegar-soured
boxes, hauled in dirty wagons or cars, and passed through dirty crushers, conveyors and
presses, may be so completely infected with injurious germs that it is impossible to
obtain a good fermentation. The most injurious forms of dirt are must, grapes, or
wine, which have been allowed to become moldy or vinegar-soured.

Dust or soil is less injurious and, if excessive, may often be removed by sprink-
ling, especially is this true if the grapes are too sweet and require dilution. Washing with
antiseptics is not permissible. A weak solution of potassium metabisulphite might be
used with benefit if it were not fop the difficulty of regulating the amount of sulphurous
acid entering the fermenting vessel.

If the grapes have to be kept for some time before crushing they should be kept
as cool as possible to delay the growth of molds. Gathering in the cool of the morning
is desirable and if grapes are gathered when warm they should be left in boxes to cool off
during the night whenever possible. If the grapes are cool when they reach the fer-
menting vat they will neutralize a certain proportion of the heat of fermentation,
accordingly the difficulty of avoiding injuriously high temperatures is diminished.

However carefully the grapes are handled, a certain amount of dust containing
germs and other injurious matters will reach the vats and presses. In the manufacture
of white wines, especially, it is desirable to get rid of these matters before fermentation.
This is best accomplished by settling and decantation.

As the juice runs from the press it is pumped into a settling tank or cask. If it is
cold, below 1 5, and of full normal acidity, the impurities may settle in twenty to forty-
eight hours. If the temperature is higher than 15 and the acidity low, molds and yeasts
will develop or fermentation will start and prevent settling. A slight sulphuring with

428 MICROBIOLOGY OF SPECIAL INDUSTRIES.

the fumes of burning sulphur or with a solution of potassium metabisulphite is there-
fore usually necessary. The sulphuring should be as light as possible with acid musts
as it tends to preserve the fixed acids. For the same reason it benefits musts of low
acidity. In from twelve to twenty-four hours the must is purged of all its gross impuri-
ties including microorganisms, dust and solid particles derived from the skins and the
stems and pulp of the grapes. It may be slightly cloudy or nearly clear. It can then
be drawn off into clean casks and fermentation started with yeast.

This defecation is of great value, ridding the must of substances that would affect
the flavor of the wine in the heat of fermentation and eliminating the excess of protein
matters that would serve as food for injurious bacteria. Centrifugal machines have
been devised to hasten the process of defecation, but their work is less perfect.

Sterilization by heat has been tried for the same purpose but with indifferent
success. High heating caramelizes part of the sugar and oxydizes the must, thus
injuring the flavor. Discontinuous heating at lower temperatures in an atmosphere of
carbon dioxide is preferable but troublesome and expensive; all methods have the
defect of extracting undesirable substances from the solid matters which are heated with
the must.

Chemical sterilization is still less practicable. No substance could be used for
this purpose except sulphur dioxide; this used in sufficient quantities would seriously
injure the flavor of the wine. The effect would be totally different from that of the
small quantities used in defecation.

All the methods discussed have for their object the diminution or elimination of
microorganisms of all kinds. With the injurious forms the true yeast is also removed.
The more perfect these methods, the more necessary it is to add wine yeast. Without
this addition, in fact, all these precautions may result in harm, for the wine yeast, being
present in much smaller numbers than many of the injurious forms, may be completely
removed while enough of other forms are left to spoil the wine.

A "starter" of some kind is therefore necessary with defecated must
and useful in all other cases.

A Starter. One method of producing such a starter is to gather a suitable quantity
of the cleanest and soundest ripe grapes in the vineyard, crush them carefully and allow
them to undergo spontaneous fermentation. Perfectly ripe grapes should be selected
and the fermentation allowed to proceed until at least 10 per cent of alcohol is pro-
duced. If imperfectly ripened grapes are used or the starter used too soon, the
principal yeast present will be S. apiculatus. Toward the end of the fermentation, 5.
ellipsoideus pre-dominates. From 4 to 12 1. (i to 3 gallons) of this starter should be
used for each 500 1. (100 gallons) of grapes or must to be fermented. Too much
"starter" should not be used in hot weather or with warm grapes; unless this precau-
tion is obesrved, it may be impossible to control the temperature.

This starter is used only for the first vat or cask. Those following are started
from the first fermentations, care being taken always to use the must only from a
tank at the proper stage of fermentation and to avoid all tanks that show any defect.

An improvement on a natural starter of this kind is a pure culture of tested yeast.

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 429

Such yeasts are being used extensively in Germany, France and Italy, usually with
excellent results. The methods of use would require too much space to describe here,
but they are simple and such as could easily be devised by anyone with some knowl-
edge of microbiological technic. They do not aim at obtaining an absolutely pure
fermentation, which is unnecessary, but endeavor to have an overwhelming proportion
of a thoroughly tested and suitable yeast which will rapidly and perfectly attenuate the
wine before the few injurious microorganisms present have time to do any harm.

DURING FERMENTATION. However carefully the injurious germs
have been excluded and the good yeast increased, fermentation will not
be successful unless conditions as favorable to the latter and unfavorable
to the former as possible are maintained.

The temperature of the crushed grapes or expressed must is of
importance. If it is below 15, unless the weather is warm, the grapes
should be warmed to 20 or 25. Unless this is done, the molds and
S. apiculatus, which require less heat than S. ellipsoideus will develop
more quickly. This is especially true when starters are not used. In
the warmer and earlier districts the grapes are practically never too
cold. On the other hand, unless there is great carelessness, the grapes
are never too warm for the commencement of fermentation. The
warmer they are, however, the more artificial cooling will be necessary
later, and the sooner it will have to be applied.

Thorough crushing is necessary in the case of white wine, to facili-
tate the expression of the juice. For red wine, the grapes are also thor-
oughly crushed and the skin, pulp and juice are fermented together.
Imperfectly crushed grapes ferment unevenly and incompletely; the
growth of mold, is much facilitated.

The must should be thoroughly saturated with air at the beginning
of fermentation to insure the multiplication of the yeast. The aeration
received in the processes of stemming, crushing and pressing is usually
sufficient for this purpose. More aeration would be harmful by in-
juring the flavor and color of the wine by over-oxidation and promoting
the growth of injurious aerobic organisms. An objection to the sterili-
zation of must by heat is the expulsion of the air and the difficulty of
replacing it in the proper amount.

The proper use of sulphurous acid in the regulation of fermenta-
tion is one of the most important and necessary but least understood
parts of the wine-maker's art. Only by this proper use can wholesome
wine of the highest quality be produced. Improper use will injure

43 MICROBIOLOGY OF SPECIAL INDUSTRIES.

or completely spoil the wine. Its beneficial effects are due primarily to
its action on microorganisms, on enzymes and on the color of the wine.

In the small quantities properly used in wine-making, it is antiseptic in a degree
varying with the amount. All microorganisms are susceptible to its action in varying
degrees. Bacteria are particularly sensitive, molds and pseudo-yeasts less so, while wine
yeast is the most resistant of the ordinary forms found in must and wine.

The result of the use of the proper amount of sulphurous acid in crushed grapes and
must before fermentation is the almost complete suppression of bacterial action, the
discouragement of molds and pseudo-yeasts and the promotion of the growth of wine
yeast which is given a clear field unhindered by the deleterious excretions of competitors.

Its action as regards enzymes is hardly less important. It would be impossible to
make the finest wines of Sauternes and the Rheingau without its use on account of the
oxidase produced by the Botrytis cinerea which is abundant and necessary on the best
grapes of these regions. In other regions where this mold and others occasionally
occur its use is also necessary. In hot climates it is especially useful, not only because
bacterial action is more intense in such regions but because of its action in preserving
the natural fixed acids of the grape, which are, there, nearly always deficient. This
preservation, according to Wortmann, is due to the suppression of acid-consuming
bacteria, but experiments of Astruc tend to show that the prevention of the action of
unknown acid-destroying enzymes is in part the cause.

Its action on the color of wines is also of importance. By the action of oxygen,
the color of red wine is gradually made insoluble and precipitated, and the greenish or
golden color of white wine is turned to brown. Both these actions are prevented or
much diminished by the use of minute quantities of sulphurous acid.

The most commonly used source of sulphurous acid is the fumes of burning sulphur.
Sulphur is burned in a cask and the must caused to take up the fumes by being
pumped into the cask through the upper bung hole. It is almost impracticable to
apply sulphurous acid from this source to crushed grapes for red wine.

The method is defective in many ways. It is impossible to tell within very wide
limits how much sulphur dioxide has been absorbed by the wine. Moreover, the sulphur
burns incompletely and the volatilized sulphur acted upon by the yeast may produce
sulphuretted hydrogen. Other sulphur compounds are also produced during the burn-
ing to some of which the so-called sulphur taste of wine is said to be due. Several
devices have been invented to decrease these defects but none remove them completely
and progressive wine-makers are adopting more reliable sources.

An improvement is the use of potassium metabisulphite (K 2 S 2 O S ) a salt which
can be obtained in the requisite purity in commerce containing 50 per cent by
weight of sulphur dioxide. The amount of potash added by this salt in the doses
used, is very small, and far within the limits of variation between different wines. By
the use of this salt, exact amounts of sulphur dioxide can be applied both to white and
red wines. Other sulphites are not permissible.

The best source of the acid, recently brought into limited use, is the liquefied gas,
which can be manufactured comparatively cheaply in great purity. By its use all the
benefits of sulphurous acid are obtained and the defects eliminated.

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 431

Some grapes, owing to their composition, especially their high
acidity, are very resistant to the attacks of injurious bacteria. Others,
owing to their low acidity or highly nitrogenous nature, are very suscepti-
ble. The addition of tartaric or citric acid to the latter has therefore a
deterring effect on some of the most dangerous forms. It is seldom
necessary, however, to modify the composition for this purpose if the
other means of control are used. The addition of acid or its decrease
by dilution should be solely for the direct improvement of the taste.

The quality and character of the wine depends greatly on the tem-
perature of fermentation. If too low, the fermentation may be unduly
prolonged, the wine yeast may have difficulty in overcoming its com-
petitors and the wine may remain inferior and cloudy. With red wine,
the desired color, tannin and body may not be secured. On the other
hand, if the temperature is too high the results are worse. The growth
of bacteria is promoted, injuring the wine by the volatile acid and dis-
pleasing flavors produced and preventing the proper action of the yeast.
Such wines may remain sweet on account of the failure of the yeast to
do its work and become unpleasantly acid owing to the volatile acids
produced by the bacteria.

Some means of controlling the temperature is therefore always needed.
Where heat is deficient it may be supplied by direct heating of the must
or part of it, or by heating the cellar. Where the heat is excessive, it
may be diminished by crushing only cold grapes, using small fermenting
vats to promote radiation and finally by the use of cooling machines
applied directly to the fermenting wine.

The best temperature for fermentation depends on the kind of wine.
For light white wines, the maximum should not exceed 25, for heavier
wines 30, while for heavy red wines where high extract and tannin are
required, it may be allowed to reach 35. Sound wines can be made at
all these temperatures.

As already explained, the ordinary processes of treatment of grapes
result in sufficient aeration for the multiplication of the yeast. With
grapes containing little sugar, this may suffice to complete fermentation.
With sweeter grapes, the fermentation usually slackens when the alcohol
reaches n or 12 per cent by volume or sooner, unless some supplementary
aeration is given. With white wine this is seldom done, with the result
that the time of fermentation is prolonged. With red wine, the necessary
stirring of the pomace to promote color extraction or the pumping over of

432 MICROBIOLOGY OF SPECIAL INDUSTRIES.

the must in the cooling process usually gives a large amount of aeration
which is sometimes excessive. Too much aeration results in extremely
rapid fermentation and consequent difficulty in controlling the tempera-
ture. It may also have a deleterious effect on the color, especially if
sulphur dioxide has not been used.

In any case, the main part of the fermentation should be over in
from three to five days in the case of red and in from seven to fourteen
days in the case of white wine. With heavy musts, however, there wall
still remain from . 5 to i or 2 per cent of sugar. With certain special
wines such as Sauternes it is desirable to retain the slight sweetness due
to this small amount of unfermented sugar. This is accomplished by the
judicious use of sulphurous acid, prompt clarification by filtration or
fining and when necessary by pasteurization. The pasteurization tends
to remove those proteins which are coagulated by heat and which are
the preferred food of bacteria.

In the case of dry wines, protection from bacteria is best obtained
by prompt and complete attenuation. Fermentation should not be al-
lowed to cease until all the sugar has disappeared. For this purpose,
one, two or more aerations by pumping over are usually necessary im-
mediately after the end of the tumultuous fermentation. The temperature
of the wine should not be allowed to fall sufficiently to check the action
of the yeast until all the sugar has disappeared.

AFTER FERMENTATION. As soon as all the sugar has been destroyed,
in the case of dry wines, or the desired degree of attenuation has been
obtained in the case of sweet wines, all the useful work of microorganisms
has been accomplished. The quality and safety of the wine then depend
on freeing it from all organisms present and preventing the entrance
and action of all others.

As soon as bubbles of carbon dioxide cease to be given off, the yeast
and other solid matters will settle to the bottom and the liquid become
clear. This often occurs before the fermentation is complete. In this
case the yeast should be stimulated by aeration as described above.

If the wine is dry it should be racked (drawn off, decanted) from the sediment

into clean casks. The first racking is usually done while the wine is still slightly cloudy

during the first month or six weeks to remove the more bulky sediment. If left too long

on the yeast the autophagy or degeneration of the latter may produce substances which

injure the brightness and flavor of the wine.

A second racking is necessary at the end of winter before the spring rise of tempera-

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 433

ture tends to renew the activity of the microorganisms which always remain in the
wine. A well made wine at this time should be perfectly bright and all solid matters
consisting of yeast and bacteria, coagulated proteins and crystals of bitartrate
should have accumulated in the sediment.

Racking should take place when possible only in settled weather, when the baro-
metric pressure is high. Low atmospheric pressures diminish the solubility of the
carbon dioxide with which the wine is saturated. Under these conditions, therefore,
bubbles of gas are apt to be given off, bringing up particles of sediment and rendering
the wine cloudy. However long wine is kept in wooden casks, it will continue to
deposit sediment owing to chemical changes due to the action of oxygen which pene-
trates slowly through the wood. Repeated rackings are therefore necessary, oc-
curring at least twice a year until the wine is bottled or consumed.

Abundant aeration is necessary during fermentation. A moderate supply of
oxygen is necessary for the proper aging of wine. Experience has shown that exactly
the proper amount of pure filtered air will obtain access to the wine for the latter pur-
pose through the wood of ordinary casks of proper size. If the casks are too small the
oxidation may be too rapid, if too large the maturing of the wine may be unduly pro-
longed. The temperature of the storage cellar is the main modifying factor. The
warmer the cellar the larger the casks should be.

With sound, completely fermented wines, all aeration, other than that due to the
porosity of the wood, should be avoided as much as possible. This is accomplished by
keeping the casks tightly bunged and completely filled. Evaporation through the wood
continually diminishes the volume of wine and the lack must be supplied by Jilting up, at
first two or three times a month and later every month or two. The drier the air of the
cellar, the more frequent the fillings necessary.

A light sulphuring of the clean casks into which the wine is racked is usual.
This should be practised with great caution. Very little is needed with sound wines,
especially if it has been used before or during fermentation and a slight excess will
injure the flavor. The amount should not exceed 1.25 g. per hectoliter for red or
2 g. for white wine. One-half to one-third of this is sufficient for old wines.
The amount can be accurately measured only when using metabisulphite or the
liquefied gas. The utility of the sulphur dioxide with perfectly sound wines is to dimin-
ish oxidation; with wines liable to disease to discourage the growth of bacteria.

All the manipulation of the wine should be conducted with strict attention to
cleanliness. This applies especially to empty casks, pumps and hoses. These should
be thoroughly cleaned immediately after use and, if of metal or other non-absorbent
material, kept perfectly dry. Utensils of wood, rubber or other porous material should
be preserved from bacterial or mold growth with sulphurous acid.

The clarification of a perfectly sound wine may be facilitated and hastened by
thoroughly stirring up the yeast immediately before racking. The yeast in settling
carries down much of the finer suspended matter, thus effecting a rough fining. Mate-
rials such as kaolin, pure silica sand, charcoal and filter-paper can be used with the
same effect. The fining, however, is never perfect and the flavor of the wine is often
injured. A very pure clay, known commercially as Spanish clay, is used largely for
clearing sweet wines where the flavor is not so delicate. From 75 to 125 mg. per
hectoliter are used for this purpose.
28

434 MICROBIOLOGY OF SPECIAL INDUSTRIES.

The best wines are nearly always fined at least once, immediately before bottling.
One or two finings may precede this to hasten aging, defecation and bottle ripeness.

The materials used are soluble gelatinous or albuminous substances which are
capable of being coagulated and precipitated by some ingredient of the wine. The best
of the commonly used substances are Isinglas (Ichthyocol) 2 or 3 g. per hectoliter,
for white wines; the white of fresh eggs, i or 2 per hectoliter for red ; and gelatin,
i o or 1 2 g. per hectoliter for either.

The proper quantity of the finings is dissolved in a little water diluted with wine
and stirred into the cask. The tannins and acids of the wine cause a gradual coagula-
tion in minute particles throughout the liquid. These particles gradually coalesce,
forming larger particles which include all the other floating solid matter of the wine as
in a net. These larger particles contracted by the alcohol then settle to the bottom,
leaving the wine perfectly bright.

The coagulum consists of a combination of the gelatinous matter and the tannin.
Some of the latter, therefore, is removed from the wine. With astringent red wines this
may be an improvement. If there is no excess of tannin present enough must be added
to combine with the finings used. With white wines which contain little or no tannin
this addition is always necessary.

The amount to use varies with the quality of the finings and of the tannin and with
the composition and temperature of the wine.

To precipitate commercial gelatin of good quality about an equal quantity of
good tannin is necessary; isinglas properly prepared requires only from one-half to one-
third this amount. Eggs require only minute quantities.

Specially prepared casein of milk is used for fining white wine. Its chief merit is
that the acids of the wine alone cause its complete precipitation and no addition of
tannin is needed. Many other albuminous substances such as milk, blood and various
proprietary preparations are also used, but they are all inferior to the three mentioned
and many of them introduce foreign matters such as milk sugar and bacteria which
are a source of danger to the wine.

Wines containing many disease-producing bacteria may be injured by the intro-
duction of finings. The evolution of gases due to the bacterial action may prevent the
settling and the protein matters introduced will favor the multiplication of the disease
organisms. By the use of 5 to 10 g. of sulphurous acid per hectoliter added to the wine
immediately before the addition of the gelatin, the bacteria may be temporarily para-
lyzed and the finings will then settle and remove the bacteria with the other floating
particles.

The bright wine should be racked from the finings very soon after the sediment has
settled, especially when disease-producing bacteria are numerous. This will be in
from ten to twenty days. If the wine is not clear in three weeks it should be filtered.

Filtering is inferior to fining in producing a perfectly bright wine. It is more
rapid, however, and is useful in clearing common wine and wines refractory to fining.

Filters of innumerable forms are used. They are of two main types. For rough
clearing of very cloudy wines some form of bag filter is usually employed in which the
wine passes through a cloth tissue. The passage at first is rapid and the filtration
imperfect. As the solid matter accumulates on the filtering surface, the filtration im-
proves but the passage of the wine is retarded. The first wine is passed a second time

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 435

through the filter and as soon as the rate of filtration becomes too slow the operation
must be stopped and the filtering surface removed.

For wines containing little sediment the filter must be primed. This is accom-
plished by putting some finings in the wine first passed through the filter. The
priming is more effective and the output of the filter much increased if a little in-
fusorial earth is used with the gelatin.

For the more perfect clearing of old wines some form of pulp filter is used. There
are various devices by which the wine is forced through a mass of cellulose or asbestos
pulp and freed from all floating matter. Some of the best of these, carefully used,
remove nearly all of the bacteria present.

BEER.

Beer is an alcoholic beverage made from certain cereal grains by
transformation of the starch to sugar, dilution with water, and fermenta-
tion with yeast. There is usually an addition of hops and sometimes of
materials containing sugar. The liquid before fermentation is called Wort.

RAW MATERIALS AND MICROORGANISMS OF BREWING.

GRAINS EMPLOYED. Barley, rice and maize are the grains most commonly used,
wheat, rye and oats but rarely. Cane and beet sugars and syrups sometimes form part
of the fermentable material.

YEASTS OF BEER. The yeast used is usually one of the many forms
of 5. cerevisicB. In some spontaneously fermented beer, other yeasts,
Torulcc and bacteria take part, but in ordinary beers most of these are con-
sidered as disease-producing organisms and injurious.

KINDS OF BEER. The principal varieties of beer are : lager beers, fermented with
bottom yeasts; ales, fermented with top yeasts (and Torulas); porters, similar to ale but
dark in color owing to the use of caramelized malt; -weisbiers, in which lactic bacteria
are abundant; and certain local types in which bacteria produce considerable quanti-
ties of lactic and acetic acids.

Typical Composition of Various Beers.

Lager

Ale

Porter

Weisbier

Temper-
ance beer

Water

OO AO

88 70

87 ?n

Alcohol (by vol.)

&. 8s

8 oo

7 J y

7 OO

3 A e

2f\f\

Extract

4 20

5e/i

6 A

45

Sugar. .

i 60

54

T 1 1

45
I 87

u o

T 7 T

95

T n8

Lactic acid

10

1 -66
2O

1.03

22

i.yj.
0*7

Ash

2?

3Q

AQ

*/

16

4

* J

o

H\j

436 MICROBIOLOGY OF SPECIAL INDUSTRIES.

OUTLINE OF THE PROCESSES OF BREWING.

INTRODUCTION. The manufacture of beer takes place in four main stages. First, a
portion or all of the grain is soaked in water, allowed to germinate and then dried.
This produces the malt which contains the enzymes necessary for the conversion of the
starch into sugar and the disintegration of the tissues of the grain. The malt is then
crushed (and usually mixed with unmalted cereals or sugar) and heated with water.
This constitutes mashing. During this process, the starch changes to maltose and
dextrins which with other matters dissolve in the water; then bacteria produce a small
amount of lactic acid. The resulting solution constitutes the wort.

The Wort, by the addition of yeast, is fermented and changed to beer. The
fourth stage includes all manipulation of the fermented beer to prepare it for consump-
tion.

MALTING : PRODUCTION OF ENZYMES. The best malt is made from barley, but
for special beers may be made from wheat or other grains. Steeping consists in soaking
in water to start germination. This requires from thirty-six to seventy-two hours and
causes an increase in weight of about 45 per cent. The temperature should be about
12.50. If higher, injurious molds will develop. If much lower germination will be
retarded. The water should contain little organic matter or chlorides, nitrates or iron
salts. A little calcium sulphate is favorable. If it contains many microorganisms it
should be sterilized by boiling. A very little sulphite of lime or of potassium may
be used to discourage molds.

During germination several enzymes appear, of which the most important to the
brewer are amylase which changes insoluble starch into soluble sugar, rendering it
available for the growth of the young plant; peptase, which performs a similar function
as regards nitrogenous matters; and cytase which helps in the disintegration of the cellu-
lose. All these are necessary to prepare for the work of the yeast. When the plumule
has grown to about two-thirds the length of the grain, sufficient enzymes have been
formed. This requires from about sixteen to twenty days.

The growth of the sprouting seed is at this point stopped by careful drying with
artificial heat in a kiln. The kilning must be sufficiently rapid to kill the germinating
seedling quickly, but not too rapid or at too high a temperature, otherwise the enzymes
will be weakened or destroyed. The enzymes are more sensitive when moist, conse-
quently the heat may be increased as drying proceeds. The process commencing at
a temperature of 30 to 35 is increased gradually to 50 or 55. In twelve to twenty-
four hours, the malt should appear dry. The temperature is again raised gradually
for another twelve to twenty-four hours to 80 or 100. The lower the temperature
the lighter the color of the malt. Higher temperatures, especially while the malt is
moist, produce dark malt.

As soon as the kilning is finished the radicles are removed by friction and screening
in special machines.

WORK or ENZYMES AND BACTERIA. The malt is first crushed by press-
ing between rollers to facilitate the work of the enzymes and the dissolving
action of the water. If unmalted grain is to be used as well, this must be
ground and the starch made soluble by heating under pressure with three

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 437

or four times its weight of water and a little malt to 80 to 85 for about
an hour.

The methods of mashing are very various. They consist in general of mixing the
ground malt with warm water, bringing the mass to a temperature of 35 to 45 which
is gradually raised to 60 to 65 by the addition of hotter water. The action of the
enzymes commences, the heated decoction of unmalted grains is added in various ways,
and the temperature controlled by additions of hot water or by heating a portion of
the mash. The whole mashing process requires from two to five hours according to
the methods used.

During the mashing the starch is transformed partly into maltose and partly into
dextrins. The ratio of these products will vary according to the amount of amylase
present and especially according to the temperature used. At about 60 the maximum
amount of maltose is produced; at higher temperature (65 to 75) the unfermentable
dextrins increase. The amount of alcohol and the amount of extract in the beer there-
fore depend to a great extent on the method of mashing.

During the first part of the mashing, while the temperature is about
45, lactic bacteria develop. If their action is too intense they will
render the beer unpleasantly acid. If moderate, the acidity they com-
municate to the wort is useful in preventing the growth of the harmful
butyric bacteria which might develop.

After mashing, the wort is separated from the solid matters by drawing off,
extracting the mash with hot water (sparging), and nitration. It is then boiled from
one to eight hours according to the result desired.

Boiling sterilizes the wort, kills all bacteria and destroys any enzymes
which remain. This occurs almost instantaneously owing to the lactic
acid present. Coagulation of protein substances is also brought about,
effecting a clarification of the wort. This requires one or more hours,
according to the nature of the wort. It is necessary also in some cases
to concentrate the wort, which is done by prolonged heating in open
kettles. This may require several hours.

The Hopping of the wort takes place during the boiling. Sometimes
the hops are added just at the end of boiling; sometimes in two or three
portions, one of which may be at the beginning and one after boiling.
Hops contain an aromatic essential oil, resins and tannin. The essential
oil is quickly soluble and volatile; to preserve its aroma in the beer, the hops
must not be boiled too long. The resins are antiseptic and help to pre-
serve the beer. They dissolve with more difficulty and require longer
boiling.

438 MICROBIOLOGY OF SPECIAL INDUSTRIES.

FERMENTAI ON: WORK OF YEAST. After boiling, the wort is separated
from the hop debris by straining. It is then cooled by means of refrig-
erators consisting usually of serpentine tubes through which cold brine
or water runs. The hot wort runs or drips over the outside of these tubes
in contact with the air. The final temperature of the wort is from 12
to 1 8 in top fermentation and 4 to 6 in bottom.

By this means the wort is thoroughly aerated, which is necessary for
the proper work of the yeast. It also effects a partial clarification by
oxidation which causes a precipitation of solid matters.

The fermentation takes place in two stages, the violent or tumultuous
fermentation in vats and the secondary or after fermentation in casks.

During the violent fermentation the temperature is allowed to reach
a maximum of 7 to 9 with light beers, 8. 5 to 10.5 with dark and 12
to 20 in top fermentations. At the end of the first fermentation the beer
is cooled gradually to 3.5 or 5.0 and drawn into fermenting casks
where the after-fermentation takes place.

The yeasts used in brewing vary very much. Besides the division
into top and bottom yeasts, various types of each are recognized. One
of the chief characteristics used for this division is expressed by the per-
centage of the total extract fermented by the yeast. The Saaz type leaves
all the dextrins and some of the maltose untouched and produces beers
light in alcohol and high in extract. The Logos type destroys all the mal-
tose and much of the dextrins. The result is high alcohol and low ex-
tract. The Frohberg type is intermediate. These differences are prob-
ably due to the differences in the amount and perhaps in the kinds of
enzymes.

The yeasts of spontaneously fermenting beers are of various species,
S. ellipsoideus, S'. pasteuriamis and others.

To produce fermentation, yeast is taken from previous vats so long
as the yeast remains sufficiently uncontaminated with foreign organisms.
The condition of the yeast is determined by the character of the fermen-
tation, the degree of attenuation, and by microscopic examination. In
breweries where modern pure culture methods are not used, the yeast
present is always of several forms or types.

In any case after a certain number of transfers, the yeast deteriorates
and finally may become thoroughly infected with bacteria. The bacteria
are revealed by microscopic examination. Where pure cultures are used,
contamination with foreign yeasts is shown by a change in the time of

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 439

spore formation. By this method a contamination of i : 200 may be
discovered.

When the yeast becomes contaminated, a new start must be made
with yeast from another brewery, which is uncertain, or by a starter of
pure yeast, which is the only reliable method.

The new start with pure yeast may be made by employing a kilogram
of pure pressed yeast or a corresponding amount of liquid yeast and
gradually increasing it to the desired amount by repeated small additions
of sterile wort. This must be done with special precautions against
contamination. Many large breweries use large pure yeast machines
which produce directly sufficient yeast to start a fermenting vat.

AFTER TREATMENT. The violent fermentation requires from eight
to eighteen days according to the temperature. It takes place in open
vats or sometimes, in top fermentation, in barrels. When sufficiently
attenuated, the beer is drawn off into large casks where the slow second-
ary fermentation takes place at a low temperature and the beer clears
by depositing yeast and other sediment. The time required for the
secondary fermentation is from six to ten weeks or, with certain types of
beer, from two to four months or longer.

A certain amount of dissolved carbonic acid is necessary for the
quality and keeping of the beer. This is obtained by tightly bunging
the casks at a suitable stage of the secondary fermentation.

The clarification of the beer is sometimes assisted by placing a quantity of chips of
beech or other tasteless wood in the casks. Top fermentation beers are often fined by
the use of isinglas or animal gelatin. Low fermentation beers are usually filtered.

The beer is then ready for delivery to the consumer and is placed in barrels with
precautions to retain the dissolved carbonic acid.

The clear beer may be put directly into bottles with the same precautions. Bot-
tled beers which are to be kept for some time or which are to be shipped to a dis-
tance are pasteurized after bottling at 60 to 65.

DISEASES OF BEER.

Beer may show defects due to imperfections in the raw material or in
the methods of manufacture. These are principally abnormal flavors
and lack of clearness.

The diseases properly so called are due to wild yeasts or to bacteria.
The disease-producing yeasts may be derived from the starter, from the
vessels with which the beer comes in contact, or from the air. They

44 MICROBIOLOGY OF SPECIAL INDUSTRIES.

develop most commonly during the secondary fermentation or in the
bottle. Some may produce a disagreeable bitterness (S. pasteurianus I)
or other unpleasant flavor (S. fcetidus) ; many produce a persistent cloudi-
ness (S. ellipsoideus, S. apiculatus, S. exiguus, S. anomalus.} They are to
be combated by preventing contamination, by proper attenuation and
by pasteurizing.

Bacterial diseases were more common before effective methods of
purifying yeasts were known.

Many forms of lactic bacteria may affect the beer, rendering it acid
and cloudy. They occur principally where the temperature is allowed
to become too high and where proper care in the cleaning and sterilization
of utensils is not exercised.

Acetic bacteria may occur under the same conditions and give a taste
of vinegar to the beer. They are more common in top fermented beers.

Various forms of Sarcina may cause persistent cloudiness, acid, un-
pleasant flavors or both. This contamination may be from the air or
water and is relatively common. The source of infection is to be looked
for in the air or water. Their growth is most rapid at 16 to 20 and is
retarded by the antiseptic properties of hops.

Several kinds of bacteria, bacilli, cocci and sarcinas may cause the
beer to become slimy or viscid and injure the flavor. This trouble is
particularly common, in spontaneously fermented beer.

Wort and beer, being organic solutions containing very little acidity,
are favorable media for the growth of bacteria, many forms of which
may cause trouble. With modern methods of using pure yeast, cleanli-
ness and the pasteurization of bottle beer, diseases can be controlled.

MISCELLANEOUS ALCOHOLIC BEVERAGES.
CIDER AND PERRY.

These beverages are made by the alcoholic fermentation of the juices
of apples and pears respectively and come next to wine and beer in the
quantities produced.

The composition of the fruit varies very much according to the variety,
especially in the matters of acidity, tannin and pectic substances. The
following analysis is that of a good cider apple:

Sugar 167 . o g. per liter.

Tannin 2 . 4 g. per liter.

Acidity (as sulphuric) : . i . 6 g. per liter.

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 441

The pectic matters vary from 2 g. to 25 g. per liter but should not be
too high. Pears contain usually about the same amount of sugar as
apples, more tannin and much less pectic substances.

The microorganisms occurring naturally on the surface of the fruit
are similar to those occurring on grapes, but special forms QiSaccharomyces
are found. Pure cultures of wine yeast are used successfully in cider
making where a perfectly dry cider is wanted. Where a small remnant
of unfermented sugar is desired, the difficulties of using pure cultures
have not yet been overcome. The wild yeasts occurring on the fruit in
large quantities usually take precedence.

Attempts to sterilize the juice by heating have not been successful
owing to the production of a persistent cloudiness. It seems probable that
a moderate use of sulphurous acid as in the case of wine may solve the
difficulty.

The principles of the control of the microorganisms, good and
bad, are the same as in wine making. The same care in gathering
and keeping the fruit and in extracting and handling the juice are
necessary.

The fermentation is similar to that of wine, but the cider should be
taken off the yeast sooner in order to promote clarification and the re-
tention of a little unfermented sugar.

Cider is subject to the same bacterial alterations as wine and requires
the same treatment. It is more difficult to keep when made in the ordi-
nary way and is usually consumed during the first year. It is particularly
subject to turning brown, owing to the large amount of oxidase present
in apple juice.

The use of sulphurous acid for preliminary defecation, pure yeast in
the fermentation, and fining, followed by pasteurization soon after the
fermentation, seem to offer the best means of improving present methods.

FERMENTED BEVERAGES OF VARIOUS FRUITS.

Many other fruits, especially those rich in sugar and with moderate
acidity, are used locally to produce alcoholic beverages. The methods
of fermentation are similar to those used in wine making, but additions
of sugar and water are usually made to correct defects of composition.
Very often distilled alcohol is also added after fermentation to preserve
the liquid, which is thus rendered unsuitable for an ordinary beverage.

442 MICROBIOLOGY OF SPECIAL INDUSTRIES.

HYDROMEL OR MEAD.

An alcoholic beverage made by the fermentation of honey and water
is much used in eastern Europe.

Honey contains from 65 to 74 per cent of reducing sugars and from
2 to 10 per cent of saccharose. It is diluted with water to reduce its
concentration to 22 Bal.* to 24 Bal. A few yeast cells are usually present
in the honey but these are of various kinds and often unsuitable. The
use of a good pure yeast is therefore advisable. As honey contains little
mineral or nitrogenous yeast food, an addition of nutritive substances is
necessary.

The following formulae are recommended by Kayser and Boullanger
to be used in one liter:

A. Dicalcic phosphate i g.

Ammonia 2 g.

Bitartrate of potash 2 g.

Magnesium sulphate o . i g.

B. Maltopeptone i . 5 g.

Bitartrate of potash i . 5 g.

Ammonium phosphate i . o g.

The same results may be obtained by mixing from 20 to 50 per cent
of grape must or apple juice with the diluted honey.

MISCELLANEOUS FERMENTED BEVERAGES.

Fermented beverages of some kind are made in practically every part
of the world. They are very numerous and varied but fall naturally into
three groups; those made from the sweet juices of fruits or other plants in
which the methods of manufacture resemble those of wine making; those
made from starchy materials in which the methods resemble those of brew-
ing; and finally those made from the milk of cows or other mammals
which are discussed in Chapter IV, Div. IV.

Belonging to the first group are numerous beverages made from the
juices of sugar cane, various palms, and tropical fruits. The best known of
these is the MEXICAN PULQUE made by the spontaneous fermentation of
the sweet juice of the agave. Little is known about the microflora con-

* "Balling" refers to the degrees of the special hydrometer for determining the specific
gravity of saccharine solutions such as must or beer wort. Its purpose is to indicate directly
the percentage of solids in solution at a temperature of 63.5 F.

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 443

cerned, but it includes alcohol-forming organisms which produce about
6 per cent of alcohol, and bacteria which cause rapid deterioration and
spoiling of the fermented product. The pulque is ready for consumption
twenty-four hours after the commencement of fermentation and cannot
be kept more than a day or two.

Of the beverages produced from starchy materials the Japanese SAKE,
RICE BEER, has been most studied. It is made from rice by the diastatic
action of Aspergillus oryzce and yeast fermentation. The process includes
three stages. First the preparation of koji which consists of steamed
rice on which the spores of the fungus are sown and allowed to grow
at 20 until the whole mass is penetrated with mycelium. The next
stage is the preparation of moto which is a thick liquid consisting of
steamed rice, water and koji in which the fungus transforms the starch
into sugar at o to 10 in a few days. Fermentation then starts sponta-
neously, alcohol being produced by the action of several yeasts and lactic
acid by bacteria, both present accidentally. In about two weeks the
moto is ready. The last stage is the principal fermentation which occurs
on mixing together steamed rice, koji, moto and water. This requires two
weeks. The liquid is then separated, cleared and stored. It contains a
considerable amount of alcohol and can be kept and aged like wine.

POMBE is a kind of beer made in Africa from millet seed by sprouting
to saccharify the starch and subsequent spontaneous fermentation in
water. It is interesting as the source of the genus Schizosaccharomyces
which appears to take the main part in the fermentation.

GINGER BEER is an acid, slightly alcoholic beverage made by the
fermentation of a 10 to 20 per cent solution of sugar containing a few pieces
of ginger root. The fermentation is induced by adding small pieces of the
so-called ginger-beer plant which consists of Bact. vermiforme and S.
pyriformis. The bacteria form a thick gelatinous sheath and seems to
live symbiotically with the yeast, each developing best in the presence of
the other.

DISTILLED ALCOHOL.

INTRODUCTION.

USES AND SOURCES OF ALCOHOL. Distilled alcohol is used as a
beverage and a medicine or for innumerable purposes in the arts and
industries. Certain methods and sources employed for the latter pur-
poses are inadmissible for the former.

444 MICROBIOLOGY OF SPECIAL INDUSTRIES.

In all cases, it is made by the preparation from saccharine or starchy
substances of a sugar solution suitable for the work of yeast, the fer-
mentation of this solution, and, finally, the distillation of the alcoholic
liquid.

Where the raw materials are sugary, methods similar to those of wine-
making, and where starchy, to those of brewing, are employed, modified
to suit the conditions of each case.

The principal potable alcohols are brandy, made from grapes, rum
from sugar cane, and whiskey from rye or other grains. Many other
sources are used and any fermented beverage will, by distillation produce
a potable spirit varying in character and quality with the source. In-
dustrial alcohol may be made from any substance capable of undergoing
alcoholic fermentation, the limiting factor in practice being, principally,
the cost of the raw material per unit of alcohol.

METHODS.

PREPARATION OF THE SUGAR SOLUTION. Saccharine Raw Materials.
When spirits are to be made from grapes or other fruit, the juice is fer-
mented in the same way as for the corresponding beverage and then
distilled. The juice, however, is diluted to 20 Bal. or less, as it is not
necessary or desirable to have too much alcohol in the fermented
liquid. The product is consumed directly as brandy or used to fortify
sweet wines. The principal fruits used besides grapes are apples,
peaches, plums and cherries.

Industrial alcohol has been made from inferior or spoiled fruits and
from cannery wastes, but the cost per unit of alcohol is usually high. The
difficulties of fermentation are great, owing to the presence of large
quantities of molds and other injurious organisms, and the extraction
of the juice is troublesome. A careful use of sulphites and pure yeast
simplify the process much.

Sugar cane and its products are used in several ways to produce alcohol. To a
limited extent the juice of the cane is fermented directly and distilled. The product is
known as Jamaica rum. Much larger quantities of alcohol are manufactured from
the cane-sugar molasses and appear in commerce as rum, tajfia, arrack or neutral
spirits.

For the making of Jamaica rum the juice is pressed from the crushed canes, and
diluted with 20 per cent of vinasses (the residue of a previous distillation) to increase the
acidity, and give the required flavor.

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 445

Cane molasses which contain from 50 to 60 per cent of fermentable sugar are diluted
with water or vinasses to 15 to 18 Bal. and partially neutralized with lime when the
acidity is excessive.

One of the principal sources of industrial alcohol is the sugar beet. This alcohol is
also used for the adulteration or imitation of potable spirits.

It may be made by the direct fermentation of the beet juice, extracted by grinding
and pressing, by methodical maceration or by diffusion. Sulphuric acid is added
during extraction. This facilitates the extraction by setting free organic acids, and
represses the growth of injurious microorganisms. The amount used should be such
that a minute quantity of sulphuric acid remains free.

Most beet alcohol is made from the coarser molasses of the sugar factories. The
molasses are diluted to 20 to 30 Bal. with water, further diluted and heated with
steam and acidified with sulphuric acid. The sulphuric acid neutralizes the lime which
has been used in the manufacture of the sugar, sets free the volatile acids and breaks up
the nitrites producing nitrogen peroxide. The liquid is then boiled for about one
quarter of an hour to drive off the volatile acids and the oxides of nitrogen which would
prevent yeast fermentation. The liquid after cooling is then fermented with yeast.

Starchy Raw Materials. In the preparation of a fermentable solu-
tion from starchy materials three methods for the conversion of the starch
into sugar may be used, depending respectively on the action of malt,
dilute mineral acids, and certain molds.

The malt used in saccharification may be made in a manner similar to that
described for brewing, from barley, oats, rye or maize. As the object in this case is to
cause complete conversion of the starch with as little malt as possible, the malt should
have the maximum diastatic power. For this reason, germination should be carried
further than for brewing and the malt used green. Drying the malt destroys half its
diastase.

The conversion may also be accomplished by boiling one part of grain in four parts
of water with hydrochloric or sulphuric acid. With the former acid, 10 per cent of the
weight of the grain is used and 5 per cent with the latter. The conversion requires
from eight to twelve hours' boiling. The starch is first converted into dextrins and then
to glucose. If the boiling is too prolonged some of the glucose may be lost by conver-
sion into caramel. The amount of acid and the time of boiling may be much reduced
by operating under 2 to 3 kg. pressure. In this case 200 liters of water are heated with
100 kg. of grain and 4 kg. of acid. Conversion occurs in from 40 to 60 minutes.

The power of certain molds, especially mucors, to convert starch into
sugar has been utilized. Mucor rottxii found in Chinese yeast, Mucor
oryzcB in Ragi, and related forms have been used for this purpose. This
is known as the Amylo Process. The grain is first soaked for a few hours,
then heated with twice its weight of water under a pressure of three and
a half to four atmospheres until soft and the starch rendered soluble.

446 MICROBIOLOGY OF SPECIAL INDUSTRIES.

The liquefaction of the starch is facilitated by slightly acidulating the
water with hydrochloric acid. The mixture is then cooled to 38 and
inoculated with a pure culture of the Mucor. A current of filtered air
is then passed through the mass for twenty-four hours, by which time
the mycelium has permeated the mass. The temperature is then reduced
to 33, pure yeast added and aeration continued for twenty-four hours
longer to promote the multiplication of the yeast. Conversion of the
starch and fermentation of the sugar then continue together. The
mucor is capable of fermenting the su^rtr and producing alcohol, but
the yeast acts more rapidly.

The malting process is the most commonly employed. The acid process destroys
a greater part of the value of the residues of distillation and the amylo process, requiring
costly special equipment and large expenditures for fuel, has not come into general use.

The starchy substances used being usually neutral or of low acidity the sugar solu-
tions produced would be very liable to bacterial invasion unless means of prevention
were used.

In the amylo process the sterilization of the solutions and the use of
pure cultures accomplish this end. In the acid process the minute quan-
tity of free mineral acid remaining in the completed solution prevents
any considerable growth of bacteria. In the malting process the injurious
bacteria are restrained by lactic acid produced by lactic bacteria, originat-
ing in the malt or in the yeast starter. The requisite bacteria are obtained
by keeping the starter or mother yeast at 50 to 58 for a certain time.
This is a favorable temperature for lactic and too high for the develop-
ment of acetic or other injurious bacteria. When the acidity of the
solution reaches 3.5 g. to 5 g. per liter the dangerous butyric bacteria
cannot develop.

Pure lactic acid may be added immediately after saccharification and
the loss of sugar, due to the action of the lactic bacteria avoided, but the
high cost of the pure acid prevents the practice.

Yeast being much less sensitive to the presence of certain antiseptics
than bacteria it is possible to control the latter by the addition of suitable
amounts of an antiseptic to the sugar solution. By gradually increasing
the amount, moreover, yeast can be accustomed to amounts of antiseptics
which render the growth of bacteria impossible. An application of this
principle is found in the use of sulphurous acid in wine-making. In
Effront's method for the preparation of distillation material, hydrofluoric
acid is used. This acid is added to the mother yeast at the rate of 10 g.

MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS. 447

per hectoliter and to the sugar solution in somewhat smaller amounts.
This results in the inhibition of lactic, butyric and other bacteria and
an increase in the fermentative power of the trained yeast.

FERMENTATION. The sugar solution properly diluted and acetified
or sterilized is fermented by the addition of a mother yeast, usually taken
from a previous fermentation.

The original yeast may be obtained by a spontaneous fermentation
as is usual in the manufacture of rum. Such a yeast is always impure,
containing various yeasts, molds and bacteria, and is therefore very
variable and uncertain in its results.

In the fermentation of beet juice and beet molasses, beer yeast of the
Frohberg type or special distillers yeasts are used. A starter or mother
yeast is prepared for each vat or the process is made continuous by
leaving one-third to one-half of the contents of a fermented vat to start
a fresh addition of the sugar solution. With the latter method the yeast
in time becomes weak and badly contaminated and a new start must
be made with fresh yeast.

In the fermentation of solutions made from potatoes, corn or other
starchy substances, each vat is started with a mother yeast. The tem-
perature should be kept below 30 by means of refrigeration, otherwise
alcohol will be lost by the multiplication of bacteria.

By the use of pure yeast, the yield in alcohol is greater as no sugar is
wasted in the production of lactic acid. The cost, however, is greater
owing to the necessity of the use of more heat in sterilization.

The fermentation of sugar-cane molasses for the production of arrack
is brought about by the use of a mother yeast called tape], prepared from
ragi or Java yeast.

Tapej is made by mixing powdered ragi with boiled rice. In two
days the rice is reduced to a semi-fluid condition and contains bacteria,
molds and yeasts. The bacteria seem to have no part in the process but
when too numerous are injurious. The mold Mucor oryza converts the
rice starch into sugar and the yeast S. vordemanni produces alcohol from
the sugar. The other molds present are more or less injurious.

CHAPTER VII.*
THE MANUFACTURE OF VINEGAR.

ACETIC FERMENTATION.

NATURE AND ORIGIN OF VINEGAR.- Vinegar is a condiment made
from various sugary or starchy matters by alcoholic and subsequent
acetic fermentation. It should contain from 4 to 8 per cent of acetic
acid and natural flavoring, coloring and other matters, varying according
to its origin.

Acetic acid (CH 3 COOH) is a monobasic organic acid the second in
the fatty acid series. It is a colorless liquid with a strong suffocating
odor, crystalizing when pure at 16.7 and at lower temperatures when
diluted with from i to 13 per cent of water. Its specific gravity is 1.08
at o and it boils at 118 under 760 mm. pressure, producing an inflam-
mable vapor. It is a solvent of many organic substances and is soluble in
water and alcohol in all proportions.

The metallic acetates are poisonous and are formed in most cases
by simple contact of metal and acid. Certain alloys of tin resist the
action of the acid.

Acetic acid is formed by the oxidation of ethyl alcohol which takes
place in two stages according to the following reactions:

C 2 H 6 0+ O =C 2 H 4 + H 2 O

Ethyl + Oxygen = Acetic + Water,
alcohol aldehyde

C 2 H 4 0+ O =C 2 H 4 O 2

Acetic + Oxygen = Acetic acid,
aldehyde

These reactions may be brought about by chemical means, but in
practice they are due to the action of certain microorganisms, mainly
bacteria. Acetic acid is also made by the distillation of wood but the
product is not suitable for consumption.

VINEGAR BACTERIA. If wine, beer or a similar organic solution con-

* Prepared by F. T. Bioletti.

448

THE MANUFACTURE OF VINEGAR. 449

taining alcohol, is exposed freely to the air, it soon becomes covered with
a film, the alcohol disappears, is replaced by acetic acid and the liquid
is converted into vinegar.

This film, the Mycoderma aceti of Pasteur, consists of bacteria coher-
ing by means of a glutinous sheath surrounding each cell, forming a
zooglea. If the film is undisturbed, the liquid remains clear until
converted into vinegar, if disturbed, portions may sink, new films form
and finally a large gelatinous zoogleic mass, "the mother of vinegar,"
may form in the liquid.

Sometimes, especially on liquids containing sugar and large amounts
of alcohol, such as sweet wines, the film formed consists, not of bacteria,
but of a yeast-like fungus, Mycoderma mni.

Wines which have been sterilized, often remain without acetifying
for a considerable time. Those containing traces of sulphurous acid
acetify slowly and with difficulty. Ordinarily at warm temperatures,
exposed wines develop a bacterial film very rapidly owing to the almost
constant presence of some acetic bacteria in all wines.

Hansen was the first to show that the vinegar bacteria included more
than one species. He isolated and described three species concerned
with the spontaneous souring of beers. Later it was shown by A. J.
Brown, Henneberg and others that several other species commonly
occurred in vinegar factories and that many more were capable of pro-
ducing acetic acid in small amounts. The species which have been
most thoroughly studied and which seem to occur most usually in vinegar
factories are Bad. aceti, Bact, pasteurianum, Bad. kutzingianum, Bad.