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Full text of "Microbiology, a text-book of microorganisms general and applied"

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, Lansing, Michigan. 

E. Fidlar, London, Ontario. 

W. D. Frost, Madison, Wisconsin. 
A. Guilliermond, Lyons, France. 
N. MacL. Harris, Chicago, Illinois. 

F. C. Harrison, Macdonald College, Que., Cana 
E. G<. Hastings, Madison, Wisconsin. 

H. W. Hill, London, Ontario. 

W. E. King, Detroit, Michigan. 

J. G. Lipman, New Brunswick, New Jersey. 



W. J. MacNeal, New York, New York. 

E. F. McCampbell, Columbus, Ohio. 

Z. Northrup, East Lansing, Michigan. 

E. B. Phelps. Washington, D. C. 

O. Rahn, Elbing, Germany. 

L. F. Rettger, New Haven, Connecticut. 

M. H. Reynolds, University Farm, St. Paul, 

Minnesota. 

, W. G. Sackett, Fort Collins, Colorado. 
W. A. Stocking, Ithaca. New York. 
C. Thorn, Washington, D.C. 
J. L. Todd, Montreal, Quebec. 
E. E. Tyzzer, Boston, Massachusetts. 



EDITED BY 

CHARLES E. MARSHALL 

Amherst, Massachusetts 

PROFESSOR OP MICROBIOLOGY AND DIRECTOR OP GRADUATE SCHOOL 
MASSACHUSETTS AGRICULTURAL COLLEGE 



SECOND EDITION REVISED AND ENLARGED 
WITH 180 ILLUSTRATIONS 



PHILADELPHIA 

BLAKISTON'S SON & CO. 

1012 WALNUT STREET 




n 



COPYRIGHT, 1911, BY P. BLAKISTON'S SON & Co. 

Reprinted with Corrections, June, 1912 
COPYRIGHT, 1917, BY P. BLAKISTON'S SON & Co. 



THK MAPI, K 



CONTRIBUTORS 



BIOLETTI, FREDERIC T., M. S. 

Professor of Viticulture and Enology, Viticulturist of Experiment Station, 

University of California, Berkeley. 
BUCHANAN, R. E., B. S., M. S., PH. D. 

Professor of Bacteriology, Bacteriologist of Experiment Station, and Dean 

of Industrial Science, Iowa State College, Ames. 
DORSET, M., B. S., M. D. 

Chief of Biochemic Division, U. S. Bureau of Animal Industry, Washington, 

D. C. 
EDWARDS, S. F., B. S., M. S. 

Formerly Professor of Bacteriology, Ontario Agricultural College, Guelph, 

Canada. Director of The Edwards Laboratories, Lansing, Michigan. 
P^IDLAR, EDWARD, B. A., M. B. 

Chief of Division of Pathology; Pathologist of London Asylum and of Victoria 

Hospital; Professor of Pathology, W. U. Medical Faculty; Bacteriologist of 

London Board of Health, London, Ontario. 
FROST, W. D., PH. D., D. P. H. 

Professor of Agricultural Bacteriology, University of Wisconsin, Madison. 

GUILLIERMOND, A. DOCTEUR fes SCIENCES. 

Professor of Botany, University of Lyon, France. 

HARRIS, N. MAcL., M. B. 

Assistant Professor of Bacteriology, University of Chicago, Chicago. 

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

Principal and Professor Bacteriology, Macdonald College (Faculty of Agri- 
culture, McGill University), Macdonald College, Que., Canada. 

HASTINGS, E. G., M. S. 

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

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

Director of Institute of Public Health; Chief of Division of Epidemiology; 
Professor of Public Health, W. U. Medical Faculty; Medical Officer of Health 
of London, London, Ontario. 

KING, WALTER E., M. A., M. D. 

Formerly Professor of Bacteriology and Bacteriologist of Experiment Station, 
Kansas Agricultural College, Manhattan. Assistant Director of Research 
Laboratory, Parke, Davis & Co., Detroit, Michigan. 



vi CONTRIBUTORS 

LIPMAN, JACOB G., PH. D. 

Dean of Agriculture, Rutgers College; Director of Experiment Station, New 

Brunswick, New Jersey. 

MACNEAL, WARD J., PH. D., M. D. 

Professor of Bacteriology and Director of the Laboratories, New York Post- 

Graduate Medical School and Hospital, New York. 
McCAMFBELL, EUGENE F., PH. D., M. D. 

Professor of Preventive Medicine, Dean of the Medical College, Ohio State 

University. 
NORTHRUP, Z., M. S. 

Assistant Professor of Bacteriology and Hygiene, Michigan Agricultural 

College; Assistant Bacteriologist and Hygienist of Experiment Station. 
PHELPS, EARLE B., B. S. 

Professor of Chemistry, Hygienic Laboratory, U. S. Public Health Service, 

Washington, D. C. 
RAHN, OTTO, PH. D. 

Formerly Assistant Professor of Bacteriology, Illinois University, Urbana. 

Now Elbing, Germany. 
RETTGER, L. F., PH. D. 

Assistant Professor of Bacteriology and Hygiene (in Sheffield Scientific 

School), Yale University, New Haven, Connecticut. 
REYNOLDS, M. H., B. S., M. D., D. V. M. 

Professor of Veterinary Medicine and Surgery, Agricultural College, Univer- 
sity of Minnesota; Chairman, Veterinary Division, Experiment Station, 

University Farm, St. Paul. 
SACKETT, WALTER G., B. S. 

Bacteriologist, Colorado Experiment Station, Colorado Agricultural College, 

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

Professor of Dairy Industry, Cornell University, Ithaca, New York; Dairy 

Bacteriologist of the Experiment Station. 
THOM, CHARLES, PH. D. 

Mycologist, Bureau of Chemistry, U. S. Department of Agriculture, Wash- 
ington, D. C. 
TODD, J. L., B. A., M. D., D. Sc. 

Associate Professor of Parasitology, McGill University. Montreal. 
TYZZER, E. E., A. M., M. D. 

George Fabyan Professor of Comparative Pathology; formerly Director of 

the Cancer Commission of Harvard University, Boston, Massachusetts. 



INTRODUCTION TO THE SECOND 
EDITION 



The continued and growing demand for "Microbiology" has caused 
the contributors to undertake a thorough revision. In this they have 
been guided by the recent developments in this branch of science, 
and also by a desire to adjust and rearrange in the light of constructive 
suggestions and criticisms. 

The primary purpose of this text-book is to place in the hands of 
college students an elementary technical treatise of the subject matter 
included. No effort has been made to review or cite literature, for to 
do either would expand the volume beyond useful limits. To provide 
an introductory text-book mainly for recitations, or for a supplement 
to lecture or laboratory courses, is about all that can be satisfactorily 
comprehended in a single project. 

The cytological aspect of microbiology has seemed to us to deserve 
some emphasis, for it has become quite definite and has been suggest- 
ively indicating much of real value in connection with the active life 
processes of the cell and microbic activities in agriculture, medicine 
and wherever microbiology is applicable. 

The significance of "Intestinal Microbiology" has required a short 
chapter for its proper presentation. 

It has also been found desirable to treat the microbial diseases of 
insects, a growing subject, in a distinct chapter. 

The study of microorganisms flounders in a fog of unsettled ideas 
for a proper designation. Whether it should be called Protistology, 
Microbiology, Bacteriology, Mycology, or something else must be left 
for the future to determine. 

CHARLES E. MARSHALL, EDITOR. 

. \\1HKKST, M \SS\CHUSETTS. 



Vll 



INTRODUCTION TO THE FIRST EDITION 



By a process of adaptation and growth, the branch of science com- 
monly 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 economic relations of the subject-matter 
has no little influence in placing limitations. In the light of such cir- 
cumstances, 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 
impurities, water supplies, sewage disposal, soils, .dairying, fermenta- 
tion industries, food preservation and decomposition, manufacture 
of biological products, transmission of disease, susceptibility and im- 
munity, sanitation, 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 instructional work of the microbiologist stops in most educa- 
tional 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. Elimina- 
tion of many features not only becomes difficult but really precarious, 
because so many avenues are open to the student that pertinency cannot 

ix 



X INTRODUCTION 

always be foreseen or determined. It is well to remember, too, that 
such aggregate subjects as Agriculture and Domestic Science, unlike 
Engineering and Medicine, because of their youth, have not developed 
to that stage in their educational history where practice and the science 
upon which practice should be founded are amalgamated. The practi- 
cal 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 dis- 
criminative 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 adjust- 
ment, 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 repeti- 
tion; besides, that very conspicuous weakness of emphasizing some fea- 
tures unduly while other features of importance are scarcely mentioned, 
confronts us. A most earnest attempt has been made to overcome 
these faults as far as possible, but a complete mastery of them cannot 
be expected in the first product, However, what is lacked in unity 
and continuity 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 demonstration be added 
or needed, we have felt that they may be easily supplied by the instruc- 
tor, who, of course, will be governed by local facilities and conditions. 
Although no space has been given to laboratory exercises, is should not 
be gathered that the authors of this book are any the less earnest in 



INTRODUCTION xi 

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. Abritrary decisions and selections have been forced in 
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 insur- 
mountable 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 
recognized as due the contributors whose unselfish aims have made it 
possible. 

CHARLES E. MARSHALL, EDITOR. 
AMHERST, MASSACHUSETTS. 



CONTENTS 



TITLE PAGE 

CONTRIBUTORS v 

INTRODUCTION (Editor) vii 

CONTENTS (Editor) xiii 

HISTORICAL REVIEW (Harrison) I 

PART I. MORPHOLOGY AND CULTURE OP MICROORGANISMS 

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

CHAPTER I. ELEMENTS OF MICROBIAL CYTOLOGY (Guilliermond) 15 

Cells and energids. Structure of the cell. Nuclear structure (general structure of 
the nucleus, centriole, value of the nucleus, forms of nuclei, theory of binuclearity), 
cytoplasm (appearance of protoplasm, chondriosomes. vacuoles, reserve products), 
membrane, locomotion. Reproduction, Various processes, nuclear division (mito- 
sis, amitosis), sexual changes. 

CHAPTER II. MOLDS (Thorn) . . 36 

Fungi in general, Bacteria, Phycomycetes, Ascomycetes, Basidiomycetes, Imper- 
fect fungi. Cytology of molds, General structure of molds, cytoplasm, nuclei, 
metachromatic corpuscles and reserve products, cell wall. Molds, Cosmopolitan 
saprophytes, molds of fermentation, parasites and facultative parasites. Considera- 
tion of groups, Mucor, Penicillium, Aspergillus, Cladosporium, Alternaria and 
Fusarium, Oidium, Monilia, Dematium. 

CHAPTER III. YEASTS (Bioletti) 59 

Morphology of certain types, Definition and bases of classification. Cytology, 
General structure of yeasts, cytological phenomena during multiplication, variation 
in the cellular structure during development, cytological phenomena of the sporula- 
tion and germination of ascospores. The principal yeasts of importance to fermenta- 
tion industries. True yeasts, pseudo-yeasts. Culture of yeasts. 

CHAPTER IV. BACTERIA (Frost) 77 

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. Cytology of bacteria, General consideration of cytoplasm and nucleus, 
minut^ consideration of cytoplasm and nucleus, life cycle of bacteria, reserve 
products, general structure of cell wall, minute structure of cell wall, general con- 
sideration of flagella, minute consideration of flagella. Higher bacteria. Classifi- 
cation. Relationship of bacteria. Cultivation of bacteria. 

CHAPTER V. INVISFBLE MICROORGANISMS (Dorset) 116 

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

CHAPTER VI. PROTOZOA (Todd, revised by Tyzzer.) 120 

Introduction. Structure of protozoa. Activities of protozoa, Locomotion and re- 
production, developmental cycle, encystment. Parasitism. Discussion of classifi- 
cation. Technic. 

xiii 



XIV CONTENTS 

PART II. PHYSIOLOGY OP MICROORGANISMS (RAHN) 

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

INTRODUCTION Principles of nutrition and metabolism, energy supply of micro- 
organisms 141 

CHAPTER I. FOOD OF MICROORGANISMS 148 

The composition of the cell, Moisture, cell wall, cell contents. Amount of food re- 
quired. Food for growth (sources of carbon, nitrogen, hydrogen, oxygen, minerals). 
Food for energy. Oxygen relations. 

CHAPTER II. PRODUCTS OF METABOLISM 158 

The chemical equations of fermentations. Physiological variations. Products from 
sugar, starch, cellulose, alcohols, organic acids, fats. Products from nitrogenous 
compounds, Protein bodies, ptomains, urea, uric acid, hippuric acid. Products 
from mineral compounds, Oxidations, reductions. Unknown products of physio- 
logical significance, Pigments, aromatic substances, enzymes and toxins. Factors 
influencing the type of decomposition. Rotation of elements in nature, Carbon cy- 
cle, nitrogen cycle, sulphur cycle, phosphorus cycle. Physical products of metabo- 
lism, Production of heat, production of light. 

CHAPTER III. MECHANISM OF METABOLISM 189 

General theory of metabolism, Metabolism, katabolism and anabolism. Intra- 
and extra-cellular fermentation, Decomposition of insoluble food, properties of 
enzymes, enzymes of fermentation. Classification of enzymes, Hydrolytic en- 
zymes (enzymes of carbohydrates, enzymes of fats, enzymes of proteins), coagu- 
lating 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 207 

Osmotic pressure (Plasmolysis), Salt and sugar solutions, colloidal solutions. Des- 
iccation. 

CHAPTER II. INFLUENCE OF TEMPERATURE 213 

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



CHAPTER III. INFLUENCE OF LIGHT AND OTHER RAYS * 222 

Phototaxis. X-rays. Radium rays. 

CHAPTER IV. INFLUENCE OF ELECTRICITY 226 

CHAPTER V. INFLUENCE OF MECHANICAL EFFECTS 227 

Pressure. Gravity. Agitation. 

DIVISION III. CHEMICAL INFLUENCES 

CHAPTER I. STIMULATION OF GROWTH 230 

Chemotropism and chemotaxis. 



CONTENTS XV 

CHAPTER II. INHIBITION OF GROWTH 232 

Poisons, germicides, disinfectants, antiseptics, preservatives. Mode of action. 
Factors influencing disinfection. Classification of disinfectants. 

DIVISION IV. MUTUAL INFLUENCES 

SYMBIOSIS. METABIOSIS. ANTIBIOSIS 241 



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

CHAPTER I. THE MICROSRGANISMS OF THE AIR AND THEIR DISTRIBUTION 247 

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. ... 252 
Air as a carrier of contagion. Organisms of the air and fermentation. Freeing air 
from bacteria. 

DIVISION II. MICROBIOLOGY OF WATER AND SEWAGE 

CHAPTER I. MICROORGANSISM IN WATER (Harrison) 254 

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, 
Temperature, light, food supply, oxidation, vegetation and protozoa, dilution, sedi- 
mentation, other causes. Interpretation of the bacteriological analysis of water, 
Quantitative standards, qualitative standards. Sedimentation, filtration and purifi- 
cation 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) 274 

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. . . . 289 
Introduction. The soil as a culture medium. Moisture relations, The amount 
and distribution of rain fall, range of soil moisture, effect of drouth and excessive 
moisture. Aeration. Mechanical composition of soils, aerobic and anaerobic activi- 
ties, rate of oxidation of carbon, hydrogen and nitrogen, the mineralization of organic 
matter. Temperature, Influence of climate and season, early and late soils, pro- 
duction and assimilation of plant food. Reaction, Range of soil acidity, causes of 
soil acidity, effect of reaction on number and species. Food supply, Organic mat- 
ter, the mineral portion of the soil. Biological factors, Fungi, algae, protozoa, 



XVI CONTENTS 

higher plants, bacteria (numbers and distribution, bacteria in productive and unpro- 
ductive soils, distribution at different depths, seasonal variations of bacterial num- 
bers and activities, morphological and physiological groups). Methods of study, 
Quantitative relations, qualitative reaction, transformation reactions, rate of oxida- 
tion of carbon, rate of oxidation of nitrogen, addition of nitrogen, reactions concern- 
ing calcium, magnesium, sulphur, phosphorus. 

CHAPTER II. DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 315 

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 decompo- 
sition. Organic acids, Sources, transformation and accumulation. Protein 
bodies, Amount and quality, carbon-nitrogen ratio. Transformation of nitrogen 
compounds, Ammonification, nitrification, denitrification. Analytical and syn- 
thetical reactions, 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.) 338 

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, resist- 
ance, immunity, and physiological efficiency, mechanism of fixation, variations and 
specialization, relation to environment. Soil inoculation, Methods of soil inocu- 
lation, Inoculation with legume earth, inoculation with pure cultures, etc. (Ed- 
wards.) 

CHAPTER IV. CHANGES IN ORGANIC CONSTITUENTS 354 

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, Avail- 
ability of phosphates, relation of phosphorus to decay and nitrogen-fixation. Sul- 
phur, Sulphur compounds in the soil, sulphur bacteria, sulfofication, sulphate re- 
duction. Potassium, The transformation of potsasium compounds in the soil. 
Other mineral constituents, Iron, aluminum, manganese, and copper. Antagonism. 

DIVISION IV. MICROBIOLOGY OF MILK AND MILK PRODUCTS 

CHAPTER I. THE RELATION OF MICROORGANISMS TO MILK. (Stocking.) (The Acid- 
forming Bacteria, by Hastings.) 364 

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, dis- 
eased 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, Gen- 
eral significance of acid-forming bacteria, groups of acid-forming bacteria (character- 
istics of the Bact. lactif 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 pepton- 
izing, pathogenic organisms. Factors influencing the developing of microorganisms 
in milk, Initial contamination, straining, aeration, centrifugal separation, tempera- 
ture, pasteurization, the use of chemicals. The normal development of micro- 
organisms in milk, Germicidal period, period from end of germicidal action to time 
of curdling, period from time of curdling until acidity is neutralized, final decomposi- 
tion changes. Abnormal fermentations in milk, Gassy fermentation, sweet curd- 
ling fermentation, ropy and slimy fermentation, bitter fermentation, alcoholic fer- 



CONTENTS XV11 

mentation, other fermentations. The commercial significance of microorganisms in 
milk, Relation of dirt contamination to germ content. Milk as a carrier of disease 
organisms, Those microorganisms which are beneficial and detrimental 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) 408 

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

CHAPTER III. RELATION OF MICROORGANISMS TO CHEESE (Hastings) 420 

General. Types of cheese, Acid-curd cheese, rennet-curd cheese. Conditions af- 
fecting 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, other groups of bacteria in cheese, 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 OF MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS 

(Stocking) 438 

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 microdrganisms, Kumyss, kefir, leben, yoghurt, arti- 
ficial buttermilk. Frozen milk. Ice cream. 

DIVISION V. MICROBIOLOGY OF SPECIAL INDUSTRIES 

CHAPTER I. DESICCATION, EVAPORATION, AND DRYING OF FOODS (Buchanan) . . . 450 
Factors that bring about changes in dried foods. Inhibition of growth of micro- 
organisms in dried food. Methods of drying, Carbohydrate foods, as fruits, maca- 
roni, 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) 457 

Historical r6sum6. Economic importance, From the standpoint of health and 
dietetics, 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 disorganiza- 
tion, normal flora and fauna). Pasteurization, Economic consideration, specific 
application (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. Home canning. Spoliation,' 
Microbiological, detection of spoiled goods. Disposal of factory refuse. 

CHAPTER III. THE PRESERVATION OF FOOD BY COLD (MacNeal) 471 

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



XV111 CONTENTS 

CHAPTER IV. ^PRESERVATION OF FOOD BY CHEMICALS (MacNeal) 479 

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, dairy products, prepared vegetables, and fruits. The nutritive value 
of preserved foods. The effects of food preservatives, Substances which preserve 
by their physical action, substances which preserve by their chemical action, inor- 
ganic food preservatives, organic food preservatives, substances added to foods to 
improve the apparent quality. The legal control of the preservation of foods by 
chemicals. 

CHAPTER V. MICROBIAL FOOD POISONING (MacNeal) 488 

General considerations. Infections of food-producing animals transmissible to man. 
Human infections transmitted in food. Food poisoning due to the growth of 
saprophytic bacteria in the food, Poisonous meat, sausage, fish, shell fish, milk, 
cream, cheese, and vegetable food. The chemical nature of food poisons. 

CHAPTER VI. MICROORGANISMS OF THE DIGESTIVE TRACT (MacNeal) 496 

Introduction. Microorganisms of certain portions of the alimentary canal, Micro- 
organisms of the mouth, microorganisms of the stomach, microorganisms of the 
intestines, microorganisms of the feces. General method of study, Collection of 
material. 

CHAPTER VII. THE MICROBIOLOGY OF ALCOHOL AND ALCOHOLIC PRODUCTS (Bioletti) . 506 
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-form- 
ing bacteria, 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 (production of enzymes) , work of enzymes and bacteria, fermentation (work 
of yeasts), after-treatment. Diseases of beer. Miscellaneous alcoholic beverages: 
Cider, perry, fermented beverages of various fruits, hydromel or mead, pombe, ginger 
beer. Distilled alcohol: Introduction, Uses and sources of alcohol. Methods, 
Preparation of the sugar solution (saccharine raw materials, starchy raw materials), 
fermentation. 

CHAPTER VIII. MANUFACTURE OF VINEGAR (Bioletti) 538 

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 IX. MANUFACTURE OF OTHER FERMENTED PRODUCTS (Bioletti) 551 

Preparation and conservation of food material, Compressed yeast, bread, yeast as 
food, vegetables, starch, sugar, tobacco. Preservation and conservation of miscel- 
laneous products, Indigo, retting, tanning. 

CHAPTER X. MANUFACTURE OF VACCINES (KING) 560 

Introduction. Actively immunizing substances (vaccines), Attenuated viruses, 
small-pox vaccine, blackleg vaccine, rabies vaccine, Dorset-Niles hog-cholera serum, 
anthrax vaccines, tuberculosis vaccine. Bacterial vaccines (bacterins), Typhoid 
fever, canine distemper, Asiatic cholera, bubonic plague. Sensitized vaccines. 

CHAPTER XI. THE MANUFACTURE OF ANTISERA, AND OTHER BIOLOGICAL PRODUCTS 

RELATED TO SPECIFIC INFECTIOUS DISEASES (King) 575 

Antitoxins, Diphtheria antitoxin, tetanus antitoxin. Antimicrobial serums, 
Antimeningococcic, antistreptococcic, antigonococcic, Dorset-Niles anti-hog-cholera, 
antirabic, antidysenteric, preservation of antiserums. Tuberculins, Koch's tuber- 
culin (old), other tuberculins, mallein. Suspensions for the agglutination tests. 
Substances used for diagnostic purposes, Luetin, antigen, Schick test. 



CONTENTS XIX 

DIVISION VI. MICROBIAL DISEASES OF PLANTS (Sackett) 

INTRODUCTION 588 

CHAPTER I. BLIGHTS 590 

Stem blight of alfalfa. Bacteriosis of beans. Blight of lettuce. Blight of mulberry. 
Blight of oats. Stem blight of field and garden peas. Pear blight. Streak dis- 
ease of sweet peas and clovers. Tomato blight. Walnut blight. 

CHAPTER II. GALLS AND TUMORS 605 

Crown gall. Olive knot. Fingers and toes of cabbages (Todd, revised by 
Tyzzer). Tuberculosis of sugar beets. 

CHAPTER III. LEAF SPOTS 612 

Citrous canker. Angular leaf-spot of cucumbers. Leaf-spot of the larkspur. 
Bacterial spot of plum and peach. Leaf spot of sugar beet. 

CHAPTER IV ROTS '617 

Black rot of cabbage. Wakker's hyacinth disease. Basal stem rot of potatoes. 
Bud rot of cocoanut. Brown rot of orchids. Rot of cauliflower. Soft rot of calla 
lily. Soft rot of carrot and other vegetables. Soft rot of hyacinth. Soft rot of 
muskmelon. Soft rot of the sugar beet. 

CHAPTER V. WILTS 627 

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

DIVISION VII. MICROBIAL DISEASES OF INSECTS (Northrup) . . 632 

INTRODUCTION. Miscellaneous insect diseases, Saprolegniaceae, Entomophthor- 
aceae, and Entomogenous fungi (Thorn). Bacterial disease of June Beetle larvae, 
Lachnosterna spp. Flacherie (silk worm). Bacterial disease of locusts. Bacillary 
septicaemia of caterpillars, Arctia caja. Graphitosis. American foul brood. Sep- 
ticaemia of the cockchafer, Melolontha vulgaris. European foul brood. Bacterial 
septicaemia of larvae of the Lamellicorna. Bacterial disease of]the gut-epithelium of 
the lug-worm, Arenicola. ecaudata. Sacbrood of bees. Wilt disease or flacherie of 
the gipsy moth caterpillar, Porthelria dispar. Pebrine. 

DIVISION VIII. MICROBIOLOGY OF THE DISEASES OF MAN AND DOMI sue 

ANIMALS 

CHAPTER I. METHODS AND CHANNELS OK INFECTION (McCainpbell) 659 

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 patho- 
genic microbic 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 infec- 
tions, dust infections, droplet 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 infections. The factors which influence the results of an infection, 
Virulence, number, avenue, resistance. The exact cause of infections, Soluble tox- 
ins, endotoxins, toxic bacterial proteins, other possible exact causes. The methods 
by which infectious microorganisms are disseminated. The methods by which in- 
fectious microorganisms are eliminated from the body. The effect of infectious 
microorganisms upon the body, The period of incubation, local reactions, general 
:<ins (metabolism, blood-forming organs, parcnchymatous tissues, epithelial ami 
emiothtlial tissues, ct -y tliroey t.i-s ami leucocytes, antibody formation). 



XX CONTENTS 

CHAPTER II. IMMUNITY AND SUSCEPTIBILITY (McCampbell) 684 

General, Definition, hypersusceptibility or anaphylaxis, predisposition and non- 
inheri^ance 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 ex- 
posed mucous membranes of the body, nasal cavity, mouth, lungs, stomach, intes- 
tines, genito-urinary tract, conjunctiva, the protective nature of inflammatory 
processes, natural antitoxins, natural antibacterial substances, normal hemolysins, 
normal agglutinins, normal precipitins) , acquired immunity (active immunity, pas- 
sive immunity). The origin and occurrence of antibodies, Antitoxins (the mech- 
anism of the neutralization of toxin by antitoxin, unit of antitoxin), lysins and 
bactericidal substances (the structure of lysins, deviation of complement, the deflec- 
tion of the complement as a test for antibodies), cytotoxins and cytolysins, opsonins 
and phagocytosis (opsonic index, hemoopsonins), agglutinins (normal agglutinins, the 
, production of agglutinins, the distribution of agglutinins in the blood, inherited 
agglutinins, the substances concerned in agglutination, structure of agglutinins and 
agglutinogens, agglutinoids, the stages of agglutination, hemoagglutinins) , precip- 
itins (normal precipitins, mechanism of the formation of precipitins, autoprecipitins 
and isoprecipitins, the phenomena of specific inhibition, antiprecipitins, the precip- 
itinogen, precipitate, coprecipitins, the forensic use of precipitins). The theories of 
immunity, Noxious retention theory, exhaustion theory, Ehrlich's side-chain 
theory, phagocytic theory. 

CHAPTER III. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 724 

Diseases caused by molds and yeasts (various authors), Pneumomycosis (Thorn), 
thrush (Thorn), dermatomycoses, barbers itch, etc. (Thorn), favus (Thorn), actino- 
mycosis (Reynolds), mycetoma (Fidlar), mycotic lymphangitis (Reynolds). Dis- 
eases caused by bacteria, Botryomycosis (Reynolds), gonorrhoea (Fidlar), epidemic 
cerebro-spinal meningitis (Fidlar), infectious mastitis (Reynolds), Malta fever (Fid- 
. lar) , staphylococcic infections (Fidlar) , streptococcic infections (Fidlar) , pneumonia 
(Fidlar), anthrax (Harrison), bacillary white diarrhoea of young chicks (Rettger), 
chicken cholera (Harrison), chronic bacterial enteritis (Reynolds), contagious abor- 
tion (MacNeal), diphtheria (Fidlar), dysentery (Fidlar), fowl diphtheria (Harrison), 
glanders (Reynolds), influenza (Fidlar), whooping cough, haemorrhagic septicaemia 
(Reynolds), leprosy (Fidlar), plague (Fidlar), swine erysipelas (Dorset), tuberculosis 
(Reynolds), foot rot of sheep (Dorset), malignant oedema (Fidlar), milk sickness 
(Harris), 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 dis- 
temper (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 (Mac- 
Neal), rabies (MacNeal), swamp fever (Reynolds), typhus fever (Dorset), yellow 
fever (Dorset). Diseases caused by protozoa (Todd, revised by Tyzzer), Amoebic 
dysentery, entero-hepatitis of turkeys, kala-azar, Delhi boil, sleeping sickness, human 
trypanosomiases of South America, trypanosomiases of animals, coccidiosis of rab- 
bits, white diarrhoea of chicks, malaria, red water, East Coast fever, oroya fever, 
anaplasmosis, sarcosporidia, myxosporidia, microsporidia, infusoria, African tick 
fever, relapsing or recurrent fever, yaws, other spirochaetal diseases, syphilis. 

CHAPTER IV. CONTROL OF INFECTIOUS DISEASES (Hill) 850 

Principles Practice Disinfection Carriage of infection by biological agents. 

INDEX OF CONTRIBUTORS 863 

INDEX OF SUBJECTS 865 



LIST OF ILLUSTRATIONS 



Frontispiece 

1. Jansen s Microscope 2 

2. Cells of Saccharomyces cerevisice 1 6 

3. Cells made up of energids 16 

4. Diffuse nuclei of bacteria 17 

5. Nuclei in Cyanophycece 17 

6. Chromidia in protozoa 18 

7. Micro- and macro-nucleus in an infusorian 19 

8. Division of micro-nucleus and chondriosomes 19 

9. Formation of chloroplasts 20 

10. Mitochondria developing into amyloplasts 21 

1 1 . Chloroplasts of different forms 21 

12. Metachromatic corpuscles 23 

13. Illustrating cyst and thread membranous walls. . " 24 

14. Organs of locomotion in bacteria 25 

15. Division of Spongomonas uvella and Monas termo 26 

1 6. Transverse section illustrating trichocysts and cilia attachments . . 26 

17. Schizogony in Amoeba polypodea 27 

1 8. Sporogony in Saccharomyces cerevisice, B. mycoides and Leucocytozoon 

lovati 27 

19. Karyokinesis in Acanthocystis aculeata and Coleosporium senecionis . 29 

20. Protomitosis in Amoeba mucicola. Amoeba froschi, Euglena splendens, 

and Amoeba diplomitotica 31 

21. Mesomitosis in Pelomyxa palustris, Urospora lagidis, and Galactima 

succosa 33 

22. Conjugation in Schizosaccharomyces octosporus 34 

23. Nuclei in mycelium of Thamnidium elegans and Mucor c ircinelloides . 41 

24. Fragments of mycelia of molds with dividing nuclei 41 

25. Filaments of molds showing chondrium 42 

26. Nucleus of Mucor in various stages of division 43 

27. Metachromatic corpuscles in Dematium 44 

28. Metachromatic corpuscles in asci 44 

29. Metachromatic corpuscles in conidia 45 

30. Metachromatic corpuscles in cell of perithecium of Pestulariajuesi- 

culosa f . , 46 

31. Mucor, general 49 

32. Mucor ', zygospore 50 

33. Penicillium expanstim 51 

34. Aspergillus glaucus 54 

35. Aspergillus fumigatus, A. nidulans 54 

36. Cladosporium herbarum 55 

37. Spores of Alternaria 55 

38. Fusarium 55 

39. Oidium lactis 56 

40. Monilia Candida 57 

41. Yeast cell 60 



XX11 LIST OF ILLUSTRATIONS 

42. Spore-bearing yeast cells 61 

43. Saccharomyces cerevisice showing vacuoles and metachromatic cor- 

puscles stained 62 

44. Saccharomyces cerevisice showing cells with nuclei, nuclear division and 

glycogenic vacuoles with grains 62 

45. Saccharomyces cerevisice showing cells stained by a special method re- 

vealing a chondriurn consisting of granular- and rod-mitochondria . 62 

46. Saccharomyces cerevisia with both nucleus and metachromatic 

granules 63 

47. Saccharomyces ellipsoideus cells with nucleus 64 

48. Copulation and sporulation in Schizo Saccharomyces octosporus. ... 66 

49. Various stages of nuclear division during sporulation in Schizosac- 

charomyces octosporus 66 

50. Cellular fusion in Schizosaccharomyces pombe 67 

5 1 . Heterogamous copulation in Zygosaccharomyces chevalieri 68 

52. Sporulation in Saccharomyces ludwigii 69 

53. Germination of ascospores in Saccharomyces ludwigii 70 

54. Wine and beer yeasts 72 

55. Wild and pseudo-yeasts 75 

56. Types of micrococci 77 

57. Types of bacilli 77 

58. Types of spirilla 78 

59. Involution forms 78 

60. The division of bacterial cells 81 

61. The formation of spores 83 

62. Location of spores in bacterial cells 83 

63. Spore germination : .... 84 

64. Division forms of micrococci 85 

65. Division forms of bacilli 86 

66. Threads of Bad. anthracis 86 

67. Plasmolytic changes 87 

68. Karyokinetic appearances in Bad. gammari 89 

69. B. megatherium in process of division 90 

70. Diffuse nucleus in Chromatium okenii and Beggiatoa alba 91 

71. B. butschlii in division 93 

72. B. sporonema in spore formation with vestiges of ancestral sexuality 94 

73. B. radicosus with nuclear appearances 94 

74. B. flexilis in division of cell and formation of spores 96 

75. Retrogression of original nucleus and formation of diffuse nucleus in 

various bacteria 96 

76. Differentiation of metachromatic corpuscles in various bacteria by 

means of stains 99 

77. Structure of bacterial membrane in section 101 

78. Capsules (Bad. pneumonia) 102 

79. Distribution of nuclear substance and various flagella . 103 

80. Monotrichous bacteria (Msp. comma) 103 

8 1. Monotrichous bacteria (Ps. pyocyanea) 103 

82. Lophotrichous bacteria (Ps. syncyanea) 103 

83. Lophotrichous bacteria (Sp. rubrum) 103 

84. Peritrichous bacteria (B. typhosus) 103 

85. Crenothrix polyspora 107 

86. Chlamydothrix hyalina no 

87. Cladothrix dichotoma 112 

88. Beggiatoa alba 112 

89. Pasteur-Chamberland or Berkefeld filtering apparatus 117 



LIST OF ILLUSTRATIONS XX111 

90. Amoeba tespertilio 121 

91. Paramecium caudatum dividing without mitosis 124 

i)2. Stages in division of Amceba poly podia 125 

93. Multiplication of Coccidium schubergi 126 

94. Herpetomonas musca-domestica 131 

95. Trypanosoma tineas and Trypansoma perca 132 

96. Trichomonas eberthi 133 

97. Lamblia intestinalis 1 34 

98. Development of sporozoits in Laverania malaria 135 

99. Amoeba proteus 144 

TOO. Influence of oxygen on microorganisms 157 

101. Crystals of bacteriopurpurin 177 

102. Carbon cycle 184 

103. Nitrogen cycle 185 

104. Sulphur cycle 186 

105. Action of light on bacteria 222 

106. Action of light on molds 223 

107. Action of light on mold colonies 224 

108. Chemotaxis 230 

109. Curve of disinfection 233 

1 10. Influence of filtered water on typhoid fever and Asiatic cholera . . . 259 
in. Section of sand filter 267 

112. Unglazed porcelain filters 269 

113. 114, 115. Location of wells on farm 271 

116. Construction of model well 272 

117. Trickling filter, sand filter, dosing tank, septic tank 285 

118. Septic tank 286 

119. Non-symbiotic nitrogen-fixing organism (B. pasteurianus) 340 

I:M>. X on-symbiotic nitrogen-fixing organism (Azotobacter vinelandi) . . . 341 

121. Ps. radicicola 345 

122. Section through root tubercle 346 

123. 124, 125. Influence of Ps. radicicola 349, 350, 351 

126. Section of cow's udder 369 

127. Bacterial colonies in dust from udder 373 

128. Bacterial colonies from cow's hair 374 

129. Bacterial colonies from dust of stable 375 

130. Small-top milk pails 377 

131. Ropy cream '. 397 

132. Ropy cream organisms 398 

133. Chart of Rochester milk supply 402 

134. Gassy cheese 422 

135. Cheese from lactic starter 423 

136. Influence of lactic organisms on casein degradation 429 

137. Swiss cheese 434 

138. Kepfir grain 443 

1 38a Tubes for f eces examination 505 

139. Bacteria of slimy wine 513 

140. Bacteria of wine diseases 514 

141. Vinegar bacteria 540 

142. Vinegar barrel 544 

143. Rapid process vinegar apparatus 547 

144. Ps. medicaginis 591 

145. Pear blight 597 

146. Walnuts affected by bacteriosis 603 

147. Crown gall 605 

I4H. Roots of cabbage plant affected with "stump-root" 609 



XXIV LIST OF ILLUSTRATIONS 

149. Plasmodiophora brassica 6n 

150. Oidium albicans 72, 

151. Oidium albicans. (Kohle and Wassermann.) 72, 

152. Trichophyton tonsurans 721 

!53- 154- Actinomyces bovis. 728,721 

155. Gonococci 73, 

156. Bact. anthracis, thread formation 75 

157. Bact. anthracis, spores 75 

158. Organisms of anthrax in capillaries 75, 

159. Bact. diphtheria 76 

1 60. Westbrook's types of Bact. diphtheria 76: 

161. Bact. mallei 761 

162. Bact. pestis 77! 

163. Bact. tuberculosis, branching forms 78: 

164. Bact. tuberculosis, from sputum 78: 

165. Bact. tuberculosis, in culture 78; 

166. B. tetani, with spores 79 

167. B. typhosus 791 

168. Msp. comma 79* 

169. Msp. comma colonies in gelatin 8o< 

170. Kidneys in hog cholera, hemorrhagic points 8oi 

171. Negri bodies 8i< 

172. Amceba coli 82; 

173. Leishmania donovani 82; 

174. Structure of trypanosome 82; 

175. Trypanosoma gambiense 82! 

176. Glossina palpalis 82* 

177. Colonization in Trypanosoma lewisi 83: 

178. Malarial parasite in human and mosquito cycles 83; 

179. Longitudinal section of Anopheles 83! 

1 80. Babesia bigemina 84* 

181. Ornithodoros moubata 84; 

182. Spirochata duttoni 84< 

183. Treponema pallidum 84) 

Colored Plate 
The Malarial parasites 835~83< 



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 whether he ever 
saw microorganisms. 

In the year 1683 Antonius vanLeeuwenhoek, a Dutch naturalist and 
a maker of lenses, communicated to the inglish Royal Society the re- 
sults of observations which he had made with a simple microscope of 
his own construction, magnifying from 100 to 150 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 infectious disease was produced, 

Prepared by F. C. Harrison. 



2 HISTORY ot MICROBIOLOGY 

that microorganisms were capable of reproduction outside of the body, 
and that they might be conveyed from place to place by the air. 

The important role that the compound microscope has played in 
microbiology calls for something regarding the invention of this in- 
strument -an invention which antedates Leeuwenhoek's discovery by 
nearly too 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- 
chromatic lenses and many minor improvements were introduced by 
the firm of Zeiss about 1880. 



I 



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

In 1786 O.JSLMiiller (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 
nineteenth, the history of bacteriology is largely the story of a con- 
troversy 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 this forma- 
tion 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 preexisting life a theory aphoristically summed 
up in the phrase "omne vivum ex vivo." Such was the doctrine of 
Biogenesis life only from life. 



HISTORY OF MICROBIOLOGY 3 

In 1668, Francisco Redi, an Italian, distinguished alike as scholar, 
poet, physician, and naturalist, expressed the idea that life in matter is 
always produced through the agency of preexisting 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- 
quarters of an hour was possible only, provided air, which had not been 
previously exposed to the influence of fire, had been admitted." Ob- 
jections were made to these experiments and the controversy went 
merrily on. Gradually experimental evidence accumulated resulting 
largely from the work of Fian^-Sdijilze, 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 spontaneous generation, or abiogenesis, could not be 
>roved. 

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 1840-60. 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 numer- 
ous wheels of the mills of Mayence." Again and again Liebig formally 
denied the correctness 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. 



4 HISTORY OF MICROBIOLOGY 

In 1841 Fuchs investigated some blue and yellow milk. He exam- 
ined it with the microscope and discovered the presence of organisms. 
He succeeded in cultivating the a 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 Bacterium syncyaneum, now known as B. cyanogenus, Ps. syn- 
cyanea and B. synxanthus, a name which is still retained in the 
literature. 

Since i86ojthe master mind of Louis Pasteur has dominated the 
realm of microbiology. His epoch-making discoveries were largely due 
to his intuitive 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 professorship 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 fer- 
mentation. 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 nourishes 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 dis- 
covered 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 microorganisms. This process, since 
called pasteurization, is now largely used, and makes it possible for 
manufacturers 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 kilograms 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 in- 



HISTORY OF MICROBIOLOGY 5 

vestigation 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 com- 
munities, but at the cost to himself of impaired health and partial 
paralysis. 

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 follow- 
ing 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, liga- 
tures, 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 is traceable 
to Lister's efforts. Lister's methods, suggested by the ideas of Pas- 
teur, have rendered possible the marvelous surgery of the present day, 
banished hospital 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 fer- 
mentations of milk, such as souring, curdling, etc. He also confirmed 
the conclusion of Robert Hall (1874), that milk could be obtained 
from the anima'l 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 re- 
sistant to heat than the rods from which they were derived. This fact 
was observed 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 



6 HISTORY OF MICROBIOLOGY 

beautiful results by Ehrlich, Koch, Gram, and others. The staining 
of microorganisms rendered it possible to obtain pictures of them by 
photographic 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 
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 protozoonof 
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 Schiitz 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, LoefHer grew the organism in pure culture. 

In 1884, Koch4xublished his results on the etiology of tuberculosis, 
in a paper which will remain as a classical masterpiece 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 188$, Nicolarer 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 organisms associated with inflammation and suppuration, 
such as the Streptococcus pyogenes and the Staphylococcus pyogenes, 
discovered by Rosenbach, and the green pus germ (Pseudomonas 
pyocyanea) by Gessard. 

*A11 temperatures are stated in Centigrade scale, unless otherwise indicated. 



HISTORY OF MICROBIOLOGY 7 

While these discoveries were taking place, largely in Germany, Pas- 
teur had be.en 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 was inoculated with virulent anthrax, and also 
twenty-five which had received no vaccine. The twenty-five non- 
vaccinated sheep died, and the twenty-five vaccinated ones remained 
healthy and in the same state as the ten control animals. This con- 
vincing experiment was followed by others; and, in the twenty-five 
years immediately following the introduction of the method, more 
than ten million animals were vaccinated in France alone, with ex- 
cellent 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 establishments 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 im- 
portance in 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 nitrify- 
ing 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 
tjaat yeasts produced diseases in beer, and established the method of 



8 HISTORY OF MICROBIOLOGY 

identifying yeasts by observing their microscopic appearance, the for- 
mation 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 cura- 
tive agent; 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 1885-94, was 9.74 per 10,000 of 
the population before the use of antitoxin; and during the antitoxin 
period of 1895-1904 it was 4.29. 

The subsequent researches on the constitution of toxins and anti- 
toxins by Ehrlich, Metchnikoff, Madsen, and others have been pro- 
ductive of a better understanding of the problems of immunity. 

In 1892 Pfeiffer discovered the organism of influenza or grippe; and 
in 1894 Yerstrrttni} 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 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 
organisms. 

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_sjpki- 
lis, a spirochaete-shaped organism, which he named Treponemapallidum, 
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 mucji still remains unknown. The 



HISTORY OF MICROBIOLOGY 9 

causes of some diseases have not been discovered. Smallpox, scar- 
let fever, yellow fever, mumps, whooping-cough, epidemic infantile 
paralysis, hydrophobia, and others offer an inviting field to the medical 
microbiologist; 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 knowl- 
edge 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 is concerned almost wholly with the field of unicellular 
life. On the one hand, the microbiologist meets the botanist and 
establishes reciprocal relations with him; on the other hand, he mixes 
with the zoologist and delves into studies of mutual interest. Pri- 
marily, the technic of the microbiologist together with, in part, the 
economic bearing of the subject seems to be the determining 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 fascinat- 
ing sea of microbiology. The graphic outlines will always be his ready 
chart. 

* Editor. 

" 



12 



MORPHOLOGY AND CULTTJRE OF MICROORGANISMS 



OUTLINE OF 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. 



Plants 



Schizophyta [ Schizomycetes (fission-fungi), bacteria. 


(fission- 






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




Chlorophyceae green algae. 




Algae 


Phaeophyceae brown algae. 






Rhodophyceae red algae. 








Characeae. 










Myxomycetes. 










Chytridineae. 








Zygomycetes (Mucors). 






Phycomycetes 






Saprolegniaceae 
(water fungi). 








Oomycetes 


Peronosporaceae 












(downy mildews) . 








Hemiasci 


(Monascus). 










Protoascineae (Sac- 


Thallophyta 








charomyces, Yeasts). 




Fungi 


Ascomycetes 


Euasci 


Protodiscineae. 
Discomycetes. 










Plectascineae (Asper- 










gillus). 










Pyrenomy ce tine ae. 






Penicittium, Fusarium, Alter- 






Imperfect Fungi, naria, 










Conidia only Oidium, Cladosporium, and 






others. 










Rusts 










Basidiomycetes Smuts 










Mushrooms. 


Bryophyta 






(mosses and 






liverworts) 






Pteridophyta 






(ferns, etc.) 






Spermatophyta 






(seed plants). 







'Charles Thorn. 



OUTLINE OF PROTOZOAL GROUPS 



OUTLINE OF PROTOZOAL GROUPS* 



"AN OUTLINE CLASSIFICATION OF THE PROTOZOA," embracing only parasitic 


and more especially the pathogenic forms. For discussion of classification see p. 130. 






Entam&ba buccalis 






Entamoeba coli 




Rhizopoda 


Entamoeba Entamceba tetragena (histolytica) 






Entamceba meleagridis 






Plasmodiophora {Plasmodiophora brassica. 






Leish mania 


Leishmania donovani 








Leishmania tropica 






Crithidia 


Leishmania infantum 








Trypanosoma gambiense 








Trypanosoma cruzi 








Trypanosoma brucei 






Trypanosoma 


Trypanosoma evansi 
Trypanosoma equinum 




Flagellata 




Trypanosoma dimorphon 








Trypanosoma lewisi 








Trypanosoma equiperdum 






Trypanoplasma 


Protozoa 




Cercomonas , _. 
Trichomonas { Tnchomonas ^ttnalis 






j, [ Trichomonas vaginalis 






Plagiomonas 






Lamblia {Lamblia intestinalis 







Gregarina 
Coccidium 


Coccidium cuniculi (Eimeria stiedas) 








Coccidium avium 








(Plasmodium vivax 








Plasmodium malaria 








Plasmodium falciparum 









Proteosoma 




Sporozoa 


Haemosporidia 


Haemoproteus 
Lankesterella (and other Haemogre- 








garines) 








Hepatozoon 








, . | Babesia bovis (bigemina) 








JjcLDCSlcl ) n T 

I Babesia cants 






Sarcosporidia { Sarcocystis { Sarcocystis miescheriana 
Haplosporidia { Rhinosporidium { Rhinos poridium kinealyi 






Myxosporidia { Myxobolus { Myxobolus pfei/eri 






Microsporidia { Nosema { Nosema bombycis 




Infusoria j Balantidium j Balantidium coli 



J. L. Todd. Revised by Ernst E. Tyzzer. 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



Parasites of uncertain 
position 



Toxoplasma 

Chlamydozoa 

Ultramicroscopic viruses 

SpirochcRta obermeieri 
Spirochata duttoni 
Spiroch&ta vincenti 
Spirochceta theileri 
Spirochceta gallinarum 
Treponema pallidum 
Treponema pertenue 



Spirochaeta 



Treponema 



CHAPTER I* 
ELEMENTS OF MICROBIAL CYTOLOGY 

CELLS AND ENERGLDS 

The microorganisms are confined to cells, such as algae, molds, 
bacteria, yeasts, and protozoa, or cytoplasmic masses with a nucleus 
associated with each (Fig. 2). Some are, however, made up of rows 
of cells, such as threads of Cladothrix, occasionally capable of branching 
out, like the mycelium of a mold (Fig. 3, A), There are also some cells 
which have a special structure. In each cell are enclosed several 
nuclei. If certain amcebse are examined, for example, Pelomyxa pa- 
lustris (Fig. 3, B), inside of what appears to be a cell there are found 
many nuclei. Such cells have not the anatomical value of true cells, 
but seem to represent as many cells as there are nuclei. Each of 
these nuclei with the cytoplasm which surrounds it, equivalent to a 
cell, may be called specifically an energid. Some algae and fungi are 
made up of threads of cells enclosing several nuclei; each cell in- 
cluded in a thread consequently represents a group of organized ele- 
ments, the union of several energids in the same anatomical unit (Fig. 
3,^)- 

STRUCTURE OF THE CELL 

A typical cell is constituted of three essential elements: the nucleus; 
the cytoplasm; and the cell-membrane. 

The general characteristics of these three elements, and, follow- 
ing this, the study of cell reproduction, may now be systematically 
presented. 

THE NUCLEAR STRUCTURE. General Structure of the Nucleus. The 
nucleus frequently takes in microorganisms the typical form which it 
assumes in the higher organisms, namely, that of a spherical vesicle 
limited by a membrane, enclosing a hyaline substance called the 
nuclear-fluid, or nucleoplasm (Fig. 21, A, a, B, a}. In this nuclear 

*By A. Guilliermond. 



1 6 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

fluid are found: the nucleolus, a spherical corpuscle made upoipyrinin 
to which the chromatin, a characteristic substance of the nucleus, fre- 
quently attaches itself; the chromatic network, the thread of which is 
made up of linin, a very slightly chromophilic substance, enclosing 
some grains, the grains of chromatin, which possess a special affinity 
for basic stains. The chromatin or nuclein is the most important 
substance of the nucleus. 

Centriole. In intimate contact with the exterior of the nucleus and 
sometimes inside is usually found a small body called the centrosome, 
or, if the dense chromatin alone is considered, the centriole (Fig. 20, 
B, a). It is a small chromophilic grain which is often surrounded by a 
clear zone of protoplasm called archoplasm. 



*> 



* * 

* * 



** 



FIG. 2. FIG. 3. 

FIG. 2. Cells of Saccharomyces cerevisia. 

FIG. 3. Cells made up of several energids. A, A portion of the mycelium of a 
mold, Aspergillus ochraceus. (After Dangeard.) B, Cell of an amoeba, Pelomyxa 
palustris (After Doflein). 

Value of the Nucleus. The nucleus is an organ indispensable to 
cellular life. It directs for the most part the physiological functions 
of the cell. It plays an active part in nutrition as is indicated by the 
fact that the greater part of the products of nutrition or of reserve 
spreads itself around the nuclear membrane. Finally, it assumes an 
important role in cellular division and in sexual phenomena. 

The experiments of Balbiani which have been repeated by other 
authors show that the cell cannot function without its nucleus. By 
cutting an infusorial cell in two portions, one of which contains the 
nucleus and the other only its cytoplasm, Balbiani found that the 
nucleated part was able to resist the wound which it had received 
and regenerate the cytoplasm which was lacking; whereas the enucleated 
portion soon perished. 



ELEMENTS OF MICROBIAL CYTOLOGY 



It does not seem probable, therefore, that cells can exist without 
their nuclei. Nevertheless, to the present time it has not been possible 
to find conclusive proof of the presence of a true nucleus in bacteria. 
The presence in their cells, however, of a great num- 
ber of small chromatin grains like the chromatin ma- 
terial of nuclei, and their evolution during the forma- :>_ >; 
tion of spores, force the observer to admit that these ; 
represent grains of nuclear substance, and that bac- a 
teria have a kind of diffuse nucleus, which is scattered 
in the form of small grains (Fig. 4) in the cytoplasm 
of the cell. 



B, Thiothrix ten- 
uis. (After 
Swellengrebel.) 



FIG. 4. Dif- 
fuse nuclei of 
bacteria. A, B. 
mycoides. (After 
Forms of Nuclei in Microorganisms. The nucleus Guiltier mond.) 

of primitive microorganisms is far simpler than in 
the higher forms, where it becomes fairly complex. 
Consequently in the Cyanophycea or blue algae, the 
lowest of ah 1 algae, the nucleus is in a very primitive state. It is 
large, not separated from the cytoplasm by a membrane, and is made 
up simply of a nuclear fluid and a chromatic network. The cyto- 
plasm is confined to a thin cortical layer 
and the nucleus nearly fills the cell (Fig. 5). 
In other microorganisms the nucleus is 
much more complex. Yet frequently this 
nucleus is found in a primitive state quite 
different from typical nuclei of higher 
organisms. In some amrebse, the nucleus 
^^ ^^ is formed simply of a poorly defined mem- 

^ K ?5* brane filled with nuclear fluid, and a large 
jt> & C body of chromatin resembling a nucleolus 
/\ called the karyosome or centriole-nucleolus 

(Fig. 21), because it acts both as a cen- 
triole and as a nucleolus. In the center of 
the karyosome is frequently seen a more 




FIG. 5. Nuclei of Cyano- 
phycea. A, Thread of Rivu- 
laria bullata with nuclei in 
process of division. B,-D, 



Fragments of threads of Colo- intensely chromophilic corpuscle' corre- 
jM^ashowingnuclear 



(pig 



Many protozoa and some algae have a 

centriole-nucleolus, but it is wholly enclosed in the nuclear fluid. 

The chromatin appears as little grains or as a network (Fig. 20, A, a). 

In the higher microorganisms (protozoa and fungi) the nucleus 



1 8 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

begins to take the form of typical nuclei. The centriole detaches 
itself from the karyosome which becomes a true nucleolus, and may 
remain either wholly intranuclear (Fig. 19, A, a, 21, A, a), or become 
entirely extranuclear (Fig. 19, B, a, 21, B, a). 

Theory of Binuclearity of Cells and Chromidia. In the infusoria, the 
nuclear structure divides into two nuclei (Fig. 7); a large one, the 
macronudeus or vegetative nucleus, which functions during the vegetative 
life of the cell, and a small one lodged in a hollow of the macronucleus, 
the reproductive nucleus or micronucleus. At fertilization, the macro- 
nucleus is disorganized and its place taken by the micronucleus which 
reproduces by division both a micronucleus and a macronucleus. 
Certain flagellates have likewise two nuclei, a large vegetative and re- 

productive nucleus, and a small micro- 
or kinetonucleus which controls the for- 
mation of the flagellum. 

Starting from these facts, a few in- 
vestigators have tried to demonstrate 

that a11 Cells haVC tW nudeL ReCent 




Fig. 6.-Chromidia in pro- 
tozoa. A, The cycle of the mi- evidence reveals that there are in the 



o most p rotozoa sma11 

hystolytica. (After Hart- mophilic granules, like the chromatin 
chromidia"' Nucleus ' chr ' material, which are supposed to emigrate 

from the nucleus during certain phases 

of development, and which are likened to the nuclear substance 
(Fig. 6). These granules are called chromidia, and all the granules 
scattered in the cytoplasm are designated as the chromidial structure 
or chromidium. Chromidia have been found in the cells of higher 
organisms. There is a theory that this chromidial system repre- 
sents a second nucleus, the vegetative nucleus, scattered in the cyto- 
plasm, and that the entire cell is provided with two nuclei, one of 
which has passed unseen up to this time because of its diffuse form. 
This theory is much doubted to-day, and it seems probable that the 
chromidium is simply a reserve material for the cell, or corresponds 
to formations which will be described later as mitochondria. 

CYTOPLASM.. Appearance and Properties of Cytoplasm Cytoplasm 
may be denned for our purposes as a semi-fluid substance, granular in 
appearance, and reacting with an acid stain. It has three essential 
physiological properties, nutrition, motility, and sensibility. Cyto- 



ELEMENTS OF MICROBIAL CYTOLOGY 1 9 

plasm appears to be composed largely of protein substances and of 
diverse lipoid substances in a state of colloidal solution. It varies 
widely according to circumstances, consequently it may be useless to 
search for any definite structure. In many microorganisms, as for 
example the protozoa, there is on the periphery of the cell a hyalin zone 
which is called the ectoplasm to distinguish it from the rest of the 
cytoplasm, the endoplasm (Fig. 16). 

Chondriosomes. Recent research has demonstrated special func- 
tioning bodies in the cytoplasm, the mitochondria, which seem to be 
the constructive elements of cytoplasm. They are a part of its struc- 
ture, and are supposed to play an important physiological role in the 
cell. These structures, visible in the living organism, but stained 







^ \ 



FIG. 7. Glaucoma piriformis, FIG. 8. Division of micronu- 

infusorian with (N) macronu- cleus and of the chondriosomes 

cleus, (n) micronucleus (ch) in Carchesium polypinum, infu- 

mitochondria, (up) pulsating sorian. (After Fattrt-Frtntiet.) 
vacuole. (After Fauri-Fre- 
mift.) 

only by a special process, are sometimes in the form of small isolated 
granules (granular mitochondria, Fig. 7, B), or of small threads (thread- 
mitochondria) or sometimes of rods much like certain bacilli (rod- 
mitochondria, Fig. J, A). These forms frequently change from one to 
the other. The granular mitochondrium is able to elongate itself into 
a rod which is itself capable of dividing up into thread-mitochondria. 

he mitochondria of one cell are called the chondrium. These 
tures seem to be made up of lipoidal substance and phosphates of 
nin. 
he mitochondria cannot generate themselves directly from the 
cytoplasm, but are formed always from preexisting mitochondria by 
division. They apparently transmit themselves, after having divided, 
from the egg to the adult individual, and from the adult individual 
to the egg (Fig. 8). 



20 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



Physiologically, mitochondria are organs of elaboration. In 
them, through some unknown physico-chemical phenomena, most of 
the products of cell activity may be formed. The product, whatever 
may be its specific nature, has its origin in a granular mitochondrium 
or in a rod-mitochondrium. It is surrounded by a mitochondrial 
exterior surface inside of which it develops slowly; the exterior surface 
remains until the product has reached its state of maturity. 

It has been known for some time that there exist in higher plants 
corpuscular elements called plastids or leucoplastids, which also possess 
a synthetic function. Some, the chloro plastids, make the chlorophyl 




v 



*/^X 





FIG. 9. Formation of chloroplasts in the young leaf of barley. A, Very young 
cells in which appear rod-mitochondria. B, Older cells in which the rod-mitochondria 
are transforming themselves into chloroplasts. C, Cells in which the chloroplasts 
are definitely constituted. 

which, by using rays of light as energy, forms starch; others, the 
amylo plastids, confine themselves to forming starch from the excess 
sugars found in the cells; still others, the chromo plastids, constitute the 
pigment bodies of plants (xanthophyl, carotins). It has been recently 
shown that plastids are nothing but mitochondria which have under- 
gone greater differentiaton and specialization than those which, at the 
expense of ordinary mitochrondria derived from the egg, have increased 
in size (Figs. 9, 10). 

Mitochondria have been found in most protozoa and fungi. In the 
latter they take part in the formation of reserve products, especially 
the metachromatic corpuscles of which more will be said later. 

Mitochondria are most highly developed in algae where they give 
origin to chloroplastids as in higher plants. On the other hand, in 



ELEMENTS OF MICROBIAL CYTOLOGY 



21 



the lower forms, no mitochrondria seem to exist, but the chloroplastids 
take on certain special characteristics. Instead of small scattered 
corpuscles is found one, or occasionally several, large chloroplastids 
filling most of the cell. They are in various shapes ribbons, spirals, 
nets, etoilated bodies (Fig. u), etc. but all appear to be made up of 
a mitochondrial substance. Their physiological r61e is much more 
general than in the chloroplastids of higher plants. They produce 
not only the chlorophyl, but other pigment bodies, the starch or para- 
mylum, metachromatic corpuscles, and globules of fat. Conse- 



O-/- 






FIG. 10. FIG. IT. 

FIG. 10. A cell from the root of a bean in which the rod-mitochondria (ch) 
form in the course of their development amyloplasts from (/>) which spring grains 
of starch (a). 

FIG. ii. A, Englena viridis with its star-like chloroplasts (chl.) at the center 
of the organism, the pyre"noid body (Py] surrounded by grains of paramylum (Par}, 
eye-spot (0), contractile vacuole (), flagellum (0, nucleus (n). (After Dangeard.} 
B, Microglena punctifera, with two elongated chromatophores arranged longitudinally. 
(After Stein.} 

quently the complex chloroplastids of the algae with their general 
function have been considered as a special form of chondrium which, 
instead of being scattered in the cytoplasm as a number of small 
structures, finds itself gathered in very compact masses. 

The CyanophycecB are the only microorganisms in which the chon- 
drium has not been found. In the Cyanophycea the chlorophyl and the 
blue pigment (phycocyanin) associated with it are diffused throughout 
the cytoplasmic area surrounding the nucleus. The very primitive 
structure of the algae explains to some extent this absence of an im- 
portant structure of the cell. 



22 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Vacuoles. There is always in the cytoplasm one (or several) rather 
bulky vesicle filled supposedly with an aqueous solution of mineral 
salts called a vacuole. Vacuoles play an important part in the ab- 
sorption of liquids by the cell. Owing to the mineral salts dissolved 
in the vacuole-fluid, the concentration of which is ordinarily higher 
than that of the surrounding medium, the vacuoles become the center 
of osmotic forces which consequently cause a part of the ambient 
liquid to penetrate the cell and determine its turgescence. 

Very curious vacuoles are found in many protozoa, namely, the 
pulsating vacuoles (Figs. 7, n). They are small vacuoles which expand 
and contract rhythmically, and which are considered as excretory and 
respiratory organs. The water that has entered the cell gathers in this 
vacuole and is expelled as it contracts. Probably in crossing the body 
this water yields its oxygen to the cytoplasm in order to charge itself 
with carbonic acid and the products of metabolism. 

Reserve Products. The cytoplasm encloses some structures differ- 
entiable by means of certain stains or chemical reagents as granulations, 
but which are not constituent elements of cytoplasm; they come 
from a secretion of the cytoplasm, and only under certain conditions. 
These grains may be found either in the cytoplasmic substance itself, 
or in the vacuoles included in the cytoplasm. Most of these granules 
are reserve products which appear when nutrition is deficient. Among 
the reserve products most common in microorganisms are the granules 
called metackromatic corpuscles (Fig. n, *A). These bodies, which 
are the object of a special study in connection with molds and yeasts, 
are made up of a substance the nature of which is still unknown, and 
are found in nearly all fungi, in most algae and bacteria, and in many 
protozoa. 

Glycogen and paraglycogen are equally well distributed in micro- 
organisms (fungi, protozoa). Among algae, glycogen is found only 
in the Cyanophycea, but it is elsewhere replaced by starch or para- 
mylum (Fig. 10), common products of chlorophyllic assimilation. 

There are also the protein substances, such as crystalloids of 
mucorin scattered in the Mucorina, or the globules of fat common 
in all cells (Fig. 12, B). 

Most of these substances seem to result from the activity of the 
chondrium structure. Recent investigation shows that the meta- 
chromatic corpuscles have their rise among the mitochondria. It 



ELEMENTS OF MICROBIAL CYTOLOGY 23 

has long been known, on the other hand, that the starch and paramylum 
are always formed in the chloroplastids. 

MEMBRANE. The cell is usually enveloped in a more or less heavy 
membrane, secreted by the cytoplasm, which acts as a protective 
organ for the cell. 

The presence of the membrane is not, however, indispensable; 
many protozoa do not have it, and are consequently naked cells. 
Motility in many microorganisms is closely associated with the mem- 
brane, for the movement of cytoplasm and the flexibility of the mem- 









' 

vy 

. 12. Metachromatic corpuscles (cm} in Sarcosporidia, Sarcocyslis lenella. 
fter Erdmann.} Fat globules (g) in Trypanosoma rotatorium. (After Doflein.) 

ie are essential factors. Cells as a rule have a membrane of 
ifferent degrees of thickness and composition. It may be albuminoid 
or chitinous (Infusoria), or it may be made up of carbohydrates, as 
cellulose, pectose, and callose (algae, fungi). Bacteria always have a 
membrane, but its nature has not yet been definitely determined. 
Often the cell membrane is able to thicken noticeably, and thus protect 
the cell from environing influences; the cell may then be regarded as 
transformed into a cyst which passes into a state of sluggish existence. 
Encystment is frequent with protozoa, and is produced when the 
environment becomes unfavorable (Fig. 13, A). 

The external layer of the membrane frequently undergoes modi- 
fications, transforming itself into a mucilaginous or gelatinous sub- 



24 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

stance as we see in many Cyanophycea, in bacteria surrounded by 
capsules, and in zooglea. The membrane then becomes extremely 
thick (Fig. 13, J5). 

LOCOMOTIVE STRUCTURE. Most algae and fungi cannot move. 
Many bacteria and all protozoa have more or less perfected locomotive 
structure. 

The Cyanophycea and many bacteria, although without loco- 
motive organs, present nevertheless oscillatory movements which seem 
due to a general movement of the cytoplasm translated exteriorly 
because of the flexibility of their membrane. With these exceptions, 
movement is effected by means of a locomotive structure. 

This structure is found in its simplest 
form in the pseudopodia of the amoeba. 
The naked cell of the amoeba pushes out 
pseudopods, simple expansions of the ecto- 
plasm arising at any part of the body, 
which take various shapes, and reenter the 
body without leaving the least trace of theii 
existence. It is a result of motility of the 
cytoplasm, one of its essential properties, 
shown here exteriorly because of the absence 
: \A.ffi> f a cellular membrane. 
geard.) B, Thread of nostoc The locomotive structure is more com- 
surrounded by a thick muci- -, . , , , T j j 

laginous case. P* ex m ther protozoa; the pseudopod 

is replaced by contractile appendages 
flagella, or mbratile cilia. 

The flagellum is a contractile appendage of definite shape and 
position which draws the body after it by means of waving movements. 
It is found on bacteria and flagellates. 

The organ of locomotion of bacteria is still little known (Fig. 14). 
It consists of a certain number of contractile appendages placed at 
one end of the cell, or at both, or sometimes distributed over the whole 
body. These appendages, called vibrating appendages, have many of 
the characteristics of flagella. Their existence, for a long time doubted, 
is now pretty well established. 

The locomotive structure of the Flagellata is much better known. 
It is characterized by one or more flagella inserted in the anterior 
extremity of the cell. In case of more, one frequently folds back 





ELEMENTS OF MICROBIAL CYTOLOGY 25 

toward the posterior end. In the lateral region of the cell it unites 
with a contractile membrane, the undulating membrane, running in 
spiral form along the length of the bady, of which it is the free end. 
Flagella are made up of one or more elastic fibers, surrounded by a 
thin cytoplasmic sheath. 

The vibrating cilia are also contractile appendages, differing from 
the flagella only in their smaller size. They cover the whole body 
of the cell, as in the case of infusoria, enabling them to move about 
very easily in liquids. 

Certain facts lead us to believe that flagella are only transformed 
pseudopods in which the cytoplasmic structure has changed and at the 
same time the kind of movement. Thread- 
like pseudopods are found with a rapid 
rhythmic movement which may serve as 
intermediate forms. Be that as it may, the 
method of forming these organs is of special 
interest. Apparently they are formed under 

the influence and at the expense of the cen- FIG. 14. Organs of loco- 
trinlp. motion in bacteria. A, B. 

subtilis. (AJter Fischer) 
In the Flagellata the flagellum is always B, Microspira comma. 

inserted in the centriole or in a similar organ ^fter Fischer and Migula.) 

C, Spirillum rubrum. 
which appears to issue from the centriole. 

It is not rare to find in cellular division some cells in which the nucleus 
is dividing with a centriole at each of its poles. Each serves as a point 
of insertion for a flagellum (Fig. 15, A, D, E). 

According to recent works, the flagellum is formed hi general 
one of two somewhat different methods. 

In the first case, the centriole divides itself by an elongation, followed 
by a contraction into two centrioles which remain united to each 
other by means of a fine thread, the centrodesmose. The centrodesmose 
then elongates, dragged by the centrioles, as far as possible from the 
nucleus, and transforms itself into a flagellum. 

In the second case, the centriole divides itself a first time just as 
in the preceding case, but the centriole farthest from the nucleus im- 
mediately undergoes a second division, thus making three centrioles. 
The one nearest the nucleus remains a centriole during nuclear division. 
The centriole situated somewhat farther from the nucleus becomes the 
point of insertion for the flagellum, and is called the blepharoplastoi basal 



26 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



grain. The centriole is united to the blepharoplast by a centrodesmose, 
the rhizoplast, which is often absorbed. Finally, the last centriole 
situated beyond the blepharoplast about equally distant, also unites 
with this cell-organ by a centrodesmose and, by approaching the 
extremity of the cell, causes the elongation of the centrodesmose which 
transforms itself into a flagellum. 

In the infusoria the vibratile cilia insert themselves in the ectoplasm 
and pass through the cuticle to reach the exterior. At the point of 




fffa 



tr ' 



end 



FIG. 15. FIG. 16. 

FIG. 15. A, Spongomonas u-oella. The nucleus is undergoing mitotic division. 
Two centrioles, each at the base of a flagellum, are located at the two extremes of 
the spindle. (After Hartmann and Chagas.) 

B, Monas termo. The cell lies in repose; a centriole (a) lies at the base of the 
flagellum; in (C) there are two centrioles, in (D) the two centrioles occupy the two 
poles of the nucleus during the process of mitosis; in (E) exists the final nuclear 
division. (After Martin.) 

FIG. 1 6. Fragments of the peripheral portion of Prorodon teres (infusorian) 
with vibratile cilia and their basal corpuscles, (ect) Ectoplasm; (end) endoplasm; 
O) trichocysts. (After Maier and Gurwitch.} 



insertion of each of these cilia is a small chromatic corpuscle or basal 
grain, a trichocyst, also supposed to arise from a repeated division of 
the centriole (Fig. 16). 

The centriole which, as we shall see later, seems to be a motor 
organ directing the internal cytoplasmic movements during cellular 
division, appears also to be a motor organ of the external movement 
of the cell. 



ELEMENTS OF MICROBIAL CYTOLOGY 



2 7 



REPRODUCTION OF THE CELL 

VARIOUS PROCESSES OF REPRODUCTION. Reproduction of microbes 
is affected by various processes; the cell may reproduce itself by trans- 
verse or longitudinal fission, binary division, schizogony (bacteria, 
flagellata, molds, Figs. 4, A; 17; 19, A}. This is by far the most fre- 
quent. It sometimes, however, divides itself by budding, gemmula- 
lion (Yeast, Fig. 2); that is, by the formation of a small protuberance 
which separates itself from the mother cell as a small daughter cell 
which, once free, grows slowly to maturity. 

Finally, a last process and a very frequent one is the formation of 
internal spores, or sporogony (Fig. 18). The nucleus undergoes a 




"" 








FIG. 17. Schizogony in Amoeba 
poly podia, with amitotic division 
of the nucleus. (After Sc/mlzr 
and Lange.) 



FH;. 18. Sporogony. A, Formation 
of spores in Saccharomyces cerevisia. B, 
Formation of spores in B. mycoldes. (After 
(inillicrniond.) C. Formation of spores in 

Lcucorytozoon lovati. (After Fantham.} 



certain number of divisions, and the cytoplasm divides itself inside the 
cell in as many small cells as there are nuclei. These cells become 
spores and are set free by a rupture in the wall of the mother cell. 
Sometimes all the cytoplasm of the mother cell divides into spores, and 
sometimes only a part of the cytoplasm is used, the rest epiplasm 
serving as nourishment to the spores during their growth. 

Whatever the means by which the cell reproduces itself, cyto- 
plasmic changes and nuclear changes take place at the same time. 
The most important of the cytoplasmic changes is the distribution 
of the chondrium structure between two daughter cells, often preced- 
ing the division of this cytoplasmic structure (Fig. 8). 



28 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

The nuclear phenomena are much more important, and better 
known. The nucleus divides in order to furnish each daughter cell 
with a nucleus containing the same amount of chromatin. 

NUCLEAR DIVISION. Nuclear division may occur in one of two 
ways, one very complex, (i) the indirect mode, karyokinesis or mitosis; 
the other very simple, (2) the direct mode, or amitosis. 

Indirect Division, Karyokinesis, or Mitosis. We shall begin with 
the indirect mode which is by far the more common, using as an example 
a Heliozoon, the Acanthocystis aculeata (Fig. 19, A). The nucleus of 
this protozoon at rest contains a large karyosome of a spongy structure, 
and a chromatic network. Outside the karyosome in the nuclear 
vesicle is a centriole surrounded by a hyaline zone, the archoplasm 
(Fig. 19, ,4, a). 

Mitosis may be divided into four steps or phases. 

The first phase or prophase begins by the emigration of the centriole 
from the nucleus outside of which it surrounds itself by cytoplasmic 
irradiations, making a star-like body, called the aster (Fig. 19, A, b). 
Following this, the karyosome dissolves in the nucleoplasm, supposedly 
conveying material to the chromatic network which enriches itself 
noticeably in chromatin. The chromatic network then relaxes, thickens 
and transforms itself into a more or less spiral cluster, the spireme 
(Fig. 19, A , c). At the same time the centriole divides into two centrioles, 
each surrounded by an aster (Fig. 19, A, c). Soon these centrioles place 
themselves at the two opposite poles of the nucleus (Fig. 19, A, d), while 
the spireme breaks itself up into a definite number of chromatic sec- 
tions, the chromosomes. While this is taking place, the nuclear mem- 
brane dissolves itself into a series of cytoplasmic fibrils, the achromatic 
spindle, resistant to nuclear stains. They appear in the middle of 
the nucleus and converge at each end to the centrioles (Fig. 19, A, d, 
c). The chromosomes group themselves in the center of the spindle 
as the equatorial plate (Fig. 19, A, e), the formation of which completes 
the prophase. Each of the chromosomes is attached to one of the 
fibrils which make up the achromatic spindle. 

The second phase or metaphase consists of the longitudinal di- 
vision of the chromosomes each of which divides itself into two equal 
chromosomes. 

In the third phase or anaphase the chromosomes equally divided 



ELEMENTS OF MICROBIAL CYTOLOGY 



move to the two poles where they make two polar plates. The cen- 

trioles located here seem to have some attraction for the chromosomes. 

Finally comes the telophase or phase of reconstitution of the two 

nuclei which terminates the process. In this phase, the chromosomes 







j 






V 



A 

5 



FIG. 19. Karyokinesis (metamitosis). A, Acanthocystis aculeata; (a) nucleus 
in state of repose with an intranuclear centriole; (b) (prophase') the centriole moves 
to the periphery and out of the nucleus and forms an aster (After Hertwig); (c) the 
division of the centriole and spireme; (d) the formation of the equatorial plates and 
the achromatic spindle; (e) equatorial plates; (/) anaphase; (g) telophase. (After 
Schaudinn.) B, In Coleosporium senccionis (Uredineae). (a) Nucleus at rest with 
its centriole extranuclear; (b) formation of chromosomes; (c) equatorial plate; (d) 
metaphase; (e) anaphase; (/) (g) (i) telophase. (After Madame Moreau.) 

form a spiral chromatic cluster making a spireme at each of the poles 
(dispireme stage, Fig. 19,^4,^); each of the spiremes is then surrounded 



30 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

by a nuclear membrane in which is included the centriole. Thus the 
two nuclei are formed in which a nucleolus soon appears. Mean- 
while the cell has elongated, become constricted in the center, and 
finally broken into two cells (Fig. 19, B, f, g, i). The achromatic 
spindle completely disappears. 

This method of division represents the typical method of karyo- 
kinesis, that which is observed in higher organisms with the single 
difference that the centriole is intranuclear, whereas in the cells of 
higher organisms it is ordinarily outside the nucleus in contact with the 
nuclear membrane. An analogous mitosis is found in the Uredinece 
(Fig. 19, B, a, i), except that the centriole is here found to be extra- 
nuclear (Fig. 19, B, a), the asters are lacking, and the nucleolus persists 
to the end of mitosis expelled in the cytoplasm. The physiological 
significance of the nucleolus in this case is not known. This method of 
division is seen in certain molds and higher protozoa, and is called 
metamitosis or perfect mitosis. 

Summing up, mitosis is a process functioning to make an absolutely 
equal division of the chromatin between the two nuclei. This dis- 
tribution is performed by the breaking up of a spireme into a definite 
number of chromosomes, a number varying according to the species 
but always constant for any single species, and then by a longitudinal 
division of the latter. The centrioles seem to play an important role 
in this phenomenon, in directing it, and in attracting the chromosomes 
once divided toward the poles of the cell where the nuclei are formed. 

It is not necessary to conclude that the processes of mitosis are 
as complex as in other microorganisms. Relatively simple in the 
lower forms, mitosis becomes complicated as it climbs the ladder, 
gaining the characteristics of metamitosis only in the most advanced 
forms. 

The simplest case is found in the Cyanophycea (Fig. 5). Here 
cellular division begins by the outline of the transverse partition 
which appears in the form of a peripheral ring. At the same time 
the chromatic network takes a definite arrangement; its filaments 
arrange themselves parallel to the longitudinal axis of the cell, thus 
giving this division the appearance of a mitotic division. The outline 
of the partition extends little by little toward the middle of the cell, 
leaving open only a small spherical space in its center to which the 
fibers of the network then contract, and the nucleus takes the form of 



ELEMENTS OF MICROBIAL CYTOLOGY 














FIG. 20. Protomitosis 



(a) Nucleus at rest; 



A, In Amoeba mucicola. 

beginning of prophase; (c) division karyosome; (d) division of centriole; (e) (f) 
equatorial plate; (g) metaphase; (i) (k) telophase. (After Chatton.) B, In Amceba 
froschi. (After Nagler.) C, In Euglena splendens. (After Dangeard.) D t In 
Amoeba diplomitotica. (After Beaurepaire Arago.) 



32 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

a dumb-bell. Soon the partition stops completely, the filaments of 
the contracted part of the nucleus break up and the two daughter cells 
appear separated by a partition. The two nuclei whose filaments have 
been sectioned by the partition are not slow in recovering their in- 
tegrity (Fig. 5, ft). 

We find in the Amoeba mucicola (Fig. 20, A) a much more char- 
acteristic mitosis, though more primitive. The nucleus of this amoeba 
when at rest is made up of a nuclear fluid surrounded by a membrane 
in which are a large karyosome and some small grains of chromatin 
localized on the periphery (Fig. 20, A). In the center of the karyosome 
is a small chromophilic centriole. The prophase begins by the elonga- 
tion of the karyosome to a rod-shaped body (Fig. 20, A , b) which then 
transforms itself into a dumb-bell (Fig. 20, A, c). The centriole 
also elongates and becomes constricted in the center (Fig. 20, A, d). 
At the same time an achromatic spindle appears all about the con- 
stricted region of the karyosome in the middle of which the grains of 
chromatin group arrange themselves peripherally to form an equatorial 
plate, but there is no differentiation of this chromatin into two 
chromosomes (Fig. 20, A, c, d). In the metaphase the karyosome 
and the centriole divide into two polar masses (Fig. 20, A, e, /), the 
equatorial plate separates into two plates which, in the anaphase, 
emigrate to the poles (Fig. 20, A, g) drawn by the centrioles. In the 
telophase the spindle elongates, disappears, and the two nuclei are 
formed at the poles (Fig. 20, A,4 t k). The nuclear membrane exists 
during the entire phenomenon. 

In other microorganisms (Amceba, Flagellata, Euglence) is found a 
similar mitosis except that the chromatin distributed in the resting 
nucleus as a network or as rod-shaped bodies forms an equatorial plate 
made up of true chromosomes (Fig. 20, B, C). 

Another form of mitosis, promitosis, is characterized by the fact 
that the centriole is included in the karyosome, by the persistence of 
the nuclear membrane, and by the simultaneous division in the meta- 
phase of the karyosome and of the chromatin gathered in an equatorial 
plate. 

Between promitosis and metamitosis are a series of intermediate 
forms. In the Pelomyxa palustris, for example, the centriole while 
remaining intranuclear is able to separate itself from the karyosome 
(Fig. 21, A, a). The prophase here begins with the usual division of 



ELEMENTS OF MICROBIAL CYTOLOGY 



33 



the centriole (Fig. 21, A,b t c), and the two resulting centriole- threads 
pass to the extremities of the achromatic spindle, while the karyosome 
cooperates in the formation of the chromosomes (Fig. 21, A, d, e). 

In other cases (various fungi, Gregarina, etc.), the centriole be- 
comes extranuclear, and the karyosome acts as a true nucleolus (Fig. 



, a 
c d 














c 

. - - - 



FIG. 21. Mesomitosis. A, In Pelomyxa palustris, (a) Nucleus at rest; (b) 
(e) division of centriole; (/) (g) equatorial plate; (k) anaphase. (After Bolt.} 
B, In Urospora lagidis (Gregarina). (a) Nucleus with extranuclear centriole and 
aster; (b) the centriole is divided and the spireme is formed; (c) spireme; (d) equa- 
torial plate; (e) anaphase. (After Brasil.) C, In the ascus of Galactima succosa 
(Ascomycete). (a) Equatorial plate; (V) anaphase; (c) telophase. 

21). Sometimes it dissolves at the beginning of mitosis, seeming to 
aid the development of the chromatin of the spireme, and sometimes 
it persists during the entire process and is expelled in the cytoplasm 
at the end of the phenomenon without any known function. The 



34 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

name of mesomitosis has been given to all the mitoses which distinguish 
themselves from promitosis by the persistence of a nuclear membrane 
throughout the phenomenon. 

Direct Division or Amitosis. This consists simply of an elongation 
of the nucleus followed by a median constriction, then by a rupture of 
this constricted part without an equal division of the chromatin be- 
tween the two nuclei which often are not the same size. It is a simple 
breaking up of the nucleus. Amitosis, then, does not necessarily in- 
sure the equal distribution of chromatin between the two nuclei. 
This rare process is found in higher organisms only in old cells that are 
degenerating, or in diseased cells. Although for a long time it was 
thought to be a primitive phenomenon, it is now considered to be 
degenerative. We see, however, in certain Amceb(E and Mycetozoa 
the karyosome enclosing all the chromatin divides itself into two equal 





FIG. 22. Conjugation in Schizosaccharomyces octosporus. (a) Two gametes in 
the process of fusion; (b) (c) nuclear fusion. 

bodies, showing the characteristics of a very primitive mitosis (Fig. 17). 
Amitosis seems to exist normally in yeasts and in certain molds. In 
the yeasts, for example, the nucleus divides by amitosis in the course 
of budding (Fig. 2), and mitosis is found only in the course of sporu- 
lation. 

SEXUAL CHANGES. In most microorganisms at certain times during 
their existence occur sexual changes, or fertilization, which seem to give 
them a new strength. It is followed by a period of very active re- 
production, whence the name of sexual reproduction given to these 
changes. This consists essentially in the fusion of two equal 
isogamous (is o gamy} or unequal, anisogamous (heterogamy] cells or 
gametes. In the latter case, the male is small and active, and the 
female large and passive. The fusion between the two cytoplasms 
and the two nuclei takes place at the same time (Fig. 22). 

If nuclear fusion were not compensated by an elimination of 
chromatin, the nucleus would increase in this substance at each fertili- 
zation. But this change is succeeded immediately in protozoa by a 



ELEMENTS OF MICROBIAL CYTOLOGY 35 

common process called chromatic reduction. The chromosomes 
in the course of the divisions which precede the formation of the 
gametes reduce themselves to half by a complex process which it 
would be superfluous to describe here. The same chromatic reduction 
takes place in the fungi and algae, but this does not always precede 
fertilization. It may follow it immediately as in the yeasts where it 
seems to produce itself during the nuclear divisions in the ascus. It 
may also occur during other stages of development. 



CHAPTER II* 

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 hyphen, singular, hypha). These hypha 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 (intercalary 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. 

* Prepared by Charles Thorn. A. Guilliermond has furnished the section on " Cytology of 
Molds." 

36 



MOLDS 37 

The formation of differentiated reproductive cells is, in general, 
characteristic of the fungi. The method of reproduction presents great 
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 reproductive 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 unfavorable 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 chlamydo- 
spores 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 of balls of thick-walled cells filled with concentrated food 
materials. These sclerotia are frequently distinctive of the species 
producing them by size and appearance. They sometimes resemble 
the sexual fruiting masses. Resting structures of either type, es- 
pecially 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 mush- 
room of commerce which is asexually produced so far as we know, yet 
represents the most perfect and most constant fruiting form produced 



38 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

by a very large group. Between the typical forms are many gradations 
resulting in many families whose relationship to one or the other 
group is difficult to determine. Probably the ancestral history 1 
(phylogeny) of the fungi, if known, would show several or many lines') 
of descent rather than one. Certain of these groups may be presented s; 
briefly. 

BACTERIA. In the scheme of plant grouping presented (page 77), 
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 asj 
Schizophyta or fission-plants because of reproduction only by the 
division of the cells. 

PHYCOMYCETES. The Phycomycetes 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 s 
are met with zygospore formation and oospore formation. The 
first, found in the Zygomycetes represented by the common mucors, con- 
sists 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 meaning yoked (Fig. 32). 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 de- 
structive 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 as'cus, 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 when sexuality is 
known is developed subsequent to fertilization, not directly from an 
egg cell. The group presents a great variety of fruiting masses pro- 
duced in connection with the asci. The simplest forms are loose webs 
of hyphae 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. 



MOLDS 39 

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 abund- 
ance. Such perithecia consist of the ascogenous 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 ad- 
jacent to the fruiting ones. 

BASIDIOMYCETES. In the Basidiomycetes there is still further reduc- 
tion 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 reduced to a fusion of the 
nuclei in certain binucleate cells. The typical structure is the basid- 
ium, a spore-bearing cell characteristically 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 hyphse 
to dense columnar areas covering the gills of the mushrooms or lining 
the cavities of the puffballs. Very few of these species occur in bac- 
teriological 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 im- 
"fect fungi and their grouping regarded only as temporary, a con- 

ience for the identification of materials. These include many forms 

economic importance, 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- 




40 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

ever, in their adaptation to environment. Many of them are not only 
parasitic but so closely adapted to 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- 
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. 

CYTOLOGY OF MOLDS* 

GENERAL STRUCTURE OF MOLDS. Three kinds of cell-structure 
formation are found in molds : 

1. Some, belonging to the Phycomycetes, show no cross- walls; they 
have a much branched, felted mycelium, but in the early stages there 
are no true transverse septa. Septa appear in many forms only when 
fruiting begins, but in the opinion of some they merely separate the 
living portions of the mycelium from those in which the cytoplasm is 
dead or degenerating. The cytoplasm in the unseptate mycelium forms 
one continuous mass; it contains a great many nuclei (Fig. 23, i and 
2). Each nucleus with the cytoplasm surrounding it, according to 
Sachs, may be considered a physiological unit acting in a somewhat 
similar capacity as a cell, or 'may be designated as an energid. This 
view is not held by all observers, however. Considered thus, the 
mycelium represents the collection of a great many indistincts cell 
which are not separated by walls. The Mucorinea, for example, belong 
to this structural type. 

2. Other fungi, especially among the Ascomycetes, have a septate 
mycelium, but one in which the transverse septa do not restrict cellular 
functions as true cells. It consists of compartments containing a 
variable number of nuclei called coenocytes (Fig. 24, i). Each compart- 
ment may be considered, not as a true cell, but as a colony of rudi- 
mentary cells, energids. 

3. Still other molds have a mycelium consisting of true cells with 
a single nucleus, as for example Endomyces fibuliger (Fig. 24, 3 and 4) 
and Endomyces decipiens. 

* Prepared by A. GuHliermond. 



MOLDS 



There are, moreover, molds which show both these last two struc- 
tural types, with transitional forms between the two. For instance, 
in Endomyces magnusii, the mycelium, ordinarily consisting of areas, 

each containing many energids, 
^>.. can in some parts progress to a 

../ uninuclear cellular structure. 

^ : y; : The conidia or spores of many 

"^.~ ^. .->.-.; molds may have either one or 

l^ : ' : ..-A ' : many nuclei, according to the 





FIG. 23. FIG. 24. 

FIG. 23. i, Part of the mycelium of Thamnidium elegans (Mucor). 2, Ex- 
tremity of a filament of Mucor circinelloides showing three swellings about to form 
sporangia. 3, A spore of the same mold. 4, Yeast forms from the same mold. 
(After Leger.} 

FIG. 24. i, Mycelial filament of Endomyces magnusii. 2, Extremity of a 
filament of the same mold in the process of growth, with a dividing nucleus. 3 and 
4, Filaments of Endomyces fibuliger. In 4, metachromatic corpuscles are seen 
in the vacuoles. 5, Filament on the way to increase, from the same mold, the 
nucleus dividing. 



species. The spores of the Mucorinea for example always have many 
nuclei (Fig. 23, 3); on the contrary, the ascospores of the Ascomycetes, 
the conidia of Penicillium and Aspergillus, contain generally but a 
single nucleus. 

The yeast forms which result from the budding of the mycelium in 
some molds, most frequently have a single nucleus (Fig. 23, 4); how- 
ever, in some, Dematium, are sometimes found yeast-forms containing 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



several nuclei. The yeast-forms of the Mucorinea, which are not other- 
wise very typical forms, are always multinuclear. 

To whichever of these three structural forms a mold belongs, it 
always represents some similar constitutional elements which we will 
now consider. 

CYTOPLASM. The cytoplasm is a semi-fluid mass, somewhat dense, j 

sometimes homogeneous and con- 
taining a more or less considerable 
number of vacuoles. Certain methods 
of fixing and staining have recently 
made possible a demonstration, in the 
cytoplasm of the most diverse molds, 
of the presence of a chondrium, very 
clear and always splendidly exhibited. 
This consists mostly of fine rod- 
mitochondria, very long and flexible, 
generally lying parallel with the 
longitudinal axis of the cell (Fig. 25). 
Sometimes also it contains granular 
mitochondria. 

The cytoplasm also has reserve 
products, of which we shall speak 
later. 

NUCLEI. The nuclei show a differ- 
entiated structure which is sometimes 
difficult to demonstrate. They con- 
sist of a nuclear membrane, a hyaline 
nucleoplasm, a large nucleolus and a 
chromatic network. The last is some- 
times indistinct, and it frequently 
happens that the nucleus appears to 
contain only a nucleolus; but a very 
careful examination always reveals 
the network (Fig. 24, 3 and 4). 

The division of the nucleus is not always easy to observe. To study 
it, one must examine the growing tips of the mycelium. In some cases 
this consists in an elongation of the nucleus which soon assumes the 
form "of a very slender dumb-bell which breaks apart at the narrow 




FIG. 25. Various molds fixed 
and stained by a special technic, 
showing their chondrium. i, Fila- 
ment of Rhizopus nigricans (Mucor). 
2-4, Filaments of Penicillium glau- 
cum. 5 and 6, Fragments of the 
conidial organ of the same mold. 
7, Filament of Endomyces magmtsii. 
8 and 9, Oidia of the same mold. 
In all these molds, chondrium is 
represented by long filaments, or 
sometimes by small grains. The 
filaments often show small vesicles 
at their crossing. 



MOLDS 



43 



portion. This is the extent of an amitotic or direct division (Fig. 24, 
2 and 5). 

Karyokinesis is usually seen only in the organs of fructification 
(asci, basidia, etc.) ; nevertheless, in the mycelium of the Basidiomycetes 
and MticorinecB, true metamitoses have been found. In the Mucorinea 
for example (Fig. 26), the nucleus loses its membrane (1-4) and gives 
rise to a spindle ending in a centrosome at either extremity, while two 
chromosomes form the equatorial plate at the center (5). Each of 




6 



FIG. 26. Nucleus of the Mucor (1-4), and various stages of its division (5-8). 

(After Moreau.) 

the two chromosomes divides and the four resulting chromosomes are 
distributed between the two poles (6-8) where they form the two 
daughter nuclei (Moreau). 

METACHROMATIC CORPUSCLES AND RESERVE PRODUCTS. The 
:uoles always contain a great many shining granules, showing 
nvnian motion and capable of being stained in the living state by 
jutral red and methylene blue. These bodies have staining qualities 
tich permit them to be easily characterized. They are stained a 
)let-red by most f the basic dyes, aniline blue or violet. They also 
e on a very pronounced reddish tinge with hematoxylin (Fig. 27). 
reason of this property of metachromatism, they have been called 
^achromatic cor pushes. These bodies, which are very common in the 
rotista, have been found in yeasts, bacteria, algae and protozoa. The 
;mical nature of the substance constituting them is still unknown, 
it the name metachromatin is often used for it. 1 Some authors, among 

Because of the priority and more exact signification, the names melachromatic corpuscles 
melachromatin are preferable to the terms grains of volutin and volulin given by Arthur 
Meyer. 



44 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



whom is Arthur Meyer, believe them to consist of a combination of 
nucleic acid, but this is a mere supposition. 

On the other hand, the r61e of the metachromatic corpuscles is now 
well known. It is evident that they are reserve substances. Their 
evolution proves it. Thus metachromatic corpuscles appear in great 
abundance in the young asci of the higher Ascomycetes (Fig. 28, i and 
2), then accumulate in the cytoplasm of epiplasm 
which is not utilized in the formation of the 
ascospores, gather all around the ascospores at 
the time of their forming (3-4), and are gradu- 
ally absorbed by the latter in the course of 
their development (5). They therefore furnish 





FIG. 27. 



FIG. 28. 



FIG. 27. Dematium species Stained by a method permitting the differentiation 
of the metachromatic corpuscles, i, Filament. 7 and 9, Yeast forms. 9, Yeast 
form starting to bud from mycelium. The metachromatic corpuscles are situated 
in the vacuoles in the form of small grains joined in chains (6) or isolated. Many 
appeer like large granules (9). n. Nucleus, v.c. Vacuole with metachromatic 
corpuscles. 

. FIG. 28. Various stages of the development of the ascus in Aleuria cerea. 
i and 2, Young asci with their nucleus and many metachromatic corpuscles. 3, 
Fragments of an ascus after the second nuclear division. 4, Ascus, still young, 
in which the ascospores are surrounded by metachromatic corpuscles. 5, Older 
ascus in which most of the metachromatic corpuscles have been absorbed by the 
ascospores. 

nourishment for the ascospores and from this standpoint behave 
exactly like glycogen and the globules of fat which are usually coexistent 
with them in the cytoplasm. We shall see, moreover, that they 
undergo a similar evolution in the asci of yeasts. Likewise in the coni- 
diophores of molds, notably in the fruiting heads of Aspergillus and 






MOLDS 



45 





Penicillium, the metachromatic corpuscles are produced in great 
abundance (Fig. 29, 26 and 30), then gradually disappear as the conidia 
from (29, 3). Here again they serve as food for the conidia. 

Metachromatic corpuscles appear not only in the vacuoles, but also 
in the perivacuolar cytoplasm. There they spring up, to diffuse finally 
in the vacuole where they increase. It is difficult to observe their 
manner of forming in the mycelial filaments, but in the preparation for 
sporulation some molds (asci of the 
higher Ascomycetes), it has recently 
been demonstrated that they start 
in the midst of the elements of the 
chondrium, which act as plastids 
similar to the plastids of the higher 
plants. They start in the interior 
of the granular-mitochondria or in 
the rod-mitochondria (Fig. 30). In 
the former case, a small corpuscle 
appears in the midst of a mitochon- 
dria, then develops gradually, while 
the mitochondrial membrane which 
envelops it grows thinner; is re- 
duced to a small capping of the 
grain on one side; then disappears 
when the latter reaches maturity. 

It is noteworthy that the corpuscles emigrate with their plastid to the 
interior of the vacuoles during their development. 

When the corpuscles start in a rod-mitochondrium, at the junction 
of these rod-mitochondria several small corpuscles are seen to form, then 
. the parts of the rod-mitochondrium which join are absorbed and the 
corpuscles, enclosed in their mitochondrial membrane, once separated, 
dergo the same evolution as above. 

Thus the metachromatic corpuscles, like grains of starch in the 

.er plants, start in the midst of the mitcohondria and develop 
gradually out of their mitochondrial matrix, and with the aid of the 
vacuolar substance. 

In molds are found still other reserve products. One often sees 
globules of fat in the cytoplasm, which are easily stained a black- 
brown by osmic acid; and glycogen which can be differentiated by iodine 



IfiS 



FIG. 29. Conidial organ of Asper- 
gillns niger with metachromatic 
corpuscles. 




46 MORPHOLOGY AND CULTURE OP MICROORGANISMS 

in iodide. of potassium. The glycogen is contained in either the 
cytoplasm or the vacuoles. It is generally very abundant. 

These products (fat and glycogen) undergo the same evolution as 
the metachromatic corpuscles, and they also accumulate in the organs 
of fructification (asci, conidial organs) to serve in the nourishment of 
spores and conidia. 




FIG. 30. Formation of metachromatic corpuscles in a cell of the perithecium of 
Pestularia vesiculosa. The rod-mitochondria form on their crossings vesicles (c) 
consisting of a metachromatic corpuscle unstained by the special method which 
served to differentiate the chondrium. Some corpuscles (a), more highly developed, 
are found in the vacuoles. still surrounded by their mitochondrial shell; others (c) 
at the completion of their development have worn through their mitochondrial 
covering. 

CELL- WALL. The cell-wall of molds is quite distinct and often 
thick. It is sometimes cutinized. According to Mangin, it consists 
of callose and pectose with which is often associated a kind of cellulose. 

SPECIFIC CONSIDERATION OF MOLDS* 

A few species are found to grow very constantly in the same situa- 
tions as bacteria. These are associated with forms of decay, fermenta- 
tion, 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 

* Prepared by Charles Thorn. 



MOLDS 47 

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 
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, derma- 
tomycoses, of both man and animals. 



THE 



GENERIC CONSIDERATION OF GROUPS* 



MUCORS OR BLACK MOLDS. The mucors or black molds con- 
stitute a large group of species belonging to the Phycomycetes or algal 

*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. 



48 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

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. 31 and 32). In the mucors, the mycelium is usually 
richly developed within and often also on the surface of the substratum; 
asexual reproduction 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 zygo- 
spores (Figs. 31 and 32). The typical mucors produce sporangia as 
capsule-like dilations at the ends of erect fertile hyphae, each con- 
taining many spores. Septa are commonly developed in the mycelium 
when sporangia begin to appear. These fertile hyphae may be micro- 
scopic 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 cosmo- 
politan species associated with the decay of many kinds of food stored 
in wet condition or in humid situations. Typical clusters of spor- 
angiophores are borne on stolons or runners, which are hyphae extending 
radially from the center of the colony and fastened to the substratum 
or to the support at intervals by root-like outgrowths. Abundant 
growth of this species is found only under 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 
becoming 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 



49 



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. 




IG. 31. Mucorinece. Mucor. From Tabula Botanicce, showing sporangia 
originating 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.) 

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 
javanicus. The fermenting power of mucors like that of yeasts varies 
greatly with the species or even with races used, approaching in some 
species the efficiency of the more active yeasts. 



50 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

THAMNIDIUM. Of related genera, Thamnidium differs from Mucor 
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 
are smaller, lack the columella, and produce few to several spores 
within an outer wall. 




FIG. 32. Mucorinecs. Mucor, Rhizopus. A, B, C, D, Formation of the zygo- 
spores from conjugating branches; E, section of D; F, mature zygospores in section; 
G, germination of zygospores; H, diagram of fruiting stolons oiRhizc 



us ngrcans; 

K, section of sporangium during spore formation, highly magnified (From Tabulae 
Botanica.) (Reduced one-half.) (By permission of A. F. Blaskeslee.) 

Thamnidium elegans, Link, produces primary and secondary spor- 
angia on different hyphae, 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 hyphse, 
branched, septate, colorless, or bright colored. The fertile hyphse 



MOLDS 



Lonidiophores) are mostly erect, arising either from submerged hyphse, 
r as branches of aerial hyphse, septate, usually branched only in the 
ruiting portion. 



Conidial fructifications consist of more or less com- 





;?;f?f In f //>// 



FIG. 33. Penicillium cxpansnm, Link, a, b, f, Branching and arrangement of 
branches of conidial fructification (Xgoo); c, d, e, conidiiferous cells and conidial 
chains (Xgoo); g, h, j, k, /, sketches of fructifications (Xi4o)',m, n, o, germination 
of conidia ( X 900) ; r, s, sketches from photographs showing in 5 loose aggregations 
of conidiphores beginning to develop into zonately arranged coremia, in r a 
coremium i mm. in height. (From Bid. 118, Bureau of Animal Industry, U. S. 
Dtpt. Agriculture.) 

plex systems of branches and branchlets, the ultimate fertile cells each 
producing a chain of conidia (Fig. 33). The whole system is usually 
grouped near the end of the conidiophore, giving the appearance of 



52 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

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 indefinite number of generations, there- 
fore 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 o: 
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 inhibitec 
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 o: 
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 
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 



MOLDS 53 

ipores which has been used physiologically to detect the presence of 
irsenic by its ability to set free arsine from such substrata. Except 
species associated with particular processes or substrata, the identifica- 
ion of the green species of Penicillium requires special methods and 
greater care than is possible aside from special study of the group. 

ASPERGTLLUS (AND SxERiGMATOCYSTis). The genus Aspergillus in- 
ludes numerous species which develop under widely different condi- 
ions. Many of these forms reach their typical development under 
xier conditions than Penicillium and Mucor, such as stored grain, her- 
Darium specimens, dried flesh, or foods containing concentrated sugars, 
uch as jams, jellies, etc. Some excite processes of fermentation, and 
few are associated with diseases. 

Characters. The vegetative hyphae are creeping, submerged in the 
ubstratum or sometimes aerial also, loose, floccose, branched, septate, 
isually colorless, and sometimes bright colored. Conidiophores or 
ertile hyphae are erect, unseptate, or few-septate, usually much larger 
n diameter than the vegetative hyphae, and gradually enlarged upward, 
nding in a more or less abrupt dilation or head which bears closely 
sacked columnar sterigmata or conidiiferous cells over the whole or a 
arge part of its surface (Fig. 34, 6). Each of these cells bears, in one 
*roup of species, a single chain of conidia, in other species (called by 
ome authorities Sterigmatocystis) three or four secondary sterigmata 
chich bear the conidial chains. Part of the species produce also thin- 
called perithecia as yellow or brown spherical bodies upon the surface 
f the substrata. These perithecia are filled with eight-spored asci 
Fig. 34, e). A few species produce sclerotia instead of perithecia, but 
nany species are not known to produce either perithecia or sclerotia. 

Important Species. Among the species constantly met with, 
Aspergillus niger is recognizable by its black or very dark brown spores 
nd in some strains by black sclerotia. Several black-spored forms are 
lescribed, but their separation is usually impossible from the data given. 
Aspergillus niger ferments sugar solutions with the production of oxalic 
icid in considerable quantity. 

Of green forms, Aspergillus* glaucus, Link (Aspergillus herbariorum, 
Wiggers), and Aspergillus repens, De Bary, both produce abundant 
fellow perithecia. These abound upon herbarium specimens, hay, 

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



54 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



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 fumigatus, Fresenius, is a green form characterized 
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/x* in diameter. This species produces a destructive 
disease of birds known as aspergillosis. The same species is sometimes 
reported as pathogenic to man. 





FIG. 34. FIG. 35. 

FIG. 34. Aspergillus glaucus. a, Conidiophore 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); d, perithecium(Xi28); 
e, ascus containing ascospores (X4So). (Original) 

FIG. 35. Aspergillus. (i) A. fumigatus, Fres; (2) A. nidulans. i and 2 show 
the simple sterigmata of A.fumigatus and the secondary sterigmata of A. nidulans. 
The conidia of these species do not remain attached in ordinary fluid mounts. 
(Original.) 

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

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

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 

'The unit of measurement is the micron (jt) or micro-millimeter (.001 mm.or Hooo in.) 



MOLDS 



55 



pale cream fruiting surfaces, A spergillus flav us produces several shades 
of yellow and green, A spergillus 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 myce- 
lium 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.* 






FIG. 36. FIG. 37. FIG. 38. 

FIG. 36. Cladosporium herbarum, showing the forms of conidiophores and conidia 
which are very, common upon laboratory culture media. (Original.) 

FIG. 37. Spores of AUernaria sp. (Original.} 

FIG. 38. Fusarium from decaying potato, a, Spores showing curvature and 
septa; b, germination of spores; c, development of spores in petri-dish culture; d, 
mass of spores as found in culture. (Original.) 



Colonies 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 
separated by some. 

ALTERNARIA AND FUSARIUM. The frequent occurrence of species of 
AUernaria and Fusarium in cultures demands that the generic charac- 
ters be recognized. Both, as a rule, produce abundant growth with a 
tendency to over-run cultures of other forms (Figs. 37 and 38). The 
spores of AUernaria are brown, Indian-club form, muriform (divided 
ito several cells by longitudinal as well as cross- walls), and are con- 
iccted together into chains (Fig. 37). The spores of Fusarium are 

1 This species has been shown to be a conidial form of Spharella lulasnei Janczewski, but 
the bacteriological student will meet only the conidial stage. 



50 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

colorless, either straight or sickle- or crescent-shaped, divided into 
several cells by cross- walls occur singly or adhere in masses on the tips 
of the fertile branchlets. The morphology of colonies in culture varies 
widely from the descriptions of the same species under natural condi- 
tions. Species of Fusarium 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. 




FIG. $g.pidium 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 Bull. 82, Bur. Animal Industry, U. S. Dept. 
Agr.} 



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 vege- 
tative mycelium entirely submerged, become powdery white with 



MOLDS 57 

spores when mature, liquefy gelatin, and produce a strong character- 
istic odor (Fig. 39) . Microscopically the species is recognized by dichoto- 
mous branching of the hyphae at the margin of the colonies, and by 
the spores or oidia which are abruptly cylindrical, varying with condi- 
tions 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 appears 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- 
tween the Mycoderma group of yeasts, Oidium and Monilia, and the 




FIG. 40. A colony of Monilia Candida. (Photographed by Z. Northrup.) 

well-fixed mold types shows a number of organisms which are found 
repeatedly in the fermentation industries (Fig. 40). 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, 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, Demalium pullidans, has 
been much studied. This is frequently found within decaying fruit as 
dark brown colonies. In culture, mycelium is sparingly produced, 
either colorless or colored, and conidia are borne in clusters and chains 



58 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

all along the hyphae submerged in the substratum. At first both myce- 
lium and conidia are colorless, later some or all of the cells develop I 
heavy dark brown walls. Although not active as an agent of fermen- 
tation, it occurs very frequently in the fermentation industries some- 
times discoloring the fermenting products. The conidia bud out from 
the cells 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.E. 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 
the destroyers of particular insects. 



CHAPTER III 
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, Torula). 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 best in the cultivated condition (S. cerevisia of beer). 

The true yeasts occur in the form of spherical or more or less elon- 
gated cells varying in normal width from 2.5^1 to i2/x. The first classi- 
fications were based on shape and size alone but these vary and depend 
so much on cultural conditions that they are of little value in differen- 
tiating 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 solu- 
tions to distinguish the various forms. 

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

* Prepared by F, T. Bioletti. A. Guilliermond has furnished the section on the " Cytology 
of Yeasts." 

59 



60 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

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

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. 




FIG. 41. Yeast cell. (Original.} 

The vegetative reproduction in the genus Saccharomyces takes place 
by budding, in Schizosaccharomyces by fission. 

The extreme temperatures for budding lie between i and 47, vary- 
ing with different species. The optimum temperature varies in the 
same way between 25 and 35. The rate of multiplication under favor- 
able conditions will range 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 con- 
ditions that discourage budding (lack of nutriment) they form endo- 
spores. 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 by cells before or after budding and may even change to asci 
and form new spores. They are generally spherical or slightly ellip- 
soidal, rarely kidney-shaped (S. marxianus) or furnished with a zonal 
ring (S. anomalus) (Fig. 42). 



YEASTS 



61 



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. 




o 



/ * 
** 



$ 



<3Q> 

<p 



^ 



FIG. 42. Spore-bearing cells. A. S. pasieurianus. (After Bioktti.) B. Sch. 
octosporus. (After Schionning.} C. S. anomalus. (After Kayser.) 



CYTOLOGY OF YEASTS* 

GENERAL STRUCTURE OF YEASTS. The structure of yeasts in no 
way differs from that of the other fungi, only it is seemingly more complex 
id consequently more difficult to interpret on account of the abundance 
the stainable granulations which sometimes accumulate in the cells 
id occasionally hinder the differentiation of the nucleus. This explains 
why it has until recently remained a subject of controversy. It is now 
fairly well understood. 

* Prepared by A. Guilliermond. 



62 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

In order to understand clearly this structure, one must observe 

young cells taken from a culture at the beginning of development. 

For this purpose we use Saccharomyces cerevisia which, because of the 

relatively large size of its cells, lends itself better than 

f,\ any other yeast to a cytological study. Examined in 

vfy the living state, highly magnified, the cells of this 

/^ yeast show a dense and homogeneous cytoplasm with 

a group of small vacuoles or a single large vacuole at 

FIG. 43. Sac- the center. In the vacuoles and also in the perivacu- 

C v isi^ myCeS youu~ olar c y to P lasm > we can clearly distinguish a great 

cells examined in many small shining granules, of varying sizes, which 

the living state man if es t Brownian motion. It is easy to stain them 

in a solution of J 

neutral red. The in the living state (Fig. 43) with a very dilute solu- 

palTred^^ontTin tion of neutral red or methylene blue. These are 
met a chromatic only metachromatic corpuscles. 

oed U d S ark S red COl ~ In fixed and stained preparations (Fig. 44, i-io) is 
seen in each cell a single, large nucleus, whose struc- 
ture is exactly like that which we have discussed in molds. This 
nucleus is surrounded by a membrane and contains a hyaline nucleo- 




b } i* 

-: /^ 4 ^ *. 

'y^y 4 ^Vi|S* &JP" 

( '' '* |% 

"e r e iz i/ ^ 

FIG. 44. FIG. 45. 

FIG. 44. Saccharomyces cerevisice. i-io, Young cells with nucleus,' showing its 
structure. 6-8, The same: division of the nucleus. 11-13, Cells after twenty-four 
hours' fermentation, with a very large glycogenic vacuole filled with lightly colored 
grains. 

FIG. 45. Saccharomyces cerevisice. Young cells fixed and stained by a special 
method revealing in the cytoplasm a chondrium consisting of rod mitochondria and 
granular mitochondria. 

plasm in which is easily seen a large nucleolus and some chromatin; 
this latter is scattered through the nucleus, sometimes found in the 
nucleoplasm in the form of a network, sometimes reduced to a num- 



YEASTS 




ber of granules smaller than the nucleolus, and sometimes even found 
gathered on the circumference of the nuclear membrane. 

The cytoplasm is dense and homogeneous. A special technic has 
Recently enabled the demonstration of a chondrium in the cytoplasm. 
This seems to consist both of granular mitochondria and of more or 
less elongated and flexible rod mitochondria (Fig. 45). 

The vacuole shows in its interior numerous metachromatic corpus- 
cles of varying sizes (Fig. 46). As in molds, these corpuscles appear not 
only in the vacuole, but also in the perivacuolar cytoplasm; there they 
start, and are next diffused in the vacuole where they finish their growth, 
then dissolve when the need is felt. It is 
difficult in the case of yeasts to determine 
their origin; nevertheless, observations 
made of fungi with larger cells than we 
have previously described, show that the 
metachromatic corpuscles start in the 
midst of mitochondrial elements, and it 
seems certain that after that the process 
is the same in yeasts. FIG. tf.Saccharomyces cere- 

In the cytoplasm of yeasts, also, have *> sta , ine . d b .y a me . thod re ; 

vealmg both the nucleus and 
been noted granulations, which can be the metachromatic corpuscles. 

stained with ferric haematoxylin, which 

I have been named basophile grains; but these formations, which are not 
well defined, seem to us to represent simply products from the altera- 
tion of the chondrium under the influence of imperfect fixing agents. 

The membrane of yeasts is quite thick and very distinct. Its 
chemical nature is still little known. According to some authors, it 
consists of a cellulose; others think that it contains only pectose. Ac- 
cording to Mangin, it is formed of callose. Finally, some authors have 
thought they discerned chitin. 

The structure we have just described is found in all the species 
(Fig. 47), only it is sometimes much less distinct because of the smallness 
of the cells. In the elongated yeasts, and in the cells composing the 
mycelial formation which are encountered under some conditions, 
especially in the films, the nucleus generally occupies the center of the 
cell; it is situated in a kind of matrix or bridge consisting of a very 
dense cytoplasm, while a vacuole filled with metachromatic corpuscles 
occupies each of the two extremities of the cell. 






64 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Summing up the elements of which a yeast cell consists are a cyto- 
plasm with a chondrium, a nucleus with clearly differentiated structure 
vacuoles containing numerous metachromatic corpuscles, a membran 
of a nature not yet clearly denned. 

CYTOLOGICAL PHENOMENA DURING MULTIPLICATION. During the 
budding of the yeasts, cytoplasm enters the young bud with some chon- 
drium; then, when the bud has reached a certain size, the cytoplasm 
forms in it a little vacuole in which appear 
metachromatic corpuscles (Fig. 46, 2-7). 

In the course of these phenomena, the 
nucleus retains the position which it occupied 
in the mother cell before the appearance of 
the bud. Only when the bud is quite large 
does the nucleus begin to divide. It is elon- 
gated so that one end penetrates the bud; the 
nucleus then resembles an elongated dumb- 
bell with the larger head remaining in the 

mother ceu and the other> smaller head> in the 

with nucleus. bud (Fig. 44, 6, 7 and 8; Fig. 46, 2, 7; Fig. 48). 

Soon the part of the dumb-bell which is 

stretched out breaks near the neck of the bud, forming two nuclei of 
unequal size, at first tapering spherical in shape, and later rounded 
off: one is the nucleus of the mother cell and the other that of the 
bud. This division is therefore effected by the direct method; it is an 
amitosis. In the Schizosaccharomyces, where the cells do not multiply 
by budding as in other yeasts, but by a transverse partition, the 
nuclear division is effected by amitosis: the nucleus, situated in the 
center of the cell, elongates along the longitudinal axis of the cell and' 
resembles a dumb-bell, ending by dividing in the middle, thus forming 
two nuclei of the same size. Soon a transverse septum appears be- v 
tween the two nuclei and separates the two daughter cells. 

We have now to note the modifications which arise in the structure 
of the cells during the different phases of development and at the time 
of sporulation. 

VARIATION IN THE CELLULAR STRUCTURE DURING DEVELOPMENT. 
In the course of development, especially during fermentation, yeasts 
reveal cytological phenomena which render their structure more com- 
plex and more difficult to interpret. Let us take for example the study 




YEASTS 65 

of the S, cerevisia. After twelve hours of fermentation, the meta- 
chromatic corpuscles become more numerous. At the same time, the 
cytoplasm forms little vacuoles which contain no metachromatic cor- 
puscles, but only glycogen, easily detected by iodo-iodide of potassium. 
These are gradually fused into a single vacuole, which enlarges much 
and modifies materially the cell structure. The glycogenic vacuole, 
increasing, pushes back to the periphery of the cell the cytoplasm, the 
vacuoles with metachromatic corpuscles, and the nucleus whose chro- 
maticity increases and which becomes homogeneous in appearance 
(Fig. 44, n). After forty-eight hours, moreover, the cell is found to 
consist of an enormous vacuole filled with glycogen which occupies 
most of it, while the nucleus, the vacuoles with metachromatic cor- 
puscles and the cytoplasm are pushed back to one side of the cell, which 
is then transformed into a kind of glycogen sack (Fig. 44, 12 and 13; 
46, 6-8). At this time the glycogenic vacuole contains a great many 
small granulations (Fig. 44, 12-13), which easily fix some staining 
materials, especially ferric hematoxylin, and whose origin and signifi- 
cance have not been determined. 

Toward the end of fermentation, the glycogen gradually diminishes 
and the glycogenic vacuole is gradually reduced, then ends by dis- 
appearing. The cell after this resumes its original structure. 

In the course of these phenomena, the membrane apparently shows 
no modification. It is known, however, that under some conditions, 
yeasts secrete gelatinous substances which englobe their cells in a kind of 
jelly and so appear like zooglcea (Hansen). It is well to add, on the 
other hand, that many pathogenic yeasts, when living in the host, have 
the ability to protect their cells against the reaction of the organisms, 
by secreting a very thick capsule of gelatinous nature: each of their 
cells is then surrounded by a large capsule. 

CYTOLOGICAL PHENOMENA OF THE SPORULATION AND GERMINATION 
OF ASCOSPORES. For a study of the sporulation, we will consider a 
representative of the species Schizosaccharomyces, the Sch. octosporus, 
in which these phenomena are easily observed and especially well 
understood. 

We know that in this yeast, as in some others, sporulation is pre- 
ceded by a sexual phenomenon consisting of an isogamous copulation. 
The ascus results from the fusion of two similar cells. The gametes are 
ordinary cells which have the structure which we have previously 






66 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

described, with one nucleus and one or more metachromatic vacuoles 
containing corpuscles (Fig. 48, a). Fusion takes place between the two 
cells which are nearest together. Each of these two cells sends out a 
tiny beak; the two little beaks thus formed anastomose and form a 

channel of copulation joining the two 
../'*) cells (Fig. 48, b, c, d). The septum 
c ~ d * separating the two gametes in the 

f\ \ middle of the channel, is quickly 

g h ~ absorbed, and the two cells then 
||^R^f } nave f ree communication. The cyto- 

; plasm of the two cells draws together 

and mingles in the channel; there the 

Ro. 48. Successive stages of two nuclei draw near to each other 
copulation and sporulation in Schizo- . 
sacckaromyces octosporus. (Fig. 48, e) and fuse into a single 

nucleus (Fig. 48, /, g, h). Next the 

zygote ends its fusion; instead of its original dumb-bell appearance, it 
assumes the form of an oval cell, then grows large (Fig. 48, i). Occa- 
sionally, however, it retains a vestige of the individuality of the two 
gametes, showing two swellings joined by a somewhat narrower middle 
portion (Fig. 48, f). 

During this time, the cell becomes filled with little vacuoles and 
assumes a more or less alveolar structure. 
These vacuoles contain a number of metachro- Q ] ^ jj^ 
ma tic corpuscles. The nucleus which occupies 
the center of the zygote begins to divide. The 
ascus, containing sometimes four, sometimes 
eight ascospores (Fig. 48, _/), will then undergo 
two or three successive divisions, as the case 
may be. These divisions are accomplished by 
karyokinesis or mitosis. In the stages preceding 
nuclear division, the nucleus is very large and 

shows a very clear structure with a nucleolus Various stages of the 
and a chromatic reticulum (Fig. 49, *) It t . isi n d 
soon elongates and assumes a special structure. 
Its membrane loses its clearness, and in the midst of the nucleoplasm 
an achromatic spindle appears, ending at each of its two poles in a 
very small centrosome and containing at its center a group of fine 
granulations representing the equatorial plate (Fig. 49, b and c). The 




YEASTS 67 

Inucleolus always persists on one side of the spindle. At a subsequent 
stage the chromatic granulations or chromosomes are divided between 
fj,the two poles of the spindle, the nucleoplasm is mixed with cytoplasm, 
then the spindle elongates, while the chromatic granulations form a 
Ihomogeneous mass at the two poles (Fig. 49 d, e, g and h). The 
Inucleolus is quickly absorbed, then the two nuclei are formed at the 
(expense of the two chromatic masses (Fig. 49, /). To summarize, 
(therefore, this division consists in mesomitoses of a primitive kind, 
which appear to take place in the interior of the nucleus, whose mem- 
Ibrane is absorbed only at the end of the phenomenon. They show 
i the characteristics of the mesomitoses which have been described in 
| the asci of the higher Ascomycetes. 






-.' . ' * a 



6 

i 

FIG. 50. Successive stages of copulation and sporulation in Schizosaccharomyces 
\pombe. 1-2, Cells just as sporulation is about to begin. 3-7, Union of the two 
gametes and nuclear fusion. 8, Ripe ascus. Cellular fusion being incomplete. 
the ascus retains the shape of the two cells joined by a channel of copulation. 

When these divisions are accomplished, the nuclei seem to be scat- 
tered in the cell (Fig. 48, i} ; they are soon surrounded by a thin layer of 
cytoplasm which is separated from the cytoplasm by a membrane; 
these are the ascospores. At first very small, these gradually increase 
| at the expense of the cytoplasm which has not been used in their forma- 
tion in other words epiplasm then reach the point where they oc- 
cupy the whole of the ascus, after having absorbed this epiplasm (Fig. 
48, j.) The metachromatic corpuscles scattered in the vacuoles of 
the epiplasm disappear during these phenomena, being absorbed by 
the ascospores. At no time during the development of the ascus can 
glycogen be seen any more than in plant cells, but this is replaced 
by an amyloid substance which is stained blue by iodo-iodide of potas- 
sium. This substance impregnates the membrane of the ascospores 
and disappears during their germination, utilized as a reserve product. 

In some Schizosaccharomyces or ordinary yeasts which bud (zygo- 



68 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

saccharomyces) the ascus comes from an egg which starts in a similar 
manner (Fig. 50.) In some species, this egg is formed by a hetero- 
gamous copulation between an adult cell (macrogamete) and a very 
young cell which has just separated from the mother cell (micro- 
gamete) (Fig. 51). On the contrary, in most species, the ascus results 
from the simple transformation of an ordinary cell without previous 
copulation. Whatever may be its origin, the ascus shows cytological 
phenomena quite similar to those which have just been described in 
Sch. octosporus, with mere differences of detail. Always in Sch. 



6 






21 22. 

FIG. 51. Heterogamous copulation in Zygosaccharomyces chevalieri, 1-3, 
Gametes sending out a beak in anticipation of copulation. 4-7, Micro- and macro- 
gametes joined by their channel of copulation. 8, The partition separating the 
two gametes is absorbed. 9-18, The contents (nucleus and cytoplasm) of the micro- 
gamete enter the macrogamete and are fused with the contents of the latter. 
19-21, Ripe asci. 22-23, Freeing of the ascospores by rupture of the membrane of 
the ascus. 

octosporus are seen only a few metachromatic corpuscles in the ascus. 
In most of the other yeasts, on the contrary, the ascus contains a very 
large number of metachromatic corpuscles, and it is easier there to fol- 
low the evolution of these bodies which present interesting singularities 
clearly demonstrating their role as reserve substances. 

Let us observe, for example, the cytological phenomena which ap- 
pear during sporulation in Saccharomyces ludwigii. In this yeast, 
which shows no sexuality in the origin of the ascus, the cells which are 
preparing to sporulate assume a finely vacuolar structure (Fig. 52, 8 
and 9) and produce a large quantity of reserve products: metachromatic 
corpuscles, glycogen and fat globules. Metachromatic corpuscles spring 
up in some vacuoles, glycogen in others; as for the fat globules, they 



YEASTS 



6 9 



are located in the cytoplasmic web. The nucleus is situated on one 
side of the cell, surrounded by a thin layer of very thick and homo- 
geneous cytoplasm which is to become the sporoplasm, at whose 
expense the ascospores are formed, the remainder that is to say the 
vacuolar cytoplasm being destined to compose the epiplasm or nourish- 
ing plasm. 

At a later stage, the metachromatic corpuscles undergo a kind of 
pulverization transforming them into small grains, and begin to dis- 





& ,& 

--* A* :^ 





FIG. 52. Sporulation in Saccharomyces ludwigii. Figs, i and 7 showing the 
evolution of the nucleus. Figs. 8-9, the metachromatic corpuscles, stained by a 
method permitting a differentiation, except in Fig. 8, are dissolving, and the sub- 
stance of the vacuole which contains them shows a diffuse metachromatic coloring 
(here gray) like the corpuscles. 

solve in the vacuoles surrounding them, the latter at this time taking, 
with aniline blue stains, a diffuse red coloring similar to that of the 
metachromatic corpuscles (Fig. 52, 9). At the same time, the nucleus 
undergoes two successive divisions, but these have not been discern- 
ible up to the present time, because of the density and the strong 
chromaticity of the sporoplasm surrounding the nucleus. They are 
manifested merely by the appearance of the two daughter cells which 
migrate to the two poles of the cell, carrying with them a part of the 
sporoplasm, which assumes the appearance of a dumb-bell and whose 



70 MORPHOLOGY AND CULTURE OP MICROORGANISMS 

slender part ends by breaking (Fig. 52, 2, 3 and 4). The cell, there- 
fore contains at this time at each of its poles a small mass of sporo- 
plasm having first one, then two, nuclei (Fig. 52, 5 and 10). After 
this, the sporoplasm condenses around each of these nuclei (Fig. 
52, 6), thus delimiting at each of the poles two small ascospores. 

During these phenomena, the metachromatic corpuscles congre- 
gate around the ascospores (Fig. 52, n and 12), then gradually dis- 
solve. The ascospores constantly increase in size at the expense of 1 
the epiplasm, which becomes disorganized and is reduced to a vacuo- 
lar liquid containing in suspension metachromatic corpuscles, fat 
globules and glycogen. They succeed in absorbing entirely the epi- 
plasm and in occupying the whole of the ascus (Fig. 52, 13 and 14). 
The metachromatic corpuscles, like the glycogen and the 'globules 
fat, are then completely absorbed by the ascospores, which indicates 
clearly that they, as well as the latter substances, act as reserve prod- 
ucts. When the ascospores are ripe, they contain in their vacuoles 
metachromatic corpuscles (Fig. 52, 14). 




FIG. 53. Germination of ascospores in Saccharomyces^ ludwigii. i, Beginning 
of the fusion of the ascospores. 2, The ascospores are joined two by two by a 
channel of copulation, but their nuclei are not yet fused. 3, The nuclei are fused. 

4, At the left two ascospores, joined, have formed at the middle of the channel of 
copulation a bud which has ruptured the membrane of the ascus. At the right, the 
two ascospores, joined by a channel of copulation have not yet fused their nuclei. 

5, Formation of the bud at the expense of the two fused ascospores. Two other 
ascospores have not yet begun their fusion. 6, The bud formed at the channel of 
copulation is already established and separated from this channel by a transverse 
septum. 

In all yeasts, at the time of budding, the ascospores have the appear- 
ance and structure of plant cells. Their germination does not differ 
from ordinary plant multiplication. In some species, however, espe- 
cially in S. ludwigii, copulation, suppressed at the beginning of sporula- 



YEASTS 71 

tion, is replaced by a compensating phenomenon which intervenes at 
the germination and consists in the fusion of the ascospores two by two 
(Fig. 53). The ascospores anastomose at their extremities by a chan- 
nel of copulation which, as soon as the nuclear fusion is accomplished, 
becomes the seat of a budding. 

THE PRINCIPAL YEASTS OF IMPORTANCE TO FERMENTATION 
INDUSTRIES 

TRUE YEASTS, SACCHAROMYCETES. The various yeasts used in 
brewing and some of those used in producing distilling material are 
grouped together as S. 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 peculiarities such as the temperature and time limits of film and 
spore formation, and the character of the fermented liquids. The vari- 
ous 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 some- 
what 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. 

Their fermentative power is considerable, some of them being capa- 
ble of producing over 16 per cent by volume of alcohol. W. V. Cruess 
has obtained 21 per cent from a Burgundy wine yeast. They differ in 
the flavors and aromas which they produce in the fermented liquid, and 
especially in the rapidity with which they settle. Some yeasts, such 
as those of Champagne and Burgundy, form a compact sediment which 
settles quickly and leaves the liquid clear. Others remain suspended 
for a long time and settle with difficulty. 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



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. 





B 






FIG. 54. Wine and beer yeasts. A, S. ellipsoideus, young and vigorous; B, S. 
ellipsoideus, (i) old, (2) dead; C, S. cerevisioe, bottom yeast; D, S. cerevisice, top yeast. 
(Original.) 

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

S. vordermanni is concerned in the manufacture of arra.ck. It fer- 
ments the sugar produced from rice by the molds, Mucor oryzce and Rhi- 
zopus oryzce. 

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. 



YEASTS 73 

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 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 elon- 
gated and in film formation soTnetimes producing a branching mycelium. 
Spores are formed easily and abundantly. 

The apiculate yeast, S. apiculatus, is very abundant on grapes and 
most acid fruits. It is very variable and undoubtedly includes many 
varieties. 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 cloudiness 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 conditions 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 to four spores are formed in a cell. These spores are often 
reduced in the fermenting liquid. The fermentative power is high and 

urge percentage 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 
endospores. They are grouped together under the name of Torula. 



74 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

They are usually small, spherical or slightly elongated. Some specie 
produce a little alcohol and some none. They seldom occur in suf- 
ficient quantities to be harmful and one form is accredited with pi 
ducing the special flavor of some English beers. 

The forms included under Mycoderma resemble yeast in shi 
but produce little or no alcohol, are strongly aerobic and do not' 
produce 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 such as wort, cider, 
grape juice, or a special medium devised for a special investigation. An example o| 
the latter is Laurent's medium: 

Ammonium sulphate, 4 . 7 1 g. 

Potassium phosphate, o.ysg. 

Magnesium sulphate, o . i o g . 

Water, i L. 

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

Pure cultures can be made, rarely, by inoculation from a naturally pure soui 
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 conditions, 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 separat- 
ing 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 steri- 
lized water until on the average every 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 into 
a mixed culture and then draw it repeatedly over the surface of a solid culture medium , 



YEASTS 



75 



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. 








B 




CDo 





FIG. 55. Wild and pseudo yeasts. A, S. pombe. (After Lindner). B. Torulce. 
(After Pasteur.') C, Mucor, (i) spores; (2) germinating spores and mycelium. D, 
S. apiculatus. E, Mycoderma vini. (After Bioldti.) 

The most useful method of separation and one which is applicable to most cases 
is that of 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 



76 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

degree of dilution is obtained. The various portions of nutrient gelatin are then 
poured, with precautions against outside infection, on glass plates or more conven- 
iently 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 percentage 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 contain- 
ing 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, 
contamination, 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 IV 
BACTERIA* 

The bacteria naturally fall into quite distinct groups or orders 
the true bacteria and the sulphur bacteria. 

A portion of the true or Eubacteria together with the sulphur form, 
are designated as the higher bacteria. The forms usually spoken of 
as bacteria belong to the group of lower bacteria, and when the 
word "bacteria" alone is used reference is usually made to the lower 
bacteria. These constitute a group of microorganisms quite distinct 
and characteristic, while the higher bacteria form links, as it were, 
between the lower bacteria and other closely related microorganisms. 
-The morphology of the two groups will need to be discussed 
separately. * 

FORMS OF LOWER BACTERIA* 

FUNDAMENTAL FORM TYPES. The forms of bacteria are exceed- 
ingly 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). 




FIG. 56. Types of micrococci. (After Williams.] 




% 

FIG. 57. Types of bacilli. (After Williams.} 
spared by W. D. Frost, with cytology by A. Guilliermond. 

77 



78 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 




FIG. 58. 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 micrococci and at another time, or, by another inves- 
tigator, as bacilli. The pneumonia germ is also another illustration 
of an organism that occupies a dual position. Migula has suggested 
a method of differentiating these which will be discussed under a 
later head. The bacilli pass almost imperceptibly into the spirilla. 
The cholera bacillus of Koch is in reality a spirillum. 




FIG. 59. Involution forms. Here are illustrated unusual forms of B. subtilis, 
water bacteria, Bact. aceti, Bact. pasteurianum, bacteroids in root nodules, Bact. 
tuberculosis, Bact. diphtherias. (After Fischer from Frost and McCampbell.) 



BACTERIA 79 

INVOLUTION FORMS. * The forms of bacteria are quite constant under 
normal conditions, but very frequently they show abnormal or bizarre 
shapes. These are known as involution forms (Fig. 59). It is some- 
times suggested that these involution forms represent another stage in 
the developmental history of the organism, and upon this supposition 
certain bacteria which very regularly show these involution forms have 
been classified as belonging to a different suborder from that in which 
the lower bacteria are placed. The ordinary view of the involution 
forms is, however, that they are degeneration forms, that they cor- 
respond, in other words, to the halt 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. In- 
volution forms are very likely to occur in artificial culture and are much 
more common with some species than with others. (See- page 98.) 

SIZE* 

The bacteria were formerly spoken of as the smallest of living things, 
but since the recognition of the ultramicroscopic organisms it is neces- 
sary to be somewhat more specific in characterizing their dimensions. 
The unit of measurement in microscopy is the micron (/*), or micro- 
millimeter. This is .001 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 in. The micro- 
cocci 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 o.2ju and a length of 0.5/4, and 
sometimes very large as, for example, the Bact. anthracis with a width 

i.2ju and a length of 5-2o/x. The spirilla average about i.o/i in 
diameter but may be as long as 30/1-40;*. 

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 rela- 

Prepared by W. D. Frost. 



8o MORPHOLOGY AND CULTURE OF MICROORGANISMS 

tive positions. In other cases the movement is vibratory, the bacteria 
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 many 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 
brownian movement is to be sharply differentiated from vital movemen 
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 o: 
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) 01 
flagella (sing, flagellum). The fact of motility or non-mo tility of an 
organism is of considerable value to the systematist. It is determinec 
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 par 
ticular organism or culture does or does not possess vital movement 
An opinion can be more definitely formed at times if some chemica 
producing an anaesthetizing effect on the bacteria is introduced int< 
the examining medium. In case the organism is actually motile its 
movement will be altered by the anaesthetic but 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, Jike a whip lash. Most 
of the bacteria possess flagella rather than cilia. The size, arrange 
ment, etc., of these flagella are constant and characteristic of a par 
ticular organism. Their structure and arrangement, therefore, will be 
discussed later. 

CHARACTER OF MOVEMENT. Different bacteria exhibit different 
kinds of movement. Some dart forward with great rapidity, others 



BACTERIA 8 1 

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 peculiar 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. 
Van 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 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 by what is known as binary fission. In addition to this a 



FIG. 60. The division of bacterial cells (diagrammatic). (After Novy.) 

number 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. 

VEGETATIVE MULTIPLICATION. This is accomplished, by means of 
Binary fission (Fig. 60). When a bacterium has reached maturity, fis- 
sion begins. Division begins by an invagination of the protoplasm 
n the middle of the cell, which proceeds until 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 

'Prepared by W. D. Frost. 
6 



82 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

at right angles with the long axis of the cell in the case of the bacrlli 
and spirilla, except in rare instances. In the case of micrococci, the 
throwing of the cell wall across one diameter is 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 elon- 
gate before division and micrococci divide before they elongate; 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 multiplica- 
tion, at least theoretically. It would be only a matter of geometrical 
progression. It is of course quite impossible for the bacteria to main- 
tain their theoretical rate of growth for any length of time, but, prac- 
tically, they grow with enormous rapidity,, as is shown in cultures and 
by 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 endos pores. 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 en- 
vironmental conditions. 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 



BACTERIA 83 

of the spore in the cell varies (Fig. 62). In some species it is equatorial, 
in others it is polar, and in still others it has an intermediate position 
[between equatorial and polar. When the spore is larger than the 
mother cell and is situated equatorially it causes the cell to bulge with 
the formation of a barrel-shaped organism, a dostridium. 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 spores 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 endosjjpres possess remarkable powers of resistance due to the 
concentrated character of the protoplasm, or to the character of the 





FIG. 61. FIG. 62. 

FIG. 61. The formation of spores. (After Fischer from Frost and McCampbell.) 
FIG. 62. Spores and their location in bacterial cells. (After Frost and McCampbell.} 

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- 
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 chem- 
icals 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 toward 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. 



84 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

When these spores are brought under favorable conditions of 
moisture, temperature, and food supply, they germinate. There are 
several types of germination (Fig. 63). In some cases the spore wall 
ruptures at the pole and the young cell emerges so that its long axis is 
in the same direction 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 another 
type the spore swells and the young cell absorbs the wall of the spore. 

In the lower 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. 



C O 

c 



d 



afl 



01 O 



FIG. 63. Spore 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 
streptococcus 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 arthro- 
spores 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 

Prepared by W. D. Frost. 



BACTERIA 85 

organism possessing them. They are of sufficient definiteness and 
constancy to be used by the systematists in characterizing 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 
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. 64). 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 



oo 

<L_/ & 





FIG. 64. Division forms of micrococci. a, Diplococcus, perfect form with 
flattened opposed surface (gonococcus) , lanceolate form (pneumococcus) ; b, strepto- 
coccus; c, consecutive 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 
lown as tetracocci, or merismopedia. Still again, the planes of division 
ty proceed at right angles to each other in three dimensions of space, 
this case packets are formed which are known as packet cocci, or 
rcina. Another group of the micrococci occurs, known as the staphy- 
i, 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 



86 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



at right angles to their long axis (Fig. 65). 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 designa- 
tion is diplobacilli. The length of the chains appears to depend not 



~ 



FIG. 65. Division forms of bacilli, a, Single; b, pairs; c, in threads. (After AWy.) 

only upon the cohesion of the bacilli but also upon the shape of the 
end; those which have square ends frequently have very long chains, 
while those with rounded ends have short chains or occur singly. 

CELL AGGREGATES AMONG THE SPIRILLA. The same kind of 
arrangement is maintained among the spirilla. 





{'/ ,--- 

l//\^^^^'% 
W,' 




j W' 
FIG. 66. Threads of Bact. anthracis. (After Migula.) 

ZOOGLCEA. 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 



BACTERIA 87 

a great many attempts have been made by systematists to make use 
of this in indifferentiating species. These zoogloeic masses usually 
assume the forms of pellicles, but their value as diagnostic features is not 
great. The formation of zoogloea is very frequently only a stage in 
the life history of an organism. 

THE CYTOLOGY OF BACTERIA 

* 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 struc- 
ture in the cytoplasm, such as nucleolus, polar bodies, etc. In addition 
to these there may be appendages, such as the cilia or flagella. In 

he case of bacterial cells, we find most of these structures present, 

uch as cell wall, cytoplasm, and appendages. 

GENERAL CONSIDERATION or CYTOPLASM AND NUCLEUS.* The 

ytoplasm of the bacterial cell is similar to the cytoplasm of other cells 

xcept that chemical analyses seem to show that it contains a higher 




a 



FIG. 67. Plasmolytic changes. (After A. Fischer.) a, Cholera vibrio; b, typhoid 
bacillus; c, Spirillum undula. (From Novy.) 

>ercentage of nitrogen. As viewed under 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 histology, fail 
to reveal the presence of a nucleus, the whole cell being usually uni- 

ormly stained with those stains generally characterized as nuclear 
stains. When these stains are applied to some bacteria, particularly 
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 
granules. 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 

Prepared by W. D. Frost. 



88 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

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 at the center. In this germ and in 
some others it is possible, by special staining, to give the granules a dif- 
ferent color from the rest of the organism. In this case these bodies are 
spoken of as metachromatic granules which are considered later under 
"Reserve Products." The presence of these granules might possibly 
be explained upon the theory that the cells are plasmolyzed (Fig. 67). 
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 explained upon this supposition. 

The cytoplasm of the bacterial cell is slightly refractive. It is 
colorless except in a few cases in which the green coloring matter, like 
chlorophyl, is present, as, for instance, Bact. viride and Bact. chlorinum. 
In the purple sulphur bacteria, the coloring matter bacteria pur purin 
is present. The bacterial cytoplasm contains vacuoles at times. 

MINUTE CONSIDERATIONS OF CYTOPLASM AND NUCLEUS.* The 
question of the cytology of bacteria has long excited the curiosity 
of biologists. It is indeed of great importance from many points 
of view. In the first place, we are interested to know whether 
bacteria are ordinary cells having a nucleus; or whether, as some 
maintain, they lack entirely a nuclear element and are an exception 
to the rule elsewhere established. Moreover, the cyfologic study 
of bacteria may furnish useful knowledge concerning the phylogeny 
and taxonomy of these organisms, a matter not yet solved. Finally, 
we may hope that it will throw light upon some problems of a physio- 
logical or pathological nature. 

Unfortunately this study is very delicate, because of the extreme 
minuteness of the bacterial cells, so that in spite of the large number of 
researches which it has incited in the last twenty-five years, it is to this 
day a matter of controversy. 

At present three theories are held by authors relative to the inter- 
pretation of the general structure of bacteria. We will examine these 

Prepared by A. Guilliermond. 



BACTERIA 



three theories one by one, endeavoring to determine which one, in our 
opinion, seems most probable. 

One of these theories claims that bacteria are cells of very primitive 
organization lacking nucleus and consisting simply of cytoplasm with 
vacuoles. The cytoplasm contains many stainable granulations, but 
these represent products of nutrition. Such an opinion scarcely accords 
with our knowledge of the constitution of the other Protista, in all of 
which the existence of a typical nucleus, or at least of chromatic 
elements replacing the nucleus, has been established. This view has 
not, therefore, had many supporters. 

Another theory maintains that bac- 
teria have a typical nucleus and are in 
no way structurally different from ordi- 
nary cells. This opinion was suggested 
by Arthur Meyer, who claims to have 
succeeded in differentiating, in a great 
many bacteria, granules which fix nu- 
clear stains, and of which one or often 
several appear in a cell. These granules 
he would consider nuclei. It seems to 
be established, however, that the ma- 
jority of the elements noted by Meyer 

are not nuclei, but reserve products 

,, ... 
common among the Protista and known 

as metachromatic corpuscles. 

Vejdowsky's efforts have resulted in much weightier proofs in favor 
of the existence of a true nucleus. In the Bacterium gammari, a 
species discovered by him in the sections of a little fresh water crus- 
tacean, Gammarus zschokkei, Vejdowsky has been able to demonstrate 
in each cell a typical nucleus which is always present. This nucleus 
appears very clearly; it consists of a colorless nucleoplasm surrounded 
by a membrane and by one or two karyosomes (Fig. 68). The author 
had the good fortune to ascertain in several cases karyokinetic represen- 
tations of the division of this nucleus (a, b, c). In short, the presence 
of this nucleus is indisputable. 

The same author discovered a similar structure in a filamentous 
bacterium found in the digestive tract of an Annelida (Bryodrilus 
ehlersi) (Fig. 68, d). 




FlG 68 ._ Bacterium gammari 
and a filamentous bacterium from 
the intestine of Bryodrilus. (After 



QO MORPHOLOGY AND CULTURE OF MICROORGANISMS 

These conclusions are positive, but the species observed by Vej- 
dowsky are not well-defined bacteria, and may be thought to belong 
to the molds rather than to the bacteria. It has also been said, 
not without reason, that Bact. gammari might be a yeast of the genus 
Schizosacchromyces and that the filamentous bacterium studied by 
Vejdowski seems to resemble a filamentous mold. 

However this may be, one of Vejdowsky's pupils, Mencl, has en- 
deavored to apply these conclusions to other bacteria, which are well- 
defined, notably B. megatherium, but has only succeeded in bringing 
forth proofs which are much less convincing of the existence of a nucleus. 
The author strived to discover a nucleus, but this organ is not constant 
and does not show the structure of a true nucleus. 

Both Kruis and Rayman have discovered a nucleus in different 
bacteria (B. myco'ides, radicosus, etc.). This nucleus appears only in 
very young cells; it is not found in older cells, and seems (like the nucleus 

noted by Mencl) to represent merely the 
incipient transverse septum which fixes 
stains well at the beginning of its forma- 
tion and in some ways resembles a nucleus. 
4 The studies of Penau, who also endea- 

FIG. 69. Bacillus megatke- vored to prove the existence of a typical 

nucleus in bacteria, were no more success- 
ful. In B. megatherium, he describes the following phases. In the 
youngest cells he observes a stage where the cytoplasm is very dense 
and uniformly stained, without a trace of differentiation. Immediately 
succeeding is a phase where the cytoplasm becomes less chromatic and is 
filled with vacuoles. At this point the author finds in each cell a tiny 
granule (Fig. 69, i), homogeneous and easily stained, situated at one of 
the poles of the cell, very near the membrane. This granule he con- 
siders to be a nucleus. Moreover, in the cytoplasmic web he observes 
a' series of stainable granules connected by slender trabeculae, thus 
forming a kind of network which he likens to mitochondrial and chro- 
midial formations. At the time of sporulation, Penau finds an in- 
crease in the size of the nucleus (Fig. 69, 2 and 3) which changes to 
a large granule; this is soon surrounded by a membrane and becomes 
the spore (4), which is therefore formed mostly of chromatin. 

The same author discovers a very different structure in Bact. 
anthracis. Here, after a stage of undifferentiated structure which 




BACTERIA QI 

! characterizes the youngest cells, follows a phase where the cytoplasm 
i becomes alveolar. At this time, at one of the poles of each cell, appears 
'a very large homogeneous granule which Penau regards as a nucleus. 
iThis nucleus, however, has only an ephemeral existence and quickly 
I undergoes a cytolysis during which it disintegrates. The disintegra- 
l tion products then impregnate the trabeculae of the cytoplasm and the 
nucleus becomes diffuse. In a last phase which corresponds to sporo- 
| genesis, the chromatin which impregnates the cytoplasm is partly con- 
densed at one of the poles, where it forms first a mass of grains, then a 
: large granule which changes to a spore. 

Nothing is less conclusive than these results, since the author cannot 
i discover an homologous structure in the different species which he 
! studies, and since the nucleus which he describes is only a transitory 
! organ not showing the distinguishing characteristics of a nucleus. 

To prove the existence of a nucleus in bacteria, it is necessary to 
i show a nucleus with a differentiated structure, the constant presence 
< of the nucleus, and to follow the division of this organ during the cellular 
{separation. So far no one has apparently been able to differentiate 
jsuch an organ in well-defined bacteria. We must conclude, therefore, 
that with the exception of the results obtained by Vejdowsky, all ob- 
servations so far gathered in favor of the existence of a typical nucleus 
in bacteria are by no means convincing. 

The third theory asserts the existence of a diffuse nucleus in bacteria. 
It was first suggested by Weigert and more carefully formulated by 
Biitschli. This author describes in a certain number of Sulfo-bacteria 
of large size, Beggiatoa, Chromatium, a kind of central body occupying 




I 

FIG. 70. i. Chromatium okcnii. 2. Beggiatoa alba. These two bacteria have 
a central body containing chromatic grains and considered by Biitschli as the 
equivalent of a nucleus. (After Biitschli.') 

nearly the whole volume of the cell and consisting of an alveolar cyto- 
plasm of highly stainable web, containing within its knots numerous 
chromatic granulations (Fig. 70). The remainder of the cell consists 



Q2 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

of a thin cytoplasmic layer, less easily stainable, surrounding the 
central body. Biitschli compares this structure with the one which 
has been demonstrated in the Cyanophycece, and claims that the central 
body represents the equivalent of a nucleus. It would be a sort of large 
nucleus occupying most of the cell, not bounded by a membrane, and 
scarcely distinct from the cytoplasm. This structure has recently been 
verified in Chromatium okenii by Dangeard. The Sulpha-bacteria, 
however, are organisms morphologically entirely distinct from ordinary 
bacteria, and are apparently directly related to the Cyanophycea. 
Such a structure is not found in other bacteria, in which it is impossible 
to demonstrate a central body and in which, one must admit, the 
nucleus is still more diffuse. 

To Schaudinn we are indebted for the most exact observations in 
favor of the theory of the diffuse nucleus. He had the good fortune 
to discover in the intestine of the cockroach, Periplaneta orientalis, a 
bacillus of very large size which he named B. butschlii. It is the largest 
bacillus known at present (4/1 wide) , and lends itself readily, therefore, 
to cytological studies. His minute observations have shown that 
there is no nucleus. The cells enclosing a finely alveolar cytoplasm, 
whose net contains many small grains which take nuclear stains 
(Fig. 71, 1-6). 

At the time of sporulation the chromatic grains increase in size 
(Fig. 71, 7-9), then gather at the center of the cell in a kind of axial 
wreath (Fig. 71, 10). The two extremities of this wreath quickly swell 
with an accumulation of chromatic grains and form two granular 
masses, one at either pole. These two masses form the beginning of 
the two spores, for each cell forms two spores (Fig. 71, n and 12). 
The grains which compose these two rudiments then condense to form* 
two large homogeneous granules (Fig. 71, 13) which strongly resemble 
nuclei and which Schaudinn considers to be such. Around these two* 
granules is soon condensed a thin cytoplasmic zone which in turn is 
separated from the surrounding cytoplasm by a membrane (Fig. 71, 
13). Henceforth the spores cannot be stained by ordinary means 
because of the thickness of their membrane which prevents the pene- 
tration of stains (Fig. 71, 14). The granules of the wreath, which 
joined the two rudiments of spores, gradually disappeared as well asi 
the cytoplasm, while the spores increased in size. Then the sporangium) 
ended by breaking and setting free the two spores. Germination con- 



BACTERIA 



93 



si^ts simply of a swelling of the spore, then the formation of a small rod 
which issues from the spore and forms a septum for itself (Fig. 71, 15 
and 16). As soon as the spore germinates, the nucleus ceases to exist 
las a morphologic entity; it is scattered in the cytoplasm in the form of 
little grains. 




I- 1 0. 

r' : ** 

9 10 II 12 13 14 

FIG. 7 1 . Bacillus bulschlii. 1-16, Vegetative cells and their division. 7-9, Begin- 
ning of sporulation: the cells about to sporulate are partitioned off crosswise; then 
the septum thus formed is absorbed, at which time sporulation begins. 'Schaudinn 
considers this partitioning off followed by fusion of the two daughter cells as a rudi- 
mentary sexuality. 10-13, Formation of the beginnings of the two spores, at the 
poles of the cell. 14, Ripe spores. 15-16, germination of the spore. (After 
\Sckaudinn.) 

In another bacillus smaller in size (B. sporonema), Schaudinn has 
found an analogous structure only at the time of sporulation; he does 

i not prove the formation of an axial filament but only the condensation 
of a portion of the chromatic grains into a large granule which forms the 
beginning of the spore (Fig. 72). 

By the fact that in these two bacilli the beginning of the spores 

j appears as a granule equivalent in some respects to a nucleus and 
resulting from the condensation of a portion of the stainable grains, 
Schaudinn is led to believe that these grains are composed of chromatin 
and represent a kind of diffuse nucleus. 



94 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

These results have been confirmed by our studies of a large number 
of endospore bacilli (B. megatherium, radicosus, mycoides, aster os poms, 
akei}. Upon examination at the very outset of their development, 
these bacteria present a homogeneous appearance and are uniformly 



'234 

5 

FIG. 72. Bacillus sporonema. i. Cell about to sporulate. 2, This cell grows 
narrow at the center, as if it were going to be divided (Schaudinn regards this pinch- 
ing together which afterward disappears (5), as the vestige of an ancestral sexuality 
like that of B. butschlii). 3-5, Formation of the beginning of the spore. (After 
Schaudinn.) 

stained with no great differentiation, explicable by the density of the 
cytoplasm or by a special condition of the membrane. At this stage 
the cells are in the process of active divisions, after which the transverse 
septa are formed as follows: On the side walls of the bacillus appear 
two small granules which take some stains (Fig. 73, i). These soon 



m 




FIG. 73. i-io, Bacillus radicosus. i, Beginning of development. 2-3, Cells 
at the end of eight hours; 4-6, sporulation. 9-10, Cells in which the chromatic 
grains are located in the middle in a mass slightly resembling a nucleus. 11-12, 
Spirillum wlutans. 

disintegrate at the center of the cell to form a thin band marking out 
the two daughter cells and forming the beginning of the transverse 
septum. This strongly resembles a nucleus and has apparently been 
considered as such by a number of authors (Rayman and Krius, Mencl). 
Toward the eighth hour of development, the cells show clearly their 



BACTERIA 95 

Structure which is changed in appearance; the cytoplasm vacuolizes and 
ends by displaying a fine alveolar structure. The web contains in its 
(knots small, highly stainable granules (Fig. 73, 2 and 3). In some 
bases (cultures on special media for example), there is noticeable a 
localization of these granules at the center of each cell, forming a 
granular region which recalls somewhat the appearance of a large 
nucleus and which is separated into two portions at the time of the 
cellular division as if it were indeed a true nucleus (Fig. 73, 7 and 10). 

These granules fix the nuclear stains, and it seems permissible to 
consider them chromatic in nature. 

At the time of sporulation there forms at one of the poles of the 
bell a small oval mass, easily stained, which is like a nucleus in appear- 
ance (Fig. 73, 4 and 5). This results from the condensation of part of 
the chromatic granules of the cytoplasm, gradually grows larger, and 
phanges to a spore. When the spore has reached a certain size, it is 
surrounded by a membrane which prevents the penetration of ordinary 
stains (Fig. 73, 6); it appears then like a large colorless sphere in the 
Stained cytoplasm of the cell (Fig. 73, 6). 

At no stage of the development have we observed the least trace of 
a nucleus. May there be a nucleus which our present technic would 
pot enable us to differentiate? That has seemed to us scarcely probable, 
ior if this nucleus existed, it would certainly be visible in a species 
ks large as B. butschlii and would not have escaped Schaudinn. The 
most reasonable hypothesis, the one which we have adopted, is to 
consider like Schaudinn that bacteria contain chromatin more or less 
mingled with cytoplasm, differentiated in the case of small grains and 
condensing at the time of sporulation to form the spore which would 
ions 1st principally of chromatin. The cells of bacteria would accordingly 
pave a very primitive structure. 

Granted the clearly demonstrated existence of this particular struc- 
ture in the Cyanophyceoe, there is no reason for not admitting that the 
nucleus, very rudimentary in the Cyanophycea, might be even more so 
n bacteria, being reduced to a diffuse nucleus consisting of chromatic 
grains scattered in the cytoplasm. 

These observations have, moreover, received a series of new con- 
firmations by the labors of a great many authors (Swellengrebel, 
Kuzicka, Ambrez, etc.) and especially by the later researches of Dobell. 



96 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

The latter investigator discovered, in the intestines of frogs and toads, 
a large bacillus (2/4 wide) almost as large as B. butschlii, and named it, 
B. flexilis. This species shows exactly the same cytological charac- 
teristics as B. butschlii (Fig. 74). 

Through a study of a number of different bacteria found in the in- 
testine of toads, frogs and lizards, Dobell has endeavored to show that 
this diffuse nucleus is not original, but derived from the retrogression 
of a more highly differentiated nucleus. 

Thus in various ruicrococci he was able to show in each cell the 
existence of a central stainable granule, dividing by constriction at the 
time of cellular division, and which he regards as a nucleus (Fig. 75, 



si I 





12 



FIG. 74. FIG. 75. 

FIG. 74. Bacillus flexilis. i, Beginning of the division of a cell about to sporu- 
late (vestige of sexuality). 2, Disappearance of the incipient division. 3, Forma- 
tion of the chromatic axial filament. 4, Formation of the beginning of two spores. 
5, Ripe spores. (After Dobell.} 

FIG. 75. Various bacteria, showing the successive types of the retrogression 
of the original nucleus and its transformation to a diffuse nucleus. (After Dobell.) 

1-5). In other cocco-bacillary species of bacteria characterized by 
spherical shape capable of elongation, Dobell discovers a similar nucleus 
n the spherical cells. When the cell lengthens and assumes the ap- 
pearance of a bacillus, this nucleus changes to a spiral axial filament 
(Fig. 75, 5 and 6). 

In various bacilli the same author demonstrates a filament which is 
ever present (Fig. 75, 7-11). The spore results from the condensation, 
at one of the poles, in the shape of a large chromatic granule, of part 



BACTERIA 97 

of the grains which compose this filament (Fig. 75, 12 and 13). An 
interesting variation of this structure is found in B. saccobrinchi. 
In this bacillus is noticed first an initial stage where the nucleus is 
represented by an axial filament quite similar to that olB.spirogyra 
(Fig. 75, 14). In the course of development, however, this filament 
resolves itself into a great many grains which scatter through the 
cell (Fig. 75, 15 and 16). The nucleus then becomes diffuse. Part of 
this diffuse nucleus next condenses at the time of sporulation into a 
large chromatic grain which forms the beginning of the spore. Finally, 
in other bacilli, Dobell finds in the whole development no more than a 
diffuse nucleus, that is, the structure described by Schaudinn and by 
Guilliermond. 

In the group of spirilla, Dobell notices these three types of structure: 
In some species he finds present a spherical body resembling a nucleus; 
other species show a zigzag or a spiral filament; still others have a 
diffuse nucleus. 

From these observations, Dobell feels authorized to conclude that 
bacteria are organisms originally containing a nucleus, but in which the 
nucleus, as a result of parasitism, has undergone a series of retrogres- 
sions which have ended by making it diffuse. 

This opinion would have the advantage of reconciling opposed 
theories. It would explain how some authors have been able to dis- 
cern a true nucleus in various forms. 

Another more weighty reasoning which might also explain these 
contradictions is the fact that under the name of bacteria are gathered 
forms perhaps very different, some of which seem to belong to the 
Sul Jo-bacteria and others might be considered as molds. 

Although we have just mentioned numerous works, the conclusion, 
to my mind, would be that while some bacteria may contain a more or 
less rudimentary nucleus whose existence is nowhere else precisely 
demonstrated, so far, in the great majority of the species, nothing more 
has been found than a diffuse nucleus consisting only of grains of chro- 
matin scattered through the cytoplasm. 

LIFE CYCLE OF BACTERIA. The Editor feels justified in adding 
to the foregoing review the very recent work of Lohnis and Smith, 
Journal of Agricultural Research, Vol. VI, No. 18, July 31, 1916, 
because, both for its suggestiveness and presentation of experimental 



98 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

evidence, it can not be disregarded in the intimate study of bacterial 
cells. Below is given in full the summary by the authors. 

Summary by Authors. A comparative study of 42 strains of 
bacteria has shown that the life cycles of these organisms are not 
less complicated than those of other micro-organisms. As represen- 
tatives of practically all groups of bacteria have been tested and all, 
without exception, behaved essentially in the same manner, in all 
probability analogous results may be expected with all species of 
bacteria. 

"All bacteria studied live alternately in an organized and in an amor- 
phous stage. The latter has been called the "symplastic" stage, be- 
cause at this time the living matter previously inclosed in the separate 
cells undergoes a thorough mixing either by a complete disintegration 
of cell wall, as well as cell content, or by a "melting together" of the 
content of many cells which leave their empty cell walls behind them. 
In the first case a readily stainable, in the latter case an unstainable 
'symplasm' is produced. 

"According to the different formation and quality of the symplasm 
the development of new individual cells from this stage follows various 
lines. In all cases at first "regenerative units" become visible. These 
increase in size, turning into "regenerative bodies," which later, either 
by germinating or by stretching, become cells of normal shape. In some 
cases the regenerative bodies also return temporarily into the sym- 
plastic stage. 

"Besides the formation of the symplasm, another mode of interac- 
tion between the plasmatic substances in bacterial cells has been ob- 
served, consisting of the direct union of two or more individual cells. 
This "conjunction" seems to be of no less general occurrence than the 
process first mentioned. The physiological significance remains to 
be studied. 

"All bacteria multiply not only by fission but also by the formation of 
'gonidia;' these usually become first regenerative bodies, or occasion- 
ally exospores. Sometimes the gonidia grow directly to full-sized 
cells. They, too, can enter the symplastic stage. The gonidia are 
either liberated by partial or complete dissolution of the cell wall or 
they develop while still united with their mother cell. In the latter 
case the cell wall either remains intact or it is pierced by the growing 
gonidia, which become either buds or branches. 



BACTERIA 99 

"Some of the gonidia are filterable. They also produce new bacteria 
either directly or after having entered the symplastic stage. 

"The life cycle of each species of bacteria studied is composed of 
several subcycles showing wide morphological and physiological dif- 
ferences. They are connected with each other by the symplastic stage. 
Direct changes from one subcycle into another occur, but they are rather 
rare exceptions. The transformation of spore-free into spore-forming 
bacteria seems to be dependent on the conditions acting upon the 
symplasm and regenerative bodies. 

"The discovery of the full life cycles of bacteria may be helpful in 
many directions. Systematic bacteriology now can be established on a 
firm experimental basis. Physiological 

studies will win considerably in con- * a * ' ;.. ' 

formity and accuracy when connected | 
with morphological investigations along m 3? 5 *6'^i 
these new lines. Several problems in ! * J t 

general biology are brought under more ; . ' 
promising aspects. Agricultural bacteri- ^ $ * * t $ 
ology and medical also will derive much Q 9 '\\j f\) 
benefit." 

RESERVE PRODUCTS. '-Besides the s t a d by^Sod wSh 

grains of chromatin which we have just differentiates only the meta- 

. , . . i . , chromatic corpuscles. 1-4 

been considering m bacteria are found Baci , lus radico ? us . s _ 6> Bac % 

other granulations which do not show lus asterosporus. 7, The same. 

. . r , ,. j , . , The cells have formed their 

the characteristics of chromatin and which spore and the me t a chromatic 

act as products of nutrition. These corpuscles outside of the spores 

. , , , . , have not yet been absorbed by 

granulations are characterized by the it s-g, Spirillum wlutans. 

reddish color which they assume with io-n, Bacillus alvei. 

most of the aniline blue or violet dyes, as 

well as with hematoxylin. These bodies, which are common to the 

majority of the Protista, are metachromatic corpuscles. 

They are found in larger or smaller numbers according to the species, 
the age of the cells, and the medium in which they are living. Some 
bacteria contain few metachromatic corpuscles (B. radicosus, megathe- 
rium, mycoides); others produce many (B. alvei, asterosporus, Sp. 
volutans, Bact. tuberculosis and diphtheria}. The metachromatic 
corpuscles appear at the beginning of development in the form of very 

Prepared by A. Guilliermond. 



100 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

small grains, which generally increase gradually in size during de- 
velopment, and finally are absorbed in the very old cells. They are 
sometimes distributed through the whole cell (Spirillum volutans) as 
grains of chromatin (Fig. 76, 8 and 9), but most often they tend to 
gather at the two poles of the cell, or line up all along the bacillus 
(Fig. 76, i to 4, 6, 10, n). In some species (B. alvei, asterosporus, 
Bad. tuberculosis and diphtheria), these corpuscles grow bigger until 
they attain relatively large dimensions, surpassing the bacillus in size. 
Thus they cause a series of swellings all along the bacillus, which in 
consequence appears somewhat like a necklace (Fig. 76, n). They 
then give the illusion of spores; one can easily understand the error 
of some authors who have confused them with spores, notably in the 
case of the Bact. tuberculosis. 

In B. asterosporus, the metachromatic corpuscles usually appear in 
the youngest cells, singly and in the shape of a small central granule 
closely resembling a nucleus and which A. Meyer seems to have taken 
for such (Fig. 76, 5). 

During sporulation, the metachromatic corpuscles exist just out- 
side of the spore (Fig. 76, 7), then are finally absorbed by it. They 
therefore act like reserve products. 

Moreover, in the cells of bacteria other reserve products, notably 
globules of fat and of glycogen, have been found. 

BACTERIAL CELL WALL. General Structure.* All the bacteria have 
cell walls and it is these that give definite form to the cell. These walls 
are rigid and elastic and are probably made up of two layers, the outer one 
of which is able to deliquesce and form capsules, or perhaps zooglcea. 
The inner part retains 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 intake and output goes on through the cell wall 
which is entire. 

Minute Structure of Cell Wall.^ In some species of large size, 
the membrane can be distinguished when strongly magnified, and 
appears with a double contour. Usually it is scarcely visible, and can 
be observed only when the contents of the cell has been contracted by 

* Prepared by W. D. Frost. 

f Prepared by A. Guilliermond. 



BACTERIA 



plasmolysis or by a suitable reagent. It is sometimes thin, some- 
times more or less thick. In the latter case, it is often possible to 
recognize two layers, an inner or cuticular layer, very thin and trans- 
parent; and the other external, not so well defined and thicker, jelly- 
like in appearance. This latter or gelatinous layer seems to result 
from a special differentiation of the peripheral zones of the inner layer. 
The outer layer ordinarily resists staining reagents and appears as a 
kind of transparent zone about the colored elements. It can acquire 
a relatively great thickness, and the formations described as capsules 
are only an exaggeration of this gelatinous layer. 

Schaudinn has been able to observe quite carefully the construction 
of the cuticular layer in B. butschlii. According to him, the membrane 
seen in profile would appear to consist of a 
series of disks alternately clear and cloudy (Fig. 
77, .1 and B). Seen from the front, it would 
give the impression of a network whose meshes 
are more refringent and stain more highly (C). 
It is laid on a peripheral zone of cytoplasm, a 
kind of ectoplasm with closer network, and is 
clearly differentiated from the rest of the cyto- 
plasm. The spore is provided with a double 
membrane and has at one of its poles a sort of 
micropyle through which germination is effected 
(Fig. 71, 15 and 16). StroctoujTif the mem- 

The chemical composition of the membrane brane and of the ecto- 
is little known. According to some authors, lutlrtiiL "c. Membrane 
this membrane consists of cellulose; according of the same bacillus, 
to others, it contains a lipoid substance; finally, ^hludinn^) 
by many authors it is supposed to be composed 

principally of nitrogenous compounds. Let us remark further that 
chitin has supposedly been detected therein. 

Capsules* A considerable number of the bacteria regularly, or 
under certain conditions, form what are known as capsules (Fig. 78). 
These are mucilaginous envelopes which in width frequently exceed 
that of the organism itself. In microscopical preparations of bacteria 
it is important to differentiate these from artifacts, since by ordinary 
staining methods the capsules are not colored but appear as colorless 

* Prepared by W. D. Frost. 




102 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

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 
bacteria which grow in the bodies of animals frequently contain these 
capsules but fail to show them when grown upon artificial culture media. 
It is difficult, therefore, to determine whether or not an organism has a 
capsule by mere examination of cultures. Some culture media, how- 




FIG. 78. Capsules. Bad. pneumonia (Friedlander). (After Weichselbaum from 
Frost and McCampbell.) 

ever, do cause a formation of capsules in the case of capsulated bacteria. 
These are blood serum, sometimes, and milk, usually. Beautiful cap- 
sules can be obtained by growing such bacteria as the Bact. pneumonia, 
Bact. capsulatum, and Bact. welchii in milk cultures. Strept. mesen- 
teroides is a bacterium which grows in the syrup of the sugar refineries 
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. 

FLAGELLA. General Consideration of Flagella* The flagella are 
very narrow thread-like structures. It is not known how narrow since 

* Prepared by W. D. Frost. 



BACTERIA 103 

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 




V 



FIG. 79. FIG. 80. FIG. 81. 

FIG. 79. Chromatium okenii; 2, Bacterium lineola; 3, 4 and 5, sulpho-bacteria; 
, Ophidomonasjenensis; 8, and 9, Spirillum undula; 10, Cladothrix dichotoma. (After 
iiitschli from Guilliermond review, Bull. Inst. Past.) 

FIG. 80. Microspira comma. Monotrichous bacteria. (After Migula from 
Schmidt and Weiss.) 

FIG. 81. Pseudomonas pyocyanea. Monotrichous bacteria. (After Migula from 
Schmidt and Weiss.) 

hem and sometimes many times that length. B. symptomatic* 

nthracis found in the soil has a flagellum sixty times its own length. 

The arrangement of the flagella on the bacteria is quite constant and 






FIG. 82. FIG. 83. FIG. 84. 

FIG. 82. Pseudomonas syncyanea. Lophotrichous bacteria. (After Migula from 
Schmidt and Weiss.) 

FIG. 83. Spirillum rubrum. Lophotrichous bacteria. (After Migula from 
Schmidt and Weiss.) 

FIG. 84. Bacillus typhos us. Peritrichous bacteria. (After Migula from Schmidt 
uid Weiss, and Frost and McCampbell.) 

s used by some authors to differentiate genera. Very few of the 
nicrococci are provided with flagella, as was indicated above, and in 
the bacilli and spirilla they may be arranged at the poles singly or in 



104 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

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. 79, SoandSi). When they 
are arranged in brushes, the arrangement is spoken of as lophotrichous 
(Figs. 82 and 83) and when they are arranged on the entire periphery, 
the arrangement is said to be peritrichous (Fig. 84). 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 
connection 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. 

Minute Consideration of Flagella* The question of the cilia or 
flagella of bacteria is not yet entirely decided. The absence of cilia 
in large bacteria capable of motion gives the idea that these are not the 
only organs of motion, and that contraction of the protoplasm certainly 
plays the most important role in the phenomena of motility. More- 
over, the nature of cilia has been debated. Van Tieghem and Biitschli, 
taking their stand primarily on the difficulty of staining cilia by the 
reagents which rapidly color protoplasm, have considered these cilia 
to be simply prolongations of the membrane, lacking all contractibility 
and locomotive power. According to Van Tieghem, when two cells 
formed by the division of the same element separate, the common por- 
tion of the transverse septum, instead of dividing neatly in two, can 
stretch out into a filament which breaks at a greater or less distance from 
each of the two daughter cells. This prolongation composes the 
vibratile cilium. 

This theory, however, does not explain the existence in certain 
bacteria of clusters of cilia at the two poles, or of cilia distributed over 
the whole surface of the membrane. Other authors, as for example 
A. Fischer, consider the cilia true prolongations of the protoplasm 
issuing through tiny apertures in the membrane. This view at present 
tends more and more to predominate, and the existence of flagella on 
bacteria appears to be demonstrated. 

Another interesting peculiarity, moreover, has recently been estab* 
lished independently by Swellengrebel and by Dangeard. According 
to these authorities, in some species (Chromatium okenii and Spirillum 

'Prepared by A. Guilliermond. 



BACTERIA 105 

volittans) the cilia have connection with one of the chromatic grains of 
the diffuse nucleus. There is a chromatic filament starting from the 
base of the cilium and ending in connection with a chromatic grain, 
similar to the organisms with flagella in which the flagellum is in 
Delation to a basal chromatic grain (blepharoplast). 

THE HIGHER BACTERIA* 

The so-called higher bacteria include some of the spiral forms, at 
Jeast the larger spirochaetes, the thread or trirkobacteria, and the 
[sulphur or thiobacteria. 

The spirochaetes and trichobacteria contain so many forms of 
Interest that their form and structure needs special consideration. 

THE LARGER SPIROCHAETES. Spirochaetes differ so much among 
(themselves that it seems necessary to divide them into two groups. 
[The members of one of these groups, the small spirochaetes, are prac- 
tically identical with the true bacteria, and naturally fall in the family of 
the Spirilliacea. Members of this group, however, so gradually approach 
the other group, the large spirochaetes, that it is difficult to draw a line 
pf separation between the two, yet the large spirochaetes resemble in 
po many essential details the trypanosomes that they are usually placed 
jas a coordinate genus with them under the flagellates a sub-class of 
the Protozoa. The larger spirochaetes are described as follows: 

Form and Size. In form the spirochaetes are long, very thin and 
flexible spirals. Their length is usually not less than twenty times their 
[breadth. Some forms are as long as 500 ju. It seems probable that 
borne of them are flattened and hence in form are more like a spirally 
ibent ribbon than rod. 

Motility. These organisms move very rapidly under normal con- 
ditions. The character of the movement may be of three kinds: 
|(i) Lashing, eel or snake like; (2) undulatory, compared to the flapping 
>f a sail in the wind; (3) rotation, similar to a cork-screw when pushed 
nto a cork. 

Reproduction. Multiplication is by means of binary fission. If 
these forms are to be considered as bacteria, the division would be 
expected to be by means of transverse partition walls. A number of 
workers, however, have described a process of longitudinal division,. 

Prepared by W. D. Frost. 



106 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

Forked forms also which are frequently seen are held to indicate longi- 
tudinal divisions. Some observers have claimed that conjugation 
occurs among the spirochaetes. If this is true their relation to the 
Protozoa would be quite likely, but accounts of this phenomenon are 
inconclusive. Several observers have described "rolled up" specimens, 
oval and ovoid forms, which have been assumed to be cysts. The 
spirochaetes break up into granules or short segments and such speci- 
mens are sometimes spoken of as "monili form." It is not definitely 
known whether these coccoid forms are simply degenerative forms or 
the equivalent of bacterial spores. 

Sheaths. A definite sheath has been described for some forms 
and the irregularity in the disposition of this around the cell may 
account for the structures that have been taken for undulating 
membranes. 

Cell Aggregates. There is apparently no definite cell grouping but 
tangled masses of these organisms have been described in several 
species. 

THE TRICHOBACTERIA. The trichobacteria are thread or fila- 
mentous forms. The cells are cylindrical and similar in form and 
may or may not vary in size in different parts of the filament. The 
individual cells are capable of independent existence, but when growing 
in the filament give evidence of differentiation in function. Some- 
times these filaments are attached to the substratum or some object in 
it; at other times they are free. In case of the sessile forms the cells 
at the attached end (base) are smaller than those at the apex. In 
other members of the group the ends of the thread are swollen or 
become club-shaped (Figs. 85 and 86) . In some forms cell division takes 
place in three directions of space, thus forming a thread of massed 
cells. 

Branching. The filaments are usually unbranched, but some 
forms show true branches, such as is found among the plants fungi 
and algae. Some again exhibit what is called false branching. This 
is due to a misplaced cell, which grows parallel or at an angle to the 
parent thread and suggests branching. 

Reproduction. The cells throughout the filament may divide to 
form spores, but the apical cells of the thread are frequently set apart 
for the purpose of reproduction, and by a process of division form 
spores or conidia. The conidia are usually round and without any 



BACTERIA 



107 



psting stage may produce new threads of cells. Sometimes spores 
terminate while still in the old thread (Fig. 85), giving a tangled 
bass of cells or whorls of new threads at intervals on the old. The 
'bnidia may be either motile or non-motile. The motility of these 
ipnidia when it exists is due to flagella. 

Slieath. The threads of cells are sometimes surrounded by sheaths 
tjf varying thickness. This sheath is a thickened and hardened mem- 




'IG. 85. Crenothrix polyspora Cohn, Brunnenfaden. 

and Weiss.) 



(After Migula from Schmidt 



rane, and forms a tube in which the different cells of the bacteria are 
ontained. This sheath is homologous to a capsule. In it are fre- 
uently deposited characteristic by-products of the cell. In Cretw- 
krix (an iron bacterium), for example, we have iron oxides. 

The best-known member of this group is the water-pest bacterium 



108 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

(Crenothrix polyspora) (Fig. 85), an iron bacterium, which has the 
power of oxidizing certain forms of iron, causing a deposit to accumu- 
late in the water pipes of cities where it may cause considerable trouble. 
It is probable also that this bacterium has had a very important part 
in the deposition of our iron ores, such as those found on the Mesaba 
range. Another member is the Actinomyces boms (Fig. 153) 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. 

THE SULPHUR BACTERIA. The sulphur bacteria are filamentous 
forms which may reach a length of many microns. They are cylin- 
drical or perhaps sometimes flat. They may be either attached or 
actively motile. The movement when present is due not to flagella, 
but to an undulatory motion like that of the spirochaetes or Oscillaria 
among the algae. " As they move forward they rotate on their own axis 
and swing their free ends. 

Spore formation is unknown in some forms where multiplication is 
accomplished by the breaking up of the threads in short segments. 
In the case of the sessile forms conidia are produced at the end of the 
thread and are motile (Thiothrix nivea). The sulphur bacteria contain 
at certain stages strongly refractile sulphur granules in their bodies. 

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 
definite determination (of species) requires so much time, so much oi> 
acumen 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, an^j 
nowadays we are finding it as difficult to decide upon a system of clas- 
sification. A great many systems have been proposed, but many ol> 
them are untenable because those who proposed them were ignorant oi> 
or unconcerned by the rules adopted by systematists in other lines, 
The only system that seems worthy of continued life is that of Migula, 

* Prepared by W. D. Frost. 



BACTERIA IO9 

vho is a trained botanist. This system, with sight modifications, is 
;iven below. In this system, the characters which separate the 
renera are morphological; while physiological characters, including 
rultural, are used for the differentiation of species and smaller groups. 
)ne of the rules adopted by systematists in other lines is the binomial 
ulc. In the violation of this rule, bacteriologists have been great 
.inners, and some of the names proposed by Migula and others follow- 
ng his system are quite different from those by which well-known forms 
lave been christened by their discoverers. 

CLASSIFICATION OF MIGULA (MODIFIED) 

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

I. Order: Eubacteria (true bacteria). 

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

I. Suborder: Haplo'bacterinae (lower bacteria). 

I. Family: Coccaceae (ZOPF) MIG. 

The cells are globular when in a free state, but in the various stages of division 
[ppear somewhat elliptical. A few species in this family are motile. Cell division. 
akt - 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 
pikes place in only one plane. Usually the cells remain united together after 
I i vision, producing chains or diplococcus forms. No endospores have been noted. 
I Genus: Micrococcus (HALLIER) COHN. 

The cells are globular and do not possess any organs of locomotion. Cell division 
lace in two planes at right angles. If the cells remain attached together after 
idl division, merismopedia plates are formed. The plates give the appearance of a 
Rigular 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 
akt-s place in three planes, all perpendicular to each other. Its cells remain attached 
:i vision; cube-like packets are formed. The composition of the medium come- 
pmes prevents this typical cube formation. 

Genus: Planococcus MIGULA. 

The cells are globular. Cell division takes place in two planes at right angles 



no 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



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 spher- 
ical bodies to very long rods. Cell division takes place in one direction in a plane 




FIG. 86. Chamydothrix hyalina Migula. (After Migula from Schmidt and Weiss.} 

perpendicular to the long axis of the cell. Some of the members of this family remaic 
attached together, forming threads, while others separate from each other soon af tei 
fission. 

Genus : Bacterium EHRENBERG. 

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



BACTERIA III 

Genus: Bacillus COHN. 

The cells are cylindrical, of longer or shorter length. The rods are sometimes 
fcval in shape. Cells are motile and possess flagella which are distributed over the 
Entire surface. Endbspore formation occurs with marked regularity. The bacteria 
In this genus are motile only during certain periods of their life. This period varies 
treatly 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- 
rtremely rare. Occasionally certain species in this genus form themselves into threads 
pr chains. 

III. Family : Spirillaceae MIGULA. 

The cells are wound in the form of a spiral or representing the portion of a turn 
iOf a spiral. In the latter case, if the cells remain attached together in the form of a 
[thread, a full spiral of several turns is produced. Cell division takes place in only 
ipne 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 
envelope, while others are free. 

Genus: Microspira SCHBOTER. 

The cells are rigid, short, and bent similar to a comma. When the cells are united 
jtogether, 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 
is 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 the 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 
jacteria 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. 



112 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



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. 86). Reproduction takes 
place by means of gonidia, which are round and arise di- 
rectly from the vegetative cell. This genus is called Lep- 
tothrix by KUTZING and Streptothrix 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 per- 
pendicular to the long axis of the cell and later by division 
in three directions of space. Only one or possibly two 
species can be placed in this genus. 
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 KUTZING, 
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. 87). 
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 sul- 
phur. The cells are colorless or pigmented rose, violet, 
or red by bacteriopurpurin. The cells are never pig- 
mented green. 

I. Family : Beggiatoaceae TREVISAN. 

Filamentous bacteria which do not contain bacteriopurpurin. The cells contain 
sulphur granules. Reproduction takes place in one direction of space. 



FIG. 87. Clado- 
thrix dichotoma 
Cohn. (After Fis- 
cher from Schmidt 
and Weiss.) 




FIG. 88. Beggiatoa 
alba. Vaucher, Trevi- 
san. (After Winogradsky 
from Schmidt and Weiss.) 



BACTERIA 113 

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 produced at the end of the threads. These gonidia are 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. 88). This genus moves by means of an undulating mem- 
brane 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: Rhodobacteriaceae (WINOGR AD SKY'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. 

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 SCHROTER. 

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 Amcebobacteriaceae. 

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



114 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

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 Chromatiaceae. 

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. 

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 diffi- 
cult to determine their exact relation philogenetically. These diffi- 
culties 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. 
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 nucleus. It is quite 
impossible to decide, furthermore, whether some forms, such as Bact. mride 
and Bact. chlorinum, 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 resemblance is 
the possession of flagella. Some of the flagellates quite closely resemble 
the bacteria in many ways, and the Spirochczta, which are usually 

* Prepared by W. D. Frost. 



BACTERIA IIS 

llieved to be bacteria, have been classed as flagellates by eminent 

lotozoologists. 

I Physiologically the bacteria are quite closely related to the fungi. 

Id are frequently classed with them under the term Schizomycetes. 

ARTIFICIAL CULTIVATION OF BACTERIA* 

I The introduction of methods of artificial cultivation marks the beginning of the 
fence of microbiology. These methods were developed by Pasteur and Koch and 
le depended upon by the microbiologist of to-day as the foundation for most of his 
fcrk. It has been the aim of investigation to discover a more general culture 
ledium. So far it has been impossible to do this, but beef broth, made after a 
Irmula suggested by Loeffler many years ago, forms the basis of nearly all of our 
llture media. This beef broth, or nutrient bouillon, is made by extracting meat 
lee from fat in water, adding a small per cent of peptone, correcting the chemical 
[action, clarifying and sterilizing. To this broth various substances are added 
Ir special purposes; gelatin and agar, in order to solidify the media, and various 
Igars and other chemical substances for the purpose of determining the physiological 
laracteristics of various bacteria. One of the difficulties with the present methods 
I the artificial cultivation of bacteria is the inconstancy of the composition of the 
ledia, due to the fact that the extract of beef, the peptone, and other ingredients, 
Iknnot be obtained chemically pure. If it should prove possible to use synthetic 
Ibstances, such as the polypeptids, it would mark a great step in advance, but it is 
robably qu'te impossible to devise a single medium upon which all bacteria will 
1-ow. Some bacteria, such as those which produce nitrification, refuse to grow on 
fdinary media containing organic material. The cultivation of bacteria in pure 
ulture is dependent upon isolation, and the method of isolation suggested by Robert 
loch in 1880, and known as the plate culture method, has given eminent satis- 
Lction. This method is dependent upon the use of liquefiable sob'd media, such 
b gelatin or agar. 
Prepared by W. D. Frost. 



CHAPTER V 
INVISIBLE MICROORGANISMS* 

The term " in visible microorganism" is used interchangeably witl 
such expressions as " ultra-microscopic organism," "invisible virus' 
and "filterable virus" to designate a group of microorganisms which 
for the most part, 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 nitrations and inoculations the disease can bci 
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 lyrnphi 
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. 

f Bovine pleuropneumonia and such others as may respond to the cultural method o^ 
Noguchi. 



INVISIBLE MICROORGANISMS 



117 




FIG. 89. Apparatus for fractional filtration, designed for use with Pasteur- 
amberland or Berkefeld filters, a, Glass mantle surrounding filter; b, Chamber- 
d filter; c, paraffin joint; d and e, rubber stoppers;/, double side-arm suction flask; 
)inchcock controlling outlet from suction flask; h, outlet tube surrounded by glass 
E:ld and attached to lower end of suction flask by means of short rubber tubing; 
lass shield fused to and surrounding outlet tube as a protection against contamina- 
i when the filtrates are drawn off; j, glass inlet tube plugged with cotton, for ad- 
tting air into suction flask; k, pinchcock governing the admission of air into flask; 
f acuum gauge; m, stopcock connected with vacuum pump. (U. S. Dept. of Agri- 
lure, Bureau of Animal Industry, BuH. 113.) 



Il8 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

pleuropneumonia, cattle plague, swamp fever or infectious 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, 0.15/4 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, j 
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 in- 
ability to imitate exactly in the laboratory the conditions which exis' 
in the animal body. 

While the invisible microorganisms possess certain qualities in com 
mon, in some respects they differ widely from one another. Some wil 
pass only through the coarsest of bacteria-proof filters, while others pas 
readily through the densest filters, thus indicating wide differences .jb 
size or in structure. Some are very susceptible to the action of germici 
dal agents, whereas others are more resistant than the ordinary bacteria 
Some produce disease in only one species of animal, while others sho\ 
little or no limitation in this respect. The diseases produced by thes 
microorganisms likewise differ markedly, some being comparative! 
benign and local in character, whereas others appear as the most prc 
found septicaemias. Some are extremely contagious, while others ca 
be transferred from one animal to another only by means of an inter 
mediate host. In fact these invisible microorganisms seem to diff( 
among themselves quite as widely as do those which are visible to u 

The existence of an invisible microorganism is determined as follow^ 

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



INVISIBLE MICROORGANISMS IIQ 

during nitration. 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. Recent suggestive developments have thrown some light on 
the possible nature of filterable viruses. The reader is referred to the 
work of Flexner and Noguchi since 1912, published in the Journal 
of Experimental Medicine; he is also requested to read the article by 
Lohnis and Smith already mentioned on page 97. 



CHAPTER VI 
PROTOZOA* 
INTRODUCTION 

Many of the diseases which are known to be due to an infecting 
agent are caused by bacteria; but others are caused by protozoa. 

The bacteria belong to the vegetable kingdom. The protozoa are 
unicellular animals; they are extremely numerous and are very widely 
distributed in nature, occurring in water, soil, and in the bodies of most 
animals. 

From a zoological point of view, the protozoa constitute an impor- 
tant sub-kingdom. It is sometimes difficult to say whether a minute 
organism is a plant or an animal. For this reason, primitive unicellular 
organisms are sometimes classified by themselves, as Protista (page 114), 
a kingdom which thus includes not only primitive organisms which 
have not yet been definitely established in either group but also certain 
unicellular animals and plants. It appears important, however, to 
determine as far as possible the genetic relationship of various or- 
ganisms and, by the study of their physiology and modes of develop- 
ment to differentiate between those which are plant-like and those 
which are animal-like in character. The protozoa are thus included 
in the animal kingdom and have been defined as "unicellular animals." 
They are to be distinguished, on the one hand from primitive forms such 
as bacteria which lacking differentiation of nucleus and cytoplasm 
do not conform to the type of structure of true cells, on the other hand, 
from unicellular organisms of plant-like character such as algae and 
fungi which are included with the Protophyta. 

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. While a large proportion of the protozoa are free-living, 
others are parasitic on animals and plants. Some of the parasitic 
protozoa are practically harmless and do no apparent injury to the 

* Prepared by J. L. Todd. Revised by E. E. Tyzzer. 

120 



PROTOZOA 



121 



iftiosts which support them; others produce severe diseases. Before 
mentioning those especially which cause disease (see page 822) it will 
be well to consider the protozoa as a class and to discuss the characters 
jwhich all have in common. 

STRUCTURE OF THE PROTOZOA 

Most protozoa are so small as to be visible only by the aid of the 
microscope but certain species are visible to the naked eye as individuals, 




FIG. 90. Amabci vespertilio. (After Doflein.} 

agglomerated masses 'of individuals. For example, the Sarco- 
sporidia, which occur in the muscles of mice and other animals, can 
easily be seen without a microscope, and the huge plasmodial masses 
if .\fycetozoa, which are sometimes seen on rotting wood or in tan 
>its, may measure many centimeters in breadth. 

Like all living things, the protozoa are composed of protoplasm (page 
t8) and its products. Protoplasm is a complex mixture of various sub- 
stances in a colloidal condition. When studied by appropriate methods, 



122 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

the protoplasm of a cell appears to be alveolar or foam-like in structure. 
This is because the protoplasm is emulsoidal in character being com- 
posed of a mixture of many more or less non-miscible substances, 
some of which are fluid in character, others more of the nature of 
solids. In such a mixture, the more viscid materials form tiny 
globules, and each of these is surrounded by a layer of softer material 
(Fig. 90). The alveolar or foam-like appearance of the cytoplasm 
of a living cell is somewhat similar to that of 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, but the outline 
of the alveoli, which are not in contact with a firm structure, is more 
nearly 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. 

Recent studies in colloidal chemistry and in the microscopic dissection 
of cells have furnished valuable contributions to the knowledge of the 
chemical and physical properties of protoplasm. The view has been 
advanced that protoplasm consists largely of material in a state known 
in colloidal chemistry as a gel, some portions being firm and viscid 
and others very soft in character. Procedures which convert such 
material into a sol or fluid state are said to cause the protoplasm to 
quickly disintegrate. Certain portions of the cell such as the limiting 
membrane, the nuclear membrane and the nucleolus are of firmer 
consistence than other portions, and some cells contain globules and 
granules of various types. 

The protoplasm of a protozoon may be divided into two main 
portions: the cytoplasm and the nucleus, Chapter I. 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 endo plasm. Denser, more resistant fibers some- 
times 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 a structure which is differ-] 
cntiated from the remainder of the cell by being more refractile ancj 
by being colored more deeply in specimens which have been stainecl 
by dyes. It stains deeply because it contains a substance called chro\ 
matin. The chromatin usually occurs in granules which may var}| 



PROTOZOA 123 

considerably in size and which are supported upon a linin framework 
that does not stain by ordinary methods. 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 termed chromidia so that, at the 
first glance, no nucleus seems to be present. Such chromidia may be 
said to constitute a distributed nucleus, although the term nucleus is 
usually applied to a well differentiated cell structure. 

The nucleus (page 1 5) is to be regarded as the most important unit 
in the structure of the cell and is apparently essential for the con- 
tinued existence of the latter. If cells are bisected portions contain- 
ing no nucleus invariably die while portions containing the nucleus 
may continue to live and eventually recover from the injury. The 
role of the nucleus is not fully understood but it seems certain that it 
is a controlling center for the cell's activities. It is concerned in the 
nutrition of the cell, frequently nuclear structures have to do with the 
motility of cells and the chromatin serves as a medium for the 
hereditary transmission of specific characteristics. Its functions, 
therefore, are at least three-fold since it is active in a trophic, kinetic 
and reproductive capacities. Usually, all these functions are subserved 
by a single nucleus; sometimes, however, as in the flagellates and 
many ciliates they are divided between two nuclei. 

ACTIVITIES OF THE PROTOZOA 

Whereas the higher animals or Metazoa are composed of a great 
number of cells, a protozoon consists of a single cell. In the former 
the various functions of the body are each carried out by a special type 
of cell; for example, movement is performed by the muscle cells, 
digestion is provided for by the cells of the alimentary tract, and urine 
is excreted by the kidney cells. A protozoon being a unicellular 
animal, these various functions must be performed within the single 
cell of which it consists. Consequently certain parts of its protoplasm 
are especially differentiated and functionate in a manner similar 
to the organs of multicellular animals. Such differentiated parts are 
termed organella and by means of these the protozoa move about, 
feed, and excrete waste products in many respects like the higher 
animals. 



124 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



The activities of a protozoon may be considered under LOCOMOTION, 

METABOLISM* and REPRODUCTION. 

LOCOMOTION. The protozoa have several different modes of mov- 
ing themselves about. Some of them move by the formation of 
temporary processes or pseudopodia; in 
this method of progression, the protoplasm 
flows out, in finger-like processes, from the 
body of the organism and, as the protoplasm 
flows into these processes, the whole organ- 
ism 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. These 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 processes, which may 
occur either in definite areas or in large 
'numbers over the whole surface of a proto- 
zoon. They produce motion by waving 
and acting together make a strong simul- 
taneous stroke in one common direction. 
The movement of all the cilia of an organ- 
ism is, however, usually not synchronous 
but proceeds in waves across the surface 
of its body so that the appearance is simi- 
lar to that produced when a breeze passes 
across a field of grain. 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 or- 
ganism. Undulating membranes consist either of a thin fold of the sur- 
face layer or of rows of fused cilia and form either fin-like organs ex- 

* Will be treated in Part II, Physiology. 







FIG. 91. Paramecium 
caudatum: division showing 
the macronucleus (N) divid- 
ing without mitosis, the mi- 
cronucleus (ri) dividing mi- 
totically. c.v 1 ., Old, and c.v 2 ., 
new, contractile vacuoles. 
(Minchin, after Butschli and 
Sehewiako/, in Leuchart 
and Nitsche's Zoologische 
Wandtaflen, No. LXV.) 



PROTOZOA 



125 



tending along the surface of the organisms or special organs for the 
intake of food. 

REPRODUCTION. The protozoa reproduce in many different ways 
and 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 union 




IG. 92. Stages in the division of A maba poly podia. (After F. E. Schulze and Lange 

from Dojlein.) 

and reproduction, though now combined in many animals, may have 
been 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. 

The simplest of the methods of reproduction is simple binary divi- 
sion, in which the organism divides into two equal parts. A modifica- 
m of this process is gemmulation, in which a small protozoon buds off 



126 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



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 pro- 
duce a large number of daughter cells simultaneously, the process is 




FIG. 93. Coccidium schubergi. A-C, asexual multiplication; D-K, sexual multi- 
plication; D, microgametes; E, macrogamete; F, G, fertilization; H, I, K, division 
and spore production. (After Schaudinn, from Dofiein.) 

called schizogony and the young parasites are called merozoites, i.e., if a 
sexual fertilization has not immediately preceded the act of division; 
if such a division, in which the parent organism disappears, takes place 
after a fertilizing act, the process is called sporogony and the young 
parasites are sporozoites. 



PROTOZOA 127 

In protozoa, as in metazoa, the essential process in fertilization is the 
union of two nuclei of opposite sex. In the metazoa the nuclei of the 
germ cells undergo before they are ready to unite repeated divisions, 
In which the number of the chromosomes is reduced to one-half the 
Msual number. In dividing, cells may go through a process called 
mitosis during which the chromatin of the nucleus is grouped into more 
br less rod-shaped masses which are called chromosomes. The number 
of chromosomes which are formed during mitosis is constant and char- 
lacteristic for each species. In the reproductive areas, during the two 
(divisions just preceding the maturity of cells which are to become ova 
or spermatozoa, the number of chromosomes is reduced to exactly one- 
half of the number which are formed during the division of cells outside 
<>f the reproductive areas of the same animals. The process by which 
ithe number of chromosomes is reduced to one-half is termed chromatic 
reduction, and the fragments of chromatin which in the female are unused 
and which are extruded from the cell during the process are called polar 
bodies. While reduction in the number of chromosomes has been 
: shown to occur prior to fertilization in a number of the protozoa, in 
[many species a more primitive process consisting of the mere extrusion 
of masses of chromatin irrespective of the number of chromosomes is 
found to occur. It is evident that the chromatin is at least usually 
reduced in amount preparatory to the sexual process. 

Although in certain of the protozoa nuclear division is accomplished 
by a process of mitosis similar to that which occurs in multicellular 
animals, in many it is affected by a much more primitive process. 
The nucleus may be resolved into scattered granules of chromatin 
chromidia which may subsequently become reconstructed into a num- 
ber of nuclei. The nucleus may divide by direct division, that is, by sim- 
ple constriction into two approximately equal parts. Between this form 
of division and the classical mitosis there is every possible transition. 
The centrioles or centrosomes are frequently intranuclear in the 
protozoa. In case of primitive nuclei without definite nuclear mem- 
brane a division simulating mitosis is termed promitosis. In other 
forms in which there is a nuclear membrane but in which the centrioles 
remain intranuclear throughout division, the process is called meso- 
mitosis. The nuclear membrane often persists throughout division 
and the chromosomes are in many forms very minute or are not 
definitely formed. 



128 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

The fertilizing processes which occur in the protozoa may be grouped 
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 there are the male or micro gametes, and the 
female or macrogametes. 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. Aniso- 
gamous copulation/ the union of two unequal cells, is most typically 
seen in the fertilization of a large macrogamete by a small microgamete. 
Copulation is the most common fertilizing process among the patho- 
genic protozoa. Conjugation, the second method of fertilization, only 
occurs among the ciliata. In it, two adult individuals place themselves 
in apposition. The nucleus of each cell first reduces 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 partheno- 
genesis, are included under the third heading. In autogamy the nucleus i 
of a single cell divides into two parts. Each of these may undergo | 
further division, during which the chromosomes are reduced or there | 
may be a simple extrusion of a portion of the chroma tin. The two 
resulting, reduced nuclei then unite, in the same cell, to form a new \ 
nucleus. Parthenogenesis is the development of new individuals from a 
female cell without a preceding fertilization; this process possibly occurs 
in many protozoa, and through it perhaps may be explained the reap- | 
pearance of malaria in patients who once suffered from that disease 
and were thought to have recovered. 

The LIFE CYCLE of a protozoon consists of the changes through i 
which it passes in the period intervening between each fertilizing act. P 
In many of the pathogenic protozoa, an alternation of generations r 
occurs; that is, cycles of development in which an asexual method of re- 1 
production occurs, alternate cycles of development in which reproduction [ 
is effected by sexual methods. The developmental cycles are com-) 
monly punctuated by binary or multiple division, by encystment, i 
and by transference to a second host as a necessary factor for the; 
completion of the life cycle. An alternation of generations occurs 
in the life cycle of one of the most important of the pathogenic protozoa, j 
the parasite which produces malaria (Fig. 177). While it is in the body j 



PROTOZOA 129 

;>f its mam::: Anan host, man, it multiplies through multiple fission or 
(schizogony; the sexual, or propagative phase of its development 
occurs 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 
Jthe parasite while it is in other stages are called intermediate hosts. 

I .NCYSTMENT. Under unfavorable conditions, such as dry surround- 
ings, 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 harmful 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 multiplication 
jor continuous existence independent 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, and, as in the coccidia, 
the presence of such a stage is often absolutely essential in the life 
pistory of a parasite. 

PARASITISM 

A parasite is an organism which is, at some time, directly dependent 
(upon another, usually, a larger organism. 

The literal meaning of the term, i.e., eating at the table of another, 
implies living at the expense of or to the detriment of another. 

Although the word parasite is often used as though it referred only 
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-produc- 
ing protozoa, and the biting insects which transmit them, are tem- 
porarily 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, by means of which food is usually 
obtained, gradually disappear; degeneration always occurs in an 
organism which assumes a parasitic mode of life. 

Organisms, such as the malarial parasite, which are wholly de- 
ll 



130 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

pendent 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. Faculative parasites 
often feed upon organic material provided by the host, and not upon 
the host itself; but they are capable of living indefinitely apart from 
the host. 

If an organism is attached to a host, and neither harms nor benefits 
it, such an organism and its host are said to be commensals. For 
example, the spirochsetes 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 may perish 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 expresses itself in derangements in the physiology of the latter 
which are known as disease. The pathogenic protozoa may injure 
their hosts in at least three ways: They may feed upon, and destroy 
cells; they may produce poisonous toxins; and their presence may do 
damage by mechanically obstructing some of the functions of its 
host. All three of these ways are well exemplified by the action of the 
malarial parasite in man (page 832). 

DISCUSSION OF THE CLASSIFICATION* 

The following grouping of the Protozoa gives a general idea of the 
position, in zoological sequence, of the individual parasites which are 
spoken of in the subsequent pages. The Protozoa are here grouped 
into four classes: the RHIZOPODA, the FLAGELLATA, the SPOROZOA, and 
the INFUSORIA; and these classes are divided directly into genera. This 
is by no means a complete classification of the protozoan families, for 
there are many orders, families 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 herpetomonas is an active organism 
moving by means of a flagellum, quite unlike its spherical form which 
is without a flagellum. Consequently, the whole life history of a proto- 
zoon must be known before it can be classified with absolute certainty,) 
The whole of the life history is known for only a few protozoa; and 

"(See p. 13.) 



PROTOZOA 



though the organisms mentioned in this classification are placed in 
the position usually given to them, it must be understood that this 
classification is not final, and that the discovery of new stages in the 
life history of some of these protozoa may make it necessary to remove 
them from the classes in which they have been placed. For example, 
before its flagellate stage was known, 
Leishmania donovani was classified with 
the sporozoa; now it is grouped with the 
herpetomonads. 

The characteristics of the different 
genera and of the unimportant parasites 
are very briefly mentioned in the follow- 
ing paragraphs; the important parasites 
are treated more fully in the pages indi- 
cated 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 with- 
out permanent organs of locomotion; it 
moves about and captures its food 
through the agency of its pseudopodia. 
Very few of the rhizopods are parasitic; 
most of those which are parasitic, belong 
to the genus Entamceba. Different 
species of parasitic amoebae may occur 
in the alimentary canals of various ani- 
mals. Certain of these produce serious 
,ses (page 822). 

The FLAGELLATA are distinguished 
iy possessing one or more flagella; 
they often have, also, a fin-like, undulating membrane extending 
along the surface of their body. Many possess two nuclei, a larger 
trophonucleus which has to do with nutrition and a smaller kineto- 
nucleus which is intimately connected with the organs of locomo- 
tion. This group has thus been termed the Binudeata by certain 
systematists. Most flagellates are free-living. Comparatively few 




B 



FIG. 94. Herpetomonas 
musca-domesticcs (Burnett). A, 
motile individual with two flag- 
ella; B, cyst; n, nucleus; M, 
kineotonucleus. (After Pro- 
wazckfrom Minchin.) 



132 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

species are parasitic, but some of these cause very serious diseases 
(page 824). 

The Herpetomonad is an elongated organism which possesses tropho- 
nudeus and kinetonucleus . The latter is situated near the flagellar or 
anterior end of the parasite, and from it arises a terminal flagellum. 
Crithidia is an organism very much resembling an Herpetomonas, with a 
pear-shaped body, and, sometimes, a rudimentary undulating membrane, 
Trypanosoma is an elongated parasite which has a trophonucleus, 
a kinetonucleus usually situated near its aflagellar extremity and an 






FIG. 95. A, Trypanosoma tineas, of the tench; note the very broad and undulat- 
ing membrane in this species; B., C., T. percce. of the perch, slender and stout forms. 
(After Minchin, X 2000.) 

undulating membrane along the border of which the flagellum extends 
to terminate in a whip-like appendage. Species of Herpetomonas, 
Crithidia and Trypanosoma are frequently found in the intestines of 
insects. One species of Herpetomonas is a frequent and harmless para- 
site in the intestine of the house fly. The genus Try pano plasma in- 
cludes 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, unimpor- 



PROTOZOA 



133 



tant flagellate organisms which have been found, occasionally in 
man in the alimentary tract, and in necrotic material from the lungs. 
Trichomonas is a pear-shaped organism which has four flagella at- 
tached to its blunt end, and an undulating membrane extending from 
the origin of the flagella at the anterior end posteriorly over the sur- 
face of its body. One of the four flagella is usually directed 
backwards and extends along the border of the undulating membrane. 




FIG. 96. Trichmonas eberthi, from the intestine of the common fowl; ///., 
anterior flagella, three in number; P.fl., posterior flagellum, forming the edge of the 
undulating membrane; chr. I., "chromatinic line," forming the base of the undulating 
membrane; chr.b., "chromatinic blocks;" &/., blepharoplast from which all four 
flagella arise; m., mouth opening; N., nucleus; a*., axostyle. (From Minchin, after 
Martin and Robertson.) 

One species 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 



134 



MORPHOLOGY AND CULTURE OF MICROORGANISMS 



sucker, by which it attaches itself to the intestinal epithelium of the 
animal in which it lives. It is said to cause diarrhoea in man, and 
also a fatal disease of the intestines in rabbits; but it is almost invari- 
ably found in the duodenum and first portion of the small intestine of 
normal laboratory animals such as mice, rats, and rabbits. 




FIG. 97. Lamblia intestinalis. A, Ventral view; N., one of the two nuclei; ax., 
axostyles;^. 1 , fl. z , fl. 3 , fl.*, the four pairs of flagella; s., sucker-like depressed area on 
the ventral surface; x., bodies of unknown function. (After Wenyon (277) from 
Minchin.) 

The SPOROZOA are parasitic protozoa which multiply by the produc- 
tion of spores at some stage of their life cycle. There are very many 
sporozoa and so, for convenience of classification, they are subdivided 
into seven orders. The Gregarina have a very distinctive shape; the 
single cell, of which they are composed, is divided into two or more 
divisions. The first of these divisions is furnished with hooks or other 
structures 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 certain cells of their 



PROTOZOA 



135 



host, for example, Eimeria stieda (Coccidium cuniculi) (page 831) enters 
the epithelium of the small intestine and of the bile ducts of the 
rabbit, while Coccidium avium enters and destroys the cells lining the 







si 

\ 






f 






FIG. 98. Sporozoits in the oocyst of Laverania malaria. A, Formation of 
nuclear points which serve as the foci from which the sporozoits develop; B, a more 
definite shaping of protoplasm and nuclei; C, D, mature sporozoits in the oocyst 
arranged about centers from which they radiate; E, a portion of one enlarged. 
(After Grassi, from Doflein.) 

intestines of the birds which it infects (page 832). The Hamosporidia 
live, for a part of their life cycle, within the red cells of the blood of 



136 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

vertebrate animals. They are a very important order. The genus 
Plasmodium causes malaria in man (page 832); while Proteosoma and 
Hcemoproteus are malarial parasites of birds (page 832). The Hcemogre- 
garina 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 gregar- 
ine, which occurs free in the blood. Hepatozob'n 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 (page 836) 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 a group of very small sporozoa of which little is known. Some of 
them are parasitic in fish; one of them, Rhino sp or idium kinealyi, has 
been found in a tumor of the nose of a native of India. The Myxo- 
sporidia (page 841) are recognized by the peculiar form of their spores; 
each spore has one or more capsules each furnished with a coiled fila- 
ment or thread which is extruded under certain conditions and probably 
serves to anchor the spore to a surface upon which further development 
may occur. Members of this order are parasitic in various tissues of 
fishes and they often produce disease in their hosts. The spores of the 
Microsporidia (page 841) are exceedingly small; a member of this 
order is the cause of pebrine in silk-worms (page 656). 

The INFUSORIA (page 841) 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. Histoplasma capsulatum (page 
842), the Chlamydozoa (page 842) and the Ultramicroscopic viruses 
(pages 116, 842) are all associated with important diseases in men and 
in animals. 

The SPIROCH^ET^E (page 843), as their name signifies, are thread-like 
organisms, which seem to be coiled in a spiral. It is probable that the 



PROTOZOA 137 



(curves of certain spirochaetes lie in one plane and, consequently, that 
their bodies are really waved and not spiral. These organisms present 
no organized nucleus but the chromatin appears to be distributed 
(throughout their bodies. 

Those parasites which are important enough to require special con- 
|sideration are described (page 822) in the order in which they are men- 
tioned in the classification (page 13). Whenever it is possible to do so, 
ja 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 
idiffer from the description of the type species. 

TECHNIC* 

The methods employed in studying the pathogenic protozoa are very similar to 
those used in bacteriology. Microscopes, with the highest magnifications, are 
Essential for successful work. 

It is of great importance in the study of protozoa to examine these organisms in 
the living condition. In no other way can their mode of locomotion be determined 
and frequently their contour is also quite different in life and in stained preparations. 
A small amount of the material in which they occur may be placed beneath a cover- 
glass on a clean slide and examined immediately with the microscope by ordinary 
daylight. In case large organisms are examined in rather thin fluid it is well to 
prevent their being crushed by interposing several minute globules of paraffin 
:tween slide and cover-glass which is readily accomplished by touching paraffin 
ith a hot needle and transferring it thus melted to several points on the slide before 
he preparation is made. When very minute forms are to be studied it is necessary 
.0 utilize what is known as the dark field illumination. This brings out very minute 
.nisms and particles which being transparent are invisible to ordinary transmitted 
ight. The dark field apparatus consists of a strong source of light such as a small 
.re lamp, a special condenser which deflects the light so that objects in the micro- 
icopic field are illuminated by light directed from the sides causing them to appear 
right on a dark background. Another method of obtaining a dark field is to mix 
>n a slide a small drop of the material to be examined with an equal-sized drop of 
ndia ink or better of saturated aqueous solution of nigrosin and then to smear this 
uixture across the surface of the slide when it may then be dried and examined at 
nee by the oil immersion lens. Only ordinary daylight is required for this method 
it it does not serve in the study of the motility of organisms. 

By special apparatus it is possible after obtaining a certain amount of skill to 
lissect many forms of protozoa. In this way knowledge is obtained of the physical 

* For a more extensive treatise of the technic applicable to the study of protozoa see Doflein, 
^ehrbuch der Protozoenkunde. Jena, Gustav Fischer; Prowazek, Der mikrochopischen Technik 
per Protistenuntersuchung, Leipzig; and Stitt, Practical Bacteriology, Bloodwork and Para- 
litology, Blakiston, Philadelphia. 



138 MORPHOLOGY AND CULTURE OF MICROORGANISMS 

properties of various portions of their bodies and it is also possible to inject various 
chemicals into their substance. This method of study is made possible by the me- 
chanical devices utilized by Barbour to whose work the reader is referred.* 

In order to make stained preparations the material may be either smeared in a 
thin film upon clean slides or sectioned after appropriate treatment. In each case 
the material requires fixation. For the preparation of stained smears the Giemsa 
method is widely used. This is briefly as follows: 

1. Make thin smears of material on a clean slide and dry. 

2. Fix immediately by covering the smear with pure methyl alcohol which should 
be allowed to act for ten to twenty minutes. 

3. Dry by waving slide to and fro. 

4. Stain for four to twenty-four hours, according to the depth of stain desired, 
in a solution made by an addition of one drop of Giemsa stain to i c.c. of distilled 
water. 

5. Rinse with distilled water. 

6. Dry and mount in immersion oil or any acid-free balsam. 

It is frequently desirable to keep stained smears unmounted as they apparently j 
retain their color for a longer period of time. They may be studied with the oil 
immersion lens but the oil should at once be rinsed off with xylol, for if left upon the 
preparation an insoluble substance is formed which produces a clouded appearance. ! 
All stained preparations should be stored away from the light when not in use. For i 
the above method it is important to have all glassware perfectly clean and without 
trace of acid. The stain must be used immediately after preparation. Certain 
materials may be smeared very readily with the platinum loop ordinarily used ir 
bacteriology. A very practical method for making blood smears is to gather t 
minute drop of freshly drawn blood from a small incision or prick in the skin on one 
edge of the end of a slide. The latter is placed in contact with the surface of anothe: 
slide and being held at an angle of 45 degrees is pushed steadily lengthwise across it: 
surface. By increasing or decreasing this angle a thicker or thinner film may b> 
made. Certain investigators prefer to use what is termed the wet method for th 
fixation of smears. In this case the smear is dropped face down immediately am 
before drying into a fixative composed of two parts of a solution of saturated HgCl 
in distilled water and one part of absolute alcohol. The technic employed in th 
staining of sections is then followed and the smear is not allowed to dry at any ste 
in the procedure. 

The preparation of stained sections requires a considerable amount of technics! 
skill. Tissue is first fixed to render its structure permanent. It is then dehydrate 
in alcohol of increasing strengths, next placed in chloroform or some other clearin 
reagent when it is then imbedded in paraffin after which it may be sectioned. F(J 
the details of sectioning and the staining of sections the reader is referred to Malloil 
and Wright's Pathological Technic, W. B. Saunders and Co., and Lee's Vade mecur I 

The cultivation of free-living protozoa is usually accomplished by keeping;! 
supply of the medium in which they live on hand. Hay infusion prepared by boilii I 

* Barbour: University of Kansas, Science Bulletin 1907-4-3; also Journal of InfectUJJ 
Diseases, 1911, 8, 248, and 1911, 9, 117. 



PROTOZOA 139 

quantity of chopped hay in water is an easy and valuable method of preparing 
ulture media. For the cultivation of amoebae, the following media is widely em- 
.oyed. It should be noted, however, that the amcebae which have been cultivated 
re regarded as free-living forms and the attempts to cultivate parasitic amcebae 
ave thus far been unsuccessful. 

MEDIUM OF MUSGRAVE AND CLEGG 

Agar 20 to 30 g. 

Liebig's extract of beef 3 to . 5 g. 

Common salt 3 to . 5 g. 

Water 1,000 c.c. 

This medium is designed to provide for slow bacterial growth in order to provide 
>od for amcebae. On a richer medium the latter are overwhelmed by the rapid 
rowth of bacteria. 

For the cultivation of trypanosomes, leishmania and other flagellates the so- 
illed triple N media is employed. This is prepared as follows: 

NlCOLLE, NOVY, MACNEAL MEDIUM 

Water 900 c.c. 

Salt 6 g. 

Agar 16 g. 

Dissolve, distribute in tubes, sterilize and add to the medium in each tube after 

Iquefying and cooling to 4o-5oC. one-third its volume of rabbit blood obtained by 

ardiac puncture. Slope the tubes for twelve hours, incubate at 37. for five days 

test the sterility of the medium and then keep them at the ordinary temperature 

the laboratory for a few days before sowing them. (The tubes should be sealed to 

revent evaporation.) 

The malaria organisms have been made to continue development outside the body 
y the following method devised by Bass. 

Bass's Method. The blood in 10- to 20-c.c. quantities is taken from the patient's 
ein and received in a centrifuge tube which contains }fo c - c - of 5 P er cent, glucose 
'lution. A glass rod, or piece of tubing, extending to the bottom of the centrifuge 
ibe is used to defibrinate the blood. After centrifugalizing there should be at least 
in.ch of serum above the cell sediment. The parasites develop in the upper cell 
yer about J^o to %Q mcn from the top. All of the parasites contained in deeper 
Ing red cells die. To observe the development, red cells from this upper %Q-'mch 
rtion are drawn up with a capillary bulb pipette. 

Should the cultivation of more than one generation be desired, the leucocyte 
Dper layer must be carefully pipetted off, as the leucocytes immediately destroy the 
erozoites. Only the parasites within red cells escape phagocytosis. Sexual 
irasites are much more resistant, and the authors think they observed partheno- 
mesis. The temperature should be from 40 to 41. and strict anaerobic conditions 
Dscrved. jEstivo-autumnal organisms are more resistant than benign tertian ones, 
trose seems to be an essential for the development of the parasites. 



PART II 

PHYSIOLOGY OF MICROORGANISMS* 



DIVISION I 

NUTRITION AND METABOLISM 

INTRODUCTION 

GENERAL PRINCIPLES OF NUTRITION AND METABOLISM. The 
utrition and metabolism of microorganisms are based on the same 
principles that regulate animal and plant metabolism; in a general 
\vay microorganisms are more closely related to animals than to 
plants, if viewed from the standpoint of their food, their mode of 
ligestion, and their general physiological nature. Only in a few in- 
stances, i.e., in the case of life without oxygen (anaerobiosis) and in 
he ability of some species to use free nitrogen gas, are there processes 
inparalleled in the more highly developed organisms. Since it will be 
lecessary frequently to refer to plant and animal nutrition in the 
:ourse of this discussion, these principles, therefore, are briefly 
discussed in the following paragraph. 

Green plants feed only on inorganic substances. They assimilate 
:arbon dioxide (CO2) from the air which unites with water, nitrates, 
potassium, calcium, and other salts of the soil and form the body sub- 
stances of the plant. The cellulose, starch, sugar, protein and all other 
:ompounds constituting the plant cells are produced from these simple 
norganic substances. This formation of organic compounds from 
norganic compounds requires a certain amount of energy. If a certain 
quantity of sugar is burned to carbon dioxide (CO 2 ) and to water (H 2 O), 
certain amount of energy is liberated in the form of heat. The heat 
given off in this case is also a distinct product of combustion. This heat 

Prepared by Otto Rahn. 

141 



142 NUTRITION AND .METABOLISM 

is always obtained and always in the same amount regardless of the 
method chosen in burning the sugar. It has been definitely determined 
to be 674 calories for i g. molecule (180 g.) of sugar. The complete 
equation of sugar combustion is therefore written 

C 6 H 12 6 + 1 2O = 6C0 2 + 6H 2 O + 674 Cal. 

Consequently the same amount of energy will be needed to produce 
sugar 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; exactly the opposite 
proceeds in the equation which should read from right to left; CQz 
and H 2 are absorbed by the plant resulting in the formation of sugar.! 
But it is evident from the equation that C0 2 and H 2 O are not sufficient j 
to produce sugar since it takes 674 calories of heat in addition. The 
radiant energy of light is transformed by the chlorophyl granules of the 
plant leaves into chemical energy which causes the formation of organic 
compounds from the simple inorganic or mineral matter. Chlorophy 
is the green coloring substance of plants, and only green plants can us< 
the energy of sunlight for their growth. 

The growth of green plants is a storing of the energy of light in th< j 
form of organic matter; their metabolism is largely synthetic, i.e.l 
building up. Plants without chlorophyl, however, like* mushrooms I 
molds, yeasts and bacteria, have to provide for their energy by somj 
other means. 

Animals construct their bodies mainly of organic matter. Thei 
body substances as protein, fat, etc., are derived from the proteir 
fat, cellulose, etc., of plants or of animals. Nevertheless, a certaii 
amount of energy is required in this assimilation process, since th 
animal protein and fat are somewhat different from the plant proteij 
and fat. Consequently, complex chemical changes and rearrangement: 
which require some energy, are necessary for growth. Energy is als 
lost. by radiation of heat and by locomotion. Animals, being entirelj 
unable to use the sunlight as a source of energy, obtain their energ 
from the digestion of organic food. The larger part of this food I 
oxidized completely; this part provides for the energy. The smallu 
part of the food is used for building the tissues of the body; it becoimi 
part of the animal itself. Animal metabolism is largely analytic, i.t\ 



INTRODUCTION 143 

j (destructive although a limited amount of energy is required for the 
chemical changes and molecular rearrangements which are essential 
ho animal tissue formation a synthetic process. Accordingly more 
i (organic matter is decomposed than is formed. Often the same sub- 
Jstance can serve both purposes; the meat eaten by a dog furnishes to 
fit energy as well as material for growth. In other cases, certain food 
'[compounds execute only one function and not the other. This dis- 
tinction between food for energy and food for growth must also enter 
|pto the interpretation of microbial metabolism. 

It might appear from this discussion that energy is needed only by 
growing cells, as the full-grown cells do not increase in size or weight 
or number. They also need energy, for in all living cells, there is 
noticed a continuous breaking down (katabolism) and rebuilding 
(anabolism) of the cell constituents. This process is commonly called 
metabolism. The katabolic processes (the breaking down) in a cell 
will continue even if the cell receives no food. The cell loses in weight, 
and the starvation which follows will ultimately result in the death of 
the cell. All living cells require food for the maintenance of life. 

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

The metabolism of protozoa which in some respects calls for dif- 
erential and special treatment is furnished by Todd and Tyzzer as 
ollows : 

"The ingestion of food is accomplished in some protozoa by 
udopodia; the protozoon simply flows around and so encloses a food 
rticle (Fig. 99). In the same way, these protozoa flow away from 
te particles which are to be eliminated. Other protozoa have defi- 
ite mouth areas for the ingestion of food, and definite anal areas for 
he discharge of residual material. Those protozoa which ingest solid 



\ 

144 NUTRITION AND METABOLISM 

food, digest it within gastric vacuoles by the aid of enzymes and of 
acids, just as is the case in many-celled animals. The most important 
of the disease-producing protozoa live within nutrient fluids, for ex- 
ample the blood, and they obtain their nourishment from the fluid in 
which they live, by osmosis; consequently, they have no definite mouth 
area, nor gastric vacuoles. 

"Some of the protozoa, for example, some amoebae and ciliata, pos- 
sess contractile vacuoles. A contractile vacuole is a clear cavity which 




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

(After Doflein.} 

appears in the cytoplasm, grows slowly, empties itself by a rapid con 
traction of the fluid which has drained into it and forms again. Th 
fluid which it ejects contains the soluble waste products resulting fron 
the metabolism of the protozoon. One function of the contractile vac 
uoles is, therefore, excretion; in some protozoa, they are probably alst 
concerned with respiration. Contractile vacuoles are frequently absen 
in protozoa which are parasitic within other animals. 

"Organisms which feed upon solid food, for example the bodies c 
other organisms, are said to be holozoic in their mode of life. Other 



INTRODUCTION 145 

Which by reason of their generic relationship are included with the 
Irotozoa are capable under the action of sunlight of manufacturing 
larch. Such are said to be holophytic. Other protozoa live upon 
rganic material in solution, the saprozoic mode of life, and finally 
feme are especially adapted to live at the expense of other animals and 
re parasitif in nature. In the latter instance food may be obtained 
torn the fluids in which they live by absorption as is the case with 
lypanosomes living in blood plasma or cells may be ingested as in the 
Ue of dysentery amoebae. 

" The process of respiration in the protozoa is in general similar to 
'pat of higher animals. Most of them require oxygen and eliminate 
arbon dioxide. The contractile vacuole which is found in certain 
prms is believed to have a respiratory function. Respiration may 
onsist of the liberation of energy through oxidation or through the 
reaking down of complex molecules. In organisms of an anaerobic 
abit the respiration is probably through internal molecular changes 
ffecting material stored in the cytoplasm. 

" In addition to the expulsion of solid undigested material from 
(be cytoplasm there is evidence that waste products other than CO* 
re excreted by contractile vacuoles. Many organisms also secrete 
naterial either of the nature of chitinous membranes on their surface 
r metabolic products in the form of globules, etc., within their bodies. 

" The most obvious evidence of liberation of energy in the physiology 
f protozoa is seen in their movement. Certain protozoa, Nocticula 
pr example, however, emit light and produce the phosphorescence 
ften observed in sea water. From analogy with higher animals it is 

be supposed that heat and electrical changes are also produced. 

" Certain chemical substances which attract protozoa are said to be 
sttively chemotactic, others which repel negatively chemotactic. Vari- 
us forms react in a definite manner to light phototaxis, heat 
rmotoxis, gravity geotaxis, etc. 

" Derangement of function may be produced associated with which 
re visible degenerative changes. It has also been found that certain 
rotozoa have the ability to recover from injury and to regenerate lost 
its." 

ENERGY SUPPLY OF MICROORGANISMS. The source of energy in 
icrobial life is always of chemical origin. The simplest processes 

the oxidations, and simplest among these the inorganic oxidations. 
10 



146 NUTRITION AND METABOLISM 

A number of different types feeding exclusively on minerals has 
been discovered during the last twenty years, and some of them are 
of great economic importance. They resemble plants in as far as they 
build their cells exclusively from carbon dioxide, nitrates and ash. 
The food used for building material is quite different from the food 
used for the provision of energy. 

Two typical examples are the nitrifying organisms in soil which I 
oxidize ammonia to nitrates. This process, according to Winogradski. | 
is divided distinctly into two phases: the Nitrosomonas oxidizes the 
ammonia to nitrous acid, 

NH 3 + 30 = HNO 2 + H 2 O + 78.8 Cal. 
and the Nitromonas oxidizes the nitrous acid to nitric acid, 
HNO 2 + O = HNO 3 + 18.3 Cal. 

These oxidation processes yield a certain amount of energy w 
enables the bacteria to build their cells from carbon dioxide, ammo: 
and certain mineral salts. Without ammonia or without nitrous acid 
respectively, these bacteria cannot grow for lack of energy; they woulc 
be like a plant without light. It is evident in this case that the food fo 
energy is also used to some extent as food for growth. The nitrogei 
necessary to the bacteria is supplied by the ammonia or the nitrous acid 
As an example distinguishing strictly between the food for growti 
and the food for energy may be mentioned the hyposulphite bacteriur 
studied by Nathanson. This organism oxidizes hyposulphites to sul 
phates and sulphur, largely following the formula 

Na 2 S 2 O 3 + = Na 2 S0 4 + S + x Cal. 

Hyposulphite Sulphate Sulphur 

Besides, some more complex compounds, like sodium tetrathionat 
(Na 2 S40e), are formed. The bacterium builds its cells exclusively froi 
nitrates, carbon dioxide, and mineral salts; organic food is rejectee 
The hyposulphite can hardly be used for the construction of the eel 
and must be considered entirely a food for energy. 

This distinction is not confined to mineral decomposition onlij 
The urea bacteria get their energy from the decomposition of urea hit 
ammonium carbonate which is hydrolysis. 

(NH 2 ) 2 CO + 2H 2 = (NHOsCOs + 14-3 Cal. 

Urea Ammonium 

carbonate 



INTRODUCTION 147 

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, and they cannot 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 favor a good develop- 
ment of the bacteria. The malic acid is used entirely for the forma- 
tion of cell substances. The energy for this formation came from the 
urea fermentation. This example shows clearly the different require- 
ments for cell growth and for the energy supply. 

With the urea fermentation, we have changed not only from inor- 
ganic to organic food, but also from oxidation processes to other 
decompositions. 

Microorganisms differ from the higher animals by their less complete 
metabolism. The food in the animal, if digested at all, is oxidized as a 
rule to the final products of combustion, CO 2 and H 2 O, the only excep- 
tion being the nitrogen which leaves the body still in organic combina- 
tion as urea. With bacteria, yeasts and molds, this is not always the 
case. Though some of these organisms will bring about complete oxida- 
tion of the food we find more commonly incomplete oxidations or 
changes which require no oxygen at all, but still yield energy to the cell. 
The biochemical side of these changes of which the alcoholic fermenta- 
tion is the best known will be discussed in the chapter on oxygen 
requirements. 



CHAPTER I 
FOOD OF MICROORGANISMS 

THE COMPOSITION OF THE CELL 

Cells under average conditions may contain certain compoun 
which are in no way essential to life manifestations; they are in 
medium in which the cell grows, and thus pass into the cell witho 
taking part in its functions. Sodium and silicon are probably elemen 
of no use to bacteria though commonly present in the cells. Most 
the compounds of the cell are, however, essential to normal devel 
ment. Some idea of the needs of the cell may be obtained by stud 
its composition. 

MOISTURE. The amount of water in the cells of microorgani 
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 
organisms 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 myce- 
lium; 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 (CeHioOs)^ though it is found in some cases. 
Other compounds, related to cellulose, are more common; chitin" 
(Ci8H 3 oN 2 Oi2), or another very similar nitrogenous compound is alsc 
found. The slime surrounding some bacteria, and the capsules 
consist largely of carbohydrates, but often contain some protein. 

* Chitin when hydrolized yields glucosamine and acetic acid. 

Ci 8 H3oN 2 Oi2 + 4H 2 O = 2CH 2 OH-CHOH-CHOH-CHOH-CHNH 2 -CHO + 3CH 3 -COOH 

148 



FOOD OF MICROORGANISMS 149 

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 r61e in metabolism (page 178) are 
produced in the protoplasm and are either secreted or retained. All 
products of metabolism will be found in the protoplasm of the cell in 
small quantities. Among other substances frequently found in micro- 
organisms may be mentioned glycogen (C 6 Hi O5) n which can be readily 
detected by the brown color it gives when acted upon by iodine. Gly- 
cogen may be considered as a reserve substance stored by the organism. 
Another carbohydrate staining blue with iodine is stored by B. amylo- 
bacter. Fat is commonly found in many bacteria. The amount of 
fat in some bacteria is surprisingly high. In the tubercle bacterium 
26.0 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 occa- 
sionally 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. Protein is sometimes ac- 
cumulated in certain places of the cell and gives a granular appearance 
in the stained cell. Volutin may be such reserve protein. 

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, manga- 
nese, and of the metalloids, nitrogen, phosphorus, and sulphur. 
Some other minerals are usually found, but seemingly are unnecessary 
to the cell, as sodium and silicon. 



AMOUNT OF FOOD REQUIRED 



he amount of food that is ordinarily decomposed by microorgan- 

and the amount that is absolutely necessary, differ widely. The 

tity 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 



150 NUTRITION AND METABOLISM 

would be possible to detect easily the organic and inorganic matter of 
the microorganisms so that it could 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 i square micron and whose height is 2 
microns (which is a high estimate) the volume of such a cell would be 
1X1X2 cubic microns = o.ooi X o.ooi X 0.002 mm. = o.ooo,- 
000,002 cu. mm. The specific gravity of bacteria 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 i liter of water containing 
100,000 bacteria per c.c. amounts to the immeasurable 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 ), hydrogen (H), and perhaps 
methane (CEU). 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 one 
per cent of lactic acid, because this is the highest acid concentration 
that this bacterium can endure. If this acid is neutralized, the in- 
hibiting 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 metabolism of the cell (page 179). 

FOOD FOR GROWTH 

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. This corre- 
sponds to about 250,000,000 bacteria. MacNeal and associates found 



FOOD OF MICROORGANISMS 151 

hat the dry matter of 550,000,000 cells of B. coli weigh o.i mg. The 
mount of food that is used as the building material for the cell is 
robably larger than the weight of the cell itself, since there will always 
e present waste products, but it is of the same order of magnitude, i.e., 
ery small and often hardly measurable. The example of the urea fer- 
icntation (page 146) illustrates this point very well. 

SOURCES OF CARBON. The compounds which can serve as building 
tones for the cell vary greatly with the species. The source of carbon 
all green plants is carbon dioxide, CC>2. Animals cannot use this, 
they all require complex compounds, such as carbohydrates, fats 
r amino-acids. Bacteria exist between the plants and animals in 
bis respect. Some bacteria have already been mentioned (page 147) 
s being able to use carbon dioxide (C0 2 ), as the only source of carbon; 
tiey are the mineral-oxidizing species. Such bacteria are called 
nitotrophic in their relation to carbon, since they use it in the inorganic 
iorm. A bacterium feeding on carbon, as such, would be called 
brototrophic; bacteria of this class are said to exist. The vast majority 
)f microorganisms are heterotrophic, using carbon in organic form. 
Organic acids and sugars are excellent sources of carbon for micro- 
organisms, although proteins and their decomposition products seem 
.0 be equally satisfactory as construction material. 

SOURCES OF NITROGEN. The sources of nitrogen are equally varied; 

he green plants use nitrates; animals must have a number of different 

amino-acids; the microorganisms again are found between them. We 

know autotrophic bacteria, and especially molds and yeasts which can 

row with nitrates or ammonium salts as the only source of nitrogen. 

There are three groups of prototrophic bacteria in their relation to 

ritrogen the B. amylobacter group, the Ps. radicicola group and the 

izotobacter group. These bacteria are of the greatest importance to 

agriculture; soil fertility depends, to a large extent, upon the last two 

groups, for they take nitrogen gas from the surrounding air, form their 

>wn protoplasm from it, and thus increase the amount of chemically 

combined nitrogen in the soil. - Details of their relation to soil fertility 

can be found in Chap. Ill, page 338. The majority of bacteria are 

heterotrophic, requiring organic nitrogen. Urea is not well adapted for 

this purpose; amino-acids or the peptones from which amino-acids are 

derived are the best compounds for most organisms. Asparagin is 

very commonly used if for some reason peptones are to be omitted. 



152 NUTRITION AND METABOLISM 

SOURCES or HYDROGEN AND OXYGEN. The sources of hydrogen are 
hardly ever discussed with bacteria since hydrogen bears such a close 
and peculiar relation in water and organic food supplies. The ulti- 
mate association of hydrogen with oxygen in the molecule of water 
(H 2 O) and with carbon in organic substances (CH 4 ) establishes its 
importance in all life processes. There are many prototrophic bacteria, 
using oxygen as such; others are able to reduce such compounds as 
nitrates or sulphates, which would be autotrophic, thus providing for 
their needs. Heterotrophic bacteria are not unusual. In this connec- 
tion it may be said that it is often difficult to distinguish between oxy- 
gen needed for cell construction and oxygen needed for energy formation. 

SOURCES OF MINERALS. The amount of mineral matter necessary 
for the construction of the cell is very small; potassium and phos- 
phorus seem to be among the most essential elements. It is customary 
to consider a tap water with 0.02 per cent of di-potassium hydro- 
gen phosphate, K 2 HPO 4 , sufficient in mineral matter of all kinds to 
provide for fair growth. Some of the common materials used in the 
preparation of nutrient media, such as meat extract and peptone, also 
contain considerable amounts of mineral matter. 

FOOD FOR ENERGY 

As all food in its decomposition results in products of some form or 
other, it may not seem justifiable to separate a paragraph on food 
from another on products. The essential difference lies in the fact that 
we consider food from the viewpoint of the cell, while products are 
commonly considered apart from the construction processes of the cell 
and only from their application, or, it may be, from the viewpoint of 
usefulness to man. 

Animals provide for their energy by oxidations, and almost exclu- 
sively by complete oxidations. Some bacteria, and most molds, do 
the same. The range of materials which can serve as food for this pur- 
pose is surprising. With animals, the food is practically limited to 
plant and animal tissue. With bacteria, we find the strangest sub- 
stances, such as hydrogen, carbon monoxide, coal, marsh gas, hydrogen 
sulphide, ammonia, nitrites, formic and oxalic acids, alcohol and thio- 
sulphates serving this purpose. The fact that many gases are used I 
as food makes us realize that oxygen is not such an extraordinary 
compound as animal physiology seems to indicate, but that it should be j 



FOOD OF MICROORGANISMS 153 

classed merely as one of the many food compounds. This is especially 
significant since it will be shown later that free oxygen is not necessary 
for microbial life, and that many organisms can exist without it. 

The oxidations are not always complete. The formation of nitrous 
acid from ammonia, the oxidation of alcohol to acetic acid are such 
examples. Some organisms are highly specialized in their food require- 
ments, especially the mineral-attacking bacteria are usually limited 
to one source of energy. The microorganisms oxidizing organic com- 
pounds have, as a rule, the ability to decompose several compounds, 
and some bacteria are .common scavengers, able to feed on organic acids, 
sugars, fats and proteins. 

OXYGEN RELATIONS 

It is characteristic of many microorganisms to provide for their 
energy without using free oxygen. One such example has already been 
given in urea fermentation. 

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

Urea Ammonium carbonate 

Very common is the decomposition of sugars without oxygen. 
The two most typical fermentations of this type are the alcoholic and 
the lactic fermentations. 

C 6 H 12 O 6 = 2C 2 H 5 OH + 2CO 2 + 22 Cal. 

Sugar Alcohol 

C 6 H 12 6 = 2C 3 H 6 3 + 15 Cal. 

Sugar Lactic acid 

In fermentations of this type, the changes take place without an 
oxygen gas partaking in the reactions. These fermentations seem to 
be essentially reactions of the oxygen atoms within the sugar molecule. 
One side of the molecule is reduced while the other side is oxidized. 
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 dis- 
tributed evenly in the sugar, is shifted to one side of the molecule in 
lactic acid. 



154 NUTRITION AND METABOLISM 

H H H H H O 

O O O O O || 

Dextrose, H C C C C C C 

H H H H H H 

H H O 

O II 

Alcohol, HC CH C Carbon dioxide, 

H H || 
O 

H H 
H O O 

Lactic acid, HC C C 

H H || 

O 

In some of the more complex fermentations, we find simultaneous 
formation of hydrogen or methane and carbon dioxide; the one is 
the end product of reduction, the other the product of complete oxida- 
tion. This also indicates that the oxidation of one part of the molecule 
takes place at the expense of the other. 

In a similar way, some organic acids, e.g., tartaric and lactic acids, 
can be fermented by certain bacteria without requiring oxygen. Some 
bacteria have the ability to attack proteins and decompose them 
completely in the absence of oxygen. 

Bacteria, having the ability to provide for their energy without 
oxygen gas, may live in the complete absence of oxygen, and may 
multiply indefinitely without it as long as there is sufficient food. But 
some microorganisms, such as yeasts, seem to grow only for a limited 
time in the absence of oxygen. Finally, they cease growing, and 
we may well assume that they need oxygen for cell construction which 
can be used in no other form except as molecular oxygen. The urea 
bacteria also belong in this group. 

A large number of bacteria and yeasts, and also a few molds, can 
provide for their energy by either oxidation or decomposition in the 
absence of oxygen. Very commonly a great variety of compounds can 
be found which may be oxidized while but very few can be intra- 
molecularly fermented without oxygen. This is easily understood: 
all organic compounds will yield heat upon oxidation, while exothermic 



FOOD OF MICROORGANISMS 1 55 

tramolecular changes require a special structure. Carbohydrates 
re the most excellent substances for such intramolecular decomposi- 
ons. S. cerevisia and B. coli can live in sugar-free broth only if ex- 
osed to the air. They provide for all their needs by oxidation of the 
rotein. If oxygen is excluded, growth depends upon sugar, or a 
milar fermentable compound. We test for the absence of sugar in a 
!ven solution by pouring it in a fermentation tube and inoculating 
ith B. coli: if the liquid in the closed arm remains clear, i.e., if B. coli 
oes not grow without oxygen, it is a good indication that no sugar is 
resent. 

It is usually assumed that in fermentations of this nature, the 
xygen atoms are shifted within the same molecule. In other cases, 
xygen is taken from one molecule and used for the oxidation of 
nother. This results in one of the molecules being reduced. Nitrates 
re reduced in this way to nitrites, or ammonia, or nitrogen gas; sul- 
jhates to hydrogen sulphide, and litmus or methylene blue to the 
rolorless leuco-compounds. Such removal of oxygen from a molecule 
equires energy, and is possible only when the bacterium by using the 
)xygen for oxidation of organic matter can obtain a larger amount 
)f energy. The following example shows such a possibility: 

2 KNO 3 + 36.6 Cal. = 2KNO 2 + 2 
C 2 H 5 OH + O 2 = CH 3 CO 2 H + H 2 O + 115 Cal. 

This process leaves an energy balance of 115 36.6 = 78.4 Cal. for 
he needs of the bacterium. 

Such decompositions are sometimes referred to as "reducing fermen- 
ations" but this term is not correct, as the reduction must always be 
iccompanied by a simultaneous oxidation process. 

The amount of energy liberated by a fermentation without oxygen 
s much smaller than that furnished by complete oxidation; the intra- 
molecular change always leaves organic compounds which contain a 
tonsiderable amount of the total energy. Yeast, in presence of very 
much oxygen, oxidizes sugar completely to water and carbon dioxide. 

C 6 H 12 O 6 + 120 = 6CO 2 -f 6H 2 O + 674 Cal. 

kvhile in the absence of oxygen it will change the sugar to alcohol and 
:arbon dioxide. 

C 6 H 12 O 6 = 2C 2 H 5 OH -f- 2CO 2 -f 22 Cal. 

The energy gained in the first process is about thirty times as large 



156 NUTRITION AND METABOLISM 

as that gained in the second process. This was demonstrated as early 
as 1 86 1 by Pasteur. He grew yeast in sugar solutions, varying only 
the amount of oxygen in contact with the medium. At the end of 
the experiment, the weight of the dry yeast and the decomposed sugar 
was determined, and the amount of sugar necessary to produce one 
part of yeast was computed. He found : 

In a closed flask, without any air i part yeast required 176 parts sugar. 

In a closed flask, with large air space i part yeast required 23 parts sugar. 

In a thin layer, a few mm. thick i part yeast required 8 parts sugar. 

In a very thin layer, in 24 hours i part yeast required 4 parts sugar. 

This experience led Pasteur to the conclusion that fermentation 
corresponded to the respiration process of animals, that fermentation 
was respiration without oxygen. 

It is quite evident that since the utilization of the food in the 
absence of oxygen is very high, the organisms have to decompose 
much more food. This accounts, to a great extent, for the enormous 
destructive power of bacteria, when comparisons of the great quantity 
of food decomposed are made with the very insignificant weights of 
cells. It has been estimated that the lactic bacteria decompose their 
own weight of sugar in one hour. 

Summing up the relation of oxygen to microorganisms, some 
bacteria, and especially the molds, are found depending upon oxygen as 
an indispensable part of their food. Three groups are recognized: 
Those, a large number, organisms in the presence of oxygen producing 
oxidations; those able to sustain life without oxygen; and those de- 
pending entirely upon decompositions which require no oxygen, 
The lactic bacteria and the butyric bacteria belong in the last group. 

In considering the oxygen requirements, it is customary to in- 
clude another influence of oxygen upon bacteria. This has reall) 
nothing to do with its food value, but deals with the poisonous qualities 
of oxygen. Oxygen in this light may well be called a poison as it wil 
kill bacteria in very low concentrations. Ordinarily it is regarded a$ 
constituting over 20 per cent of our atmosphere. But if a study it 
made of its effect upon bacteria, it is necessary to measure it in th< 
same way food is measured, and consider the concentration in wirier 
it is offered to the cell. Microorganisms obtain their oxygen not ai: 
gas, but as dissolved oxygen. The solubility of oxygen is very small 
about 0.0009 P er cent at 2O ' Practically all bacteria die readily if th< 



FOOD OF MICROORGANISMS 157 

Ifeygen concentration is raised to thirty times the atmospheric pressure. 
'his would mean a concentration of 0.027 P er cent. It shows that 
Jxygen is about as poisonous as formaldehyde or bichloride of mercury. 

Some bacteria are extremely sensitive to oxygen, and will die if 
xposed to ordinary atmospheric oxygen. They grow only if oxygen 
k almost completely removed. These organisms are called the 
Irictly anaerobic or obligate anaerobic bacteria. They are contrasted 
Hth the facultative anaerobic bacteria which thrive with oxygen as well 
s without, and the strictly aerobic bacteria which have to have oxygen 
or their normal life processes. 

No strict limits can be drawn between aerobic and anaerobic 
>acteria. Even the most sensitive of organisms will be able to tolerate 
traces of oxygen, while the strictly aerobic bacteria can multiply also 
E the oxygen concentration is below that of a saturated solution. The 
imits of growth for the anaerobic bacteria are the limit of tolerance of 
he poisoning oxygen; the lower limit of growth for the aerobic bacteria 
5 a question of too scanty food supply. -The relation between bacteria 
,nd oxygen is graphically represented in the following diagram, after 
Cruse : 

o.\ CM o.b of 10 2.0 3.0 



FIG. 100. Influence of oxygen upon microorganisms. 
The lines indicate the oxygen concentrations where growth, is possible. Line 

is a strict anaerobe; 2 is not quite so strict; 3 is still less sensitive though it 
:annot grow if exposed to direct influence of the atmosphere; 4 is a facultative 
bacterium such as B. coli; 5 is another one which can tolerate still more oxygen; 
> can grow only with oxygen but can get along with very little: it might be one 
>f the urea bacteria; 8 is more dependent upon oxygen and the line would corre- 
pond to average molds; 7 is a peculiar type needing oxygen and yet being very 
ensitive to it. The sulphur bacteria, e.g., the Beggiatoacea, belong to type 7. Type 

is said to be representative of B. abortus. 



CHAPTER II 
PRODUCTS OF METABOLISM 

GENERAL CONSIDERATIONS. The great difference in the meta- 
bolism of animals and of bacteria, even though they feed essentiall} 
on the same foods, is the incomplete metabolism of most bacteria 
contrasting sharply against the very complete oxidation of food in tht 
animal body. The food of the animal is decomposed by the body cells 
to carbon dioxide, water and urea. It is the most complete decom 
position possible, excepting urea which, however, is very near the fina 
decomposition product, ammonium carbonate. Microorganisms, or 
the contrary, are characterized by incomplete metabolism. They d( 
not commonly oxidize their food to the end products but many of their 
produce organic compounds which are not farther decomposed b) 
them. It is this partial decomposition of organic matter which makes 
bacteria play such an important role in life and industries. Ou: 
modern bacteriology is dated from the time when Pasteur showed tha 
the alcohol in the beer fermentation, the lactic acid in the souring o 
milk, the acetic acid in the vinegar fermentation are products o: 
microbial activity. The existence of bacteria had been known fos 
nearly 200 years, but they were considered largely as a curiosity 
as soon as they were recognized as the cause of fermentations, anc 
of toxins, they received at once the greatest attention. Not al 
bacteria cause incomplete decompositions; some oxidize as com 
pletely as animals do. Others, again, form first intermediary products 
which they later decompose completely; among these, are found man) 
molds, the sulphur bacteria, and some species of the vinegar bacteria 

THE CHEMICAL EQUATIONS OF FERMENTATIONS 

The metabolism of all organisms is considered to be a chemica 
process which follows in all respects the laws of chemistry. That w 
are not familiar with all the changes taking place in the cell is no 

158 



PRODUCTS OF METABOLISM 159 

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. Since a chemical process can always be expressed by a 
chemical equation, we should expect the same with the fermentations 
and decompositions caused by microorganisms. 

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 organic 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 products. There are several possibilities for dis- 
crepancies; accurate equations can be given only for the simplest fer- 
mentations, the products of which can be analyzed more or less exactly. 

The best studied microbial process is the alcoholic fermentation. 
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 -f- 2CO 2 

180 92 88 

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 

I 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 



l6o NUTRITION AND METABOLISM 

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, and we 
always find fusel oil. 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 suc- 
cinic 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. Fusel oil is now believed to be a waste product of cell 
construction. 

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 

Ci 2 H 22 On ~}- H 2 O = 4C 3 HeO 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 de- 
termination. Small discrepancies will also be found in fermentation 
of urea and in the nitrifying process, where small amounts of the 
nitrogenous material are used for 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 
always constant. In the butyric fermentation of dextrose by B. 
amylozyma, Perdrix tries to account for this change by assuming three 
different phases of the process at various ages of the cultures, repre- 
sented by the following equations: 

First stage : 56C 6 Hi 2 O 6 + 42H 2 O = 1 1 6H 2 + i i4CO 2 + 3oCH 3 COOH + 

Dextrose Acetic acid 

36CH 3 CH 2 CH 2 COOH. 

Butyric acid 

Second stage: 46C 6 Hi 2 O 6 + i8H 2 = ii2H 2 -f 94CO 2 + i5CH 3 COOH + 
38CH 3 CH 2 CH 2 COOH. 



PRODUCTS OF METABOLISM l6l 

Fhird stage: C 6 H 12 6 = 2H 2 + 2 CO 2 + CH 3 CH 2 CH 2 COOH. 

Kruse has called attention to the fact that these complex equations 
can well be explained as the simultaneous occurrence of the following 
simple fermentations: 

C 6 H 12 O 6 = 2H 2 + 2 CO 2 + CH 3 CH 2 CH 2 C0 2 H 

C 6 H 12 6 = 3 CH 3 C0 2 H 

C 6 H 12 O 6 + 6H 2 = 6CO 2 + i2H 2 



I 



e first fermentation continues when the others have already ceased, 
jand thus the last stage of Perdrix's equations is very simple. Brede- 
jmann also found that the proportion of the various products formed by 
B. amylobacter varies greatly with the conditions, and the same has been 
recently established in the fermentation of B. coll. 

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 com- 
pounds 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 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 
substances 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 



1 62 NUTRITION AND METABOLISM 

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 mis- 
take is quite commonly made with microorganisms. 

The best illustration for the variability of cultivated microorganisms 
is the enormous number of varieties of Saccharomyces cerevisice. 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 I 
and optimum temperature of spore-production, by the appearance or 
the budding yeast in the hanging drop, and also in other respects. The 
cultivated organisms are not alone in showing this tendency toward 
variation. The transferring of a soil or water bacterium into the ordi- 1 
nary laboratory media is a complete change of conditions; the different' 
cells of the same species may react differently and give several varie- 
ties. A lactic bacterium on meat medium without sugar does not thrive I 
well in the first generations, but it gradually becomes able to grow or 
this medium. By this treatment, it loses gradually the power of pro- 
ducing acid and does not thrive as well in milk. The attenuation o: 
pathogenic bacteria by cultivation on media, as potato, very differen 
from the blood and muscle upon which they grow most naturally, o: 
by growing them at low temperature, or above the maximum, furnishe 
another example. The decrease and finally the entire loss of patho 
genicity is caused by a change of metabolism, by a loss of the power ti 
produce toxin. 

As by certain diet the metabolism can be changed, so certaiij 
physiological properties of bacteria can, by proper cultivation, b 
increased. By the frequent transferring of an organism on gelatin, itj 
liquefying qualities can be increased, provided it had some at the start) 
By continued passing of a bacterium through an animal, its virulenci 
can be increased. Strains of bacteria which will produce a very hig 
acidity can be bred; this is illustrated by the quick- vinegar proces! 



PRODUCTS OF METABOLISM 163 

and by the strong alcohol-producing yeasts of the distillery process. 
By continued cultivation of an organism upon a certain medium, it 
will become so acclimatized that it degenerates readily when the con- 
ditions become unfavorable. Such specifically trained strains of 
microorganisms are used in alcoholic and lactic fermentation, in patho- 
jgenic bacteriology and in the inoculation of leguminous plants with 
(nitrogen-fixing bacteria. 

PRODUCTS FROM NITROGEN-FREE COMPOUNDS 

SUGARS. It would be entirely beyond the limits of this book to 
give an account of all the different ways in which sugars and other 
compounds can be decomposed by microorganisms. It is much more 
important, for the beginning bacteriologist, to acquaint himself with 
the main types of sugar fermentations, and with the characteristics 
of the organisms which bring about these changes. 

In the action of microorganisms many distinguish somewhat crudely 
six 'common types: 

Complete oxidation. 

Partial oxidation. 

Alcoholic fermentation. 

Lactic fermentation. 

Acid gas fermentation. 

Butyric fermentation. 
Most of these types have been mentioned previously. 

Complete oxidation of carbohydrates is observed most commonly 
among molds and mycodermas, and also in a few bacteria, e.g., in Azoto- 
bacter. It is possible only where there is a ready oxygen supply, as, 
e.g., in soils of an open texture, in trickling filters, and on the surface 
of decaying fruits. 

The incomplete oxidation is, as a rule, more common in nature. 
Frequently microorganisms produce first an incomplete oxidation, but 
later oxidize the intermediate products completely. The molds are 
typical examples. Aspergillus niger is noted for its formation of oxalic 
acid. If it is grown in a sugar solution, it will bring about at first a 
rapid increase in acidity, but after a while, it decreases again, when the 
acid is oxidizing completely. The following processes may be noted: 






C 6 H 12 6 + 90 = 3(C0 2 H) 2 + 3 H 2 

Oxalic acid 

(CO 2 H) 2 + O = 2 CO 2 + H 2 O 



1 64 NUTRITION AND METABOLISM 

The intermediate product can be accumulated by precipitating it with 
lime which neutralizes the acidity. This principle is used in the com- 
mercial manufacture of citric acid by Citromyces, a mold closely 
related to the genus Penicillium. This mold oxidizes sugar to citric 
acid according to the following equation: 

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

Citric acid 

This fermentation is much more complicated than this equation indi- 
cates, on account of the entirely different chemical structures of citric 
acid and dextrose. The practical yield in the factory is only about one- 
half of the theoretical, since complete oxidation cannot be avoided 
altogether. 

The oxidation processes, just recited, can take place only in the 
presence of oxygen; the other four types of carbohydrate decomposi- 
tion require no oxygen, and take place as well in the absence of oxygen; 
the butyric fermentation is brought about only in the absence of oxygen. 

Alcoholic fermentation is caused only by yeasts and a few molds; 
no bacterium produces alcohol according to the well-known equation 
mentioned above. Alcohol is formed by several bacteria but only in 
small quantities and always together with several acids; this is a 
distinctly different type of decomposition. 

In the above groups and the following groups of microorganisms, 
there appears to be a close agreement between the morphological 
characters of the organisms involved and the specific type of fermenta- 
tion. Practically all the alcoholic organisms are yeasts, and the lactic 
acid-producing organisms are streptococci or closely related bacteria. 

The lactic bacteria as they are briefly named, such as are responsible 
for lactic fermentation, are readily recognized by their scanty growth 
on agar, and their excellent growth in milk, bringing about a solid 
curdling in one to three days. They change sugar to lactic acid only. 

C 6 H ]2 O 6 = 2C 3 H 6 3 

No gas and no volatile acids are formed by these bacteria. The best- 
known representative of this group is the organism which causes the 
normal souring of milk. It was originally called Bacterium lactis acidi, 
but on account of its very close relation to the streptococci, it is more 
commonly now named Streptococcus lacticus. Many streptococci will 
produce the true lactic fermentation. 



PRODUCTS OF METABOLISM 165 

The last two groups of organisms, alcoholic and lactic, represent 
omplex fermentations. There are several products formed, and as has 
Iready been pointed out in the paragraph on the equation of fermen- 
ations, the entire fermentation cannot be described accurately by one 
quation, for different fermentations operate independently and simul- 
taneously in the same cell. Under slightly different experimental 
onditions the one or other of these simultaneous fermentations may be 
avored, accordingly a varying proportion of the products are formed. 

The typical representative of the acid-gas forming group of micro- 
rganisms which cause acid-gas fermentation are B. coli, and its near 
elative, Bad. aerogenes. Many of the gas-formers in nature belong in 
his group; the bacteria of the fermentations of pickles, sauerkraut, 
alt-rising bread, the gassy fermentation of milk are some of the many 
Representatives. They are distinct rods, with good surface growth, 
ind do not liquefy gelatin. They are commonly spoken of as the coli- 
terogenes group. Some of them have peritrichiate flagella, while 
)thers are not motile. 

The fermentation of dextrose brought about by these organisms 
las been described originally by Harden in the equation : 

!>C 3 H 12 6 + H 2 = 2C 3 H 6 3 + CH 3 C0 2 H + C 2 H 5 OH + 2 CO 2 + 2 H, 

Dextrose Lactic acid Acetic acid Alcohol 

rlarden himself stated later that this equation holds only for one 
strain, and that we have several different strains distinguished by a 
proportion of products quite different from the one suggested by the 
Aquation. Recently Kamm has shown that a good mineral food 
r probably phosphates are the essential agent) favors a formation of 
;as and of volatile acids, while a scant supply of minerals causes the 
Bacteria to produce mainly lactic acid. We must assume, therefore, 
t least two simultaneous independent fermentations: 

C 6 H 12 6 = 2C 3 H 6 3 
nd 

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

"he first equation is already known to us; it is the true lactic fermenta- 
ion. The second equation may be divided still further into several 
impler equations. 

B. typhosus, causing typhoid fever, is closely related to B. coli, but 



1 66 NUTRITION AND METABOLISM 

does not form gas. It forms, however, formic acid, HCO 2 H, which, if 
decomposed, would give H 2 + C0 2 . 

The last type of sugar fermentations is the butyric fermentation, 
in which butyric acid is the most conspicuous, but not the only fermen- 
tation product. Acetic acid, hydrogen and carbon dioxide, and, with 
some organisms at least, ethyl and butyl alcohols are formed along with 
butyric acid. As already mentioned in the paragraph on the equation of 
fermentation, Kruse believes this fermentation to consist of several 
simultaneous fermentations, of which the most interesting at this stage 
is the one showing the formation of butyric acid. 

C 6 H 12 O 6 = 2H 2 + 2 CO 2 + C 4 H 8 O 2 

The organisms producing butyric acid are mostly strictly anaerobic 
spore formers with a tendency to form spindle-shaped cells; they stain 
bluish-black with iodine and Bredemann gave the clostridium group 
one species name, B. amylobacter, as he found no distinct and char- 
acteristic differences between the many strains which he studied. 
Many members of this group have the ability to fix nitrogen, i.e. 
to build up their protoplasm without using any sources of nitrogen 
other than nitrogen gas. Most of the so-called "Clostridium" species 
belong in this group. Butyric acid is also formed by B. tetani and by 
B. botulinus, the latter of which causes the most dangerous kind of meat 
poisoning. 

Of other sugar fermentations may be mentioned here only by name, 
the slimy fermentations, as manifested in ropy milk and the mannit 
fermentation. The latter is one of the very few reduction processes 
brought about by bacteria, and one which causes trouble in wine. 

What has been stated broadly for sugars holds to some extent true 
also for the alcohols derived from sugars, including glycerin. Many 
bacteria fermenting dextrose can also ferment mannit and glycerin 
with a slight variation of the products, but some do not do this. 

Among disaccharides there is a great variation of fermentation. 
Some groups ferment lactose readily as the coli organisms and Strept. 
lacticus, while among yeasts, fermentation of lactose is rare. Practi- 
cally all yeasts ferment saccharose, however, and among the lactic 
bacteria and the coli group many strains cannot ferment saccharose. 

STARCH. Quite different is the fermentation of the insoluble carbo- 
hydrates of which we can mention only starch and cellulose. Insoluble 






PRODUCTS OF METABOLISM 167 

compounds can be fermented only after being made soluble by an 
enzyme, the amylase (see mechanism of metabolism). Amylase is 
produced by most molds, by none of the fermenting yeasts, by a few 
torulas, and perhaps mycodermas, and by a great many of the bacteria. 
The sugar thus produced from starch is decomposed according to the 
main types mentioned under sugars. The lactic bacteria and the coli 
bacteria do not attack starch, but some acid-gas fermentations of 
starchy foods do take place. Butyric fermentation of starch is com- 
mon. Alcoholic fermentation can be accomplished only by some of 
the ^fucors, and A spergilli. 

CELLULOSE is decomposed only by very few organisms; these must 
be very active and very numerous, to judge from the enormous amounts 
of cellulose produced and destroyed every year on earth. Molds and 
higher fungi play probably the main role in its decomposition; the 
products have not been determined, but we may well assume a complete 
oxidation, since no intermediate products have ever been mentioned. 
; No yeast is known to decompose cellulose, and among the bacteria we 
find but very few species. Some species have recently been isolated 
which decompose cellulose in the presence of air; the products have not 
been determined; we can, however, assume a partial oxidation, eventu- 
ally a complete oxidation. Besides the aerobic fermentation, we have 
two types of anaerobic fermentation which are ordinarily described as 
the hydrogen fermentation and the methane fermentation. In these 
fermentations the gases mentioned, together with carbon dioxide, are 
liberated, and butyric and acetic acids are formed at the same time. 
The marsh gas of the marshes originates in this way. 

Summing up all the products formed from carbohydrates, we find 
several acids, among them lactic and acetic acids most commonly, 
and ethyl alcohol, rarely other alcohols, besides carbon dioxide, 
hydrogen and water. The variety is not so great, but with these few 
compounds, a number of different combinations are possible, and the 
complication of the study of such fermentations lies mostly in the 
simultaneous formation of several of the compounds. 

ACIDS AND ALCOHOLS. The organic acids and alcohols can be 
decomposed further by bacteria and molds, also by some yeasts, to 
simpler compounds. Ordinarily, this decomposition consists in the 
complete oxidation. Thus, Oidium lactis will destroy the lactic acid 
of sour milk and of soft cheeses by complete combustion. 

C 3 H 6 3 + 60 = 3 C0 2 + 3 H 2 



1 68 NUTRITION AND METABOLISM 

By the same process, the acidity of sauerkraut, ensilage, pickles is 
reduced by mycoderma species. Another Mycoderma is known to 
destroy acetic acid and thus spoil vinegar or fruits and vegetables kept 
in vinegar; the yeast grows in a thin, dry white scum over the surface, 
and oxidizes the acetic acid. 

CH 3 CO 2 H + 40 = 2C0 2 + 2 H 2 O 

The oxidation of alcohols is not always complete. Especially ethyl 
alcohol is usually oxidized first to acetic acid; this is the common vinegar 
fermentation. Many different kinds of vinegar bacteria are known, 
some forming gelatinous masses of cell membranes called mother-of- 
vinegar, while others remain as separate small cells. They all oxidize 
alcohol first to acetic acid. 

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

But most of them will oxidize later the acetic acid completely to carbon 
dioxide, after the alcohol is all exhausted, unless the oxygen supply is 
shut off. This behavior reminds one of the formation and destruction 
of oxalic acid by Aspergillus, mentioned previously. It may be re- 
marked here that the vinegar bacteria cannot attack the sugar directly 
to any appreciable degree, and the manufacture of vinegar from sugar 
requires two agents, the alcohol-forming yeast, and the alcohol-oxidizing 
bacterium. 

Some of the acids can also undergo an anaerobic fermentation. 
This is possible only with hydroxy-acids. The fermentation of the 
calcium salt of tartaric acid has been the first anaerobic fermentation 
observed by Pasteur, and the fermentation of lactic acid to butyric 
acid has a reputation for its chemical peculiarity. A compound with 
four carbon atoms is formed from a compound with only three carbons, 
a very unusual thing in fermentation. 

FATS. The decomposition of fats is comparatively simple. All fats 
are glycerides of organic acids, and if they are attacked at all by micro- 
organisms, they are first split into glycerin and free acid. 

H 2 C - O - CO - Ci 5 H 3 i HOH H 2 COH HO 2 C-C 15 H 3 i 

I I 

HC - O - CO - C 15 H 31 + HOH = HCOH + HO 2 C-C 15 H 31 

I I 

H 2 C - O - CO - Ci 5 H 31 HOH H 2 COH HO 2 CC 15 H 31 

Fat Water Glycerin Acid 



PRODUCTS OF METABOLISM 169 

This brings about the liberation of three molecules of free acid from 
neutral fat molecule. It is customary to test for the splitting of fat by 
letermining its acidity. The glycerin is readily used up by the micro- 
jjrganisms, while the fatty acids are oxidized but very slowly. 

The number of organisms which can attack fat is quite small, 
[ost molds can destroy it; one torula has been found in butter which 
tttacks it, and perhaps a dozen species of bacteria will do the same, 
imong them B. fluorescent and B. prodigiosus , which cause occasionally 
the rancidity of butter. 

PRODUCTS FROM NITROGENOUS COMPOUNDS 

On account of the complexity of the protein molecule, the products 
of protein decomposition by microorganisms are little known. Some 
products are conspicuous through their odor, others can be told by cer- 
tain color reactions, but as we cannot, at the present, give the structural 
'ormula of proteins, there is no possibility of stating protein decomposi- 
tions in equations similar to those of carbohydrate fermentations. 
The discussion must be limited, for this reason, to the enumeration of the 
ost important products, and to the general types of decomposition. 
As in the carbohydrates, soluble compounds are more easily de- 
composed than the insoluble. The keratin bodies of hair, epidermis 
End horn are- slowly attacked by a very few organisms. Gelatin, 
asein and serum albumin are more readily decomposed, though their 
olubility is quite limited. Peptones which are readily soluble are 
Msed by the vast majority of microorganisms. Of interest in this con- 
fection is the fact that the fresh white of egg is poisonous to most bac- 
teria, and fresh blood and animal tissues as well as freshly drawn milk 
lave also germicidal properties which are lost by heating or upon 
tanding. 

PROTEIN BODIES are as numerous as plants and animals. Each 

pecies 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 individ- 

bal in different parts of the body. The constituents, carbon, oxygen, 

ydrogen, nitrogen, and sometimes sulphur and phosphorus, can be 

letermined in their relative amounts without, however, furnishing any 

nowledge of the structure of the molecule. The molecular weight of 



' 



1 70 NUTRITION AND METABOLISM 

proteins is estimated to be at least 10,000, while the weight of the very 
large molecule of saccharose is only 342. The protein molecule can be 
broken up into smaller molecules. This cleavage is generally believed 
to be a hydrolytic process similar to the decomposition of starch to 
maltose. 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 a 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 com- 
pounds, 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 percentage of the total nitrogen. Thus the j 
terms "water-soluble nitrogen," "acid-soluble nitrogen" and others I 
originated, meaning the nitrogen of the compounds soluble in 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 I 
that of the protein bodies. We speak of peptonisation of proteins, | 
e.g., in the clearing of milk or the gelatin liquefaction, meaning that the 
insoluble protein has been made soluble. 

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, j 
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 ) COOH. Others 
are of a more complex nature, such as the tyrosin or hydroxy-phenyl- 
aminopropionic 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(NH2)| 
COOH. 

Of other nitrogenous products which are not amino-acids, a fewi 
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 oij 
skatol (C 8 H 6 N-CH 3 ). Indol gives a rose color with nitrites in acidi 
solution, and this convenient reagent is used in the identification ol 



PRODUCTS OF METABOLISM 1 71 

bacteria. Another group are the amins. 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. 

The ptomains (page 491) are alkaloid-like bodies of basic character 
iand 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 called ptomains because they were first discovered in 
putrefying corpses. The best-known compounds of this character 
are the putrescin or tetra-methylen diamin [NH 2 (CH2)4NH2] and the 
cadaverin or penta-methylen-diamin [NH s (CHi)|NHj, which can 
scarcely be considered poisonous. The methyl-guanidin 

NH 2 
HN = C/ 

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. 

Ammonia is the end product of protein decomposition, as far as 
the nitrogen-containing fragments of the protein molecule are con- 
cerned. That ammonia is formed by many bacteria, is well known. 
In some decaying proteins, e.g., in old Camenbert cheese, ammonia 
can be very easily detected by the smell. As all proteins contain 
many amino-groups as well as acid-amid groups, it is easily understood 
how the ammonia originates through the hydrolysis of protein. In 
the complete oxidation of proteins, the nitrogen is always left as NH 3 
or (NH 4 )2CO 3 respectively, never, so far as known, in any other 
form. No bacterium is known to produce urea, as most of the higher 
animals do. 

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 parts of the molecule will contain no nitrogen. 
Many organic acids, like acetic, butyric, capronic, benzoic and phenyl- 
acetic acids are quite generally found among the products of putre- 






172 NUTRITION AND METABOLISM 

faction. Alcohols too, especially benzene derivatives like phenol and 
cresol, are not unusual. Gas is often formed in putrefaction ; especially 
carbon dioxide and hydrogen; occasionally these gases are mixed with 
traces of nitrogen and methane. 

Many protein compounds contain, besides the organic elements, 
larger or smaller amounts of phosphorus and sulphur. The phos- 
phorus compounds may be changed to phosphine (PH 3 ), which is a gas 
of a strong disagreeable garlic odor. Generally, however, the phos- 
phorus of protein after its degradation is found as phosphoric acid 
(H 3 PO4). Very little is known about the phosphorus of organic 
compounds and the changes it may undergo in the putrefactive process. 

The sulphur of proteins is commonly changed to hydrogen sulphide 
(H 2 S). Some microorganisms are able to form mercaptan (CH 3 SH), 
a compound of very foul penetrating odor. 

After this enumeration of the products, the main types may be 
considered briefly; since much less work has been done on protein 
decomposition than on carbohydrate decomposition, the groups are 
not so well denned. We might consider the following types: 

Complete Oxidation. This is brought about by many molds, by 
yeasts if they depend upon proteins only, and by many bacteria, of 
which the large, aerobic spore-forming rods, such as B. mycoides, are 
the main representatives. The products of oxidation are CO2, H 2 0, 
NH 3 and H 2 SO 4 . The nitrogen is never changed to any oxidation 
product, but is found as NH 3 , while the sulphur is oxidized. 

Incomplete oxidation is caused by other bacteria, and perhaps molds 
and yeasts. Quite a large number of organisms live on sugar-free 
media if they have oxygen, but they do not oxidize their food com- 
pletely. We can distinguish at least three different groups of micro- 
organisms here. 

B. proteus is the collective name for a number of closely related 
forms which belong to the most common organisms found on decaying 
organic matter, especially when protein is abundant. They produce 
leucin, tyrosin and tryptophane, but no skatol, or phenol. Indol 
and hydrogen sulphide are formed in certain media. Less important, 
but also very common are the pigment-forming rods among which B. 
fluorescens, B. prodigiosus, Ps. pyocyanea are the best-known repre- 
sentatives. Their metabolism is a little different; amins and ammonia 
are formed, while hydrogen sulphide, phenol and indol are absent. 



PRODUCTS OF METABOLISM 173 

s a third group, B. coli may be mentioned which forms indol, but no 
immonia from peptone, and whose proteolytic powers are very weak 
is it does not even liquefy gelatin. 

Anaerobic decomposition of proteins is limited to very few species; 
there is a great difference in the availability of proteins and of carbo- 
lydrates as a source of energy, protein being available only to a few 
species, most of these preferring carbohydrates if they are present 
together with protein. B. putrificus is the main representative, but 
other forms exist. B. putrificus is strictly anaerobic, and a spore former, 
very common in nature. Among the products are skatol, hydrogen 
sulphide, ammonia and other very offensive compounds. 

UREA, URIC ACID, HIPPURIC ACID, are the end products of protein 
metabolism of the higher animals. The decomposition of urea to 
ammonium carbonate has been mentioned in several places, mainly 
on page 146. It is a simple hydrolysis 

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

This change can be brought about by only a few bacteria which are 
commonly grouped together as "urea bacteria." These organisms 
have hardly anything else in common, however, and the group is not a 
well-defined one. There are rods and coccus forms, motile and non- 
motile organisms, spore-formers and non-spore formers, and even molds 
ihave recently been found to hydrolyze urea. All urea bacteria can 

ive without urea, feeding on organic matter like other bacteria, but 
t of them require an alkaline medium. 
Hippuric acid is split by certain bacteria to benzoic acid and 

mino-acetic acid which can be oxidized completely. Uric acid can be 

:hanged in several ways. In some of these changes, urea is found as 

n intermediary product. 

PRODUCTS FROM MINERAL COMPOUNDS 

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



tl 



174 NUTRITION AND METABOLISM 

sulphites to sulphates, of hydrogen sulphide to sulphur and of sul- 
phur to sulphuric acid, of ferrous salts to ferric .salts. All these 
microbial changes are simple processes and can be followed by chem- 
ical analysis much more easily than organic fermentations. The 
organisms which cause these changes, do not, as a rule, thrive in 
organic substances and for this reason pure cultures can be obtained 
only with difficulty. Their activity is of great importance in soil 
fertility. 

REDUCTIONS of minerals, too, are of great significance. As a typical 
example, nitrates may be reduced to nitrites, to ammonia, to nitrogen 
gas, and, rarely, to nitrogen oxides. The reduction may be performed 
either by the direct removal of oxygen, or by the formation of free 
oxygen. The reduction of nitrates to nitrites can be written in the 
following three ways: 

KN0 3 - O = KN0 2 

KNO 3 = KNO 2 + O 

KN0 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(N0 3 ) 2 - 50 = CaO + 2 N. 

Nitrates are reduced through the efforts of the organism to secure a 
supply of oxygen. The denitrifying bacteria have strong oxidizing 
properties; they take oxygen from all sources possible. If cultures of 
denitrifying bacteria are well aerated, as in soils with a proper mois- 
ture 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. 

Sulphates are reduced in a very similar way to hydrogen sulphide 

H 2 S0 4 - 4 = H 2 S. 

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



PRODUCTS OF METABOLISM 175 

While only a few bacteria reduce sulphates, many reduce sulphites or 
lulphur to hydrogen sulphide. The potassium and sodium salts of 
elenic and telluric acid (H 2 SeO 4 and H 2 TeO 4 ) are reduced by certain 
ftrganisms and not by others. The reduction results in a colored 
ftrecipitate; this reaction has been suggested as a diagnostic means to 
distinguish different species. The reduction of arsenious oxide to 
Irsin (AsH 3 ) is used as a very delicate test for arsenic; it is applied in 
Shhe detection of arsenical poisoning. The material to be tested is 
sterilized and inoculated with Penicillium brewcaule (page 52, the 
'('arsenic mold"). This will reduce most arsenious compounds to arsin 
[I (As H 3 ) or to diethyl arsin, As H(C 2 H 5 ) 2 , both of which are easily 
Recognized by their very pronounced 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 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 original 
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 corre- 
sponding 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 



176 NUTRITION AND METABOLISM 

the mycelium. Pigments cannot be considered as reserve substances, 
since many pigments 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 j 
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 i 
individuals themselves are colorless and may under certain conditions 
cease to form pigments. To this class belong B. prodigiosus, B. cyano- 
genes, Ps. pyocyanea, and others. 

3. Parachrome bacteria, which form the pigment as an excretory prod- j 
uct but retain it within their bodies, as B. janthinus and B. molaceus. 

When the pigment is soluble in water, as those produced by Ps. 
pyocyanea and the fluorescent bacteria, it diffuses through the medium. 
When the pigment is not soluble, it either lies within the cell wall or 
between 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- 
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 tc 
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 shorl 
exposure to daylight in order to produce their pigment, while cultures \ 
grown in absolute darkness may remain colorless. Strong sunlight j 
however, will check pigment production in the same degree as da 
antiseptics and other harmful influences. 



PRODUCTS OF METABOLISM 177 

The chemical nature of microbial pigments is little known. They 
distinguished according to the solubility in various liquids, water, 
J:ohol, ether, chloroform, benzol, and other solvents, and according 
t the change of color caused by acid and alkali. A group of 
tnil in bodies, named because of their similarity to the pigment 
c carrots, the prodigiosin bodies, named after B. prodigiosus, the 




]&. 101. Bacteriopurpin, from a Rhodo spirillum, crystallized from a chloroform 
solution. (After Molisch.) 

lorescent pigments and perhaps a few other groups are distinguished, 
it their chemical nature is rather vague as yet. The absorption of 
stinct lines of the spectrum by solutions of these pigments is claimed 

be a very reliable means of distinguishing the pigments of different 
ecies. 

AROMATIC SUBSTANCES constitute another group of metabolic prod- 
ts. The chemical analysis accomplishes more with these com- 
unds than with pigments, since they are frequently well-known 
mpounds. The main difficulty arising in their identification is in 
e very minute quantities of the products available. Some substances 
th strong, mostly very disagreeable odors have already been men- 
Dned: indol, skatol, hydrogen sulphide, mercaptan, the amins and 
rimonia, butyric acid, and some of the higher alcohols. There re- 
ain to be mentioned certain oils and esters giving rise largely to 
easant aromas. The formation of aromatic oils has been established 
though their nature is entirely unknown. The same is true with the 
ters. The substance causing the fishy flavor in butter is volatile 
ith steam and is neither of an alkaline nor acid nature. The strong 
lor of freshly plowed earth is caused by an Actinomyces; the odor 
12 



178 NUTRITION AND METABOLISM 

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 leasl 
understood products of microbial action are the enzymes and the toxins 
These two groups are related in many respects. The enzymes will b< 
discussed extensively in the following chapter and toxins are treatecj 
more extensively on pages 575, 676. Toxins and enzymes are formecj 
by the cells in such small quantities that they would never have beei j 
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 am, 
enzymes are chemically unknown. It is assumed that they are chemica 
bodies, but even this has not been proved. A pure toxin has neve 
been obtained and we have no criterion for its purity. The presenc! 
of a toxin is recognized only by an animal test and in this way the con| 
parative concentration can be determined approximately. Sue 
standardization of toxin solutions is only comparative, however, an 
gives no clue as to the actual amount of toxin present. Not all an| 
mals are sensitive to all toxins. It is quite possible that all bacteri! 
produce compounds with chemical qualities similar to toxins, and onlj 
a few of them happen to react upon men or animals. 

Toxins are not always the product of microbial action. Vegetab 
toxins or phytotoxins are known, among which the ricin of the casto 
oil bean is perhaps the most studied representative. The best-kno^j 
zoo toxin is the rattlesnake poison. These non- microbial compountj 
have the same quality as the microbial toxins they are extreme | 
poisonous. Toxins are the cause of disease in diphtheria, tetanus ai 1 
botulism. If a culture of these organisms is filtered through a porcela 
filter which removes all bacterial cells, the filtrate injected into \\ 
animal will cause the disease with all its accompanying symptori 
though there are no microorganisms introduced into the animal bod| 



PRODUCTS OF METABOLISM 179 

I the filtrate is heated, however, no effect will take place after the in- 
Iction, because heat destroys the toxin. The amount of toxin that will 
[111 an animal is extremely small. .000005 m - f tne purest tetanus 
|)xm will kill a mouse, .0007 mg. of ricin will kill a rabbit, less than 
83 mg. of tetanus toxin will kill an adult man. The body of an animal 
T man forms an anti-body against the toxin which neutralizes its 
oisonous action. Anti-bodies are also formed against enzymes 
njected into an animal. 

Toxins are very sensitive to heat. A short exposure to temperatures 
etween 80 and 100 will inactivate them. They are also very sensi- 
ve to light. While some toxins are secreted, others are retained within 
le cells of microorganisms, and never leave them until the cells die or 
isintegrate. Ptomains, which are also metabolic products of micro- 
rganisms and sometimes cause poisoning, differ from the toxins in their 
esistance to heat and light (page 171). Ptomains differ in no way 
bsentially from ordinary organic compounds; the animal or human 
lody produces no anti-ptomains to counteract their poisonous effects. 
There is no chemical relation whatever between toxins and ptomains, 
nd the physiological effects are also quite different, though they both 
lause poisoning. 

Toxins are not essential products of the metabolism of pathogens, 
trains of pathogenic bacteria can be bred which do not produce toxins 
9 chromogens can be bred without pigment, or lactic bacteria which 
o not produce acid. The strains which lose their pathogenicity grow 
etter on artificial media but are less able to produce disease in the 
nimal. They may regain the power of producing toxin if passed 
irough the body of the animal. The real object of toxin production 
y microorganisms is not known; the microorganisms derive no ap- 
irent benefit. 

FACTORS INFLUENCING THE TYPE OF DECOMPOSITION 

In the chapter on products of metabolism, it has been shown 
hat the same compound can be decomposed in many different ways, 
nd the question may well be asked what decides the type of decomposi- 
on. Since bacteria are widely distributed, it must be expected that 
here are certain conditions which are most favorable to a given type 
f fermentation, while under changed conditions, other types are more 



l8o NUTRITION AND METABOLISM 

likely to dominate. The fact that sugar in cider nearly always under- 
goes alcoholic fermentation, while in milk it undergoes lactic fermen- 
tation, has its reason in the physiology of the bacteria, and in their 
reaction upon the environment. 

Cider is acid, and acid is not well suited for the growth of most 
bacteria. The vinegar bacteria can grow in fruit juices, and a few other 
bacteria, especially those causing trouble in wine, are not retarded by 
fruit acids, but the common types attacking proteins and causing 
organic decay are not able to grow on fruits. Yeasts, however, and 
molds thrive well only in acid media. They can exist in neutral 
solutions if in pure culture, but in nature they are easily crowded out 
by bacteria. Acidity of the medium is therefore one of the most 
important factors regulating the type of microbial decomposition. 
This principle is commonly utilized by preserving foods of all kinds in 
vinegar, and by making butter from sour cream rather than sweet 
cream; the keeping qualities of hard cheeses depend upon their acid 
content. 

In acid environment, the two most common types of decompositior 
are oxidation, complete or incomplete, and alcoholic fermentation 
The oxidation is brought about by molds or organisms closely allied t< 
yeasts. The latter are very common on all sour foods, especially or 
foods containing lactic acid, such as cottage cheese or sauerkraut 
The kind of acid decides the type of mold; wherever there is lactic acid 
there is Oidium, while malic and tartaric acids favor Penidllium am ] 
Aspergillus. 

If the decaying materials contain no acid, the type of decomposi 
tion depends mainly on the presence or absence of carbohydrates 
especially sugar. It is an old experience, recently verified througlj 
a large number of experiments by Kendall and Walker, that practicall; 
all bacteria will decompose sugar in preference to proteins. If a lea 
contains sugar and protein (cabbage) the sugar decomposition will b 
conspicuous, and the protein is not attached very readily. Putrefac 
tion in the presence of sugar or of acid does not take place. Meat wi 
not putrefy if mixed with sugar, while milk putrefies readily if the suga | 
is removed by dialysis. The three types of sugar decomposition whic 
come into consideration in neutral media, are the lactic, the acid-gas an j 
the butyric fermentations. The latter is a strictly anaerobic ferment^ 
tion, and thus limited to special conditions. Of the other two, the acici 



PRODUCTS OF METABOLISM l8l 

rmentation is the most common, and the souring of vegetables 
f all kinds is due to this type of fermentation (pickles, sauerkraut, 
nsilage, salt-rising bread). Sometimes the acid-gas fermentation 
P followed by a butyric fermentation. The true lactic fermentation 
I not common, and is limited almost entirely to milk. This is ex- 
(lained by the circumstance that the organisms causing this decom- 
osition are parasitic in their habits, causing disease or living in the 
itestine of animals. In the absence of acid and sugars, putrefaction 
B the most common type of decomposition. 

Many factors aside from the chemical composition of the medium 
re essential. Oxygen has already been mentioned as preventing buty- 
ic fermentation. It will also prevent the acid-gas fermentation if too 
bundant. Ensilage is trampled and pressed down to avoid air spaces 
s much as possible, for molds will outgrow the acid-forming bacteria 
: air has free access. Absence of oxygen will prevent mold growth, 
nd for this reason, jelly is paraffined, and butter wrapped tightly into 
mpermeable paper. The influence of oxygen upon the type of protein 
j,nd of cellulose decomposition has been pointed out previously. 

The moisture content is of great importance. As will be shown 
ater, not all organisms have an equal need of moisture; some molds 
vill grow on foods too dry for bacteria and yeasts. Molds are es- 
pecially adapted for growing on dry media, as only part of their cell 
mbstance is immersed in the medium. Their thread formation enables 
hem to search a dry medium, such as flour, for moisture, the extreme 
if adaptation being Rhizopus, and the construction of the fruiting 
)odies shows that they are destined by nature to be spread by air and 
*dnd. It is no wonder that damp organic matter, if it can be de- 
omposed at all, will show molds, and nothing else, regardless of the 
hemical composition, for there is no competition. Flour, moist seeds, 
ncompletely dried fruit, damp milk powder will always become 
noldy. The same holds true with very concentrated sugar solutions 
uch as syrups, jellies and jams, while in concentrated salt solutions, 
nolds cannot thrive, and the torula yeasts are best adapted to such 
:onditions. 

A very important part is also the structure of the material. Micro- 
>rganisms act mainly upon organic matter, and since this comes 
rom living organisms, it has usually definite structure, exceptions 
:>eing milk and blood. The structure of all living organisms is such 



182 NUTRITION AND METABOLISM 

as to prevent the intruding of microorganisms. The body of plants 
and animals is surrounded on the outside by tough and dry layers of 
epithelial cells, and the cavities of the animal body also have their 
protective membranes. Microorganisms cannot enter the tissues 
if these membranes are perfectly sound, and we know that, as a rule, 
the tissues of healthy plants or animals are free from bacteria. Thus, 
a healthy apple or potato or egg will not be infected and decomposed 
by microorganisms if handled carefully, meat will begin to decom- 
pose on the outside, and the inner parts may be still good when the 
outer layer is already in a state of decay. 

In the plants, each cell is surrounded by its special cell membranes 
which are a barrier to infecting organisms. If we prick the skin of a 
healthy apple with a pin infected with yeast, the infection will not I 
spread though we know that yeast will grow most abundantly in cider;! 
in the apple, however, it has no means of spreading from one cell tc 
the other. Molds possess this means; they can puncture cell walls 
and forcing their way from one cell to the other, they will soor 
bring about the rotting of the entire fruit after it once becomes 
infected. This protection seems especially necessary in the plant's 
roots which are greatly exposed to injury from insects and other animals j 
in the soil and surrounded by billions of microorganisms. They ar<! 
attacked only by fungi which can force their way from cell to cell! 
or by bacteria which can dissolve the membranes by means of enzymes j 
and thus cause a softening of the root tissue. The bacteria causing th< 
various rots of vegetables belong to this type. 

There is, then, a great variety of factors deciding the type o 
decomposition of organic matter in nature, and by knowing the chemica 
composition as well as the structure and other physical conditions 
it is possible to foretell which group of organisms is most likely t 
attack the compounds in question. 

Another quite important factor, the temperature, will be dis' 
cussed in more detail in one of the following chapters. 

ROTATION OF ELEMENTS IN NATURE 

All organic matter on earth is undergoing continuous change. Oi| 
ganisms grow and decay. The same carbon and nitrogen atoms whic 
constitute the organic world of to-day constituted it thousands <j 



PRODUCTS OF METABOLISM 183 

ears ago. The amount of carbon, nitrogen, hydrogen and of all 
ther elements of life on earth is limited, and the same atoms will 
ie used for the future generations of life that constitute the present, 
.[here must be continuous destruction to enable new construction. 
Construction is mainly the task of green plants, enabled by the chloro- 
hyl to use the energy of sunlight in building up organic substances 
rom minerals, water and carbon dioxide. Destruction is caused 
nainly by animals and other organisms which have to break down 
irganic matter in order to exist. These two factors keep the atoms of 
ine organic world in perpetual rotation. 

In this circulation of the elements it is necessary that all compounds 
if organic nature be decomposed finally to a form available for plant 
iood. If this were not the case, the indestructible compound would 
looner 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 
iecomposed 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 con- 
tinuously. The enormous quantities of nitrogen taken out of cir- 
culation 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, 
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 
the study of its cycle might be begun with its simplest compound, 
the carbon dioxide of the air. It is absorbed in this condition by the 
green plants, and is changed by the chlorophyl granules of the leaves to 



184 NUTRITION AND METABOLISM 

organic compounds of various types, either to carbohydrates (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 com- 



Gt 

rrote/ 




ouo 
/'at, Protein 

FIG. 102. Carbon cycle. 

pletely oxidized to carbon dioxide. Even the marsh gas which might! 
be liberated in this process will find organisms that oxidize it to carbon j 
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 j 
life. Fig. 102 gives a schematic illustration of the carbon cycle; the I 
microbial activity is marked by heavy lines. 



PRODUCTS OF METABOLISM 



NITROGEN CYCLE. Nitrogen shows the same continuous change 
as carbon. Plants take up nitrogen in mineral form usually as nitrates. 
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. 103). 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, 



Dead 
Organisms 




tftrates 



Prot 



FIG. 103. Nitrogen cycle. 



ammonia. Ammonia is then oxidized to nitrites and nitrates, when 
the nitrogen cycle is completed. 

There is, however, one discrepancy in this cycle. It has been 
mentioned 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 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 nitrates would be produced from decaying organic matter and 
would eventually be destroyed. After a certain time, this continuous 



1 86 NUTRITION AND METABOLISM 

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 continu- 
ously. Finally, the plants would cease to grow because the nitrogen in 
the soil would be exhausted. 

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 proto- 
plasm. Thus, organic nitrogen is produced from nitrogen gas and the 
continuance of organic life is guaranteed. 



-Sulphide 



an/sms 




FIG. 104. Sulphur cycle. 

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 contain- | 
ing a certain amount of sulphur (Fig. 104). These bodies are either j 
digested by higher animals or broken down by putrefaction to the I 
final 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 i 
out completely, but from the discussion in the last pages, it is plainly j 
seen that a simple cycle very much like the ones above must exist. It ' 



PRODUCTS OF METABOLISM 187 

s probably much simpler because phosphorus does not enter as easily 
into organic compounds as nitrogen. 

PHYSICAL PRODUCTS OF METABOLISM 

PRODUCTION OF 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 142. Decomposition of organic matter means a liberation of 
energy which is used for the continuation of life processes; the utiliza- 
tion is, as a rule, incomplete, and a part of the energy 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. 

Much heat is produced in the vinegar fermentation. In the quick- 
vinegar process (page 546) the temperature rises sometimes as high as 
10 to 15 above the temperature of the room and the vinegar manu- 
facturer uses the heat produced by the bacteria to keep the generators 
at the optimum temperature. If the process is not controlled 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. 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 



1 88 NUTRITION AND METABOLISM 

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 not of bacterial origin, but due 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 into contact with oxygen again, they produce light 
immediately, and this sudden flashing is used occasionally by physiolo- 
gists as a very delicate test for oxygen. The light appears to be pro- 
duced 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 com- 
pound is formed in the cell which generates light when in contact 
with oxygen. 

The life processes of the photogenic microorganisms are not neces- 
sarily 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 
pigmentation also, no bearing upon the development of the cell, and the 
light-giving compounds may be regarded as incidental waste products. 
Certain 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, KATABOLISM, METABOLISM. In the introduction to the 
Physiology of Microorganisms, it was stated that microorganisms need 
food for at least two different purposes: building material and building 
energy. They may need it for other purposes also, e.g., for motion. 
The sum of all changes which the food undergoes in the body, including 
the deterioration of the cells, is called metabolism. Metabolism con- 
sists of several separate functions: One of them is the construction 
of new cells, or parts of cells, called anabolism, another the deteriora- 
tion of cells, called katabolism, and the most important quantitatively 
is the fermentation or respiration. The fermentation or respiration 
processes are fairly well understood; many of them can be produced 
in the chemical laboratory without microorganisms. Katabolism is 
the sum of many processes some of which are well understood while 
others are still unknown. The synthetic, anabolic processes of the 
cell, however, are almost entirely unknown, and we can only speculate 
regarding the various means by which the cell grows. The explana- 
tions of the different cell activities began, as in most other fields of 
theoretical bacteriology, with a close analogy with animal and plant 
metabolism, but owing to the comparative simplicity of the micro- 
organisms, they led to the establishment of new facts and theories which 
proved afterward useful for the understanding of the metabolism of 
the more complex organisms where the multiplicity of facts prevented a 
clearer insight into the separate processes. 

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, 

189 



1 90 NUTRITION AND METABOLISM 






depend upon soluble food since they have no means of incorporating 
insoluble compounds into their protoplasm. The protoplasm, however, 
must be considered the center of metabolism, and the digestion of food 
and the formation 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 by secreting certain agents with 
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 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 protein bodies; there is ereptase 
for the peptones, lipase for the fat, imertase for the saccharose, and 
many other enzymes. The object of all these enzymes is apparently 
to prepare the food for passing through the membrane into the proto- 
plasm of the cells, where the final changes which liberate energy take 
place. The same processes occur with microorganisms but in a more 
simple manner. Surrounded by a liquid medium, they secrete enzymes; 
these dissolve certain insoluble foods which then diffuse through the 
cell wall to be decomp.osed 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 HioO 5 ) n + nH 2 O = nCi 2 H 22 Oii. 



MECHANISM OF METABOLISM IQI 



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



Oa + 3H 2 = C 3 H 5 (OH) 3 + 3 

Tristearin Glycerin Stearic acid 

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. Iniertase decomposes 
saccharose into dextrose and levulose: 

Ci 2 H 22 On + H,0 = 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 
compounds 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 
enzymic 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. Rennet will coagulate many hundred 
times its weight of casein, and still the whey will contain rennet. Con- 
sidering 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 179). 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 



I Q2 NUTRITION AND METABOLISM 






can, like coagulated albumin, by no means be brought back to their 
original state. This temperature 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 
secreted it. Light will also affect enzymes considerably. The great 
similarity of enzymes and microorganisms in these respects, the simi- 
larity of their reactions' and the extreme minuteness of the bacteria 
render it explicable why the chemists of eighty years ago could not 
determine the difference between microorganisms and enzymes, and 
called them both "ferments." 

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 en- 
zymes 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 in- 
fluences. Mineral salts seem essential for their action, because con- 



MECHANISM OF METABOLISM 193 

inued dialyzing weakens the activity which can be restored only by 
idding salts. 

ENZYMES OF FERMENTATION. It has been demonstrated in the 
ibove paragraph that food is prepared for digestion or fermentation by 
nzymes. The final decomposition, the process which yields the energy 
or cell life, must take place within the cell. 

The difference in importance of food preparation and fermenta- 
ion may be illustrated by the example of Rhizopus oryzce. This 
nold attacks starch, changes it, by means of diastase, to maltose, 
he maltose to dextrose, dextrose to alcohol and carbon dioxide. The 
mold grows well in a starch medium, without sugar; it grows equally 
well in maltose, and equally well, or better, in dextrose; it does not 
grow at all with alcohol and carbon dioxide. The last change, dex- 
trose to alcohol, is absolutely necessary for this organism; it is the 
^ource of its life; the others are incidental processes, not absolutely 
jnecessary under all circumstances, in fact greatly suppressed if dextrose 
is given together with starch. The fermentation must take place in 
the cell; the preparation of food may take place in the cell or outside; 
t is not essential where it happens. 

The investigations of recent years have demonstrated that fermenta- 
tions also are caused by enzymes. It has been proved beyond doubt 
that in the alcoholic, lactic, acetic and urea fermentations the fermen- 
tation process may continue after the death of the fermenting cells. 
In the case of alcoholic fermentation, the fermenting agent was 
separated first by Buchner from the lacerated cells and was 
filtered through porcelain filters without losing its ability to act. 
This proves the enzyme-nature of the fermenting agent which, once 
being formed, remains and acts independent of the cell. These en- 
zymes 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 pa- 
tience 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 biochemical changes taking place in the final dissociation 
of food within the cell are 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 

13 



194 NUTRITION AND METABOLISM 

oxidations and many reducing processes have been recognized as caused 
by enzymes, and it is quite probable that the whole process of intra- 
cellular food decomposition in all organisms is accomplished entirely 
by means of enzymes. 



CLASSIFICATION OF ENZYMES 



jely 



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 en- 
zymes. This definition is not quite exact, since the urea fermenting 
enzyme is also a hydrolyzing enzyme, and the acetic fermentation is 
caused by an oxidizing enzyme. The distinction between endo-enzymes 
(infra-cellular) and 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), erep- 

sin (ereptase). 

(6) coagulating (coagulases) : 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, methylene 
blue, litmus. 

Several different names have been given to some of the enzymes; 
these are found in parenthesis in the above classification. "]j| 

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



MECHANISM OF METABOLISM 1 95 

HYDROLYTIC ENZYMES 

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

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

, considering that the cellulose molecule is really many times 
}Hi O 5 , the formula will be more accurately written 

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

hich indicates at the same time that one cellulose molecule gives 
any sugar molecules. 
Cellulase is an enzyme which is quite difficult to obtain. Though 

must be produced by all the cellulose destroying molds and bacteria, 
^periments have failed in some instances to prove its presence. It 

found in some wood destroying fungi and in some of the bacteria 
lusing the rot of vegetables. The organisms of certain plant diseases 
>rce their way into the cell by dissolving the cellulose membrane by 
a enzyme, while certain molds are able to puncture the cell wall 
echanically. 
Diastase, or amylase, is the starch-dissolving enzyme which is one 

the most common enzymes in nature. It is found in all green plants, 
nd it forms during the sprouting of starchy seeds. Many molds 
ad a few bacteria produce this enzyme, while yeasts generally cannot 
ecompose starch for lack of diastase. Starch has the same formula 
5 cellulose, and it is broken up into soluble sugars in the same way. 
luch attention has been paid to this process by the chemists, and it 

found that the process is a gradual one, giving first dextrins, and 
rially maltose (Ci 2 H 22 On). The hydrolysis of starch expressed in 
icmical symbols may be presented as follows: 

2(C 6 Hio0 5 )n + nH 2 O = nCi 2 H 22 On. 

Starch Maltose 



IQ6 NUTRITION AND METABOLISM 

The disaccharides or double sugars, having the chemical formula 
Ci2H 2 2Ou are broken up into single sugars, monosaccharides, by the 
following process: 

C 12 H 22 Ou + H 2 = C 6 H 12 6 + C 6 H 12 6 . 

The two molecules of C 6 Hi 2 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 par- 
ticular sugar and none of the others; like a key, made for one lock, 
it will not open another lock. Maltase will split only maltose mole- 
cules, not lactose, while the lactase cannot attack the maltose. In- 
vertase (or sucrase) will decompose nothing but saccharose. This 
decomposition of the complex sugars into the simple sugars was be- 
lieved to be necessary because only sugars of the type CeH^Oe 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 
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. Recent 
experiments have shown that bacteria exist which ferment lactose 
and saccharose but not dextrose or levulose. An explanation for this 
cannot be given. 

Invertase is, like diastase, a very common enzyme in green plants. 
It is also produced by most molds and yeasts, and 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 especially 
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 monosacchar- 
ides. If these are decomposed further, the resulting product is no 
longer a carbohydrate. The simplest sugars are decomposed by zy- 
mases, inside the microbial cell, into compounds which are generally 



:alled 



MECHANISM OF METABOLISM 1 97 



ed fermentation products; these may result from alcoholic, lactic, 
DUtyric fermentations or some other. 

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

C 20 H 27 OnN + 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 
Deen found in yeasts. Glucoside-splitting enzymes play an important 
r61e 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 168. It is possible that there are several fat-splitting enzymes, 
jbut 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 exclusively substituted by the more significant word lipase. 
Occassionally they are called lipolytic enzymes which expression is 
analogous to the proteolytic enzymes; in the same way, the term 
amylolytic enzyme is used for diastase. 

ENZYMES OF PROTEINS. The enzymes composing 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 enzymes which accomplish the digestion of protein food. Naturally 
most experimenting had 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 experi- 



198 NUTRITION AND METABOLISM 

ences 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 formalde- 
hyde 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) i 
the proteolytic enzyme of the animal stomach, and from trypsin (tryp- ' 
tase) which is found in the small intestine of animals. This classifi- 
cation 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 tryp- 
sin 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 on the 
decomposition as far as amino-acids and traces of ammonia. Another 
class recently recognized is ereptase (erepsin) which cannot decom- 
pose 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 the 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 -f 
sign means proteolysis, a sign means no action. 



MECHANISM OF METABOLISM 



199 



Milk 


Gelatin 


Serum 


Egg 


Pihrin 


Coag. Digest. 






album. 




Bact. anthracis -f + 
Microspira comma 4- 4- 
M. pyogenes var, aureus 4- 4- 
Pscudomonds pyocyanea -f- 


4- 


4- 


4- 


+ 


B. violaceus 


4- 








B. mycoides + + 
B. prodigiosus 4- 
Aspergillns niger 4- 4- 
Aspergillus oryz(C 4" 




; 


; 


I 



Apparently not all organisms which liquefy gelatin are able to de- 
compose egg albumin; we must conclude that the enzyme liquefy- 
ing gelatin is different from the proteolytic enzyme dissolving egg- 
white. 

COAGULATING ENZYMES. The blood-clotting enzyme (throm- 
base) 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 precipitated by the acid. If enough acid is 
added, the milk curdles immediately; 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 addition 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 
Bacteriologists uses the German word "lab" 



200 NUTRITION AND METABOLISM 

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 fermenting 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 .' . . 80 calories 

Lipase 4 calories Alcoholase 120 calories 

Maltase, invertase 10 calories Urease 230 calories 

Lactase 23 calories Vinegar-oxidase 2,500 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 
insignificant. 

The first zymase known was urease, the enzyme which changes 
urea to ammonium carbonate. The actual investigation of the 
zymases did not start until Buchner had demonstrated that yeast can 
be ground with infusorial earth until all cells are lacerated, and then 
can be pressed and the juice filtered without losing the power of alco- 
holic fermentation. Such fermentation, cannot be due to anything 
but a soluble compound of the yeast cell. Thus the alcoholase was dis- 
covered. 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, lactad- 
dase. 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 fermentations 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 un- 
stable 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. 



T<1. 



MECHANISM OF METABOLISM 2OI 



The vinegar oxidase is an enzyme which remains in the cell of the 
Acetic bacterium, oxidizing alcohol to acetic acid. Its independence of 
he living cell has been demonstrated by killing the cells with acetone. 

The PROTEOLYTIC ENDO-ENZYMES of yeasts, only, have been studied 
xtensively. That such enzymes exist is recognized by the observa- 
ion that certain microorganisms do not liquefy the gelatin until 
liter they are dead and the proteolytic enzymes diffuse out through 
he deteriorating cell membranes. That yeast in the absence of 
ugar loses in weight, and that leucin and other cleavage-products of 
>rotein are formed, was the first indication of a proteolytic process in 
he yeast cells. By pressing the juice out of the ground yeast cells, 
k liquid is obtained which liquefies gelatin, digests casein, albumin and 
ibrin. The living yeast cell does not attack these compounds, be- 
pause they cannot diffuse into the cell and the enzyme cannot diffuse 
Dut. The proteolytic endo-enzyme of yeast is called endo-tryptase. 
[ts object is apparently the regulation of the protein-content of the cell 
md perhaps it has some bearing on the formation of cell plasma. 
The possible relation between enzymes and growth is discussed in a 
"ollowing sub-chapter. 

If yeast is mixed with a weak antiseptic (chloroform, toluol) 
:he proteolytic process takes place quite rapidly. This process is 
called autolysis (self-digestion). Similar autolytic enzymes are found 
in other microorganisms. Autolysis is a well-known process in the 
ligher animals. To this is due the ripening of meat. 

Proteolytic endo-enzymes must be expected in all microorganisms 
^vhich depend upon protein as food material only. These organisms 
secrete certain enzymes which decompose the insoluble protein 
nto bodies which diffuse easily into the cell. Here, proteolytic endo- 
;nzymes further decompose these products. Such an endo-enzyme is 
:he amidase discovered by Shibata in the mycelium of Aspergillus 
riger which forms ammonia from urea, acetamid, oxamid, biuret. 
Endo-erepsin and amidase were also found in Penicillium camemberti 
)y Dox. 

Similar to these proteolytic enzymes is the urease which is formed 
n large quantities in the so-called urea bacteria, but it is also present 
n the mycelium of some molds. An endo-enzyme, splitting hippuric 
icid into benzoic acid and glycocoll, is found in the mycelium of a few 
nolds. 



202 NUTRITION AND METABOLISM 

OXIDIZING ENZYMES 

The most typical example of an oxidizing enzyme is the mnegar- 
oxidase, because its chemical action is well known. Most of the oxi- 
dases 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 test- 
ing 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. Hydro- 
chinon 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 oxidarJihs 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 
bacteria. Among these exceptions is the Strept. lacticus. 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 intra-cellular 
oxidations. 

A number of other reductases are known. Nearly all of the re- 
ductions 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 



MECHANISM OF METABOLISM 203 

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 produces 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 alcohol, this means that th*alcoholase of this yeast cannot 
I tolerate more than 8.5 per cent of alcohol. The inability of the cell 
i 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 observed in the lactic, acetic and alcoholic fer- 
mentations, and, perhaps, occurs in many others. 

Probably, all cells produce several enzymes. Microorganisms 
; feeding upon various foods must form various enzymes. Frequently 
i several enzymes are necessary for the decomposition of one com- 
pound. Rhizopus oryzcB 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. Asper- 
gillus niger has the reputation of forming almost all enzymes which 
i have ever been found in microorganisms. Penicillium camemberti 
i produces (after Dox) erepsin, nuclease, amidase, lipase, emulsin, 
lase, inulase, raffinase, invertase, maltase and lactase. It has 
believed for a long time that certain enzymes are regular products 
e cell while others are formed only if the substance upon which 
act is present. According to Box's investigations with Peni- 
ium camemberti, there is no evidence that enzymes not normally 
ed by the organism in demonstrable quantities can be developed 
>y special methods of nutrition. The addition of a particular 
food compound does not develop an entirely new enzyme, but stimu- 



204 NUTRITION AND METABOLISM 

lates the production of the corresponding enzyme which is normally 
formed, although in small amounts, under all conditions. 

THEORY or KAT ABOLISH 

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, 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 separate from the products of food digestion which 
would obscure the results of experiment on 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 men- 
tioned in the above paragraph. Unquestionably, the katabolic processes 
are similar to enzyme processes, since katabolism is checked by heat 
or poison just like enzyme processes. 



THEORY or ANABOLISM 






ANABOLISM AND INTRA-CELLULAR ENZYMES. All changes dis- 
cussed in the previous chapters are processes in which organic or 
inorganic compounds are broken up to smaller molecules. These 
processes are exothermic, i.e., liberating heat or energy in other forms. 
The opposite is true of the anabolic processes which build up complex 
molecules from simple compounds. These synthetic processes are 
endothermic, absorbing heat or other energy. 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 com- 
pounds necessary for cell life. Of. all the substances found in the cell, 
practically none are contained in the food, and it is wonderful thai 
in such a small unit as a microbial cell, there are contained the power? 
of making protoplasm, enzymes, nucleaf bodies, chroma tin bodies, 
the substance of the cell wall and probably many other unknowrj 



MECHANISM OF METABOLISM 205 

ompounds. All these complex substances are generally made from 
imple food compounds as amino-acids, carbohydrates and others. 

These synthetic processes of the cell will, like most endothermic 
processes, take place only if energy is provided. This condition is 
sually fulfilled in the living cell, due to the fermenting processes 
oing on continuously. There is a strange interaction between 
f nabolism and mtra-cellular fermentation proceeding in the pro- 
ioplasm and this linking together of destructive and constructive 
eaction is the basis of life processes. The life processes decompose 
:ertain substances, the energy liberated allows the formation of proto- 
plasm, which again liberates energy. Thus a continuous formation of 
orotoplasm is secured. 

An explanation of anabolism based upon chemical experiments is 
lot possible at the present time. In the study of intra-cellular destruc- 
tion it is possible to trace most processes back to enzymic action. 
There our knowledge ceases because the nature and mode of action 
f enzymes is unknown. In the study of anabolism our knowledge 
las not even progressed so far. The most promising explanation at 
)resent is based upon the reversibility of enzymic action. 

REVERSIBILITY OF ENZYMIC ACTION. Chemical reactions be- 
ween organic compounds proceed quite rapidly at first, then become 
lower and slower until the reaction stops entirely. The reaction is 
lot complete at the time it reaches an equilibrium. If the equilib- 
rium 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 
broceed either way, depending upon the relative concentrations of 
the reacting substances. The standard example is esterification. As- 
jcetic acid plus alcohol gives ester plus water, 

CH 3 COOH + CH 3 CH 2 OH^CH 3 COOCH 2 CH3 + H 2 O. 

Acetic acid Alcohol Ester 

The proce'ss 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 



206 NUTRITION AND METABOLISM 

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 com- 
plex 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 specula- 
tion about assimilation and growth. It seems reasonable to suppose 
that the cell forms its protoplasm from ammo-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 speculation, and the only fact to support it is the reversi- 
bility of certain enzymes. The conditions under which chemical reac- 
tions 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 understanding of all 
these reactions. Thus, our knowledge 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 
ily bacteria, but every microscopic and macroscopic being requires a 
nsiderable amount of moisture. Living organisms contain on the 
^erage between 70 per cent and 90 per cent of water, and only 10 per 
nt to 30 per cent of solid matter. Microorganisms which live 
itirely submerged in liquids need water not only within but without 
ic cells. Bacteria, yeasts, molds, and some protozoa obtain their food 

diffusion through the cell-membrane; their food-substances must 
e soluble and dissolved. No other liquid can take the place of water. 

The amount of water required by microorganisms cannot be stated 
rieSy. Several factors have to be taken into consideration, as the 
smotic pressure, the insoluble and the colloidal substances, the species 
f organisms, temperature, and perhaps others. 

OSMOTIC PRESSURE. In the organic world we find very commonly 
icmbranes which will allow water to pass through but retain some 
ompounds dissolved in the water. Such so-called semi-permeable 
embranes are found surrounding the protoplasm of cells. They are 
ot the cell wall, but separate the protoplasm from the cell wall, 
imilar properties are found in parchment paper, pig's bladder, and 
ther organic membranes. 

If a salt solution is poured in water, the two liquids will mix in a 
hort time and soon every smallest portion of the mixture will have the 
a me concentration. If a salt solution and water are separated by a 
lembrane which does not allow the salt to pass, the water will go 
irough the membrane toward the salt with a certain amount of 
ressure. This pressure depends upon the nature of the dissolved 
ubstance as well as upon its concentration. 

207 



208 PHYSICAL INFLUENCES 

The pressure increases in direct ratio with the number of molecules 
in solution. Therefore, a compound with large molecules (cane sugar) 
will produce a lower osmotic pressure than one with small molecular 
weight (glycerin) if we compare solutions of equal concentration. 
The osmotic pressure of protein, starch and peptone solutions can be 
measured only with the finest instruments, while the pressure of a 30 
per cent dextrose solution is 22 atmospheres.* 

PLASMOLYSIS. If a cell is brought into a strong solution of a sub- 
stance 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 pres- 
sure 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 Fig. 67, page 87. 

While plasmolysis is easily demonstrated with the cells of higher 
plants, microorganisms do not show it so readily. In fact, many bac- 
teria, like B. subtilis, Bad. anthracis, cannot be plasmolyzed by any 
concentration of salt in solution. Others, as B. coli, B. fluorescent, 
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 whatevei 
is explained in the same way. These organisms probably have plasma- 
membranes so constructed that the salts diffuse through nearly as fast aj 
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 diffusior 
through the membrane. 

The resistance of various microorganisms against concentrate( 
solutions depends upon the organism as well as upon the dissolved sub 
stance. The sodium and potassium salts of the common mineral acid j 
act upon a culture nearly in proportion to their osmotic pressure, bu 
the potassium salts always retard growth a little less than the sodiunj 
salts. The effect of salts upon microorganisms is therefore not due t 
the osmotic pressure only; the chemical constitution of the salts alsl 
plays an important r61e. 

* One atmosphere equals the pressure of i kg. per square centimeter or about 15 pouncj 
per square inch. 



MOISTURE 209 

The different functions of life are influenced in different degrees by 

ntrated solutions. Some bacteria will multiply but not form 

:s in salt solutions. Molds will sometimes show a good growth in 

titrated sugar solutions but fail to produce spores. Bact. anthracis 

its virulence in sea water. Often, the form of microorganisms is 

tlected by concentrated solutions. Some bacteria grow more spherical, 

thers become elongated or distorted. The deforming influence is not 

ue to the osmotic pressure only, but depends mainly upon the chemical 

haracter of the salt; magnesium salts especially have a tendency to 

produce such involution forms. 

Salt and Sugar Solutions. Most experiments on the influence of 
pncentrated solutions have been carried on with sodium chloride, be- 
ause 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 to 10 per cent. At 1 5 per cent only few cocci develop 
slowly, while some species of Torulce grow without a very noticeable re- 
tardation. Above 20 per cent the Torulcs, are practically the only 
organisms which can develop. They are, therefore, found in all food 
)roducts which are preserved by salt, as salted pork, beef, fish, butter, 
md pickles, often in nearly a pure culture. It seems that they are 
easily overpowered by other organisms in the absence of salt, but in 
;alted food, this competition is eliminated. 

The selective influence of salt is used in some fermented products to 
prevent undesirable fermentations. This is true in sauerkraut and 
Drine 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 
hould 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. Its preserving effect is indeed a little higher than 
that of saccharose, but the proportion is not nearly 1:2. The common 
molds are extremely resistant to strong sugar solutions, about 60 to 70 
per cent of cane sugar seems to be the limit of growth for Penicillium 
and Aspergillus species. Yeasts can also grow and ferment in very con- 



210 PHYSICAL INFLUENCES 

centrated solutions while bacteria in general do not tolerate solutions 
higher than 15 to 40 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. prodigio- 
sus 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, namely, colloidal 
bodies which have a very large molecular weight. Their osmotic pres- 
sure 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 decora- 
posed. The above statement refers only to solutions, while in natural 
media as dried foods or soil, a combination of solid and dissolved 
substances is involved. Butter is an excellent medium for many bac- 
teria, 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 solid 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 microorganisms in the droplets of buttermilk 



MOISTURE 211 

ibuted all through the butter. The concentration in these small 
ets is the deciding factor. If the growth of microorganisms in 
utter is to be prevented by salt, it is unnecessary to give any attention 
o the fat; the bacteria live only in the water and not in the fat globules, 
n adding 3 per cent of salt to a butter with 15 per cent of moisture, a 
)rine of 3 parts of salt in 15 parts of water is produced; in other words, 
20 per cent brine, because salt does not dissolve in the fat. Similar 
onsiderations will come up in the preservation of fruit, vegetables, meat, 
ilk, and other food substances by drying or condensation. 

DESICCATION. Microorganisms do not die immediately after the 
moval of the water, and they do not die all at once after a given time, 
eath through drying is a slow and regular process. Paul and his 
Associates founded that the number of bacteria dying in the unit of 
jtime is, under constant conditions, proportional to the number sur- 
viving. If we had 1,000,000 cells per gram in the beginning, and the 
death rate were 90 per cent per day, there would be, at the end of each 
day, 10 per cent of the original number surviving. This would give the 
following numbers for one week: 

Beginning 1,000,000 cells per gram. 

After i day 100,000 cells per gram. 

After 2 days 10,000 cells per gram. 

After 3 days 1,000 cells per gram. 

After 4 days 100 cells per gram. 

After 5 days 10 cells per gram. 

After 6 days i cell per gram. 

After 7 days o.i cell per gram. 

This table shows graphically the mode of death of dried bacteria. The 
number of cells approaches zero without ever (at least theoretically) 
reaching it. From one cell per gram after six days we do not come to 
lo on the seventh, but to one cell in 10 g. and on the eighth day one 
cell in 100 g. The total number dying in the first day is much larger 
than that dying on the sixth day, but the rate is constant, 90 per cent 
of the number surviving. This regularity has been found with bacteria 
dying from various causes, and it is commonly compared with the 
simplest chemical processes, the monomolecular reactions. 

Paul and his associates found further, that the death through drying 
I is caused by an oxidation process; in pure oxygen bacteria died much 
(faster. The poisonous effect of oxygen upon moist bacteria has already 
jbeen pointed out on page 156. 



212 PHYSICAL INFLUENCES 

Most resistant to drying are the spores of bacteria; mold spores, 
too, show considerable resistance, while some bacteria, e.g.,B. carotarwn 
and Ps. radicicola, are readily killed. 

The resistance of microorganisms is influenced greatly by the me- 
dium 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 char- 
coal kept as long as ten years; Ps. 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 con- 
taining an average number of 17,000,000 bacteria per gram 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 cul- 
tures of microorganisms are commonly sold for several purposes, as 
dairy-starters and the so-called "magic yeast" and "yeast foam" used for 
bread-making. Such cultures are dried on milk, sugar, starch, flour or 
similar porous and absorbing material. Starters 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 consider- 
able importance in the spreading of contagious diseases. Many patho- 
genic 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 fac- 
ors of life. It is so important that the most highly developed animals 
rotect 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 
causes the metabolism of warm-blooded animals to be different from 
that of 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 at lower tempera- 
ture. All other organisms, reptiles as well as bacteria, have the tem- 
perature 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- 
tures. Growth and metabolism of microorganisms will increase with 
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 mini- 
mum 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 
temperature is too high. The temperature requirement is largely de- 
pendent upon the natural habitat of the organisms. The bacteria of 

213 






214 



PHYSICAL INFLUENCES 



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. 

The great majority of bacteria and related organisms, in fact of al 
living organisms, except in a few instances, has its optimum tem- 
perature between 20 and 40. The optimum temperature of an 
organism is generally somewhat higher than the average temperature 
of its natural habitat. 

The following table shows the data obtained for a few microor- 
ganisms. 



Temperatures 





Minimum 


Optimum 


Maximum 


Penidllium glaucum 


I tf 




i- 6 


Aspergillus niger 
Saccharomyces cerevisicg I 
Saccharomyces pasleurianus I 
Bacterium phosphoremn 
Bacillus subtilis 


0-5 
below o 
6 


33-37 
2 8-30 

30 


40. 
34 
28 


Bacterium anthracis 
Bacterium ludwigii 


10 

50 


3o-37 
5S-57 


43 
8o c 



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 at 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 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. Bacteria are found to multiply in it as long as 
it is not entirely solid. A certain yeast multiplied slowly in salted but- 
ter kept at about 6. 



INFLUENCE OF TEMPERATURE 21$ 

The number of microorganisms that developed at the freezing- 
win t 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 

han the optimum. The development of microorganisms above the 

>ptimum temperature is not quite normal; there is a great tendency 

oward involution forms. The mycelium of molds grown near the 

maximum temperature 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 bac- 
teria. Most bacteria do not grow above 45, but with some of the 
maximum temperature is considerably lower. Bad. phosphoreum dies 
if exposed for a few hours at 30; others may require still lower tem- 
peratures. 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 thermophilic 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 or- 
ganisms are found in soil, sewage, ensilage and occasionally in milk. 
They find the temperature suitable for their life only under extra- 
ordinary circumstances, as in fermenting manure piles, in silos, in 
self-heating hay and similar organic material that develops a high 
temperature by fermentation. Some hot springs have a very remark- 
able flora of thermophilic bacteria. 

The range of temperature within which growth is possible, is very 
uniformly 35 to 45; the starting points and end-points of this range 
vary greatly, while the total range is quite constant, except fcr some 
bacteria adapted to special conditions, such as some pathogenic bac- 
teria. The temperature relations of bacteria can be shown graphically 
by using as ordinate the rate of growth, as abscissa the temperature. 



2l6 PHYSICAL INFLUENCES 

BIOLOGICAL SIGNIFICANCE OF THE CARDINAL POINTS OF TEMPERA- 
TURE. The importance of the temperature requirements of certain 
organisms 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 Aspergilli and Mucorinece. 
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 pre- 
dominate, while the remainder are retarded or inhibited by unfavor- 
able 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 appar- 
ent change; no acid or deterioration 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 Oidium 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 quantity of whey 
is extruded, and much gas is produced by Bad. aero genes and B. coli. 
The odor is disagreeable and later butyric 'acid is produced; eventu- 
ally the lactic acid increases further by the action of Bad. bulgaricum* 
If kept above 50 the milk either keeps permanently, or a decomposi- 
tion by thermophilic bacteria begins which is either an acid fermenta- 
tion followed by digestion or a complete putrefaction, depending upon 
the species of thermophilic organism that happens to be in the milk 
sample. Thus there can be induced in the same substance, contain^ 
ing the same organisms at the start, four entirely different types 
of decomposition merely by the difference of temperature. 



INFLUENCE OF TEMPERATURE 21 7 

This indicates the importance of temperature regulation in the fer- 
nentation industries. Even pure cultures may give different products 
[ working at different temperatures. Cream ripened with a pure 
ulture starter at too high a temperature will have a sharp acid flavor. 
"he cold curing of cheese has become a very common practice because 
f the much improved flavor. Bioletti claims that the value of the dry 
California wines would be doubled if the fermentation were carried 
n generally at a lower temperature. 

END-POINT OF FERMENTATION. Another question is" the relation 
etween the end-point of fermentation and the temperature. Of the 
sw data existing, many indicate that at a lower temperature the final 
iermentation goes farther than at a higher temperature. Miiller- 
Thurgau found that under exactly the same conditions with the tem- 
perature as the only varying factor the following final amounts of 
jlcohol 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 differ- 
nce in the end-point, while others obtained results similar to the re- 
ults with alcohol. With three strains of Bad. lactis acidi were ob- 
dned after thirty-four days, by C. W. Brown: 

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 0.81 per cent of lactic acid. 

At 18 i. 08 per cent i. 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. 

iese results are quite logical and perhaps can be explained by 
ic recognized experience that all products of fermentation tend to 
icck the process of fermentation, and that any chemical product 
|r substance acts the more vigorously upon any life process the higher 
ic temperature. The same amount of alcohol that will still allow a 

fermentation at 10 may check the fermentation entirely at 20. 
Jaturally the rate of fermentation in the beginning will be higher at 
ic higher temperature but the end-point is lower. The end-point of 
ic lactic cultures A, B, and C at 6 is probably not final, because 



2l8 PHYSICAL INFLUENCES 

thirty-four days is a short time of growth at so low a temperature 
Above the optimum, the rate of decomposition will decrease rapidl) 
with the rising temperature and the end-point will also be lower. 

FREEZING. The discussion of the relation of temperature tc 
microorganisms has so far considered only the temperatures withir 
the limits of growth. However, the temperatures below the minimuir 
and above the maximum are also of greatest importance. If bacteria 
are cooled below their minimum temperature they do not die immedi 
ately. They- remain alive in a dormant condition ready to multiph 
as soon as the temperature rises. Even the freezing of a liquid wil 
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 then- 
will remain dormant much like dried organisms, and like them thei] 
number will decrease very slowly. An example is given in the follow 
ing table relevant to the number of bacteria in frozen milk (aftei 
Bischoff). The decrease in numbers is not very uniform, since there art 
many different bacteria in milk, but the general tendency is the same 
as in the dried bacteria. 

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 72,300 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 40,500 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 b> 
freezing, but as long as it is frozen it will keep; there in no possibilit) 
of any microorganisms decomposing a frozen liquid, for the organisms 
need water above all. If food substances change in cold storage 
(and some food products do deteriorate), this must either be due tc 
changes other than microbial or the material was not completel} 
frozen as is probably the case with salted butter. 



INFLUENCE OF TEMPERATURE 2 19 

After bacteria are once frozen, they do not seem to be affected by 
iy lower temperature. Macfadyen and Rowland found that they 
lerate very low temperatures remarkably well. Many bacteria 
ere not killed by a twenty hours' exposure to the temperature of 
}uid hydrogen ( 252). Yeasts are not quite so resistant and the 
ycelium of most molds is easily destroyed by freezing, while the spores 
e hardier. 

THERMAL DEATH-POINT. Heating above the maximum tempera- 
re is quite harmful to bacteria, and the amount of injury increases 
ith the temperature. Recent experiments have shown that heat does 
>t kill bacteria instantaneously, but that we have an orderly process 

in the case of death by drying. This can be observed only in a 
jry narrow range of temperature, however, since the death rate rises 
;ry rapidly with the increase of temperature. 10 increase may make 
e death rate ten to one hundred times as great, and death is almost 
stantaneous. For most practical purposes, it is sufficient to state 
e time and temperature neccessary to bring about complete sterili- 
ition. It has become customary to define, as the thermal death- 
Dint, the lowest temperature at which a culture will be killed in ten 
inutes. As most bacteriologists will use very nearly the sametech- 
c, they will have fairly uniform numbers of cells to start with, 
id therefore obtain fairly uniform results. 

The thermal death-point does not depend upon the species and 
e temperature only. It varies with the age of the culture since 
der cells are less resistant than younger ones especially if heated in 
eir own products. The medium in which the organisms are heated 
also of great significance. The fact that acid liquids, as fruit juices, 
e more easily sterilized than neutral meat or vegetables is largely 
ae to a chemical (poisonous) action of the acids upon the bacteria. 
ut the greater resistance of tubercle bacteria in the sputum compared 
th those suspended in salt solution cannot be so readily 
counted for. 

A necessary factor for the prompt destruction of organisms by 
;at is the presence of moisture. The resistance of dry organisms 
remarkably higher than that of the same organisms in a liquid cul- 
re. The following table shows the death-point of yeast cells and 
ores in a dry and moist state. 



220 PHYSICAL INFLUENCES 
THERMAL DEATH-POINT or DRY AND MOIST YEAST 


Variety of yeast 


Cells 


Spores 


Moist 


Dry 


Moist 


Dry 


Pale ale yeast 


6 5 

55 
5o-55 


95-I05 
8 5 - 90 
ioo-io5 


6 5 -70 
65 
60 


II5-125 
II5-I20 
115 


Hofbrau yeast 


Sdccharomyces pasteurianus 



RESISTANCE or SPORES. The organisms most resistant to heat arc 
the spores of certain bacteria. In the chapter on moisture require 
ments attention has been called to the great resistance of spores t( 
drying. We find the same exceptional resistance to high temperatures 
Boiling heat will not kill spores readily. Some bacterial spores car 
stand the temperature of 100 for several hours. In order to kill spore; 
in one heating the temperature must rise to about 110 for fifteen t( 
thirty minutes; this can be accomplished only by heating under pres- 
sure. This is not always advisable for sterilizing food substances 
While vegetables are usually sterilized under pressure without losing 
much of their palatability, other foods like milk are changed materiall) 
in taste and appearance. 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 I 
few spores of bacteria. After cooling, these spores will germinate undei : 
suitable conditions and the vegetative cells thus appearing instead of the j 
resistant spores are easily killed in a second heating. A third heating 
is necessary in order to kill any vegetative cells which may have devel- 
oped from spores not yet germinated before the second heating. It i< 
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. 



INFLUENCE OF TEMPERATURE 221 

ry spores will resist still higher temperatures than moist spores, 
me dry spores survive an exposure to 140 or 150 for ten minutes, 
requires a very high temperature to sterilize glass, cotton, gauze, and 
truments with dry heat. A discontinuous sterilization of dry mate- 
is useless, since the spores will not germinate without moisture, 
jerefore their resistance remains unaltered. 

The spores of molds are more resistant than the mycelium, but if 
joist, they all die at 100. The dry mold spores can tolerate asome- 
'uit higher temperature, but not as high as the spores of many bacteria. 
east spores and yeast cells are very much alike in their resistance to 
tat. The table on page 220 shows hardly any difference between their 
itsistance. 



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 con- 
tact 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. 




FIG. 105. These plates were heavily inoculated with B. coll and B. prodigiosu. 
respectively and then were exposed, bottom side up, to the direct rays of the sun 
for four hours. On the instant of exposure, a figure O cut from black paper wai 
pasted to the plate shading the bacteria underneath. After one, two and three hour; 
the corresponding figures were pasted to the plates. The above picture was taken it 
hours after exposure, proving that three or four hours of direct sunlight weaken am 
and may even kill bacteria. B. prodigiosus proved more sensitive than B. coli 
(Original.} 

The influence of light varies with its intensity. Direct sunlight 
has a very harmful effect upon microorganisms. Most bacteria an! 
killed by direct sunlight in a few hours; the time depends upon thfj 
organism as well as upon the intensity of light; this again varies wit! 



INFLUENCE. OF LIGHT AND OTHER RAYS 



223 



e amount of moisture and dust in the atmosphere, with the time of 
e day and with the season; an absolute measure for the action of light 
nnot be fixed, therefore, as easily as with the action of heat in the ther- 
al death-point. The different colors of the spectrum do not act 
ike; the part of the spectrum from red to green is practically without 
fluence upon microorganisms, while the blue light acts strongest 
id the intensity decreases in the violet and ultra-violet. In carrying 
i experiments with the influence of light, it must be remembered that 
ass absorbs ultra-violet rays, and further that the heating of the 
edium by direct radiation must be avoided (Fig." 105). 




o. 106. Phototropsim of Rhizopusnigricans. The mold is grown on gelatin with 
diffused light coming from right side. (Original.) 

Yeasts, molds, and bacteria and probably Protozoa are equally sensi- 
to light. Even the spores of most^bacteria do not show a greater 
sistance to light, while the mold spores are an exception. The col- 
ed spores of the Penicillium, Aspergillus and Mucor species can be 
posed to light for a long time without being killed, but the colorless 
ores of Oidium and Chalara show no increased resistance. It is sup- 
sed that the pigment in mold spores is a protection against light. This 
not true with the pigment of bacteria. The colored and colorless 
rains of pigmented bacteria show no difference in their resistance to 
;ht. The only exceptions are the so-called purple bacteria. These 
culiar organisms, many of which feed on hydrogen sulphide, seem to 



224 PHYSICAL INFLUENCES 

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 phototaxis or heliotaxis. The pigment, bacteriopurpurin, does 
not take the place of chlorophyl, however, since the bacteria do not pro- 
duce 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. 107. 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. Ultra-violet light is 
used in the sterilization of water and of milk. 

Diffuse light is not nearly as harmful to microorganisms as direct j 
sunlight. Long exposures to diffuse light will kill most bacteria, while j 
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 allj 
green plants, is called heliotropism or phototropism (Fig. 107). It haS| 



INFLUENCE OF LIGHT AND OTHER RAYS 225 

, 1 en found that molds produce mycelium mostly in the dark, while in 

ylight sporangia are produced mainly. This difference in the devel- 

ment during the day and during the night accounts for the concentric 

igs which are quite commonly found in older mold colonies, and 

jiich indicate the age of the culture (Fig. 107). Similar rings are 

jcasionally found with yeast and bacterial colonies, and are possibly 

ie to the same influence of light. 

X-KAYS. Of other rays, the invisible X-rays and the radium rays 
Ive attracted the attention of bacteriologists and physiologists. It 
i known that the X-rays will destroy living tissue by long exposures; 
Microorganisms cannot be considered less resistant. X-rays are used 
j the treatment of microbial diseases of the scalp and skin. 
i RADIUM RAYS are not so well known, and their bactericidal action is 
oubtful. The treatment of certain bacterial diseases has been 
^tempted, but it has not been applied as generally as yet as the X-ray 
jethod. The sterilization of milk and possibly other foods by this 
lethod has been suggested, but the practical application is at present 
aite improbable because of the cost and the uncertainty of the results. 



CHAPTER IV 
INFLUENCE OF ELECTRICITY 

The influence of elecrticity upon microorganisms is much less th* 
one might perhaps expect, if the electriticy as such is considered, 
direct electric current passing through a nutrient medium will, of cours 
cause electrolysis which is usually manifested by the formation of ac 
on the positive pole and of alkali on the negative pole. The acid ar 
alkali will kill microorganisms, as is discussed in the chapter on chemic 
influences. In this case, it is not the electricity itself that destroys tl 
bacteria. It is also possible to kill bacterial cultures by passing j 
alternating current through the medium for some time. No electrolye 
takes place in this case, still it is not the direct action of the current th 
acts upon the organisms, but rather the heat produced by the curre: 
passing through a medium of high resistance. If the culture is cool< 
properly the influence of the current is insignificant if at all noticeabl 
Whenever electricity is applied against microorganisms the effect is co 
sidered electrochemical. 

The electrical current is used in a very small way in the purificatk 
of sewage. The sewage passes between two iron plates which represei 
the two poles of a strong current. The electrical sterilization of mi 
has been patented. Wines are improved by electricity. The steriliz 
tion of drinking water by ozone is also an application of electricit 
though of course the ozone once formed by the current acts as a cher 
ical compound independently of its source, and the same effect wou 
be produced if the ozone were manufactured chemically. 



226 



CHAPTER V 
INFLUENCE OF MECHANICAL EFFECTS 

PRESSURE. The resistance of microorganisms to mechanical pres- 
iires is very great. Pressures of 3,000 atmospheres* will .not kill the 
ajority of bacteria in four hours. They are, however, weakened and 
3me species will die. A specific difference between the molds, yeasts, 
ind bacteria in this particular does not seem to exist. Of the organisms 
frposed to 2,000 atmospheres for ninety-six hours, Bact. anthracis, Bad. 
\seudodiphther ice, M. pyogenes var. aureus, Oidium lactis and Saccharo- 
tyces ceremsicB survived, while seven other organisms lost the power of 
multiplication. Some of these were not dead, however, since they 
stained their motility for several days. It is noteworthy that high 
ressure will destroy one quality (multiplication) and not effect another 
inotility). Pigment-production and virulence of pathogenic bacteria 
ere either diminished or lost completely. The resistance against 
igh pressure is necessary for the organisms which cause the decay 
f organic matter at the bottom of the oceans. Vertebrates breathe 
xygen in the form of gas or have at least an organ filled with gas (fish 
ladder) ; the volume of gas is changed considerably by slight changes 
i pressure; this will affect organisms depending on gas. Microorgan- 
ms do not require gas as such. They can absorb gases only in 
^lution. A change of pressure therefore will not cause a change of 
olume, since liquids have a very small coefficient of compression. 

The situation is entirely different if the liquid is not exposed to the 
ressure directly, but to compressed air. In this case, the chemical 
ffcct of the gas is the deciding agent. The higher the pressure, the 
lore gas will be dissolved in the culture medium. The fatal pressure 
nder these conditions will vary as much as the fatal dose of an antisep- 
c; it depends upon the chemical qualities of the gas, upon the pressure 
oncentration), upon the temperature, and upon the organism. 

'One atmosphere is i kg. pressure per square centimeter (or about 15 pounds per square 
ch). 

227 



228 PHYSICAL INFLUENCES 

Some data have been given already in the chapter on oxygen require- 
ments. It was mentioned in that connection that Bad. butyricum can- 
not tolerate more than 0.65 per cent of the total oxygen content in aii 
(0.2 atmosphere); mother words, an oxygen pressure higher thano.ooi. 
atmosphere will kill the organism. The maximum pressure for E 
prodigiosus was found to be about 5.4 to 6.3 atmospheres. Very fev 
experiments have been made with other gases. Carbon dioxide at E 
pressure of 50 atmospheres retards the growth of bacteria in water anc 
will sterilize it in twenty-four hours. Suspensions of pure cultures o: 
B. typhosus and Msp. comma are killed by 50 atmospheres carbon dioxidt 
pressure in three hours. Milk cannot be sterilized by his pressure bu 
bacteria do not multiply. Carbonated milk has been recommended a: 
a refreshing drink by several investigators. The ordinary market mill 
will keep about two days longer under the pressure of 10 atmosphere 1 
(150 pounds) than without pressure. If pasteurized it is said to keej 
for a week. 

GRAVITY. Gravity would have a great influence upon the growth o 
microorganisms in liquids if their specific gravity were much greate 
than that of water. This does not seem to be the case however. It ha 
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 centrifuginj 
bacteria in salt solutions of varying specific gravity, but these data ar< 
not exact since the salt solution will diffuse into the cells and thus in 
crease their weight. The specific gravity being very nearly that of tb 
culture medium, it is plainly seen that gravity has but little influence 
The microorganisms will live suspended in the liquid and sediment ou 
very slowly. The slightest current in the liquid will carry then 
around and distribute them through the medium. The motility is o 
minor importance; the actual distance covered by motile bacteria ha 
been measured, and under the most careful exclusion of currents in th 
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 watej 
moved by changes of temperature or other incidental agents. 

Yeast cells and other gas producers use the carbon dioxide as a ve 
hide. The gas bubbling up in the fermenting liquid keeps it constantl;! 
in motion and moves the yeast cells against gravity toward the surfac 
where the gas escapes and lets the cells fall back to the bottom. 

The production of scums and pellicles on the surface by organism! 



INFLUENCE OF MECHANICAL EFFECTS 22Q 

hich are heavier than the liquid they float on, is often accomplished by 
nail gas bubbles between the cells (Mycodermce). In other instances, 
may be just the floating of cells having oily surfaces. 

The growth is influenced by gravity very little. The sporangia of 
olds are the only exceptions, growing decidedly away from the center 

gravity (negative geotropism). 

AGITATION. For the majority of microorganisms, the quiet, undis- 
arbed growth of the laboratory culture is the normal or the ideal one. 
uch cultures, if shaken for a considerable time, show a decrease of liv- 
g organisms, and it is possible to sterilize cultures by continued shak- 
g. The effect is not a simple mechanical breaking or tearing of the 
lls. The bacteria break up into the finest particles. This is also the 
se if cultures are exposed for several days to the trembling motion 
i used by the working of very heavy machines. There is no grinding or 

ring effect but the cells break to pieces just the same. 

A slight and slow agitation seems to be advantageous for many cul- 
.es, only continuous heavy motion proves harmful. Different organ- 
ms 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 they will stimulate the develop- 
ment. Other substances too, which are not food, can also act as 
stimulants. It is a recognized fact of long stand- 
ing that many poisons in very small doses will 
stimulate. This applies to the most highly 
developed animals and plants as well as to micro- 
organisms. Raulin noticed in 1869 that Asper- 
gillus 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 
constituent of the mold cells. Alcoholic fermenta- 
: .V tion can be stimulated by metallic salts. It is be- 
lieved by some physiologists that, as a law of nature, 
FIG. 108. Chem- every substance that is injurious in a certain con- 
o taxis. (After centra tion is a stimulant in a lower concentration. 
A similar action of certain chemical compounds 
upon enzymes has been noticed, retarding in high concentrations, 
stimulating in weaker solution. 

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 microorganisms may be accounted for largely by chemical or osmotic 

230 



STIMULATION OF GROWTH 



231 



rces. In a solid medium the hyphae of molds will grow toward the 
st source of food supply. This growth on account of chemical 
mulation is called chemotropism, analogous to the phototropism 
growth toward light. If some injurious compound is offered, 
e hyphae will grow away from it. Thus we have to distinguish 
tween positive and negative chemotropism. The motile organisms, 
cteria as well as protozoa, demonstrate their preference for certain 
od compounds by swimming toward them. This is called chemotaxis 
ig. 1 08). Here also a positive and negative chemotaxis must be 
stinguished, the latter taking place if injurious substances are present. 



CHAPTER II 
INHIBITION OF GROWTH 

POISONS, GERMICIDES, DISINFECTANTS, ANTISEPTICS, PRES 
TIVES. A great number of inorganic and organic bodies will destroy 
life in comparatively weak solutions. These substances are callec 
poisons if they are considered in their effect upon man and animals. Ii 
their application to microorganisms they are generally called germicide. 
(germ-killers), or disinfectants if the emphasis is laid upon the preventior 
of infection rather than upon the actual killing of the microorganisms 
Analogous to the general term germicides, the terms bactericide anc 
fungicide are used occasionally. The term antiseptic means a preventior 
of sepsis which may be accomplished by checking the growth withou' 
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 origir 
while the word antiseptic is largely used in relation to microbial diseases 
A strict line cannot be drawn between any of these definitions. A dis 
infectant, if diluted, becomes an antiseptic. A strong salt solution is ar 
antiseptic for some organisms and a disinfectant for others. Of thf 
above expressions, germicide is the most definite, but is nof 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 tc 
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 (HgC^) so that they will no longer de-i 
velop if transferred to a fresh medium. If the mercuric chloride is re- 
moved from the cell by means of hydrogen sulphide, some of the organ- 
isms may be revived. 

The mode of death through poison is the same as that through! 

232 



INHIBITION OF GROWTH 



233 



or drying. The number of cells dying in a given time interval is 
roportional to the number of cells surviving. In the last five years, 
his has been tested and found true with practically all disinfectants, 
ig. 109 shows the curves plotted^ rom data obtained with Bad. anthracis, 
he full-drawn line representing the number of live spores in .21 per 



3500 



3000 



1000 



I 500 



IQOO 



500 






\ 



0\ 




min. 



IG. 109. Curve of disinfection. Spores of Bad. anthracis in mercuric chloride 
solution. (After Chick.) 

ent of mercuric bichloride, the dotted line the same in .11 per cent 
lution. 

The (apparent) resistance of the few remaining cells is of great im- 
ortance in those applications of disinfection where a thorough kill- 
g of all bacteria is intended, e.g., in the treatment of drinking water, 
ur ideas of the efficiency of a disinfectant would depend, therefore, 



234 CHEMICAL INFLUENCES 

upon the accuracy with which we can prove the presence of a certain 
bacterium. 

FACTORS INFLUENCING DISINFECTION. The efficiency of a dis- 
infectant 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 50 to 70 per cent solution. The necessity of moisture 
is further demonstrated in the sterilization with gases, as with formal- 
dehyde. The effect of formaldehyde gas without the provision of a 
very moist atmosphere is surprisingly weak. 

The temperature is also quite an important factor in the study 
of disinfectants. 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. As a matter 
of fact, the death rate through poisoning is usually doubled or trebled | 
by a temperature increase of 10. Above the optimum temperature, ! 
where the growth is not very vigorous, and when the disinfecting 
power of the poison is increased considerably by the higher temperature, 
a very small amount of poison will have a very strong germicidal effect. 
The combination of high temperatures with a disinfectant has been j 
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 probable 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 diffi- 
cult to poison bacteria in blood, pus, or similar material. The sensi- 
bility of the microorganisms in pure water is remarkable. Very small j 
doses which would not be considered efficient under any other condition, ! 
will destroy microorganisms in pure water. The concentration of| 
chloride of lime which is sufficient to sterilize drinking water, does! 
not at all suppress the development of bacteria in sewage. 

The influence of the number of cells is evident from the above ex- 
planations of the mode of action, and from the curves of disinfection. 
The concentration of the poison is of course of greatest importance. 



INHIBITION OF GROWTH 235 



236 CHEMICAL INFLUENCES 

the degree of dissociation, there is a specific effect due to the chemical 
structure, as is 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 mer- 
curic chloride (HgCl 2 ) and decrease also its disinfectant power. Mer- 
curic chloride dissolved in absolute alcohol is not dissociated. Ir 
this case, it has almost no action upon bacteria. 

Acids are not commonly used as disinfectants, except in the house- 
hold, but they play a certain role in nature. The common fruits con- 
tain so much acid that bacteria cannot easily attack them; the decay- 
ing of fruit is almost exclusively due to molds which have a preferena 
for acid media. The acid in the stomach of man and animals plays ar 
important role as a sterilizing agent for the food. Many microorgan 
isms are killed in the stomach. In the household, the natura 
acidity of fruit helps in keeping canned fruit, preserves and jellies 
Especially in heating, the acid together with the high temperatun 
has a very strong germicidal effect. Vinegar is often used to pre 
serve fruit and vegetables; in some parts of the country, meat is kepi 
in buttermilk. Benzoic and salicylic acids are often used in the pres- 
ervation of fruit and vegetables. Their poisonous influence is noi 
so much due to the acid reaction but to the specific chemical charactei 
of these compounds. 

Of the alkalies, only one is used extensively, namely, lime; quick 
lime (CaO) is considered a valuable disinfectant for excreta in priv> 
vaults; it is universally applied as a whitewash in stables, barns 
poultry houses and similar buildings. Quite commonly, it is used a: 
"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 carbor 
dioxide of the air and thus gradually loses its disinfecting power. 

Of the metallic salts, many are well-known germicides. The mos 
powerful disinfectant is mercuric chloride (HgCl 2 ) which is one of th< 
standard disinfectants. It is generally used in a dilution i : looc 
which is sufficient to kill all vegetative cells as well as spores in a fev 
minutes. Quite commonly, hydrochloric acid or salt is added, t( 
prevent coagulation or precipitation of slimy or albuminous matte: 
which would protect the enclosed bacteria from immediate contac 
with the poison. The addition of hydrochloric acid or any chloridi 



INHIBITION OF GROWTH 237 

ecreases somewhat the disinfectant value for bacteria suspended in 
istilled water because it decreases the electrolytic dissociation. 

Another disinfectant of remarkable strength is silver nitrate; it 
5 not used commonly because of its high price. It also decomposes 
asily and leaves dark spots on the skin and clothes. Of the other 
netallic salts, copper and iron sulphate are not used extensively, 
hough recommended for the disinfection of feces. Zinc*sulphate may 
e applied to mucous membrane the same as silver nitrate. Many 
ther salts may be used occasionally for disinfecting purposes, though 
he expense or undesirable qualities prevent their common application. 

The alcohols are well known for their poisonous effects, but the 
of ethyl alcohol as a disinfectant is usually overestimated. It 
akes quite strong alcoholic solutions, more than 20 per cent, to kill 
ertain yeasts and the spores of some bacteria in less than a day, 
tnd a complete sterilization by alcohol in a few minutes cannot al- 
vays be guaranteed even with 50 to 60 per cent solution. It has 
dready been mentioned that desiccated organisms are very resistant 
:o concentrated alcohol, more so than to a 50 per cent mixture. 
VLethyl alcohol is weaker, the higher alcohols, especially amyl alcohol, 
ire stronger disinfectants than ethyl alcohol. They all give good 
esults in the presence of water while the absolute alcohols have 
.carcely any effect upon desiccated bacteria. None of these alcohols 
n whatever concentration they may be used, can be relied upon to 
all bacterial spores. 

Stronger germicidal effects can be obtained by the alcohols of the 
group, of which phenol or so-called carbolic acid (CeH 5 OH) 
s the simplest representative. Phenol, like ethyl alcohol, is not as 
effective as is commonly believed. It is applied in solutions from .5 per 
ent 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 
nsoluble in water, and not as effective in solutions as they are in 



238 CHEMICAL INFLUENCES 

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 con- 
stitute a large part of coal gas, have very little if any effect upon bac- \ 
teria; gas is used occasionally in place of hydrogen for growing anae- 
robic 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 comon anaesthetics, ether 
and chloroform. The high prices of these agents forbid their general 
use, but they are sometimes used for laboratory work. 

The essential oils have a little more practical importance. Some of 
these are the main constituents of mouth washes, especially the oil oi 
peppermint (menthol), of thyme (thymol), and of eucalyptus (eucalyp- 
tol). Their action is very weak, however. The volatile oils of spices 
have to be considered in the preserving of fruit, pickles, catsups, and 
other food products. Though the antiseptic value in general is insigni- 
ficant, certain microorganisms are sensitive to certain spices. The 
bacteria of the mesentericus 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 powder of black and white pepper i 
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, j 
Formaldehyde (HCOH) is a gas, soluble in water to the amount of 40 j 
per cent at room temperature; it does not attack metal, clothing, wood-i 
work, and is, therefore, preferable to many other disinfectants for steril- 
izing rooms. It kills spores of bacteria in a short time in a i : 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; thisj 
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 tc 
formaldehyde gas by gentle heating. This paraformaldehyde is com- 



INHIBITION OF GROWTH 239 

monly used instead of the liquid, because it is more easily handled and is 
jquite 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. 
IThough 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 (Os). 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 C>2) resembles ozone in its chemical reactions; 
it changes readily to H 2 O -f O, and this oxygen atom in the nascent 
istate 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 dis- 
infectant a still higher concentration is required. It loses its disinfect- 
ing 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 
ihypochlorite and free chlorine; these substances are good germicides 
and chloride of lime is used in the disinfectant of privy vaults, and other 
places in which it may be employed without injury. Hypochlorite is 
now used with great success for rendering safe drinking water and 
ewage; it has also become the basis of some commercial dis- 
infectants. 

Potassium permanganate is only incidentally used as a disinfectant. 
Its chemical qualities prevent an ordinary use. 

Sulphurous acid, or sulphur dioxide (SOj) was for a long time a 

tandard 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 

infect properly, and under these circumstances it will attack metal, 
yes of clothing and even the fiber itself. 



240 CHEMICAL INFLUENCES 

In addition to these disinfectants which are used outside of the 
human body, or applied to its surface only, there have come into use 
during recent years, several disinfectants which are injected into the 
body to kill the microorganisms in the blood. Among these might 
be mentioned the colloidal metals, mainly colloidal silver which is sold 
under various trade names, e.g., collargol. It is given especially in 
pneumonia, but its action upon the bacteria directly is very insignificant, 
though it greatly stimulates phagocytosis. Further, there is to bei 
mentioned ethoxyl, given against the protozoon of sleeping sickness, | 
and the latest and most discussed of all, salvarsan, an organic arsene 
compound, against syphilis. 



DIVISION IV 
MUTUAL INFLUENCES 



INTRODUCTION. 

The biological relations of microorganisms are of the greatest im- 
rtance in nature. Pure cultures in nature are very rare and of excep- 
onal occurrence; they are hardly ever found except in certain diseases 
f man, animals and plants. Generally, nature works with mixed cul- 
ures. All natural fermentations, decompositions and putrefractions 
re accomplished by a number of different species among which perhaps 

dominates, but is influenced by the rest. The study of the mutual 

tions of microorganisms is in the very first stage as yet; practically 
.11 laboratory work is done with pure cultures. The experiences obtained 
vith pure cultures are not sufficient to explain all microbial activity in 
.ature. 

There are many possibilities of mutual influence between different 
rganisms. Generally three main cases are distinguished: symbiosis, 
jvhere two organisms profit by the combination; metabiosis, where one 
Profits by the other's action without benefiting the other in return, and 
niibiosis, where one organism injures the other. These cases cannot be 
eparated strictly. The relations are not always constant through the 
ntire development of the cultures; an originally beneficial influence 
nay change to an injurious one in a few days. Many terms have been 
oined to designate all these various possibilities, but in order to avoid 
his multiplicity of more or less indefinite names for the various relations, 
he general term "association" has come into use, especially when the 
elationship is not well understood. 

SYMBIOSIS. 

Symbiosis is not very common among microorganisms, and it is 
ifficult to find examples where true symbiosis exists through the entire 

16 241 



242 MUTUAL INFLUENCES 

development of both organisms. The association of lactic bacteria anc 
Oidium lactis in milk is, for a certain period at least, a symbiosis. Th( 
bacterium will produce only a certain amount of acid, and then it car 
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 turr 
removes the excess acid which otherwise would check the bacteria 
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 carbo 
hydrates provided by the plant and furnishing the plant nitrogen frorr 
the air which the plant cannot assimilate directly. The typical exam 
pie in the animal kingdom is B. coli in the intestine of animals, bein 
nourished by the food of the animal and rendering the food more easil) 
digestible. 

METABIOSIS 

Metabiosis may be considered a one-sided symbiosis ; two organisms 
live together, but only one is benefited, the other remains uninfluencec 
or later may be injured by the association; the latter case is the mosl 
common. In this relation, one usually prepares the food for the other 
It has previously been mentioned that the metabolic products of om 
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 oi 
microorganism. Consequently, 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 nitrite 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 tht 



tMICBOBIAL ASSOCIATIONS 243 

tion of lactic bacteria with certain protein destroying organisms, 
ctic bacteria often develop much better if the protein bacteria 
;row together with them or have grown previously in milk. Meta- 
)iosis does not require the growth of the two associated organisms at 
he same time. The effect will be the same if first the one and later the 
>ther develops, and even after the first organism is killed or removed, 
ts effect upon the pure culture of the second will still be noticed. This 
loes not occur in the case of symbiosis. 

One species can favor the development of another by other means 
han food provision or preparation. Certain bacteria cannot live in 
icid media, and molds or mycodermas 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 organ- 
.sms. 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 satisfac- 
torily explained. Though the aerobic bacteria continuously remove the 
oxygen from the water a certain amount will remain, sufficient to pre- 
vent the growth of the anaerobic bacteria under ordinary conditions. 
There seems to be a certain protective influence derived from the aerobic 
bacteria, the nature of which is unknown. 

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 begin 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 saturate 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. 



244 MUTUAL INFLUENCES 

In the lactic fermentation of milk, Bad. lactis acidi combats all 
other organisms by a rapid production of lactic acid. Though it is pres- 
ent in fresh milk only in very small numbers, its rapid growth and the 
formation 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, 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 considered as such only from the stand- 
point of the lactic organism. The protein bacteria are killed by the 
acid formed by the rapidly growing lactic bacteria. From the view- 
point of the protein bacteria, the relation is antibiosis. Another illus- 
tration 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 produced, 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. 

RELATIONS BETWEEN CELLS OF THE SAME SPECIES 

That cells of the same species will also influence each other, may well 
be assumed. The simplest relation will be the competition for food. 
This will be the case in nature more commonly than in laboratory media 
which are, as a rule, so rich in nutrients that development ceases before 
all food is used up. 



MICROBIAL ASSOCIATIONS 245 

The cause for cessation of growth in a culture is of great theoretical 
and practical interest. Apparently there are various factors concerned 
in this. Lack of food, or of one single essential food compound, may be 
the cause. This is found sometimes in media where it would be least 
expected. Some strains of Strep, lacticus are supposedly limited in 
milk by the lack of available nitrogen ; they cannot attack casein readily 
and albumin; besides these proteins, nitrogen compounds are not plenti- 
ful. Addition of peptone increased the maximum number of cells from 
0.7 billion to 2.5 billions per c.c. More commonly, however, growth 
is checked by the accumulation of metabolic products. Yeasts are 
checked by the alcohol, and acid-formers by the acid, urea bacteria 
by the alkali. In many of these cases, the removal, or neutralization, 
of the inhibiting product will bring about new development. 

The harmful products accumulating are not always of such simple 
nature. Some very interesting observations have been made during the 
last ten years. .Eijkmann, as the first, found that B. coli reached its 
maximum growth in gelatin at 37 in a few days, and that this gelatin, 
after hardening at 20, would not support growth after streaking with 
a young culture of the same organism; but after this gelatin had been 
heated at 60 for half an hour, B. coli grew on it as well as on fresh 
gelatin. Broth in which B. coli had grown became fit again for growth 
of the same bacillus after filtration through porcelain. The inhibition 
of growth is, in this case, due to a compound which resembles a toxin 
in many respects. The importance of such investigations to general 
physiology is evident. 



PART III 

APPLIED MICROBIOLOGY 



DIVISION I* 
MICROBIOLOGY OF AIR 



CHAPTER I 

'HE MICROORGANISMS OF THE AIR AND THEIR DISTRI- 
BUTION 

The atmosphere is not the normal habitat of bacteria, for growth and 
nultiplication cannot take place in it under ordinary conditions. The 
)hrase "microorganisms of the air" is therefore somewhat ambiguous, 
"he small size of microorganisms enables them to remain suspended for 
onsiderable periods when physical forces have separated them from the 
ubstrata on which they have developed. 

MICROORGANISMS PRESENT IN THE AIR. Molds, bacteria, and yeasts 
re all found in the air under certain conditions. The first two are usu- 
lly relatively abundant, the latter are less common. 

The common molds have adapted themselves for the most part to 
vind distribution. They bear spores that are small in size and possess a 
urface that is not readily moistened. These spores are resistant to 
lesiccation and light and remain viable for a considerable tine even 
inder unfavorable conditions. Furthermore, the fruiting bodies of 
nany, though not all molds, show a distinct negative hydrotropism,*'.e., 
he mycelium remains in contact with the moist substratum while the 
hreads which bear the spores rise at right angles to it. These latter are 
o sensitive that they can detect slight differences in the moisture con- 
ent of the air and grow in the direction which will bring the spores into 

Prepared by R. E. Buchanan. 

247 



248 MICROBIOLOGY OF AIR 

the driest 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 distribu- 
tion 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 pulver- 
ized substratum on which they have been growing. Not all dust par- 
ticles 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 some- 
times sprayed into the air with water. Droplets containing bacteria' 
are thrown off in the saliva in coughing or in speaking, and from the i 
surface of fermenting liquids on which bubbles are bursting. Wher 
the drop is small enough, the air currents keep it in suspension and the 
water soon evaporates and frees the organism. This brings about th( 
condition first discussed, free bacteria in the air. The decrease ir 
weight and size incident to this loss of water probably accounts for th< 
fact that the so-called "infectious droplets" are sometimes carried fo: 
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 tin 
organism is passive and is freed only by air currents or by mechanica 
agitation. Some molds, as has been stated, release their spores even ii 
the presence of moisture, so that complete desiccation is unnecessary fo 
their dispersal. Bacteria and yeasts, on the other hand, are not usuall; 
given off from moist surfaces. Only when dry and pulverized can th 
bacterial medium be readily blown about. Hansen found that in th! 
immediate vicinity of a heap of decaying malt, the air was comparativel; ! 
free from bacteria. Winslow has shown that sewer air is frequent!;! 
practically free from bacteria although the surface with which it come; 
in contact teems with bacterial life. Mechanical agitation often throw' 
large numbers of organisms into the air. Moving hay and stra\\| 



MICROORGANISMS OF THE AIR 249 

rooming animals, sweeping a floor or carpet will multiply the dust and 
(acterial content of the air many times. In a similar manner, tiny, 
erm-holding droplets may be scattered by the splashing of sewage or of 
jrmenting or putrefying liquids, and in speaking, sneezing or coughing. 
CONDITIONS FOB SUBSIDENCE OF BACTERIA. The length of time 
uring which an organism may remain suspended in the air is dependent 
pon several factors. Small particles settle out more slowly than large 
>r the reason that as the size of an object is decreased, the surface area 
ecreases less rapidly proportionately than the volume. The lifting 
!ect of air currents depends upon the ratio of surface area to volume 
nd specific gravity. The smaller the object, therefore, the greater is 
resistance to subsidence. Consequently, bacteria usually settle 
ut of air very slowly if free in a quiet atmosphere. The time of sus- 
ension is determined also by the velocity of the air currents. While 
onsiderable velocity may be necessary to dislodge microorganisms and 
ring them into suspension, a very slight air current will sustain 
hem. Winslow has found that a current of 17 inches per minute is 
ufBcient to sustain B. prodigiosus. The relative humidity of the air is 
Iso an important factor. In a supersaturated air solid particles, such 
s bacteria, become foci of condensation for water and quickly settle 
>ut. When dust is present in considerable quantities, and certain elec- 
rical or moisture conditions exist, flocculation occurs and the larger 
)odies so formed subside rapidly. The character and abundance of 
urfaces with which the suspended particles may come in contact also 
lay an important part. Moist surfaces are much more effective in 
etaining particles than those which are dry. 

DETERMINATION OF THE NUMBER OF BACTERIA IN THE AIR. The 
lumber of bacteria in the air is frequently determined by exposing open 
>etri dishes of gelatin or agar in different places for definite periods, 
'his is a comparative quantitative method only. The number of colo- 
ies developing upon these plates will give the number of dust particles 
aving living spores or cells upon them that fall in the given area under 
he conditions of the experiment. Evidently this is of value only for 
ough comparative work as constantly shifting currents of air usually 
ntroduce great errors. A somewhat more accurate method is to draw 
rieasured volumes of air into a flask, the bottom of which is covered 
ith a layer of gelatin or agar. The colonies which develop represent 
he number of organisms which settle out from the givenVolume. _More 



250 MICROBIOLOGY OF AIR 

accurate results still may be obtained by drawing measured vol- 
umes of 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 suffi- 
cient contact of air and water to remove all dust particles. A propor- 
tionate 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 i 
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 I 
the composition of the medium used in order to favor the development i 
of certain types of organisms desired, for example, a higher precentagt I 
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 th< 
air is determined by a variety of conditions. The velocity of air cur 
rents and the nature of the surface with which these currents will com< 
into contact, are probably most important. Bacteria are usually more 
abundant on quiet days in the air of buildings than out of doors, but 01 
windy' days the reverse is true. They are often more abundant in citie: 
than in the country. Fewer are found at high altitudes and over largi : 
bodies of water. Frankland found that there are fewer in winter thai! 
in summer. They are washed from the air during rains. Bright sun 
light destroys many. The nature of the soil and the vegetation cover 
ing it has a marked influence. The following figures from variou 



MICROORGANISMS OF THE AIR 



251 



? thors are appended to serve as an index to what may be expected in 
te air content of bacteria. 



Locality 


Number of organisms 
per cubic meter 


Observer 


tdoor air, Boston 

en air 
en field 
acoast 
buntain altitude, 200 meters 
bnt Blanc 


100-150 bacteria. 
50- 75 molds. 
100-150 bacteria. 
250 

100 

o 
411 


Sedgwick and Tucker. 

Fischer. 
Uffelman. 
Uffelman. 
Pasteur. 
Ellis 


itzbergen (Arctic Regions) 


o 


Levin 


iddleof Paris 
ris Street 
.ilor's Room in Whitechapel 
ot Workshop 


4,000 
3,Soo 
17,000 
25,000 


Ellis. 
Fischer. 
Ellis. 
Ellis. 



SPECIES OF ORGANISMS IN THE AIR. Penicillium is the most com- 

iDn mold isolated from the air. Next in importance are Mucor, 

hizopus, and Aspergillus in the order given. In addition to these a 

msiderable number of species of hyphomycetous molds are occasion- 

< y found. Torula, but not true yeasts, are usually common. Bac- 

tria are either spore-bearing soil bacilli or cocci. Of the former, B. sub- 

tis, B. mycoides, and related forms are ubiquitous. Sarcina lutea and 

iircina aurantiaca and certain other chromogenic cocci are to be found 

almost every plate exposed. Since the air does not have a true flora, 

e species as well as the number of bacteria present must depend en- 

ely upon the character of the environment. 



CHAPTER II 

MICROBIAL AIR INFLUENCE INFERMENTATIO] 
DISEASES, ETC. 

AIR AS A CARRIER OF CONTAGION. There are many popular mi 
conceptions of the influence of air upon health. Experience ear 
taught that exposure to the night air in certain localities or to swan 
air during certain seasons was generally followed by disease. Natu 
ally, the air itself was held responsible. We know now that certa 
fevers, malaria, etc., are caused in every instance by infection wi 
specific microorganisms and that these organisms are not usually ca 
ried by the air but by insects, such as the mosquito, in water and foe j 
Nor can the emanations from decaying organic matter or sewer gas its 
be held to produce disease directly. Before the establishment of t 
germ theory of disease, leading sanitarians held that sickness \\ 
induced by the gases from the decaying organic matter, by the efflux 
from cesspools and by sewer gas. However important the places nam 
may be in harboring disease microorganisms, we have learned that t 
air itself rarely acts as a carrier. Sewer gas has been shown to be i 
usually free from bacteria. Hazen says, " After many years of exp 
ience and long-continued investigation, there is not the slightest rea< 
to believe that infectious diseases are carried by the air of sewers." 

Undoubtedly the air does play some part in the carrying of disc 
germs. In certain diseases, as the exanthemata (smallpox, meas 
etc.), the infecting agent may be present on the dry skin and may 
blown about and inhaled. This means, however, is not establish . 
In certain nasal, tracheal, and pulmonary infections, the organic 
may be spread through speaking, sneezing, and coughing, for the in.' - 
tious droplets, as has been seen, remain suspended for a time in ,e 
air. Pyogenic cocci are present in the mouth and care must be use< ;i 
surgical operations that the mouth is so protected that none of tl e 
organisms gain entrance to wounds. Rarely, if ever, are intest il 
infections, as typhoid or cholera, spread through the air. We_may th : 

252 



MfROBIAL AIR INFLUENCE IN FERMENTATION, DISEASES, ETC. 253 

fc; conclude that air is of secondary importance as a carrier of infection. 
Ilnay be of importance in a crowded workroom, but even under these 

ditions it is probable that transmission of infection comes about 
njre frequently through actual contact or through food and drink. 

ORGANISMS OF THE AIR AND FERMENTATIONS. A uniform inocula- 






with soil bacteria such as produce the nodules on the roots of leg- 



es is obtained over considerable areas through the action of the wind 
ir|)lowing dust particles. The bacterial flora of milk is to some extent 

endent upon air currents as is also the development of the molds 
ircessary to the proper ripening of cheese, such as the Camembert. 
Aitic, butyric, and other organisms are likewise distributed in this 
rrnner. The organisms responsible for putrefaction and decay, the 
n ding and spoiling of foods are wind-borne. 

FREEING AIR FROM BACTERIA. Air is most commonly freed from 
bpteria by sedimentation, for this is the ultimate fate of most dust par- 
tijes. We have seen that they gradually subside in a quiet atmos- 
pbre. When large quantities of pure air are required, dust and bac- 
t<jia may be removed by passage through a spray of water or through 
vrious types of filters, such as cotton, glass, wool, etc. A familiar 
e:,mple of this type of filtration is the laboratory use of cotton plugs in 
tft-tubes. It is sometimes necessary to resort to fumigation to destroy 
t oiganisms of the air when an undesirable species is present. 



DIVISION II 
MICROBIOLOGY OF WATER AND SEWAGE 



CHAPTER I* 
MICROORGANISMS IN WATERf 

Water is necessary in the life of man. Besides its use as a beverage j 
for cooking, and all domestic purposes, it is largely used in many maim 
facturing industries; therefore, the study of its chemical and biological 
content is one of the most important features of modern hygiene. Al 
natural waters contain microorganisms, which gain entrance from man; 
sources. 

Under the influence of the sun, sea water evaporates and forms 
water vapor, which we call clouds; and these, driven by the wind ove 
the land, are precipitated as rain and in the form of snow or hail. 

Most of this water collects from vast areas into brooks, creek I 
rivers, lakes, or in subterranean streams, and finally reaches the se 
whence it came. 

The water vapor arising from the sea or land contains no organism 
but as soon as the vapor is precipitated microorganisms find their wa 
into it. These come from the air and from the soil. Some of them fir 
in water sufficient nutriment for their life and growth; and, because 
their constant presence and evident ability to thrive in water, they a 
sometimes spoken of as belonging to the "water flora." Others, such ; 

* Prepared by F. C. Harrison. 

t For specific details regarding methods of analysis and a fuller presentation of the subje i 
readers may consult any of the following excellent books: 

1. Savage, W. G.: The Bacteriological Examination of Water Supplies, London, H. 
Lewis, 1906. 

2. Horrocks, W. H.: An Introduction to the Bacteriological Examination of Water, Lond 
J. and H. Churchill, 1901. 

3. Prescott and Winslow: Elements of Water Bacteriology, 2d Ed., New York, Wiley 
Sons, 1913. 

254 



MICROORGANISMS IN WATER 255 

e soil bacteria, are found only at certain seasons, as after rain or dur- 
?g flood-time, and flourish only for a time; while some few, such as 
nal organisms that find their way into water, survive for only 
.short period. 

CLASSES OF BACTERIA FOUND IN WATER 

The bacteria found in water are here roughly divided into: (a) natu- 
ll water bacteria; (b) soil bacteria from surface washings; (c) intes- 
nal bacteria, usually of sewage origin. But there is no strict divid- 
g line between these three groups; for some organisms belonging to 
le water flora are found in the soil, and vice versa. Water draining 
om manured land frequently contains intestinal organisms. The 
vision, however, is sufficient for all practical puropses. 

NATURAL WATER BACTERIA. The natural water bacteria are gen- 
ally regarded as harmless to man. These organisms are frequently 
jimerous in river, lake, and all surface waters; certain species predomi- 
ite at one season, and disappear at another. Some of the best known 
e mentioned below. Several investigators have grouped the bacteria 
und in water into classes according to their biochemical properties. 
'here groups are subsequently referred to, the classification is that 
&ed by Jordan and followed by many other workers. 

B. flnorescens liquefaciens, Group V, together with some closely allied 
irieteis, is probably more frequently found in water than any other 
rm, and is easily recognized by the green fluorescence and liquefaction 

produces in gelatin. 

B. fluorescent non-liquefaciens, Group VI, as the name implies does 
)t liquefy gelatin, but produces characteristic colonies with a fluores- 
nt shimmer, is often very abundant in river waters, and is representa- 
ve of a group comprising B. f. longus, B. f. tennis. B. f. aureus, and 
i. /. crassus. 
Certain organisms which liquefy gelatin and acidify milk classed by 

rdan in his Group VIII are quite common at certain seasons. 

me of these are soil organisms and are closely related to the proteus 

oup; and some of them are B. liquefaciens, B. punctatus, B. circulans. 
Chromo genie bacilli and cocci (Groups XIII, and XIV) are often 

esent in water. Of those producing red coloring matter, the well- 

lown B. prodigiosus is the type of the group; others are B. ruber, B. 



2$6 MICROBIOLOGY OF WATER AND SEWAGE 

indicus t 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, particulary in river and brook waters, organisms 
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. coemleus. 

The chromogenic cocci produce either orange or yellow pigment, and 
as a rule are not numreous 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 . corona- 
tus 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 ill 
natural waters; and occasionally certain species persist for a consider! 
able time. Among the commonest species is B. mycoides, with it 
characteristic rhizoid colony; also B. subtilis, B. mengatherium, and B 
mesentericus vulgatus, with its allied varieties; likewise B. m.fuscus am 
B. m. ruber all belonging to Jordan's Group VII, and having man 
characters in common, such as characteristic colonies, followed b 
liquefaction when growing in gelatin, production of spores, etc. 

Cladothrix dichotoma, one of the thread bacteria, easily recognize 
on gelatin plates by the brown halo that surrounds the colony, is ofte 
found in fresh and stagnant water, and in most soils. It seems t 
flourish wherever there is much organic matter. 

These are the soil organisms most often found when beef peptor 
gelatin is used for isolating purposes; but if other media are used, 
different flora appears, and we find nitrifying organisms, yello 
chromogens, etc. 

INTESTINAL BACTERIA, USUALLY OF SEWAGE ORIGIN. Prote: 
Group. There are several groups of sewage organisms found in impuj 
water; some of these are very abundant in crude sewage, but are n! 
found in such relatively large numbers in contaminated water. Jc 
dan's Group III contains the organisms belonging to the large prote 
group, the principal species being B. vulgaris, B. zenkeri, B. mirabil 
B. zopfii, the sewage proteus of Houston, and B. cloaca. All these a 
frequently found in impure water, and in sewage. In the latter Hoi 



MICROORGANISMS IN WATER 257 

has found as many as 100,000 per c.c. All these organisms are mo- 
, liquefy gelatin, and produce gas in dextrose and saccharose broth, 
I little or none in lactose; reduce nitrates, curdle milk, produce indol, 
I give a fecal, disagreeable odor in broth or other media. 
Sewage streptococci. The streptococci found in sewage are probably 
-nilar to those found elsewhere; but their appearance in contaminated 
iter may be regarded as indicative of recent sewage contamination, 
:cause the bulk of the evidence available seems to show that they are 
ilicate organisms, which rapidly die outside of the body. While it is 
,sy to ascertain their presence in polluted water, it is almost impossible 
enumerate them; and they do not furnish such good evidence of sew- 
;e pollution as the colon bacillus. They may be said to furnish valu- 
)le confirmatory evidence of sewage contamination. 

B, enieritidis sporogenes. This resistant, spore-bearing organism is 
mally present in the intestinal tract of man; is found in sewage, milk, 
id dust; and occurs in foodstuffs, such as wheat, oatmeal, rice, etc. 
n account of its ubiquity and the resistance of its spores, it cannot be 
nsidered a good indicator of excretal pollution. 
B. coli. The presence of this organism in potable water is gener- 
ly accepted as the best bacterial indicator of sewage pollution. It 
ust be remembered, however, that there are many varieties of this 
ganism, to which certain investigators have given specific names, even 
hen the differences from the type organism have been very slight. It 
lay be well to mention some of these, to avoid confusion in the mind of 
ic reader. The true colon bacillus, B. coli, or B. coli communis, or B. 
ni communis verus, is a short bacillus with rounded ends, motile, forms 

spores and is Gram negative, does not liquefy gelatin, produces 
pidity and coagulation in litmus milk, gives rise to acid and gas in 
lucose and lactose media, causes canary-yellow fluorescence in neutral 

' pd media, and produces indol when grown in peptone water. The term 

iVxcretal B. coli" has been suggested as a convenient designation of an 
ganism which possesses the above characteristics. 

1 A saccharose fermenting variety of B. coli has been named B. com- 
i unior; and we have a whole series of organisms which differ more or ess 
p various biochemical reactions, or lack some of their positive reactions. 

o some of these the name " para-colon" has been given; and the name 
para typhoid" has been applied to those which more closely approxi- 
mate to the cultural peculiarities of the typhoid bacillus. 

'. 



258 



MICROBIOLOGY OF WATER AND SEWAGE 



For practical purposes in the analysis of water, these distinctions are 
unnecessary. 

Bact. 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. 

B. typhosus. Very few instances are recorded in bacteriological 
literature of the direct isolation of the typhoid bacillus from infected 
water. The organism is not long-lived, even in pure water (eight 
or 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 ir 
water, it is well understood at the present time that the purification oi j 
the water supply of a town or city produces a marked decrease in th( 
number of cases and in the mortality from typhoid fever, as the following 
table shows: (See also Fig. no.) 

DEATHS FROM TYPHOID FEVER PER 100,000 PER YEAR 



Place 


Purification 
by 


Date of 
change 


Five years 
before 
change 


Five years 
after change 


Percentage 
of 
reduction 


Hamburg 


Filtration 


18923 


47 


7 


85 


Zurich 


Filtration 


1885 


76 


10 


87 


Lawrence, Mass 
Albany, N. Y. 


Filtration 
Filtration 


1893 
1899 


121 
IO4 


26 
28 


79 
73 















Not only has such a marked improvement followed the purificatioi 
of public water supplies in the case of typhoid fever, but it has beeij 
shown by statistics that "where one death from typhoid fever has bee;| 
avoided by the use of better water, a certain number of deaths, probabl; 
two or three, from other causes have been avoided." 

In the routine examination of water, no particular effort is made t 
isolate this organism, owing to the difficulty of the task. The tests tha; 
the present-day investigator has to satisfy are extremely thorough; an! 
unless the suspected organism conforms to the whole of these necessar j 
tests it cannot be accepted as true B. typhosus. 

Msp. comma. The spirillum, or vibrio, of Asiatic cholera i 
an intestinal organism; and the disease it produces is spread largel 
by water. Epidemics of cholera are more easily traced to the 



MICROORGANISMS IN WATER 



259 



AVERAGE ANNUAL DEATH RATE FROM TYPHOID FEVER PER 100.000 OF THE POPULATION. 

20 30 ^0 SO 60 



BERLIN 

ZURICH 

HAMBURG 

PARIS 

LONDON 

CLEVELAND.O. 



PATERSON.NJ. 

WATERTOWN.N.Y. 
0. 



SEATTLE .WASH. 



CHICAGO.ILL. 

ST.LOUIS.MO. 

MINNEA 

PHILADELPHIA,^. 

PITTSBURGH. PA. 

NEW ORLEANS.LA. 

NEWYORK.NY. 

SPRINGFIELD.HASS. 

BINGHAMPTON,N.Y 

ALBANY, NY. 

LAWRENCE, MASS. 

RICHHOND.VA. 

BALT1MORE,MD. 

MILWAUKEE.WIS. 

TOLEDO.O. 

ATLANTA.GA. 

BIRMINGHAH.ALA. 

WHEEUNG.W.VA 

HEMPHIS.TENN. 

ATLANTA.GA. 




An instructive contrast between Altona and Hamburg before the latter filtered 
ts water, having learnt its lesson from a sharp outbreak of cholera. 

A FEW 

SCATTERED CASES OF CHOLEflA- 
ALTONA __ 

'HAMBURG. 

POPULATION: 600.000 
CHOLERA CASES. 17.000 
DEATHS: 8.6OO 




FIG. no. (After G. E. Armstrong.} 



260 



MICROBIOLOGY OF WATER AND SEWAGE 



source 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. no). 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 illustrate. 

NUMBER OF BACTERIA PER LITER OF 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 




8 OOO 


July.. 


=;,6oo 


February 


1, 32O 


August 


8,300 


March 


2 02O 


September 


5,770 


April . . 


2,140 


October 


3,22O 


May 


2 44.O 


November 


3,250 


Tune 


s,6oo 


December 


4,330 











Yearly average 5,300 per liter 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 L.| 
With a yearly rainfall of 609.6 mm. (24 inches), the rain washes: 
down during the year some 5,000,000 organisms to the square yard.j 



MICROORGANISMS IN WATER 261 

SNOW. The results obtained from snow are similar to those ob- 
ained from rain; but as a rule the numbers are larger, a result doubtless 
ue to the larger particles of the snow flakes. One investigator has 
ound from 334 to 463 bacteria per c.c. of snow water. On the sum- 
nit of high mountains snow is practically sterile, Binot not finding 
single organism in 8 c.c. of water from mountain-top snow. 

Water issuing from glaciers is of remarkable purity, containing 
Inly from three to eight organisms per c.c. ; but the numbers are larger 
Ls the distance from the glacier increases. 

HAIL. Hail stones usually contain large numbers of bacteria, 
arying from 628 to 21,000 per c.c. of water obtained from the melt- 
ng hail. Fluorescing bacteria have been found in some samples; 
md the presence of these microorganisms suggests that surface water 
s sometimes carried up by storms and congealed. The presence of 
nany molds in hail is due to contamination from the air. 

DEEP WELLS. Deep well water and spring water contain as a 
ule but few organisms, usually less than 50 per c.c. on gelatin at 20, 
nd less than 5 per c.c. on agar plates at blood heat. In a series of 
;ests 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. for the gelatin and 1.5 per c.c. for the agar counts. These tests 
lave 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 loca- 
ed and constructed, the number varies with the amount of rainfall, 
ind is often large. In polluted wells, very high numbers of organisms 
ire 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 
tree of contamination gave an average count of 400 per c.c. 

Polluted wells often give counts approximating the higher numbers 
mentioned above; but, of course, the character of the bacterial flora 
is quite different. 



262 



MICROBIOLOGY OF WATER AND SEWAGE 



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, tempera- 
ture, rain fall, vegetable debris, etc., influence the microbial popu- 
lation. 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 mile . 


Above 
Below 


305 
0,387 


4,786 


About 2.7 miles 


Below 


13,503 




About 6.0 miles . . . ... 


Below 


8,764 


30,432 


About 1 2 o miles 


Below 


47Q6 


12,460 


About 15.0 miles 


Below 


3,602 


Q.<QZ 


About 26.0 miles . . 


Below 




7,86o 



In the Chicago drainage canal, Jordan found 1,245,000 bact 
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 number rises 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 rivei 
water. Near the shore, the bacterial content is higher than farthei 
out, showing the contaminating influence of habitation. Thus Lakf 
Geneva contains as many as 150,000 bacteria per c.c. near the shore 
and further out only 38 per c.c. Other figures are as follows: Loci: 
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 frorr 
the coast; but near the shore and in the neighborhood of seaport; 
there may be large numbers. 






MICROORGANISMS IN WATER 263 



j Examples: 350 M. from Naples, sea water contained 26,000 bac- 
'ria per c.c. At a distance of 3 KM., only 10. Samples taken from 
epths of 75 to 800 M. at distances from 4 to 15 KM. from shore were 
>und to contain from 6 to 78 bacteria per c.c. in surface water, and 
[om 3 to 260 at various depths below. 

CAUSES AFFECTING THE INCREASE AND DECREASE OF THE 
NUMBER OF BACTERIA IN WATER 

There is a number of causes which influence the multiplication 
r diminution of microorganisms in natural 'waters; and while it is 
ecessary to discuss each of these causes in detail, it must be remem- 
ered that a number of them may be simultaneously influencing the 
icrease or decrease. 

TEMPERATURE. In natural waters, a low temperature probably 
cts injuriously on parasitic bacteria, reducing their numbers; but 
ihe bacterial content of water during the hot summer months is gener- 
lly not so large as during the cooler seasons. Water collected for 
xamination should be analyzed at once; otherwise, contradictory 
esults as to numbers will be found. Usually, in most waters, there is 
. reduction in numbers for a few hours, followed by a large increase. 
/ery much polluted waters, however, show a marked decrease of 
ntestinal organisms, if the samples are kept cool. 

LIGHT. Although the germicidal effect of sunlight is well known, 
ret it has not such powerful effects on the bacteria in water. 
Vluch depends, no doubt, on the turbidity and speed of the cur- 
ent, the maxmium killing effect being produced in shallow, clear 
md slow-moving water. It has been found by experiment that the 
^erm-killing power of light extends to a depth of 3 M (about 9.84 feet). 
\s 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 
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 
orest lands and is characterized as an upland peaty water carrying a 
rather high percentage of organic and volatile matter, contains through- 



264 MICROBIOLOGY OF WATER AND SEWAGE 

out the year a larger number of organisms to the cubic centimetei 
than the water of the river St. Lawrence, which is much clearer anc 
contains much less organic matter. Sewage water is rich in organic 
matter, and proportionately rich in bacterial life; and bacterial purifica 
tion is synchronous with a diminution of organic matter. 

Jordan remarks in this connection that "in the causes connectec 
with the insufficiency or unsuitability of the food supply is to be founc 
the main reason for the bacterial self -purification of streams." 

OXIDATION. On the surface of waters, in rapids, falls, and tidai 
rivers, much oxygen is absorbed, and much impure matter is 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 j 
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 i 
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 mate- 
rial is thus diminished; the space occupied by a definite number of bac- 
teria 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 L. (100 gallons) 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 L. (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- 
moving rivers the influence of this factor is also quite pronounced; 
and, according to Jordan, "The influences summed up by the term 
sedimentation 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 



MICROORGANISMS IN WATER 265 

the self-purification of the Chicago drainage canal illustrates Jordan's 
ntention. 

OTHER CAUSES. There is a number of other causes, not well 
lown nor of sufficient practical importance for more detailed com- 
ent, which may increase or decrease the number of bacteria in water, 
ch as the inhibiting action of microorganisms and their products 
. one another, the effects of pressure, etc. 

A peculiar fact, which has never been satisfactorily explained, is the 
ick death (in three to five hours) of the cholera vibrio in the waters 
the Ganges and Jumna. When one remembers that these rivers 
e grossly contaminated by sewage, by numerous corpses of natives 
[ten dead of cholera), and by the bathing of thousands of natives, 
seems remarkable that the belief of the Hindoos, the water of these 
^ers is pure and cannot be defiled, and they can safely drink it and 

Ithe in it, should be confirmed by means of modern bacteriological 

isearch. It is also a curious fact that the bactericidal power of 
mna water is lost when it is boiled; and that the cholera vibrio 

]opagates at once, if placed in water taken from wells in the vicinity 

( the rivers. 



INTERPRETATION OF THE BACTERIOLOGICAL ANALYSIS OF WATER 

In making any analysis of water, all data, such as the kind of 
nter and the particulars regarding collection, transmission, sampling, 
i nfall, etc., should be given, as these are a great help in interpreting 
te results. One analysis is rarely sufficient; examinations should 
t regularly and systematically made. 

QUANTITATIVE STANDARDS. No absolute guide can be given to 
c termine the potable quality of water from the- number of micro- 
qanisms in it. It may, however, be safely assumed that high bacte- 
1 1 counts indicate a large amount of organic matter. The number of 
( ^anisms growing in beef peptone gelatin at 20 to 22, and termed 
t; "gelatin count," should be given. For deep wells and springs, 
ts should not exceed 50 per c.c.; and for shallow wells and rivers, 
it over 500 per c.c. After rains or floods, these figures might be 
e:eeded, and would not necessarily indicate dangerous pollution. 

The number of organisms which develop on beef peptone agar 



abated at blood heat, commonly termed the "agar" or "blood- 



266 MICROBIOLOGY OF WATER AND SEWAGE 

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 ol 
specific disease organisms, such as typhoid and cholera microbe? 
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, wher 
it may be necessary to attempt the isolation of these pathogenic 
bacteria, the presence of the colon bacillus (B. coli} in small amount: 
of water, is generally looked upon as significant and indicative of sew 
age pollution. The technical methods used in this isolation am 
enumeration are many, and may be found in the works cited; but then 
is considerable difference of opinion as to the number of B. coli whicl 
should condemn a sample of water. Prescott and Winslow stati 
that if the colon bacillus is in "such abundance as to be isolated in ; 
large proportion of cases from i c.c. of water, it is reasonable proo 
of the presence of serious pollution." Savage suggests that B. coll 
should be absent from 100 c.c. in the case of water from deep well 
and springs, and should be absent from 10 c.c. in surface waters, sud| 
as rivers used for drinking purposes, shallow wells, and upland surfac 
waters. 

The streptococcus examination is next in importance as an indi 
cator of sewage. Streptococci should be absent from the amount j 
of water mentioned above for B. coli; and B. enteritidis sporogene\ 
should not be present in 1,000 c.c. of water from deep wells, nor i: 
100 c.c. from surface waters. 

SEDIMENTATION, FILTRATION, AND PURIFICATION OF WATER 

As areas become more and more thickly settled and towns an 
cities increase in population, the problem of obtaining sanitary cor 
trol over the water supply increases in importance. Very few town 
and cities are fortunate to obtain their water supply from an unpo 



MICROORGANISMS IN WATER 



2 6 7 



lied area. Consequently expensive installation must be made, in order 

purify a suspiciously contaminated water by freeing it from organ- 

s injurious to health. There are several methods of accomplish- 

such purification; and these will be briefly mentioned. 

SEDIMENTATION AND FILTRATION. This method of purifying water 

h; been used for nearly a hundred years; but the great impetus given to 

tls hygienic measure was due to Koch, who showed in 1893 that the 




Sana 





lic of 



FIG. in. Section of a sand filter. 

filtration of Elbe water saved the town of Altona from an epi- 
cholera which devastated Hamburg as a result of drinking un- 
fiered water. In this system of purification, the water is first stored in 
hge reservoirs, where the effect of sedimentation and storage reduces 
cjisiderably the number of bacteria. From the reservoir, the water is 
ed through sand, gravel, and pebbles, etc., arranged as shown 
ig. in. This filtration removes from 97 to 99.5 per cent of the 
roorganisms. 

The action of the fiter bed is due to the mechanical obstruction of 
urities, to oxidation of the organic matter, and to nitrification due 



268 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 
16,138 
1,400 
79,000 
186,986 


7,820 
1,067 


6 3 

400 


London, Chelsea Works 


Berlin, Lake Muggel 
Paris Marne 


Paris, Seine 







to the living bacteria in the scum which forms on the top of the layer 
sand. Of these, the last is the most important; for until this gelatine 
layer forms, the filter does not act properly in fact, it has little filte 
ing action, as the following figures show: 

BACTERIAL CONTENT OF WATER BEFORE AND AFTER CLEANING THE SAND FILT 

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 t 
fore the filtered water is used for domestic purposes. 

The rate of filtration must be regulated; for if the water is allowed 
exceed a certain rate (101.6 mm. or 4 inches per hour), inefficien 
follows. 

COAGULATING BASINS AND FILTRATION. This method of purific 
tion consists in adding a coagulant, such as basic sulphate of aluminui 
by means of a mechanical device which regulates the quantity, as t 
water is pumped into the coagulating basins or reservoirs, where it i 
mains for six to twenty-four hours. The aluminum sulphate is decoi 
posed by the lime in the water and forms insoluble aluminum hydrat 
and the sulphuric acid combines with the lime. The hydrate of alun i 
num is precipitated in large flocculent masses, entangling all particl 
of soil or organic matter; and these, being deposited on the surface of i 



MICROORGANISMS IN WATER 



269 



sj d, form the filtering layer. Such filters are very efficient; they re- 
n ve from 97 to 99.8 per cent of the bacteria from the water. 

POROUS FILTERS. (Fig. 112.) These filters are either made from 
uglazed porcelain or baked diatomaceous earth; the former are known 
a Chamberland, and the latter as Berkefeld filters. These filters 
a usually candle-shaped, require considerable pressure to force water 
t ough them, and can be used only when a small supply of water 
isieeded. Water which is forced through these filters is at first sterile; 
b: with repeated use they allow bacteria to pass through the pores and 




Fj. ii2. Unglazed porcelain filters. Chamberland system; A, without pressure; 
B, fitted to main water supply; C, section of a porous porcelain filter. 

tjis the filtering efficiency is impaired and will remain so, until the fil- 
ls are cleaned and baked to red heat in a muffle-furnace. Unless this 
lone regularly, no dependence should be placed on these filters, as 
3y only put those who use them off their guard against the danger to 
|ich they are exposed. 

PURIFICATION BY OZONE. The antiseptic properties of ozone are 
II known. It is used in the purification of the water supply of some 
vns Nice, Chartres, etc. Ozone used for this purpose is usually 
led by means of the electric current; and a flowing film of water is 



270 MICROBIOLOGY OF WATER AND SEWAGE 

brought into contact with an upward current of air charged with ozom 
which current makes the water almost completely sterile. This methoi 
of purification is efficient, but rather expensive. 

PURIFICATION BY HEAT. By bringing water to the boiling point, a 
harmful bacteria are destroyed; a few spores may resist this treatmeni 
but they are harmless. Boiled water is of a flat, insipid taste, due to th 
driving out of the contained gases. The taste may be improved b 
cooling and shaking. The boiling of water is often resorted to as a h) 
gienic measure in times of epidemic, and for the supply of armies in th 
field. 

PURIFICATION BY CHEMICALS. The addition of a small amount 
calcium hypochlorite, or potassium iodide, etc., purifies water; but thes 
methods are seldom used, except for the use of soldiers on campaigij 
Hypochlorite, however, is now used more commonly in municipal wat(| 
supplies where they can not be otherwise controlled. 



LOCATION AND CONSTRUCTION OF WELLS 






Farms in many sections of this country are practically all supplie 
with surface water collected in shallow wells. Hence farmers shoul 
understand the principles involved in the location and constructic 
of wells. 

Many farm wells are badly located too near such sources of coi 
tamination as outhouses, cesspools, stables, or barnyards; and tho: 
who locate them give too little attention to the slope of the ground, ar 
the nature and slope of the subsoil. There should be at least 22 
30 M. (75 to 100 feet) between the well and all probable sources 
contamination; and this distance is too small, if the soil is very porou 
or if the surface and subsoil drainage is toward the well, or if the w< 
is sunk in fissured rock as it is obvious that there are serious chanc 
of contamination in each of the above circumstances. 

In all cases, the surface drainage should be away from the well; ani 
as far as possible, the subsoil drainage also should be from the well. 

Sketches 113, 114, and 115 illustrate these points, the upper part 
each drawing showing the plan and the lower portion a section throuj 
the dotted line marked on the plan. Fig. 113, shows that the surfa 
drainage is from the house, privy, stables, and barnyard to ward the we 
The section through the line "A" shows the relation of the impervio 



MICROORGANISMS IN WATER 



271 



6 \S 




'"'E* 




A- 



FIG. 113. 



/ El/ 

-g 



/ / 




FIG. 115. 

FIGS. 113, 114, and 115. In each figure plan above section through A B below. 

'Soil; B = Impervious subsoil or strata, i, House; 2, well; 3, outhouse; 4, 
,i?2ry' t 5, stables; 6, stable yard; 7, hen house; 8, sheep stable. Arrow heads 
licate direction of water flow. (Original.) 



272 



MICROBIOLOGY OF WATER AND SEWAGE 



subsoil " B " to the drainage. Water falling on the surface of the ground 
would penetrate 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, improper!} 
located. A better place for the well would be at the letter "X". 

In Fig. 114 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 | 




FIG. 116. Construction of a model well. On the right is brick construction, o: 
the left stone construction, as illustrated. (Original.) 

except the house, is in the direction of the well; and thus contaminatioi 
of the water supply is liable to occur. 

Fig. 114 shows a well properly located as regards both surface an<; 
subsoil drainage. Such a well will supply pure water, if it is properlv 
constructed. 

Fig. 115 shows the proper construction of a well with brick or stone 
Large vitrified drain pipes with cemented joints will answer equally we! 
when there is an abundant supply of water; but in case the supply o 



MICROORGANISMS IN WATER 273 

er is limited, a large area is needed, and a stone or brick well is 
njessary. 

[.eference to the illustrations will show that every endeavor is made 
>revent surface water from entering directly into the well. The walls 
impervious; and the earth or clay is well rammed against the outer 
fc of the wall. The curb is carried well above the surface of the 
gJund. The waste water is conducted by means of a sloping platform, 
trb, and drain, away from the well; and the well opening is properly 
cjered. All water entering such a well must percolate through a con- 
shrable depth of soil, and undergo purification by means of the aggre- 
gjjions of living bacteria in the soil spaces. Thus the soil around a well 
fills the same function in purifying the surface water as the scum 
laer that forms on the surface of gravel filters. 



CHAPTER II* 
MICROBIOLOGY OF SEWAGE 

THE BACTERIAL FLORA OF SEWAGE 

COMPLEXITY or FLORA. Sewage is made up of the miscellaneoi 
and varied wastes of human life and activity, and the bacteria which a 
found therein are the result of a haphazard and chance admixture 
substances of diverse origin and character. The resulting flora is m 
only of great diversity and variability, but it is with few exceptions no; 
characteristic. In brief, the medium with which we have to deal h; 
had an origin too indefinite and a history too short to have permits 
the establishment of anything approaching a constant or characterist 
bacterial flora. 

TYPICAL FORMS. Our interest in this sewage flora is a very practic 
one, being confined to those organisms which carry on the work of bi 
logical purification and to certain pathogens which for obvious reasoi 
require special treatment. We are interested chiefly in what these ba 
teria do rather than in what they are, and our classification is influena 
accordingly. It is based, not upon the species or the genus nor ev< 
upon the group or type, that proves so convenient in general bacteri 
classification, but upon a sort of physiological or functional type, havir 
to do solely with the activities of the organisms in sewage and in its pui 
fication. Bacteria performing a common function or producing a cor 
mon result are members of one type. Individuals may belong to sever 
of our types and there are doubtless a great many that belong to nor 
These latter simply have no place assigned them as yet in the role 
sewage purification, because they possess none of the recognized typic 
functions. 

Apparent exception may be taken to these general principles : 
the case of such organisms as the 'B. coli, sewage streptococci ar 
B. enteritidis. These are, to a certain extent, characteristic sewaj 
bacteria. But interest in them as individuals is confined to wat 

* Prepared by Earle B. Phelps. 

274 



MICROBIOLOGY OF SEWAGE 275 

bacteriology. If they have any functions in the bacterial changes of 
sewage, they receive attention as members of a corresponding type, not 
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 place natur- 
ally in sewage after anaerobic conditions have become established. It 
involves 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 pur- 
poses 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 oxy- 
gen is added to the molecule, the product always containing more oxy- 
gen 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 objec- 
tive rather than subjective functions, in sewage, is made up of patho- 
genic 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 modi- 
fied throughout in accordance with this assumption. 

With these definitions in mind we may proceed to a more detailed 
study of the bacterial types themselves. 






276 MICROBIOLOGY OF WATER AND SEWAGE 

PUTREFACTIVE AND ANAEROBIC BACTERIA. Putrefaction or anae- 
robic fermentation involves the withdrawal of oxygen from one molecule 
or part of a molecule and the subsequent oxidation of another molecule 
or 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, they are 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 = (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 oxidation under aerobic conditions. 

The Liquefaction of Protein. One of the most clearly denned 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 gas- 
tric 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 corre- 
spondingly high. Nevertheless, aside from our knowledge of analogous 
processes in digestion and in bacterial putrefaction of albuminous sub- 
stances, 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 out the initial reaction, 
from which point other groups carry it through successive stages. Ab- 
sence 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 activities within a septic tank, for example, 



MICROBIOLOGY OF SEWAGE 277 

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 chemical 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 complex symbiosis as well as bac- 
terial antagonisms that are involved in the reactions with which we 
are dealing. 

During these reactions proteins and albumins are hydrolyzed by sec- 
cessive stages to albumoses, peptones, amino-acids, amines, and finally 
to ammonia, carbon dioxide, methane, hydrogen, etc. Simultaneously 
ammonia, amines, and carbon dioxide are eliminated at each stage as 
products. The tendency then is toward simple, soluble and gas- 
eous side 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 
vored by total immersion but the effect in both instances seems to be 
to fungus growths which are more active in the moist wood. 

The anaerobic fermentation of cellulose is that which is found typ- 
ically 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 
tiseptic intercellular substances are present. 

Chemically considered the action is hydrolvtic and can be imitated 



iA/u< 

fav< 

due 



278 MICROBIOLOGY OF WATER AND SEWAGE 

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, al- 
though 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 fermen- 
tation is probably a series of side reactions produced during the fermen- 
tation of the nitrogenous material rather than a definite reaction upon 
which the metabolism 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 organisms. 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 split- 
ting 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 
most noticeable in the case of long sewers in which considerable vel 
ties are maintained. In quiescent tanks there is a tendency for the 
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 dis- 
charge,, but in the main the fatty material has become 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 proteoly tic 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. It has also been 
demonstrated that bacterial activity is commonly associated with fat 



clKC 

lich | 
fats ! 



MICROBIOLOGY OF SEWAGE 279 

s onification and decomposition. Whether specific enzymes are pres- 
which assist in this final process or not has never been determined. 
s significant to note, however, that where sewages are slightly acid, 
iltered fats are much more abundant, even though the acidity is 

iufficient to prevent vigorous putrefactive changes in the sewage 



The Fermentation of Urea. The fermentation of urea has already 

n referred to as a typical and simple case of anaerobic decomposition. 

s reaction has great significance in sewage chemistry since a consider- 

2 proportion of the nitrogen of sewage is present initially as urea. 

Oing to the ease and rapidity with which the reaction takes place, 

hvever, no special effort is necessary to bring it about in sewage 

tiatment and it therefore receives brief attention in discussions of the 

dmistry of sewage. The change to ammonia takes place in the small 

s4 r ers of the system and it is difficult and generally impossible to detect 

tit presence of urea in sewage. It has even been suggested that certain 

eiymes present in fecal matter are instrumental in bringing about this 

cinge and that the bacteria are only indirectly concerned. It is 

k)wn, however, that a large number of bacteria of general occurrence 

hire the power to produce this fermentation. Of these the Bact. urea 

(jiquel) may be cited as an example. 

' The Reduction of Sulphates and Nitrates. The production of sul- 
piiretted hydrogen during the anaerobic decomposition of sewage 
iszommonly noted. This substance may arise in at least two ways. 
Sphur, being a constituent of most protein substances, is split off 
fib the molecule in this form during certain types of fermentation. 
I formation in these cases is analogous to that of ammonia from 
tern. The amount so produced is small and is usually neutral- 
\ and precipitated by the small amounts of iron and other metals 
ays present in sewage. There is therefore no liberation of the 
itself and it is often said that sulphuretted hydrogen is not formed 
mally in a septic tank. This conclusion is readily disproved by 
imple test of the black residue found at the bottom of such tanks. 
A second and more important source of this substance is the sul- 
ite normally present in many sewages. Throughout many parts 
the country the water supply contains material quantities of mag- 
n ium or calcium sulphate, and upon the sea coast the sewage gener- 
receives more or less salt water. 



280 MICROBIOLOGY OF WATER AND SEWAGE 

In these cases the reduction of sulphates to sulphuretted hydr< 
gen is not only of interest bacteriologically but probably exerts a 
influence upon all the reactions that are going on simultaneous! 
In fact this example serves excellently to illustrate the great comple 
ity of these anaerobic reactions and the mutual interdependence 
each upon all the others. Sulphates, under anaerobic condition 
are a source of oxygen and it is upon oxygen that the course of all the 
reactions depends. Therefore the presence of sulphates and tl 
possibility of their yielding oxygen may alter the course of the oth 
reactions involved. The products of the protein hydrolysis for e 
ample may be profoundly modified by the presence of this addition 
source of oxygen. 

The effect upon the bacteria themselves is also to be considen 
as a factor quite distinct from the purely chemical effect just d 
scribed. It has frequently been observed, and in fact would be e 
pected, that the products of anaerobic putrefaction are themselv 
detrimental to the activity of the organism producing the chang 
in question. The nature of sulphuretted hydrogen makes it appe 
quite probable that we are dealing here with a toxic substance th 
would at least inhibit the activities of certain bacteria and in this wi 
further modify the final result. 

The same might be said of almost all the reactions with which i 
have to deal but this example is cited as a typical one. 

It is known in practice that the presence of sulphates in a sewa 
does lead to a distinct type of anaerobic change which is characteriz 
by the marked blackening of the sewage, the formation of seconda 
reaction products which precipitate after the removal of the suspend 
matter of the sewage, the evolution of hydrogen sulphide, an excess! 
amount of mineral or non-volatile residue in the sludge and the forir 
tion of free sulphur upon subsequent aeration of the sewage. 

Here again, as in the other types of reaction, it is useless for the pn 
ent to attempt to ascribe this reaction to any particular species. 
desulphuricans and B. sulphureus have been isolated. A non-liquefj 
ing anaerobic bacillus, which reduced sulphates strongly, was isolat 
from Boston sewage in the writer's laboratory by G. R. Spauldir 
Others have been described and there is undoubtedly a large group 
organisms capable of bringing about the reaction. 

Just as the reduction of nitrates is a function performed by man 



MICROBIOLOGY OF SEWAGE 281 

jerhaps most, anaerobes, so the reduction of sulphates, although a 
bss common function, is still common to many forms. In fact ni- 
irates, sulphates, and phosphates form a series in regard to their 
educibility and the effect of their presence upon the reaction as a 
(vhole. The phosphates so far as has been recorded are not ordinarily 
[educed. 

OXIDIZING BACTERIA. The Production of Nitrate and Nitrite. A 
Jong series of investigations upon the organisms which oxidize 
bitrogen began with the Franklands and Winogradski, and has 
ontinued to the present day. These have given us much in- 
ormation concerning the habits and functions of the nitrifying 
)rganisms. Winogradski's original types were Nitrosomonas and 
\itrobacter, the former oxidizing ammonia to nitrite, the latter 
:ompleting the oxidation to nitrate. Work upon these organisms 
constitutes such an important factor in soil bacteriology to-day 
uhat more detailed discussion of this nitrifying function is left for 
(mother place. 

In the earlier days of sewage purification great stress was laid upon 
:he 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 
las been found to be of only minor and incidental importance. 

Other Oxidizing Reactions. The great groups of aerobic and facul- 
tative bacteria are in general concerned in the oxidation of organic 
matter. There is nothing specific in this reaction and very little that 
is characteristic 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 care- 
ful study of the complex reactions involved in the oxidation of sewage 
would show a certain sequence in the order of events and certain definite 
work being accomplished by definite groups. In other words, symbio- 
sis and specialization 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 



282 MICROBIOLOGY OF WATER AND SEWAGE 

decompositions be normally 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 antag- 
onisms 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. 

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 pos- 
sible 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. Further- 
more, if, as we are coming to believe, a definite proportion of the popu- 
lation 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 suffering 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 



MICROBIOLOGY OF SEWAGE 283 

reJrality can be extended to sewage is debatable. Our best informa- 
io leads to the belief that any reduction in numbers of typhoid 
jajeria which may take place within the sewer before discharge is of 
ni >r importance and of slight sanitary significance. 

Discussion of other pathogens must be in even more general terms, 
[nrmation is almost wholly lacking and it can only be assumed for 
mloses of safety that, in so far as organisms of these various types are 
iisnarged into the sewer, they will persist to a certain extent in the 
Jge until it is finally disposed of. If such disposal be by discharge 
nq a stream without purification; then the waters of that stream 
)eJme polluted with infectious material. Studies recently made by 
5e( wick and McNutt have indicated the possibility that many dis- 

, other than the oft-quoted typhoid fever, may be transmitted 

is way. 

,ife in Septic Tanks and Filters. With the introduction of the 
ief c tank at Exeter, England, in 1893, the question of the fate of 

ogenic bacteria in such a tank was raised. It was even suggested 
bacteria, such as the typhoid organism, might multiply in the 



an. The question was investigated by Professor Sims Woodhead, 
rhf concluded that no organisms capable of setting up morbid changes 
limals were discharged from the tank. This negative evidence, 
3ver, has little weight in the light of more recent experiments, 
icard introduced an emulsion of typhoid bacteria into this same tank 
noted only a gradual decrease. After fourteen days he was able 
tect i per cent of the initial number. He also reported a removal 
:> per cent of the typhoid organisms introduced into a contact 



These data must be interpreted in the light of two established 
The typhoid organism tends to die at a rapid but diminishing 
at under any but the most favorable conditions. This results in a 

1 decrease at first, with a prolonged survival of a few individuals, 
fh process takes place in sewers, in streams, and, in fact, under most 
irt cial conditions. The second fact of importance is the difficulty 

covering the typhoid organism under experimental conditions like 
ho described. 

. t borough study of the bacteriology of sewage and of filter effluents 
ed louston to conclude that the biological processes at work in a filter 

nk were not strongly inimical, if hostile at all, to the vitality of 



ogenic germs. 



284 MICROBIOLOGY OF WATER AND SEWAGE 

A conservative study of all the evidence bearing upon this imj 
tant question including the vitality and fate of certain non-pathog< 
species, such as B. coli, leads to the conclusion that the remova.f 
pathogenic bacteria in purification methods is due to two allied can 
the efficiency of which can be approximately determined. Ther s 
first the time element and the known rapid decrease in the number 
certain bacteria such as B. typhpsus when placed under conditions ' 
preclude multiplication. The rate of decrease varies but is rou{ 
about 50 per cent in twenty-four hours. 

The second factor, acting in reality in conjunction with the i 
is the mechanical hindrance that is offered to the free passage of 
pended materials through the body of a filter. Even fine sand o: 
little straining action as such, since the open channels are thousj 
of times as big as the bacterial cell, but surface tension phenon 
tend to make all solid material adhere to the medium and thui 
passage is delayed. This action is prominent although of less im 
tance in coarse-grained filters. Actual experiments by the writer 1 
indicated that while the liquid may pass through a trickling 1 21 
in half an hour, small suspended particles such as ultramarine an 3. 
prodigiosus cells require an average of over twenty-four hours, 
this way the actual time of passage is greatly delayed even when cc 
broken stone is the filter medium, and the times that are now kn r n 
to be necessary for the passage are ample in themselves to accoun 
the reductions that have been noted. 

It may therefore be stated as a conservative view of the effici 
of purification processes in the removal of pathogenic bacteria, 
there are no* strongly inimical processes at work in the tanks or fil s, 
and that the rate of decrease is not materially greater than woul 
observed in the same period of time under the conditions of a run 
stream. 

THE CULTIVATION or SEWAGE BACTERIA 



There are two general methods employed for the cultivatio of 
those bacteria which are of assistance in sewage purification, 
may be cultivated in so-called filters of sand or coarser materiaior 
in specially constructed tanks such as the septic or the hydrolytic t'k. 
In the former case the bacterial growth occurs upon the special me<jni 
provided, the sand or stone; in the latter, it takes place in the lijid 



MICROBIOLOGY OF SEWAGE 



ts< and a continuous life history within such a tank is possible only 
nrh i the rate of flow is sufficiently slow to permit of the inoculation of 
the ncoming stream by the contents of the tank. 

ILTERS. The filtering media most commonly employed are sand 
r rushed stone or other coarse material. In natural sand beds a 




IG. 117. Sewage Experiment Station, Mass. Inst. Technology. Trickling 
iltJ m front, sand filter just behind filter, dosing tank just behind sand filter, and 
&epc tank just behind dosing tank. 

brf period of treatment with sewage suffices to produce an active 
5tae of "nitrification." By this term is indicated alt the complex 
prtesses of oxidation one index of which is the formation of nitrates. 
Aftr such a filter has once become active in this way it will continue, 
wi i proper care, to oxidize sewage almost indefinitely. Improper care, 
sin as an overdose of sewage or continued flooding of the surface due 
to oor drainage, will soon destroy the activity of the filter. The addi- 
tic of germicidal substances has a similar effect and cold weather some- 



286 



MICROBIOLOGY OF WATER AND SEWAGE 



what reduces the efficiency. From all this it is apparent that a filt 
is a biological culture medium upon which the various types of bactei 
are growing and carrying out their functions and that such a mediu 
requires careful control and is sensitive to unfavorable changes 
environment (Fig. 117). 

The other niters are similar to this and illustrate the true function 
filtration. In the case of the sand filter it might be maintained th 
filtration or straining was an essential element in the process, but in t 
case of these coarse-grained media straining action is eliminated. He 
there is nothing but a pile of stones, varying from i to$ in dies 
more in diameter, upon the surface of which the bacteria grow. T 




FIG. 1 1 8. Sketch of septic tank. (Original.) 



sewage trickles slowly over the surfaces, or is held in contact with tin 
temporarily, according as we are dealing with trickling or contact filte 
Solids adhere to the stones or settle upon them, and soluble materia 
"absorbed" by the surface growth and removed from solution. Wit! 
these gelatinous growths to which the air also has free access, the pr 
esses of oxidation take place and the products, the semi-oxidh 
organic material, are later "shed" from the stones appearing again 
the effluent as humus or stable organic matter. 

ANAEROBIC TANKS. The cultivation of bacteria in anaerobic tar 
is not quite as simple a matter as that which has just been describ 






MICROBIOLOGY OF SEWAGE 287 

."he sewage is allowed to flow slowly through the tank and after some 
ime, from a few days to a month or more, a normal and constant 
lora will have become resident there. This flora will soon have be- 
iome so well established that the incoming sewage laden with a flora 
f its own mingles with a liquid in which the established flora is so 
reatly in excess that the former in large measure gives way to the 
Ltter. In this way, while the sewage itself moves onward and is 
bne within a few hours, the flora is constant and persistent. A further 
fid in preserving this constant flora is the sludge at the bottom, in 
|rhich the bacteria lodge and multiply and from which they are carried 
Ipward by the ever moving eddies and constantly re-inoculate the 
iquid above (Fig. 118). 

THE DESTRUCTION OF SEWAGE BACTERIA 

BY BIOLOGICAL PROCESSES. Reference has already been made 
p the effect of biological processes of purification upon pathogenic 
acteria. What was stated in regard to the pathogens is equally true 
f the sewage bacteria as a whole. Their destruction is due to time and 
b environment unfavorable to growth, rather than to any specific 
uise. Further evidence of these facts may now be given. Bacteria 
s a whole do pass even the fine-grained filters in large numbers. 
Careful analyses of their types show them to be a haphazard mixture 
j-om the original sewage flora with little or no observable selection, 
louston pointed out the relative abundance of the streptococci, sup- 
osedly delicate organisms, and found on the whole that the relative 
bundance of the different kinds of bacteria seemed to be much the 
ame in the effluent as in the crude sewage. 

; On the whole we may conclude that the biological processes remove 
acteria not by any specific antagonistic action but by delaying their 
assage and permitting the natural decrease that occurs when multi- 
lication is prevented. The more efficient the mechanism of the 
Iter in producing this delay the more complete will be the removal. 

BY CHEMICAL PROCESSES. A much more reliable and economical 
icthod for bacterial destruction is now available in chemical disin- 
:ction of sewage effluents. The writer's studies at Boston, Baltimore 
nd elsewhere have shown that the application of hypochlorite of 
alcium in amounts depending upon the character of the effluent, and 






288 MICROBIOLOGY OF WATER AND SEWAGE 

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 possible 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 purification into its component parts 
each part can be carried out at a maximum of efficiency and economy. 



DIVISION III* 
MICROBIOLOGY OF SOIL 



CHAPTER I 
MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 

INTRODUCTION 

Rational views on soil fertility were first presented, in a systematic 

ty, by Justus von Liebig in 1840. In his " Organic Chemistry in its 

plications to Agriculture and Physiology" he developed important 

Tories on the circulation of carbon and nitrogen in nature, and on 

t ; function of the so-called mineral constituents of plants. 

When Liebig's book appeared, many of the leaders and students of 
ariculture still believed that humus, the partly decomposed residues of 
pints and animals in the soil, was the direct food of crops. They 
Uieved that soils could yield poor or rich harvests in proportion to the 
aiount of humus present in them; they believed, in other words, that 
pints, like animals, used organic substances as food. 

Liebig rendered a great service to agriculture in emphasizing the 

snificance of decay processes. He made it evident that humus* as 

sj:h is of no use to plants, and that it becomes valuable only in so far 

it is resolved into the simple compounds carbon dioxide, ammonia, 

ric acid and various mineral salts. To be sure, he regarded the 

imposition of organic matter as a phenomenon purely chemical, 

:ertheless he succeeded in showing that decay, putrefaction and 

fomentation are fundamental facts, connecting links between the 

Id of the living and the world of the dead. 

The research of the following decades brought to light the intimate 
ration existing between microorganisms and the decomposition of 

repared by Jacob G. Lipman with exception of sub-chapter on " Soil Inoculation " 
:h has been prepared by S. F. Edwards. , 

19 289 



2 go MICROBIOLOGY OF SOIL 

organic matter. In the realm of soil fertility the new discoveries re 
vealed the vastness of the task assigned to soil microorganisms ii 
providing available food for crops. It was shown that under the attacl 
of bacteria and of other microorganisms the various organic de*bris ii 
the soil is split into relatively small chemical fragments; that th 
carbon is restored to the air as carbon dioxide; that the nitrogen i 
changed into ammonia, nitrites and nitrates. It was shown, further 
that in this breaking down of organic matter the various cleavag 
products, and, particularly, carbon dioxide, hasten, to an amazin 
extent, the weathering of the rock particles and make available thereb 
the mineral portion of plant food. It was shown, likewise, that apar 
from accomplishing the transformation of unavailable into availabl 
plant food, microorganisms are concerned also in the addition c 
nitrogen compounds to the soil. The evidence gathered slowly b 
many investigators made it plain, therefore, that microbes are a 
important factor in the growing of cultivated and uncultivated plant: 
Hence, the important place assigned to microorganisms in the study c 
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 specie 
Variations as to moisture, temperature, aeration, reaction, food suppl 
and biological relations are important, in each case, in determiniE 
the survival or disappearance of any particular species. For th| 
reason, the study of soil microorganisms must reckon with the mechai 
ical composition of soils, their ability to retain water and their contei 
of inert and soluble plant food. 

MOISTURE RELATIONS IN THE SOIL 

AMOUNT AND DISTRIBUTION or RAINFALL. Precipitation : 
different regions of the earth's surface varies from practically nothirj 
to more than 1,524 cm. (600 inches) per annum. A portion of th| 
water runs off the surface into the nearest stream, another portion 
rapidly changed into vapor and is returned to the atmosphere, and tl 
remainder passes downward, into the soil and becomes the mediu 
in which plant food is dissolved. It is estimated that only about h 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 2QI 

tal rainfall percolates through the soil. Where the soils are 
o.'n and nearly level the proportion of percolating water is relatively 
gater; where the soils are fine-grained and more or less impervious, 
othe topography broken, the proportion is relatively smaller. 

I Bacteria and other microorganisms, as well as the higher plants, are 
djectly influenced by the amount of moisture available for their various 
ntds. Hence soil microbial activities are affected not alone by the 
aiount of rainfall, but also by its distribution. It is obvious, for 
hjtance, that an annual rainfall of 762 mm. (30 inches) distributed 
n|her uniformly throughout the year would produce different soii- 
irjisture relations than the same amount of precipitation confined to 
ojy two or three months. As is pointed out by Abbe, a daily pre- 
ditation of 2 mm. (.079 inch) distributed throughout the three 
simmer months would be quickly changed into vapor, and would 
h dly wet the soil; whereas the total quantity of 180 mm. (7 inches) 
qnly divided into ten or twelve rains would penetrate the soil to a 
csiderable depth, and would furnish very favorable conditions for 
rrrobial development. In a similar manner it is pointed out by Hil- 
gid that Central Montana, and the region in the vicinity of the bay 
oSan Francisco, have each a total precipitation of 610 mm. (24 inches). 
Bt while in Montana the rainfall is distributed over the entire year 
a::l irrigation becomes necessary, the precipitation near San Francisco 
isimited to the portion of the year that nearly coincides with the 
giiwing season, and crops are enabled to mature without irrigation. 

RANGE OF SOIL MOISTURE. Any given volume of dry soil consists 
ol-olid particles separated by empty spaces. The sum of these spaces 
ismown as the "pore-space." It varies from about one-third of the 
eiire volume in coarse sands to more than two-thirds in pipe clay. In 
P' t. and muck it may amount to as much as 80 or 90 per cent of the 
eiire volume. Under air-dry conditions each soil grain is surrounded 
b a very thin film of moisture designated as hygroscopic water. When 
aidry soil is moistened the films around the soil particles become 
tl :ker and finally cease to be isolated. A continuous liquid membrane, 
a;t were, is stretched from particle to particle, and the surface tension 
tlit thus comes into play is capable of lifting large amounts of water 
e surface. The continuous film of soil water that can hold its 
O'i against the pull of gravity is known as capillary water. Finally, 
wen the liquid films around the soil grains increase in thickness be- 



2Q2 MICROBIOLOGY OF SOIL 

yond a certain point, the attraction between the molecules in the s 
grains and the more distant molecules of water is no longer gre 
enough to overcome the force of gravitation, and the excess of wal 
percolates downward. The water more or less readily moved 
gravitation is called hydrostatic water. 

For any given conditions of the soils the amount of hydrostat 
capillary and hygroscopic water is directly dependent on the mediant 
structure. It is evident that the aggregate surface of the particles 
a fine-grained soil is much greater than that in a coarse-grained sc 
Actual determinations have shown that the aggregate inner surfa 
of .02832 c.m. (i cu. ft.) of coarse sand may be but a fraction of 
acre; whereas the same quantity of the finest clay may have 
inner surface equivalent to 1.2141-1.6188 hectares (3 or 4 acre 
These differences are to be expected, since, as is shown by Lyon a 
Fippin, i g. of fine gravel may contain 252 particles; i g. of medii 
sand, 13,500 particles; i g. of very fine sand, 1,687,000 particles; i 
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 a 
varies in amount with the fineness or coarseness of the soil, and sir 
the quantity of plant food going into solution is determined largt 
by the amount of water in contact with the soil particles, it follows tl 
clay soils will, under the same conditions, contain more plant food 
solution than loam soils and still more than sandy soils. From t 
standpoint of soil microbiology this is important, for the microorganisi 
live and multiply in the film water surrounding the soil particles. T 
concentration of salts in this film water as well as their compositi 
must of necessity affect bacterial activities. In the same way, methc 
of tillage and cropping affecting the concentration and compositi 
of the film water will modify the chemical changes caused by bacte: 
and other microorganisms. 

EFFECT OF DROUGHT AND OF EXCESSIVE MOISTURE. Optimi 
conditions for plant growth and the development of many importa 
soil bacteria are furnished when about half of the entire pore space 
filled with water. In light sandy soils the optimum moisture conte 
may be reached when the wet material contains sarcely more than 
to 10 per cent of water by weight; while in silt and clay soils ti 
optimum may reach 1 6 to 20 per cent, or even more. 

Continued depletion of soil moisture by plant roots and evaporati 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 293 

a the surface causes the film of capillary water to stretch more and 
re. Finally it becomes very thin, breaks, and ceases to be con- 
nous. The soil then becomes air-dry and contains only hyrgo- 
sjpic water. It is estimated by Lyon and Fippin that, under average 
ditions of humidity, light sand will contain 0.5 to i per cent of 
rroscopic moisture; silt loam, 2 to 4 per cent; and clay, 8 to 12 per 
t. The amount of water present in air-dry muck or peat may range 
to 40 per cent, or even more. According to Hall the film of hygro- 
sjpic moisture is about 0.75^ (0.00003 inch) thick. As the soil 
out bacterial activity is suspended and many vegetative cells 
u|loubtedly perish. Nevertheless, it will be seen that the moisture 
even in air-dry material is deep enough to allow the bacteria a 
r|sonable degree of protection. This will account for the survival 
o lon-spore-bearing bacteria in dry soil for a long time. Indeed, in- 
nces are on record of the isolation of Azotobacter and Nitrosomonas 
fin soils that had been kept in a dry state in the laboratory for 
s eral years. It may be noted, in this connection, that in the process 
Irying the soluble salts in the soil the moisture may be sufficiently 
centrated in the films to cause plasmolysis and the destruction 
o individual cells. 

On the other hand, excessive moisture in the soil is not only directly 
uiavorable to aerobic species in that it limits their supply of oxygen, 
bj is objectionable because it encourages the formation of reduction 
pi ducts that are toxic to these species. It is apparent, therefore, that 
fforable conditions for the formation of available plant food by 
b teria are created when a certain relation is established between the 
vlumes of moisture and air in the soil. The shifting of this relation in 
o direction or another is bound to react on species relationships and 
nnbers. 



! MECHA: 



AERATION 



NICAL COMPOSITION OF SOILS. Soil ventilation is an impor- 
tijt factor in crop production. It provides for the proper supply of 
nentary oxygen so essential to decomposition processes in normal 
s; for the supply of elementary nitrogen required by nitrogen-fixing 
ies; for the removal of excessive amounts of carbon dioxide; and 
fc the destruction of various toxic substances. The intimate relation 



2Q4 MICROBIOLOGY OF SOIL 

existing between soil ventilation and the mechanical composition of t 
soil material is bound to react on the microbial factors involved. It 
well known that the rate of flow of air through soils is inversely propc 
tional to the fineness of the material; in other words, the fine-grain' 
soils, notwithstanding their greater pore space, will not allow air 
pass through them as rapidly as coarse-grained soils. King shows, f 
instance, that 5,000 c.c. of air passed through a column of fine gra\ 
in thirty-seven seconds, whereas in similar columns of medium san 
fine sand, loam and fine clay soil the same amount of air required for i 
passage 1,178, 44,310, 282,200, and 2,057,000 seconds respectively. 

AEROBIC AND ANAEROBIC ACTIVITIES. The more rapid diffusi- 
of gases from open soils naturally leads to a more frequent renewal 
their oxygen supply. In its turn, the latter affects the ratio of aerob 
to anerobes; it follows, therefore, that in clay soils and clay loam so 
the activities of aerobic species are retarded to a greater extent th; 
they are in sandy loams or sandy soils. It follows, also, that in fir 
grained soils the activities of the aerobes are confined to a shallow 
soil layer than in coarser grained soils. The reverse is true of anaerol 
species. Methods of soil treatment tending to improve soil ventilati< 
react both on the amount of chemical change produced by defini 
species, as well as the numerical ratio of different species to one anoth< 
Among such methods may be included drainage, liming, manuring ai 
tillage. 

RATE OF OXIDATION OF CARBON, HYDROGEN AND NITROGEN. 
Experiments carried out by Wollny proved conclusively that the pr 
duction of carbon dioxide in soils is directly affected by the amount 
oxygen supplied; that is, by the more or less thorough aeration of t 
soil. In one of these experiments air containing varying proportio 
of oxygen and nitrogen was passed through columns of soil. Wh 
this air contained 21 per cent of oxygen there were produced for eve 
1,000 volumes of air 12.51 volumes of carbon dioxide; while with 2 p 
cent of oxygen in the entering air there were produced only 3. 
volumes of carbon dioxide. Similar observations were made 1 
Schloesing in connection with the formation of carbon dioxide and 
nitric acid. Deherain and many others have recorded the favorat 
influence of aeration on the rate of nitrate formation, while Lipmii 
and Koch have observed an increased fixation of nitrogen by Azotobad 
consequent upon a better supply of oxygen. 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 



2 95 



THE MINERALIZATION OF ORGANIC MATTER. Conditions that favor 

t intense activities of decay bacteria lead to a relatively rapid restora- 

ti of the phosphorus, sulphur, calcium, magnesium and potassium 

1 1 had been made fast in plant tissues, to the stock of available plant 

fid in the soil; indeed, in extremely well-aerated soils the decomposition 

^mic matter and its ultimate mineralization proceed too fast. It 

happens that the farmer is unable to maintain a proper supply 

o mmus in these soils because of their openness and is forced to adopt 

nisures that will retard soil aeration. He resorts therefore, to rolling, 

ruling, manuring and green manuring. 

On the other hand, heavy, fine-grained soils are not sufficiently well 

i'd to allow a rapid mineralization of the organic matter. Under 

ne conditions the decomposition processes do not keep pace with 

tl process making toward the accumulation of organic matter, and a 

or less considerable increase in the amount of the latter takes 

p :e. This occurs in low lying meadows, and, more particularly, in 

:md swamps. Hence the farmer attempts to intensify aeration 

he resulting mineralization of the humus by more thorough 

ti ige, drainage, liming and manuring. 

TEMPERATURE 

INFLUENCE OF CLIMATE AND SEASON. An illustration of the differ- 

ihat may exist in the soil temperatures of different regions is given 

b i comparison of the mean temperatures of 1901 recorded at Moscow, 

I< ho, and New Brunswick, New Jersey. The soil temperatures were 

ticn to a depth of 152 mm. (6 inches). 

SOIL TEMPERATURE,* 1901 





Jan. Feb. 


Mch. Apr. 


May 


June 


July 


Aug. 


Sept. 


Oct. 


Nov. 


Dec. 


ow, Idaho. . . 


32 .0 30.0 


35.0 40.0 


52.0 


58.0 


68 .0 


72.0 


57-0 


50.0 


40.0 


34.0 


Brunswick, 


31-5 28.6 35. 3 47.9 


57. 9 72.1 76.4 


73.4 


68.5 


56.0 


41.1 


33-4 



AIR TEMPERATURE,* 1901 



T, Idaho. .. 30.0 30.5 38.3 44.0 



56.9 55 o 65.5 



Brunswick, 30.8 24.8 39.1 08.3 59.2 70.9 77-4 74-6 67.6 



69.6 



50.3 



50.5 39.53<;'> 
54 6 38.632.6 



Recorded in Fahrenheit scale. 



2g6 



MICROBIOLOGY OF SOIL 



It will be observed that in the months of November to March tl 
soil temperatures in the two places were nearly the same. On tl 
other hand, in April to October the average temperatures at Ne 
Brunswick were for soil 14.5 (58F.) and for air 22.5 (72?.),! 
spectively; and in July they were 20.0 (68F.) and 24.5 (76.4! 
respectively. It will also be observed that there is an unmistakat 
relation between the corresponding air and soil temperatures. 

As a further illustration of the relation of climate to temperature 
comparison may be made of the average daily mean temperatures 
Bismarck, North Dakota, for the period 1873-1895, and at Key We: 
Florida, for the period 1872-1895. 

DAILY MEAN TEMPERATURES* (AIR) 





Jan. 


Feb. 


Mch. 


Apr. 


May 


June 


July 


Aug. 


Sept. 


Oct. 


Nov. D 


Bismarck, N. D... 

Key West, Fla.... 


4-5 
69.7 


9-5 
71-4 


22.6 

72.7 


42.1 
76.1 


54-2 
79-4 


63.8 
82.5 


69.5 
83.9 


67.5 
83.9 


57-0 
82.5 


43.8 
78.5 


25.9 i. 

74.2 7' 



It is obvious from the figures given here that, because of the i: 
portant temperature variations of different soil regions, the mia 
biological activities must be profoundly modified. But apart from t 
climatic variations already indicated there are seasonal variations 
any particular locality that are of great moment for soil microbiologi* 
activities. Such differences are demonstrated by the temperatui 
of 1898 and 1902, taken to a depth of 152 mm. (6 inches), at N 
Brunswick, N. . J. 

SOIL TEMPERATURES* 





Jan. 


Feb. 


Mch. 


Apr. 


May 


June 


July 


Aug. 


Sept. 


Oct. 


Nov. E 


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 3 


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 3 



In this instance, the season of 1898 was not only earlier, but 1|: 
temperatures of June to September were sufficiently higher to fav 
more intense bacterial growth and activity. 

* Recorded in Fahrenheit scale. 



CROORGANISMS AS A FACTOR IN SOIL FERTILITY 



297 



EARLY AND LATE SOILS. Under any given climatic conditions the 
arming up of soils in the spring will depend on their chemical and 
lechanical composition, color, tillage and topography. Because of the 
gh specific heat of water, fine-grained soils containing a relatively 
rge amount of moisture will warm up more slowly than coarse-grained 
dls containing a relatively small amount of moisture. The differences 
the specific heat of humus, sand, clay and chalk are less important, 
et they introduce appreciable variations in the soil temperature 
:cording to the proportion of each present. The topography of the 
>il introduces a factor of some importance for it affects the inclina- 
on toward the sun's rays as well as the drainage conditions. Tillage 
Derations are of considerable moment, since they influence the rate 

vaporation, that is, the rate at which heat is lost from the soil by 
le transformation of liquid water into vapor. Finally the color of 
ils exerts an influence on their temperature in that it affects the 
Dsorption and reflection of heat. 

Taking all of the factors together, it is found that sandy soils and 
.ndy loams are early soils, because they part readily with their excess 
: water. Clay soils and clay loams are, on the other hand, late soils; 
means, therefore, that in the more open soils microbial activities be- 
)me intense earlier in the spring. Market gardeners usually attempt 
> improve matters still further by the use of large quantities of readily 
rmentable manure that develops enough heat to raise slightly the 
)ii temperature. 

PRODUCTION AND ASSIMILATION OF PLANT FOOD. It was already 
pserved by Moller that slight amounts of carbon dioxide may be 
solved from frozen soil. Kostychev could detect a considerable pro- 

tion of carbon dioxide at o to 5. In a series of experiments carried 
y Wollny the amounts of carbon dioxide produced were as follows: 

CO 2 IN 1,000 VOLS. OF AlR 



Water in soil 


10 


20 


30 


40 


so 


~ 
79 per cent 


2 O3 




6- 86 




2 c T7 


.79 per cent 
.79 per cent. 


18.38 

2 tr 07 


54-22 

6 1 40 


63-50 
82 12 


80.06 
91 86 


8l.S2 
O7 4.8 


















he increased production of carbon dioxide at the higher temperatures, 
.own in the above table, correspond with the observations that had 



298 MICROBIOLOGY OF SOIL 

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 ob- 
servations of numerous investigators on the multiplication of in- 
dividual 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 activities become more intense as gauged by the 
formation of carbon dioxide. 

Coming down to specific groups of soil bacteria, it may be noted thai 
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 to 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 tem- 
peratures are approached. The organic matter itself, apart from serv- 
ing as a source of plant food, furnishes carbon dioxide and various 
organic acids that help to attack the rock fragments and to rendei 
available compounds of phosphorus, potassium, calcium and mag- 
nesium. It is likewise evident that in warm countries bacteria' 
activities are not only more intense at any one time, but they continue 
through a longer period. For this reason, the soils of the South car 
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, there- 
fore, assimilated and more moisture used up until the very rank growth 
of the crops hastens the depletion of the soil moisture. In this mannei 
the soil may be dried out sufficiently to retard seriously the growth oi 
soil bacteria and to retard thereby the decompositon of organic matter 
under such conditions, moisture, rather than temperature, becomes 
the controlling factor of growth. 

REACTION 

RANGE or SOIL ACIDITY. Acid soils are very common in huraic 
regions. The older soils of Europe include extensive areas whosejime 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 299 

ontent has been restored repeatedly by the application of wood ashes, 

arl, oyster and clam shells, and various grades of burned or crushed 

imestone. In the United States acidity is becoming prevalent in many 

If the cultivated soils, as is shown by the investigations of the Rhode 

sland, Ohio, Illinois, Oregon and Florida experiment stations. These 

ivestigations, confirmed by experiments in other states, show that 

ere is a marked removal of lime and of other basic materials from the 

il as cultivation and the use of commercial fertilizers become more 

orough. Knisley shows, for instance, that 38.75 per cent of the 

)regon soils examined were acid, and that 16.25 per cent were strongly 

cid. Similarly, Blair found that of 189 samples of different Florida 

oils and subsoils, examined, 68.22 per cent of the former and 51.35 

er cent of the latter were acid. He also found that virgin soils were 

'. i ss acid than cultivated soils. 

CAUSES OF SOIL ACIDITY. Soil acidity may be due to acids or acid 
ilts, both inorganic and organic. Under ordinary conditions the 
itter are of much greater importance than the former as a cause of 
)il acidity. This is demonstrated by the extremely acid conditions 
[ peat and muck soils that are particularly rich in organic acids. In 
)ils left to themselves the formation of basic substances in the break- 
ig down of silicates and other compounds keeps pace with their 
eutralization by acid and their removal in the drainage water. When 
>ils are placed under cultivation, lime and other bases are removed 
iore rapidly and the inert humic acids are left behind. The loss of 
ases is intensified by application of .acid phosphate, potash salts and 
mmonium sulphate, commonly used as fertilizers. This accounts 
r the less extensive acidity in and among virgin soils as compared 
ith cultivated soils. Arid soils lose scarcely any of their basic sub- 
i.nces by leaching and are seldom acid. Residual limestone soils 
ay be alkaline, neutral or acid, according to the loss of bases they 
ive suffered by leaching. Low-lying soils, including meadows 
id swamps may accumulate large amounts of organic acids because 
their imperfect aeration. 

EFFECT OF REACTION ON NUMBERS AND SPECIES. Some of the 
iportant groups of soil bacteria including nitro, azoto and ammonif y- 
g species will develop slowly or not at all. when the amount of acid in 
ie medium is increased beyond a certain point. Hence it is realized 
progressive farmers that a proper supply of lime is essential for the 









300 MICROBIOLOGY OF SOIL 



satisfactory decomposition of organic matter in the soil, and the abuncj- 
ant supply of available nitrogen compounds, as well as of other con- 
stituents of plant food to growing crops. The influence of lime on the 
multiplication 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 limied and cultivated for severa ] 
years the 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 oi 
organic matter is practically devoid of bacteria. To the fresh and th( 
partially decomposed organic matter (humus) the soil organisms must 
look for most of their food and energy. Being largely of plant origir 
this organic matter contains starches, fats, organic acids, higher al 
cohols, proteins and amino-compounds. Because of the differen 
relations that these vegetable substances bear to the several species o 
soil bacteria, a high or low proportion of starch, of cellulose, or proteii 
must necessarily modify both numbers and species relationships. Fo: 
instance, observations have been made by Coleman and others tha 
small amounts of dextrose favor nitrification, whereas larger quantitie: 
retard it; similarly, it has been noted that in the spontaneous de 
composition of protein bodies bacteria are prominent and molds absen 
or relatively few in numbers. But where dextrose is added to thi 
decomposing proteins molds soon appear in large numbers. Then 
may also be cited, in this connection, the observation of Hilgard tha 
humus should contain at least 4 per cent of nitrogen if it is to furnisl 
a sufficient quantity of available nitrogen compounds; otherwise, tb 
soil bacteria seem to be unable to decompose it, so as to meet th 
needs of the growing plants. Many similar facts could be cited t 
show that as a culture medium the soil is influenced by green manures 
barnyard manure, commercial fertilizers, lime, tillage and any othe 
treatment that will modify the quantity as well as the quality of it 
organic matter. 

THE MINERAL PORTION OF THE SOIL. The moisture films sur 
rounding the soil grains contain in solution substances derived fron 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 301 

iese soil grains. A particle of calcium carbonate will be surrounded 
f a moisture film containing some calcium bicarbonate. In the 
me way particles of feldspar may give rise to a solution of potassium 
carbonate; particles of apatite to a solution of calcium phosphate; 
articles of selenite to a solution of calcium sulphate; particles of 
rotein to a solution of ammonia, etc. In view of the fact that these 
actions are more or less localized and diffusion slow, there are, un- 
Dubtedly, in the soil minute zones where individual species are more 
eminent than they are in others. For example, Heinze has found it 
invenient to isolate Azotobacter by inoculating suitable culture solu- 
ons with particles of calcium carbonate picked out from the soil, 
vidently these organisms were present in much greater abundance 

1 these particles than on others of non-calcareous origin. Indeed, 

2 occasionally obtained in this manner Azotobacter membranes that 
mstituted almost pure cultures. The more general significance of 
lis relation is apparent when it is remembered that nitro-bacteria 
e particularly favored by magnesium carbonate; tubercle bacteria 
v- gypsum and calcium carbonate; Azotobacter by calcium phosphate 
id calcium carbonate; photo-bacteria by sodium chloride, etc. 

Considerable as must be the local differences in any one soil, they 
e undoubtedly even more pronounced when different soils are com- 
ired. Extreme conditions are met with in certain irrigated soils 

which a marked concentration of salts occurs. In so far as crop 
eduction is concerned, it is stated by Hilgard that the upper limit is 
act ically reached when the concentration of soluble salts in the irriga- 
jon water is about 4.55 g. (70 gr.) per gallon. Nevertheless, in Egypt 
jid the Sahara region irrigation water is occasionally used that con- 
ins more than 13 g. (200 gr.) of soluble salts per gallon. Further 
fferences are introduced by the quality of these salts, e.g., the pro- 
>rtion of sodium sulphate, magnesium sulphate, sodium chloride, 
'dium carbonate, etc. Again, instances are on record, as in the investi- 
nions of Headden in Colorado and California, where the concentration 

nitrates in the soil water is so great as to kill even relatively resistant 
ants like alfalfa. It is to be shown by future investigations what the 
feet of the concentration and composition of such salts may be on the 
il bacteria. 

In humid soils conditions are less extreme, yet even here the variable 
ncentration and composition of the soil solution are of direct moment 






302 MICROBIOLOGY OF SOIL 

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. Distribution. While the study of the lower bacteria in the 
soil has attracted the attention of many investigators, that of fungi and 
actinomyces received until recently, but scant consideration. Fungi oc- 
cur in all soils, cultivated as well as uncultivated, rich or poor in organic 
matter, heavy or light in texture. Most of them are obligate sapro- 
phytes, although facultative parasites are found in large numbers ir 
the soil, especially where single-cropping or short rotations favor the 
survival of the particular organisms. The isolation of soil fungi has 
been accomplished either by the dilution method, where a sample o; 
soil was shaken with water, and only a certain dilution was used foi 
inoculation; and by the direct method, where a clump of soil was inocu 
lated into a sterile medium, and the fungi developing on it were isolated 
About 150 different species of fungi have been isolated from different 
soils, and the data accumulated by investigators in this country anc 
in Europe seem to point to the fact that many of these fungi an 
universal in their habitat, since the same species are recorded to hav< 
been isolated from different soil types and in different localities. Mos ! 
of the work done refers to the classification of the organisms isolated 
The largest group of soil fungi belong to the following genera: Mucor 
Aspergillus, PenicilUum, Cladosporium, Fusarium, Trichoderma 
Cephalosporium, Alternaria, Zygorrhynchus, Monilia, Rhizopus, anc 
Acrostagmus. Many other genera have been isolated, but to a mor< 
limited extent. As to the individual species occurring in the soil 
Hagem, having isolated about 30 mucors from the soil, states tha 
certain Aspergilli occur in larger numbers than all the mucors takei 
together. As to quantitative relations, no exact data are availablej 
Some investigators report only several hundred fungi per g. of soilj 
while others record as many as 1,000,000 per g. of soil; that is th 
total number of spores and pieces of mycelium that develop on suitabl'i 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 303 

iedia. As to the numbers and types in relation to depth, Goddard 
included that there does not seem to be any appreciable variation in 
imbers at the different soil depths. Unpublished data of the New 
rsey P^xperiment Station bring out the fact that there are very few 
ingi in the soil below 8 inches, and that one of the most common forms 
t these depths is Zygorrhynchus vuilleminii. It was formerly thought 
mt soil fungi are abundant only in acid soils, but recent investigations 
lake it appear that also limed and well-cultivated soils have an 
bundant fungus flora. 

Am monification. Miintz and Coudon, and after them Marchal, 
orking with pure cultures, proved conclusively that fungi decompose 
rganic matter and cause an accumulation of ammonia in the soil, 
/ilson and McLean found that the forms of Monilia&rz the most active 
mmonifiers among the several groups of organisms studied, while the 
spergilli showed the least ammonifying power. More recent work 
as confirmed the earlier findings and has proved that fungi may 
lay an active part in the decomposition of organic matter, and the 
:cumulation of ammonia. 

Nitrogen-fixation. Experiments on nitrogen-fixation by fungi were 
irried on by Jodin as early as 1862. He observed a rich fungus growth 
a nitrogen-free media, supplied with sugar, tartaric acid, or glycerin, 
erthelot, Saida, Ternetz, and others also reported fixation of atmos- 
heric nitrogen through the activities of fungi, such as Aspergillus 
iger, Alter naria tennis and several species of Monilia, Penicillium, 
lucorini and others. But other investigators among them, Wino- 
radsky, Czapek and Heinze were unable to confirm these observa- 
ons. The careful work of Goddard has also given negative results, 
he entire question is therefore still an open one with the weight of 
yidence on the negative side. 

Cellulose Decomposition. The destruction of cellulose in the soil is 
ue to a large extent, to the activities of soil fungi, as has been demon- 
.rated by several investigators. Cellulose decomposition by fungi was 
observed in the study of plant diseases. Van Iterson used filter 
r for the isolation of fungi, by exposing this medium to the air for 
r elve hours. Thirty-five species of fungi were isolated thus proving 
at a large number of cellulose-destroying fungi may be present in 
e air. Appel found that certain species of Fusarium destroyed in 
urteen days 80 per cent of the filter paper used. Marshall Ward 



304 MICROBIOLOGY OF SOIL 

and others recorded that a number of fungi are economically impor- 
tant as wood-destroyers. Spores of a pure culture of Penicillium sowr 
on sterile blocks of spruce wood, germinated and grew normally 
Sections of the wood showed that the hyphse had entered the starch 
bearing cells of the medullary rays of the sapwood and consumed th< 
whole of the starch. MacBeth and Scales found that when the mediuir 
is slightly alkaline, certain aerobic bacteria will play the principa 
r61e in the destruction of cellulose. When the medium is acid, molds 
and higher fungi become the active agents of destruction. They als( 
found that the cellulose-destroying forms multiply with great rapidit} 
in alkaline soils when cellulose in the form of filter paper is added. Th( 
power to destroy cellulose is reported for a number of species of Penicill 
ium, Aspergilli, Trichoderma and other organisms which belong to thi 
common soil forms. Though the fungi may play an important par 
as cellulose destroyers also in alkaline soils, in acid soils where th 
activity of bacteria is greatly inhibited, fungi probably play a pre 
dominant role. This fact led Marshall to conclude in 1893 tha 
fungi take an active part in the mineralization of the organic matte: 
in acid humus soils. 

Mycorrhiza. Apart from the so-called soil fungi, there exists anothe 
group known as mycorrhizal fungi. These live symbiotically on thi 
roots of the higher plants. Many roots of forest trees, when examinee 
carefully, show that there is a union between the mycelium of certaii 
fungi, usually belonging to the fleshy fungi, and the root of the plant 
This union is called a "mycorrhiza." The fine filaments of the fungu: 
enter the cells of the root. These organisms were thought at firs 
to supply the roots with water and soluble plant food from the soil 
The power to fix atmospheric nitrogen has been ascribed to these organ 
isms by several investigators. But aside from these useful so-callec 
endotrophic Mycorrhiza, there are also the ectotrophic Mycorrhizc 
which probably live only parasitically upon the roots of plants. 

Actinomyces. The study of soil Actinomyces is nearly all of ven 
recent origin. Several years ago but two soil Actinomyces had beer 
definitely described, viz., Act. albus and Act. chromogenus. The worl 
of Krainsky, of Conn and of Waksman and Curtis has demonstratec 
that Actinomyces are widely scattered in cultivated soils. The last 
named investigators have shown that while the absolute numbers o 
Actinomyces decrease with depth of soil, their relative numbers ar< 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 305 

r tcrially increased so that if at a depth of 25 mm. (i inch) there 
a^ only 6 to 10 per cent of Actinomyces and 82 to 93 per cent of 
tcteria, at a depth of 750 mm. (30 inches) the Actimonyces form 
4 to 80 per cent of the total micro-organic flora of the soil. The 
rmbers of Actinomyces in the surface soil vary greatly with the types 
csoil and abundance of plant food. In one instance 1,300,000 A cti- 
tnyces were found in a total of 15,000,000 bacteria per g. of rich 
radow soil. As to the activities of Actinomyces in the soil, Beyer- 
i k has shown that the Act. chromogenus produces an oxidizing sub- 
e, quinon (C 6 H 4 O 2 ) which may play an important part in the 
odation of organic matter in the soil. Munter, Krainsky and Scales 
Ive demonstrated that many Actinomyces are able to decompose cellu- 
1 e in the soil, and that in some instances this ability is very marked. 
lainsky records that soil Actinomyces need very little nitrogen for 
t ir life activities, and that they can get it from any available source. 
I nitrates are present, these are reduced first to nitrites, and then 
ulized. Waksman and Curtis working with soil sterilized by steam, 
d not find any great accumulation of ammonia through the activities 
o Actinomyces, although different species seemed to show marked varia- 
1 i in their power to accumulate ammonia. 

\LGJE. At times the influence of algae in changing the character of 
t soil as a culture medium for bacteria is quite considerable. As 
c orophyl-bearing organisms they are enabled to manufacture sugar 
a 1 starch with the aid of sunlight, and to favor thus the development 
o 1 zotobacter and of other microorganisms dependent for their energy 
o the organic matter in the soil. Investigators both in France and 
ii Germany have found that the fixation of nitrogen in sand used for 
p culture experiments occurs in the surface layer possessing a growth 
o algae. The advocates of bare fallows attribute the greater pro- 
d tivity of fallowed land to the growth of algae, the accumulation of 
nrogen through their influence and to other changes affecting the soil 
b teria. 

PROTOZOA. It has been shown for a long time that certain species 

>rotozoa are common in soils and that their food consists of bacteria. 

1 what extent protozoa play a part in soil fertility has not yet been 
f'y explained, even though Russell and Hutchinson and of the 

imsted Experiment Station have maintained that these minute 

a mals are extremely important in that they maintain a certain bac- 
20 






306 MICROBIOLOGY OF SOIL 

terial equilibrium in the soil. Their claim is mainly based on the fac 
that partially sterilized soils (either by means of heat or antiseptics 
soon come to contain enormous numbers of bacteria. 

It is, therefore, assumed by them that this abnormal increase i 
made possible by the destruction of the protozoa (which have a lowe 
power of resistance to heat and antiseptics than bacteria) that normall 
check the increase beyond a certain point. Under the conditions n 
corded a causal relationship obtains between an increase in numbers c 
bacteria and the rate of ammonia production, which is considered to t 
an index of fertility. 

This theory has been the basis of considerable investigation, muc 
of which has failed to corroborate the above conclusions. The fa< 
that there is an increase in bacterial numbers and in consequenc 
enhanced fertility of the soil may not be due to the elimination < 
protozoa but may rather be ascribed to such effects of the parti 
sterilization process as (i) increase in available food for bacteri: 
(2) rendering soil toxins insoluble; (3) destroying bacterio-toxin 
(4) acceleration of the biological processes. 

It has even been noted in some instances that partial sterilizatk 
has been responsible for a decrease rather than increase in the produ 
tion of ammonia. Such considerations, among others, have been i 
strumental in stimulating investigation in another branch of soil f ertilit 
namely, soil protozoology. There has been difficulty in establish!] 
suitable methods and technic, as for example the development 
media favorable for isolation and the culture of soil protozoa; althouj 
blood meal solution, hay infusion and soil extract have been used 
advantage. The organisms, have been counted in the same manner 
bacteria, namely, by the dilution method, or by means of a standa 
platinum loop. An adaptation of the apparatus used in the count! 
of blood corpuscles has been successfully employed by Kopelc 
Lint and Coleman. 

A study of the morphology and life history of soil protozoa reve; 
the fact that encystment occurs under most conditions which are n 
immediately favorable, as for example slight variations in moisti 
content, or food. In point of fact this period of the protozoan life cy< 
which is analogous to the spore-forming stage of bacteria forms t 
basis for the question which arises as to the existence of protozoa, 
their trophic form, in field soils. Of the well-defined groups of p 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 307 

(page 130), namely, flagellates, ciliates and amoebae, many types 

[e been described. Among those occurring frequently are: Colpoda 
Stilus, Boda ovatus, Colpidlum colpoda, Amoeba terricola, Monas, etc. 
T: requirements for maximum development in the soil for these organ- 
is s are: (i) A high degree of moisture, closely approximating saturation ; 

(an abundant supply of organic matter; (3) moderate temperature, 
thermal death point of active forms has been found by Goodey 

he 40 to 50, and for the cyst forms of the same organisms about 
1\ The optimum temperature for most forms is about 22. 

ystment of protozoa occurs within wide limits in an alkaline medium 
cctaining up to .18 per cent NaOH, and in the presence of an acidity 

esented by .09 per cent HC1. 

Protozoa are found in many greenhouse soils, due no doubt to the 
far that they contain a high degree of moisture and organic matter. 

vever, in dealing with field soils some investigators have failed to 
is ite active forms of protozoa, whereas others record the presence of 

e numbers of these organisms. Their distribution appears to 

jillel that of bacteria, namely, the greatest number of protozoa occurs 
wjiin the upper 100 mm. (4 inches) of soil, with a decrease down to 
3d mm. (12 inches), which represents the lower limit of their activity. 

;As regards the occurrence of the various groups of soil protozoa, 
fljellates are found to be dominant over ciliates and amoebae. G. P. 
K:h has found that the development of soil protozoa in artificial 
ciure solutions varies (i) with the kind of media employed; (2) the 
ity of soil used for inoculation; (3) drying of the soil; (4) different 
Mis of soil and different soils of the same kind; (5) the temperature 
oiicubation. 

While it is generally accepted that protozoa feed upon bacteria, 
u;il the relation that obtains between the various types of protozoa 

the different species of soil bacteria has been more fully investigated 

direct effect of protozoa upon bacteria must remain, to a degree, 

^terminate. 

Soil sterilization has had a practical application in eliminating 

ious diseases in greenhouses and infested fields. Partial steriliza- 
tiji as employed by Russell and Hutchinson while not so drastic, 
iiplves serious changes in the soil, which might be considered in much 
til same light as the phenomena attending complete sterilization 
b;| means of heat and antiseptics. It is an established fact that 



308 MICROBIOLOGY OF SOIL 

sterilization is responsible for increased plant growth, and to expla 
this phenomenon the following theories have been advanced: 

1. R. Koch's theory of direct stimulation to plant growth- 
physiological effect of the sterilizing agency. 

2. Hiltner and Stormer's theory of indirect stimulation an alter 
tion of the bacteriological equilibrium resulting in a marked develo 
ment of numbers after decimation. 

3. Liebscher's view that soil sterilization may be regarded in t 
same light as a nitrogenous fertilizer. 

4. Russell and Hutchinson's protozoan theory of soil fertility. 

5. Pickering and Schreiner's contention that the alteration 
chemical composition is largely responsible for increased plant growt 

6. Greig-Smith and others adhering to the bacterio-toxin hypothes 
HIGHER PLANTS. Higher plants modify the soil as a cultu 

medium for bacteria in at least three ways. The root-hairs coi 
into contact with the moisture films surrounding the soil grains a 
not only modify the composition of the film water, by withdrawing 
portion of the dissolved matter, but also change its character by seci 
tions from the roots. The changes thus effected must, necessari 
modify the character of the soil and the soil solution as a cultv 
medium. Again, the rapid removal of water from the soil by growi 
crops causes the film water to become more concentrated in so far, 
least, as some salts are concerned. Modifications, are, also, introduc 
thereby in the proportions of oxygen, nitrogen and carbon dioxide 
the soil air. Finally, higher plants modify the soil environment i 
bacteria by their root and stubble residues. For example, residues 
leguminous plants, being richer in nitrogen and possessing a narrov 
carbon-nitrogen ratio than the corresponding residues of non-legum 
will affect the soil somewhat differently than the latter. 

BACTERIA. Occupying, as they do, the leading role, bacte 
demand a more detailed consideration, in fact, most of the biologi 
discussions of soil are based upon a knowledge of these organisms. 

Numbers and Distribution (Bacteria in Productive and Unproduct 
Soils).- The numbers of bacteria in soils well supplied with orga: 
matter usually range from 3,000,000 to 15,000,000 per g., as sho i 
by the agar plate method. These numbers vary from soil to soil, ail 
from season to season for any particular soil. The numbers of fui, 
are also variable and may reach a total of 1,000,000 per g., althou. 



A 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 309 

ii till remains to be demonstrated whether the large numbers thus 
f ( id represent organisms which lead an active life in the soil or only 
sires of fungi brought in by external agencies. The numbers of 

inomyces may reach 1,000,000 or more per g. of soil. The fungi 

ost disappear below 20 to 30 cm., while the actinomyces do not 
el rease rapidly at depths lower than 30 cm. 

Distribution at Different Depths. Most of the soil bacteria are found 
ii he stratum in which the organic residues are concentrated, that is, 

he surface soil. Immediately at the surface the rapid evaporation 

the germicidal effect of direct sunshine act as disturbing factors, 

h ce the numbers in the uppermost 25 to 50 mm. (i to 2 inches) are 

iller than in the layer of soil immediately below. Beyond the 
d th of 20 cm. or 22 cm. (8 or 9 inches) the numbers diminish rapidly. 

terial from a depth of .6 m. to .9 m. (2 to 3 feet) is nearly sterile in 
h nid regions. Differences occur, however, in keeping with the 

:hanical composition of the soil. In light, open soils the bacteria 

not only carried down to greater depths by the percolating water, 
can also multiply there, thanks to better aeration. On the con- 
try, fine-grained compact soils are more effective in holding back 
sJpended material and do not allow the bacteria to pass downward as 
rdily. Moreover, the less thorough aeration of these soils and the 
a,umulation of toxic reduction products in the subsoil serve as an 
e: ctive check in the increase of bacteria in the deeper layers. 

In irrigated soils of the arid and semi-arid regions bacteria are dis- 
tnuted at much greater depths. Their occurrence 2 m. to 3 m. 
(hr 10 feet) below the surface is made possible not only by the better 
action of these soils, but by the penetration of roots to great depths 
ail the accumulation there of considerable amounts of organic matter. 
TJ practical significance of distribution appears, among other things, 
ir he use of soil for inoculation purposes; for instance, it is reported by 
S r.rom that in making peat soils arable the addition of small amounts 
o/ertile loam increases to a very marked extent their crop-producing 
pj^er. The efficiency of the inoculating material decreases as it is 
tilen from the deeper soil layers. Similarly, in the use of alfalfa soil 
f( the inoculation of new fields the most efficient material is secured at 
a pth between 7.62 cm. and 17.78 cm. (3 and 7 inches). 

\Seasonal Variations of Bacterial Numbers and Activities. Conn has 
norted an apparent increase of bacteria in frozen soil. This increase 



C,i 



310 MICROBIOLOGY OF SOIL 

seems to be due to an actual multiplication of the organisms rather th; 
to a mere lifting of the bacteria from lower depths by capillary actio 
The greatest increase was found to occur during the winter in the slcr 
growing bacteria and not in those that liquefy gelatin rapidly or in t 
Actinomyces. Conn tries to account for the phenomenon by assumi: 
the existence of two groups of bacteria, winter and summer bacter I 
The latter, he thinks, prevents the former from multiplying rapid 
in warm weather. Hence, the increase in frozen soils is not to 
ascribed directly to the low temperature, but to the depressing effr 
of the cold upon the summer bacteria. Brown found that the s 
bacteria diminish during the fall season with the lowering of ti 
temperature, but, when the soil is frozen, an increase in numbi 
occurs. He also found frozen soils to possess a much greater ammo 
fying, denitrifying and nitrogen- fixing power than non-frozen sol 
According to him, the lowering of the freezing-point of the capilla 
water, due in part to the concentration of salts at the time of freezii 
may account for the abnormal bacterial activities. 

Morphological and Physiological Groups (Morphological Groups] 
Rod-shaped organisms are numerically the most prominent among s 
bacteria. They occur at times to the extent of 80 or 90 per cent '. 
the total number. Spherical organisms usually constitute less ti. 
25 per cent of the bacterial flora. Spirilla and sarcinae are present t 
slight numbers. Conditions may occur, however, when the proport 
of spherical organisms is markedly increased. This happens, p 
ticularly, when large quantities of composted manure (rich in spheri 1 
organisms) is added to the soil. 

Among the rod-shaped species B. mycoides, B. subtilis, B. mes 
tericus, B. tumescens and other members of the subtilis group are qi 
prominent. Members of the amylobacter group are seldom abse 
Members of the proteus group and various fluorescens are alw 
present, while Bad. osrogenes and allied species are common inhabita 
of the soil. 

(Physiological Groups). In the decomposition of organic mattei 
the soil certain important changes in both nitrogenous and non-nil 
genous material are accomplished by definite groups of bacteria, 
breaking down of protein substances is accomplished by the fori 
tion of ammonia, nitrites and nitrates. These in turn may be tra - 
formed back into more complex amino-compounds, peptones, and j - 



OORGANISMS AS A FACTOR IN SOIL FERTILITY 31 1 

ins, or they may be destroyed with the evolution of free nitrogen, 
joreover, there are groups of bacteria capable of joining non-nitro- 
jnous organic matter to elementary nitrogen and of producing thus 
jtrogen compounds. Again, there are groups of bacteria bearing 
.stinct and important relations to the decomposition of cellulose, or 
|e transformation of its cleavage products, methane and hydrogen, 
jiere are, likewise, definite groups of bacteria concerned in the 
information of sulphur and its compounds, and of ferrous compounds. 

METHODS OF. STUDY 

QUANTITATIVE RELATIONS. Since the early work of Koch in 1881 
liny investigators have determined the number of bacteria in soil 
mples, by means of the plate method. It is well known, however, 
at on ordinary gelatin or agar plates kept under aerobic conditions 
jit a fraction of the soil organisms produce visible colonies. The 
i.aerobic species do not appear, nor do aerobic Azotobacter, and nitro- 
i.cteria, while other common soil organisms form colonies sparingly 
not at all. By employing synthetic agar media instead of beef broth 
latin or agar, Lipman and Brown have succeeded in securing the 
.;owth of a much larger number of colonies from any given quantity 
I soil, yet even these larger numbers were incomplete for reasons 
jentioned above. 

H. Fischer recommends a simple medium of agar to which nothing 
Is been added but soil extract (prepared by extracting with a .1 
IT cent solution of Na 2 CO 3 ) and potassium phosphate. Following 
|e path of Lipman and Brown in reducing the content of organic 
latter, Temple employed i g. of peptone per 1. as a culture medium 
Id obtained satisfactory results. Brown has further modified the 
jrtnula of Lipman and Brown by replacing the .05 g. of peptone with 
|g. of albumin, and obtained results which were somewhat superior, 
comparison of culture media, Conn considers the former media 
irable for quantitative purposes because they contain substances 
lefinite chemical composition, and offers an agar medium con- 
ig no organic matter except agar, dextrose and sodium asparag- 
ite, and also a soil-extract gelatin which is valuable for qualitative 
irposes. Another medium that has been suggested, after a com- 
of all of the above-mentioned media, is the urea-ammonium 



312 , MICROBIOLOGY OF SOIL 

nitrate agar of R. C. Cook. It is evident, therefore, that the result 
secured in the counting of soil bacteria have only a relative value 
With the same media and methods some information may be secure< 
concerning the influence of fertilization, tillage, liming, etc., on certaii 
of the soil bacteria. But even this information must be properb 
discounted, since equal numbers do not necessarily mean equal amount 
of chemical work accomplished; for example, there is no certainty tha 
1,000,000 of decay bacteria derived from one soil will accomplisl 
exactly as much decomposition as the same number of similar organ 
isms from another soil. Otherwise stated, individual cells differ ii 
their physiological efficiency from other cells of the same species. 

QUALITATIVE REACTIONS. By modifying the composition of th 
culture media different physiological groups may be favored in thci 
development. In this manner the silica jelly medium proposed b 
Winogradski, or the gypsum plates proposed by Omelianski may be em 
ployed for making numerical comparisons of nitro-bacteria in differen 
soils. In like manner Beijerinck's mannit agar may be used for th 
numerical comparison of Azotobacter, and other media could be adapte 
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, possesse 
defects already noted in connection with the more strictly quantitativ 
method. Apart from the vast amount of work involved in the preps 
ration of a large number of media and in the counting of colonies o 
many plates, this method fails to indicate differences in physiologic; 
efficiency. Furthermore, the colonies of the specific organisms sough 
are almost invariably accompanied by those of foreign species nc 
always easily distinguished. With these limitations properly recognize 
and with further improvement in the constitution of special media th 
method may be made useful in supplementing data secured by oth( 
methods. 

TRANSFORMATION REACTIONS. Instead of counting soil bacteria i 
accordance with colonies produced in general or special media, so 
investigators have attempted to measure the bacteriological functions ( 
soils by comparing more or less definite quantities of the latter undt 
known conditions. This method was employed by Wollny and othei 
in studying the factors that affect the formation of carbon dioxide i 



MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 313 

It was also used by Schloesing and Mlintz and their followers in 
ilar studies on nitrate formation. A method somewhat similar in 
nciple but different in its application was proposed by Remy in 
2. He suggested the use of special media for the quantitative 

timation of different physiological reactions; thus, making a i per 

( it solution of peptone and inoculating with equivalent quantities of 
1, he caused the decomposition of the peptone and the formation of 
imonia, and secured comparisons of the ammonifying power of 

( iVrcnt soils. In a similar manner he used special solutions for com- 

jring quantitatively the transformation accomplished by nitrifying, 

c litrifying or nitrogen-fixing bacteria. 
Remy's method has been extensively tested by Lohnis, Ehrenberg, 

I>man and others. It has been shown to possess a serious defect in 
it it deals with conditions unlike those occurring in the soil itself. 

Ir this reason more recent investigations have been carried on in 
ighed portions of soil rather than in culture solutions inoculated with 
per cent of soil as is done in Remy's method. 
RATE OF OXIDATION OF CARBON. The rate of decomposition of 

hmus or of other organic matter in the soil may be measured, as was 

c ic by Wollny, by determining the amount of carbon dioxide evolved 

i]weighed quantities of material kept under definite conditions. The 
uence of temperature, moisture, aeration, organic matter, anti- 
tics, etc., has been determined in this manner. The same method 

r y be used in studying decay, and factors influencing decay, in soils 

ii.he field. 
More recently Russell and his associates have modified the method 

i that they have determined the rate of oxidation of carbon not by 

r asuring the carbon dioxide evolved, but by estimating the amount of 
i absorbed. In either case decay is measured from the carbon 

Mdpoint. The method based on this principle should find wide 

aDlication in future soil fertility investigations. 
RATE OF OXIDATION OF NITROGEN. Another method or series of 

r thods for studying decomposition processes in the soil may be based 
e determination of nitrogen compounds formed in the breaking 

d vn of proteins. Two of the derivatives of protein, namely, ammonia 

a 1 nitrate, have been used successfully to gauge the decomposition of 
ic matter in the soil. The recent results secured by Lipman and 
-ociates demonstrate that ammonia formation from dried blood in 



314 MICROBIOLOGY OF SOIL 

weighed quantities of soil may serve as a very accurate measure < 
decay from the nitrogen standpoint. Corresponding determination 
nitrates may similarly be employed in tracing protein cleavage ar 
transformation as influenced by the various factors of season, s< 
and cultivation. 

ADDITION OF NITROGEN. At least one other bacteriological fact 
in soils should be mentioned here as deserving attention in a systemat 
study of soil fertility from the nitrogen standpoint. It is known th 
Azo-bacteria are widely distributed in arable soils, and that they a 
more prominent in some regions than they are in others. The stude 
of soil fertility finds it desirable, therefore, to study azotofication 
different soils, and employs (for this purpose) mannit solutions li 
those proposed by Beyerinck, sand cultures supplied with sugar sol 
tions like those proposed by Fischer, or weighed quantities of soil 'mix 
with sugar as suggested by Koch. 

The methods referred to above make possible thus the study 
ammonification, nitrification and azotofication under controlled cc 
ditions and permit, thereby, the measure of bacteriological factors 
soil fertility from the nitrogen standpoint. 

REACTIONS CONCERNING CALCIUM, MAGNESIUM, SULPHUR A 
PHOSPHORUS. In addition to the purely chemical methods available : 
the study of these constituents, microbiological methods have also be 
suggested. In some of his still unpublished experiments with Azc 
bacter Lipman employed solutions of mannit in distilled water, provic 
with small quantities of sterile soils which were to supply the organis 
with the essential mineral constituents. In this manner interest: 
data were secured on the availability of phosphorus compounds 
different soils; similarly, Christensen has suggested the use of Azt 
bacter for determining the lime requirements, of soils, and Butkev 
has experimented with cultures of Aspergillus niger in determining 
availability of the mineral constituents. 



(CHAPTER II 
DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 

CARBOHYDRATES 

Origin. The sugars, starches, vegetable gums and allied pectine 
ibstances, as well as the cellulose, contained in roots and other crop 
sidues add large quantities of carbohydrates to the soil. The crop 
sidues are augmented still further by green manures and animal 
anures whenever these are used. A good growth of timothy, for 
cample, may increase the content of organic matter in the surface 
>il by 250-500 kg. (500 or 1,000 pounds) per acre, and three-quarters 
this consists of carbohyrdates. In the same manner, a green ma- 
are crop, or an application of barnyard manure may add to the land 
5 much as 1,500 pounds, or even more, of carbohydrates per acre. 
hese carbohydrates contain a large proportion of cellulose. 

The Decomposition of Cellulose. Pure cellulose (page 167), 
^eHioOs^ is a rather inert substance, as exemplified by the resistance 
cotton and flax fiber to decomposition processes. It is well known, 
ivertheless, that even cellulose is in the end decomposed and resolved 
to simple compounds. Plant roots, leaves and stems, as well as the 
unks of fallen trees, gradually disintegrate and vanish. Under favor- 
Die conditions this may proceed rapidly, as is indicated by the process 
tic tanks, or in manure heaps on the one hand, and in open 
soils on the other. The disappearance of cellulose may be ac- 
.ed by (a) anaerobic organisms, (6) by aerobic organisms, (c) 
trifying bacteria, and (d) by molds. 

'he Production of Methane and Hydrogen. The decomposition 
cellulose and the formation of methane and hydrogen mixed 
th other gases was first noted by Popov in 1875. Some years 
ter Tappeiner and also Hoppe-Seyler confirmed Popov's observa- 
that nearly pure cellulose in the form of Swedish filter-paper, or 
fiber may be fermented by bacteria with the evolution of 
e, carbon dioxide and occasionally also of hydrogen. These 
315 




316 MICROBIOLOGY OF SOIL 

investigators ascribed the decomposition of cellulose to an organisrr 
found by Trecul in decomposing vegetable materials, and named b} 
him Amylobacter in 1865, because of the blue color assumed by it wher 
stained with iodine. 

Subsequent investigations by Omelianski begun in 1894 and con 
tinued through a period of years demonstrated the presence of specifi<> 
anaerobic organisms in decomposing cellulose. He described two dis 
tinct species of long, slender bacilli, assuming the clostridium form wher 
in the spore stage. Morphologically the organisms can hardly be dis 
tinguished, but physiologically they show important differences in tha 
one causes the fermentation of cellulose with the production of gase 
consisting of carbon dioxide and methane, while the gases produced b 
the other consist of carbon dioxide and hydrogen; hence the one is desig 
nated by Omelianski as the methane bacillus and the other the hydro 
gen bacillus. These organisms do not stain blue with iodine, and do no 
belong, therefore, to the butyric bacilli designated as Amylobacter b 
earlier investigators. Omelianski's investigations make it appear tha 
the butyric organisms are not capable of fermenting cellulose proper. ! 

In culture solutions containing mineral salts and nitrogen in the forn 
of ammonium compounds the decomposition of filter-paper and othe 
forms of cellulose proceeds with considerable rapidity. Calcium car 
bonate must be added to neutralize the acids formed, otherwise th 
fermentation soon comes to a standstill. In one of Omelianski's experi 
ments begun in October, 1895, and ended in November, 1896, 3.347 
g. of cellulose was decomposed by a nearly pure culture of hydroge : 
bacilli. The products consisted of 2.2402 g. fatty acids, .9722 g. carbol 
dioxide and .0138 g. of hydrogen, a total of 3.2262 g. which nearl 
accounts for all of the cellulose destroyed. The fatty acids were mad 
up of butyric and acetic acids with a slight proportion of some highe 
homologue, probably valerianic acid. 

In a similiar experiment with an apparently pure culture of th 
methane bacillus, begun in December, 1900, and ended in April, 190; 
fermentation began after an incubation period of about one month, an 
the entire volume of gas gradually evolved was 552.2 c.c. This mb 
ture consisted of 190.8 c.c. methane and 361.4 c.c. carbon dioxide. Th! 
products formed from the 2.0065 cellulose consumed include! 
1.0223 g. fatty acids, .8678 g. carbon dioxide and .1372 g. of metha 
or a total of 2.0273 g. The slight difference in weight in favor of t 






DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 317 



jrmentation products falls within the limit of error. These experi- 
ents show that about one-half of the fermentation products is 
Iseous and that the other half consists of acetic and butyric acids. 

The Oxidation of Methane, Hydrogen and Carbon Monoxide. Aside 
bm cellulose, methane may also be produced from various other carbo- 
rdrates, organic acids and proteins. Large amounts of methane are 
us contributed to the atmosphere by swamps, manure heaps and low- 
ing meadows. In a purely chemical way methane may also be set 
ce from volcanoes and mineral springs. The constant additions of 
ethane, ethane, hydrogen and carbon monoxide represent a consid- 
able amount of potential energy. It is important to know, therefore, 
let her these materials are at all utilized. 

That methane may be utilized by bacteria as a source of energy was 
1st shown by Sohngen in 1905. He isolated an organism named by 
m B. methanicus that showed itself capable of growing in inorganic 
ilutions confined over an atmosphere of methane, oxygen and nitrogen. 
jie methane gradually disappeared and there were formed considerable 
lantities of organic matter. The ability to oxidize methane has been 
limed for a number of other organisms by Sohngen and others. 

Early observations on the ability of moist soil to cause the oxidation 

hydrogen are credited to de Saussure (1838). Many years later 
892) Immendorff called attention to the same fact. It was not, 
>wever, until 1905 that the oxidation of hydrogen was shown to be a 
aecific biological process. In that year papers by Sohngen and Kaserer 
iported experiments wherein inorganic solutions confined under an 
^mosphere of hydrogen, oxygen and carbon dioxide and inoculated with 
jry small quantities of soil developed a bacterial membrane at the 
:;rface. The hydrogen was oxidized and organic matter produced at 
le expense of the energy set free. The observations just noted have 
(en confirmed by other investigators, by means of mixtures and single 
jecies of soil bacteria. Finally it should be added here that B. 
gocarbophilus previously isolated by Beijerinck and Van Delden is 
according to Kaserer, to oxidize also carbon monoxide. 
E CLEAVAGE AND FERMENTATION OF SUGARS, STARCHES AND 
. Sugars (page 163) are a very acceptable source of food and 
for soil bacteria. A culture solution containing suitable mineral 
ts and sugar ferments readily when inoculated with a small amount 
soil. When no combined nitrogen is added, Azotobacter, or B. 



318 MICROBIOLOGY OF SOIL 

(Clostridium} pasteurianus (or both), may come to the fore, The cleav- 
age 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 
number 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-buty 
alcohol are common cleavage products. 

In the case of starch, pectins and pentosans, similar conditions hole 
good. Diastatic enzymes seem to be produced by various bacteria 
as well, as molds and streptothrices. Members of the subtilis grouj 
and B. fluorescent seem to be able to transform starch into sugar with 
out difficulty. It needs hardly be added here that the vast quantitie 
of organic acids and of carbon dioxide thus formed must play an im 
portant role in the breaking down of the mineral constituents in th 
soil. 

FATS AND WAXES 

Origin and Decomposition. Plant substances contain varyin 
proportions of fats and waxy materials. In the dry matter of grasse 
and cereal straw crude fat is usually present to the extent of 1.5 t 
2.0 per cent. In hay made from clover and other legumes the propoi 
tion of crude "fat is rather more than 2 per cent. In cereal grains i 
may range up to 4 or 5 per cent while in soy beans the content c 
crude fat. is 19 per cent, in germ oil meal 22 per cent and flax see 
meal 34 per cent. 

. Under the influence of enzymes produced by molds, yeasts an 
bacteria the fatty acids occurring as glycerides are decomposed int 
glycerin and fatty acids.' The extent of fat decomposition, brougl 
about largely by molds in the opinion of some, is shown by numeroi 
experiments with peanut cake, olive press cake, cottonseed mea 
almond oil, corn meal, etc. In a number of these experiments Aspe 
gillus niger seemed to be particularly efficient in decomposing fat 
Analogous decomposition processes may occur in the soil as proved t 
the experiments of Rubner. 



DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 319 

ORGANIC ACIDS 

Source. The cleavage products of proteins include large quantities 
amino-acids. The latter are still further transformed and yield a 
riety of fatty acids. The carbohydrates being present in larger 
rantities than the proteins are still more important as a source of 
cjanic acids. Finally, the fats, gums, and higher alcohols contribute 
^ditional quantities of the latter. Among the more simple acids, 
aitic, propionic, butyric, succinic and lactic are common. The extent 
dacid production was already indicated in connection with cellulose 
composition by the methane and hydrogen bacilli. Apart from these 
c;anisms, organic acids are formed by nearly every important species 
csoil bacteria; moreover, the tissues of dead plants and animals are 
rt the sole source of organic acids in the soil. According to Stoklasa 
Editions may occasionally occur in the latter, especially when 
qnospheric oxygen is excluded, that favor the excretion by plant roots 
< appreciable quantities of acetic acid. 

Transformation and Accumulation. Salts of organic acids are 
stable as food for a wide range of soil bacteria. Azotobacter will 
r.dily make use of acetates, propionates and butyrates. A number of 
nitrifying bacteria will grow vigorously with citrates as the only 
girce of organic nutrients. The fermentation of lactates by butyric 
tjcteria has been known for a long time. The decomposition of 
mlates, succinates, tartrates and valerates may be accomplished by 
vrious species, and even simple compounds like formates may yield 
fid and energy to certain soil bacteria, among them B. methylicus 
sidied by Loew and his associates. It is evident, therefore, that 
cyanic acids are not liable to accumulate in* well-ventilated soils. 
bids, as well as bacteria, destroy them rapidly, and carbonates, 
drbon dioxide and water are the final products of the decomposition 
c non-nitrogenous organic matter. 

: Notwithstanding the ready decomposition of the more simple 
ci;anic acids in the soil, it is well known that arable soils are frequently 
ad. This acidity is largely due to the so-called "humic acids," 
tpnic compounds whose composition is not well understood. They 
|j composed, to some extent, of rather complex organic acids or of their 
z;.d salts. Continued cultivation seems to favor the accumulation of 
fee acid compounds, partly on account of the diminished supply of 
lie and of other basic materials in older soils. When these soils are 



$20 



MICROBIOLOGY OF SOIL 



limed the humic acids and acid humates are changed into neutral con 
pounds and are then subject to more rapid decomposition by mien 
organisms. According to the investigations of Blair the average ac: 
soil in Florida requires 1,500 pounds of lime (CaO) per acre to neutrali; 
the acidity to a depth of 84 mm. (9 inches). This means an acidil 
equivalent to more than one ton of hydrochloric acid per acre. ] 
peat and muck soils the acidity is equivalent to many times th 
amount of hydrochloric acid. 

% 
PROTEIN BODIES 

Amount and Quality. The protein content of farm crops th; 
leave residues in the soil is variable, but in all cases quite considerabl 
Dried corn stalks contain 5 per cent of protein, timothy hay 6 per cer 
red clover hay 12 per cent or more, alfalfa hay 15 or 16 per cent. Ev< 
wheat and rye straw may contain as much as 3 per cent of protei 
Cotton-seed meal and other oil cakes, tankage, ground fish, hair ai 
wool waste and dried blood (all used more or less extensively as sourc 
of nitrogen to crops) are made up in a large measure of prote 
compounds. 

Being derived from plant residues, from microorganic, insect ai 
animal remains, and from fertilizers and manures applied, the nitrog 
in the soil humus exists, for the most part, in the form of protein coi 
pounds. Hilgard reports the following humus and nitrogen contei 
as based on the analyses of a large number of samples of humi 
semi-arid and arid soils. 





(Humus), 
per cent 


(Nitrogen in 
humus) , 
per cent 


(Nitroger 
soil), 
per cent 


Arid uplands 


O QI 


I< . 23 


0.135 


Sub-irrigated arid soils 
Humid soils from humid and arid regions 
(California) 


1. 06 

2 4- 1 ? 


8.38 
*> 2O 


o.opg 
o. 13 = 


Humid soils from other states . 


7 .01 


3.78 


0.29- 











Taking the weight of an acre-foot of dry soil at 2,000,000 1 
(4,000,000 pounds) and multiplying the nitrogen by 6.25 (the faci 
usually employed for converting nitrogen into protein) we find t 
protein content of these soils to range from about 11,339 kg. (25,0 



DUOM POSITION OF ORGANIC MATTER IN THE SOU, 321 

ds) per acre to nearly three times as much. Similarly, the 
nois Experiment Station reports quantities of nitrogen equivalent 
3,175 to 4,989 kg. (7,000 to 11,000 pounds) per acre to a depth of 
.6 cm. (40 inches) in gray silt loams, of the lower Illinoisan glacia- 
t'n. In the brown silt loams the amount of nitrogen to the same depth 
h|isually more than 4,535 kg. (10,000 pounds) per acre; occasionally 
more than 9,071 kg. (20,000 pounds) per acre. In one instance a 
bick clay loam of the late Wisconsin glaciation is reported to have 
a|)Ut 13, 154 kg. (29,000 pounds) of nitrogen per acre, to a depth of 
.6 cm. (40 inches). This would be equivalent to more than 81,646 
(180,000 pounds) of protein; of course, not all of the nitrogen in the 
exists in the form of protein, some of it occurring as amino-com- 
pinds, and a small portion as ammonia and nitrates. Nevertheless, 
b far the greatest part of it occurs as protein compounds. 

JThe protein compounds of the soil humus must be considered from 
til standpoint of quality as well as from the standpoint of quantity. 
Its well known that fresh plant residues are attacked more readily by 
mroorganisms than older plant substances. For this reason soils 
fluently supplied with fresh organic material supply greater amounts 
of vailable food to crops than similar soils whose organic matter con- 
si 5, largely of older residues. 

Carbon-nitrogen Ratio. The decomposition of organic matter is 
rdlily influenced by the relative content of nitrogenous and non-ni- 
tr ;enous compounds. Substances of animal origin yield relatively and 
ablutely more available nitrogen in a given length of time than sub- 
ices of plant origin. The difference noted is due largely to the 






ter proportion of protein in the animal materials; in other words. 



to he narrower carbon-nitrogen ratio. On this basis Hilgard attempts 
to xplain the adequacy of the small proportion of humus in arid 
an semi-arid soils. Because of the narrower carbon-nitrogen ratio 
th humus compounds in these soils are decomposed with greater 
p ity and yield a sufficient amount of ammonia and nitrate to supply 
th needs of the crop. 

3ut when plant substances alone are considered the statement just 
e requires qualification. It is true that cotton-seed meal or linseed 
I. having a narrower carbon-nitrogen ratio, will decay more readily 
corn-meal or wheat flour. It is also true that any given plant sub- 
as it undergoes decay, will lose in proportion more carbon than 



322 MICROBIOLOGY OF SOIL 

nitrogen. Older humus has, therefore, a narrower carbon-nitroge 
ratio than humus of recent origin. The former is more resistant 1 
decay, however, than new humus. In a concrete way, on the oth< 
hand, it may be stated that fresh vegetable material of a narrow ca 
bon-nitrogen ratio will decay more rapidly than fresh vegetable materi 
of a wide carbon-nitrogen ratio. The reverse, nevertheless is true 
vegetable materials in advanced stages of decay. Under any give 
climatic conditions and in any given soil type, the carbon-nitrogt 
ratio may give important indications only as to the availability of tl 
humus nitrogen. Lawes and Gilbert, as quoted by Hall, found tl 
following carbon-nitrogen ratio in the organic matter of different soil 

Cereal roots and stubble 43 .o 

Leguminous stubble 23.0 

Dung 18.0 

Very old grass land 13.7 

Manitoba prairie soils 13 .o 

Pasture recently laid down 11.7 

Arable soil 10. i 

Clay subsoil 6.0 

Hall concludes, therefore, that humus with a wide carbon-nitrog 
ratio is more valuable than humus with a narrow carbon-nitrogen rat 
since the latter will be attacked more easily by the soil bacteria. Bro 
and Allison indicate that there might be a possibility of applying n 
terials of a wide carbon-nitrogen ratio to supply the deficiencies 
organic matter on the basis that the former may have the same 
better effect on bacterial activities such as azofication, or non-symbic 
nitrogen fixation. 

THE TRANSFORMATION OP NITROGEN COMPOUNDS 

AMMONIFICATION. Experimental Study. By ammonification 5 
meant the production of ammonia by bacteria out of protein substan s 
or their cleavage products. That ammonia production in the soi 
a biological process was first demonstrated by Miintz and Coudor i 
1893. These investigators showed that no ammonia is formed in ste 
soils. They also showed that ammonia may be produced out of nil 
genous organic matter by molds as well as by bacteria. Marchal 
only confirmed these observations, but proved that various mi< - 
organisms differ markedly in their ability to produce ammonia, 
the several species of bacteria tested by him, B. mycoides (one of 



DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 323 

imon soil bacteria) proved itself particularly efficient in the breaking 

of nitrogenous materials and the production of ammonia. 
Since the publication of these experiments a large number of investi- 
tors, both in Europe and America, have studied ammonia production 
:ulture solutions as well as in the soil itself. It has been shown that 
ler favorable conditions the breaking down of protein compounds and 
formation of ammonia may be very rapid; for instance, in some ex- 
iiments carried out by Lipman and his associates the following pro- 

T -lions of nitrogen were transformed into ammonia in the course of 

s days: 

Dried blood 16 . 74 per cent 

Concentrated tankage 56.66 per cent 

Ground fish 47 . 16 per cent 

Cotton-seed meal 4.95 per cent 

Bone meal 16 . 65 per cent 

Cow manure, solid and liquid excreta 32.60 per cent 

Cow manure, solid excreta 5 . 39 per cent 

The experiments were carried out in equal quantities of soil and with 

divalent quantities of nitrogen in the different substances. It will 

-erved that more than 56 per cent of the nitrogen in the con- 

c;t rated tankage was transformed into ammonia, whereas under the 

onditions cotton-seed meal yielded less than 5 per cent. 

Mechanism of Ammonia Production. The relatively large protein 
nlecules are readily broken into larger or smaller fragments. This 
ny be accomplished by purely chemical means, as, for instance, by 
bling with acids or alkalies, or by biological activities. Among the 
fit cleavage products albumoses and peptones are quite prominent. 
lese in turn undergo further cleavage and the various amino-acids 
a 1 their derivatives, as well as ammonia, make their appearance. In 
as the different species of bacteria are concerned, ammonia pro- 
d ion seems to depend, to a marked extent, on the ability to secrete 
P'leolytic enzymes. With the aid of such enzymes the proteins are 
n re readily hydrolyzed and further changed into amino- and hydroxy 
a is, ammonia and carbon dioxide. 

Influence of Soil and Climatic Conditions. Ammonia production in 
t soil is affected by (a) its mechanical and chemical composition; by 
U the amount and distribution of rainfall; by (c) the prevailing tem- 
p atures; by (d) fertilizer treatment; and by (e} methods of tillage and 




324 MICROBIOLOGY OY SOIL 

cropping. The mechanical composition of the soil influences the pr< 
portion of aerobic and anaerobic species, while the chemical compos 
tion, particularly that of the humus, influences the rate of multiplic 
tion and the character of the chemical transformation accomplishe 
It is well known, for example, that additions of fresh organic matt 
intensify the rate of decomposition of the soil humus, and, likewis 
ammonia production as has been already demonstrated by Breal. In 
more general way it was proved by Lipman and his associates tha 
with a constant bacterial factor, ammonia production in soils varies wi 
the chemical and mechanical composition of the latter. In some 
these experiments 100 g. portions of different soils were each mixed wi 
5 g. of dried blood, sterilized in the autoclave, cooled and inoculati 
with equal quantities of infusion from fresh soil. The followii 
amounts of ammonia nitrogen were produced in six days: 

Soil Ammonia nitrogen found 

A- 31.62 mg. 

B 68 . 29 mg. 

C 1 1 7 . 06 mg. 

D 107 . 16 mg. 

E 156-47 mg. 

With all other factors constant, chemical and mechanical differenc 
in the soil used were responsible for striking variations in ammor 
production, as indicated by the figures given above. 

The influence of temperature and moisture conditions is fully 
important as that of the chemical and mechanical composition of t 
soil. The following data secured by Lipman may be cited in tl 
connection as showing the effect of moisture : 

One-hundred-gram quantities of air-dried soil were each mixed wi 
3 g. of dried blood and varying amounts of water added. The ammoi 
formed was distilled off and determined at the end of eight da 
The amounts of ammonia nitrogen found were as follows: 

Water added Ammonia nitrogen found 

C.C 4-13 mg. 

1 C.C. 4-13 mg. 

3 c.c 5 40 mg. 

5 c.c . 10.64 mg. 

7 c.c 26 . 37 mg. 

10 c.c 49-57 mg. 

12 c.c 70. 71 mg. 

15 c.c 93.90 mg. 



^COMPOSITION OF ORGANIC MATTER IN THE SOIL 325 

It appears, therefore, that ammonia production in soils rises or falls 

the rainfall or irrigation is increased or decreased, or as the soil water 

i|more or less thoroughly conserved by proper methods of tillage. In 

same way, seasons of high temperature favor ammonification while 

sons of low temperatures discourage it. This point is well illustrated 

I; the observations of Marchal that at o to 5 only traces of ammonia 

vre formed in his culture solutions; that at 20 ammonia production 

; quite marked, and that at 30 the maximum was reached. More- 

r, apart from the seasonal variations in any one locality, there is a 

ie range in ammonia production, as we pass from the torrid to the 

t operate and from the latter to the frigid zones. 

Species and Numbers. Ammonia production is a function common 

most soil bacteria. Already in the earlier experiments of Marchal, 

enteen out of the thirty-one species tested were found capable of 

P>ducing ammonia. Prominent among these ammonifiers were B. 

oidcs, B. (Proteus') vulgaris, B. mesentericus vulgatus, B. janthinus, 

al B. subtil is. Of a considerable number of soil bacteria tested by 

C ester all but one were observed to produce ammonia. In Gage's 

e>eriments with sewage bacteria, seventeen out of twenty species 

tjted proved to be ammonifiers. Similarly, a number of species tested 

b the writer, among them B. coli, B. cholera suis, B. (Proteus) vulgaris, 

Lvibtilis, B. megaterium, etc., all produced ammonia in meat infusions. 

Amass of additional data, accumulated by different investigators, 

finish further proof that ammonia production is a common function 

o-oil bacteria. 

The more prominent ammonifiers, including members of the B. 
styiiis group and certain streptothrices, are numerically important in 
aiarable soils. Their numbers are affected, however, by the amount 
aJ'. composition of the soil humus. It has been found, for instance, 
additions of straw and of strawy manure increase markedly the 
bers of B. subtilis and of other members of the group. An increase 
in he numbers of certain ammonifiers is caused also by additions of 
lii.2 or of green manure. For example, in experiments carried out by 
Lman and his associates portions of fertile soil inoculated with B. 
woides were found to contain, a month later, 2,000,000 of bacteria per 
g. f soil. In similar soil portions that had also received additions 
ofrrass tin- number \va< 1 \\itc as 




326 MICROBIOLOGY OF SOIL 

Relative Efficiency of Different Species. In Marchal's experimen 
already referred to, the species employed showed marked differences 
their ability to produce ammonia out of egg albumin. The followir 
proportions of the protein nitrogen were converted into ammonia 
twenty days: 

B. mycoides 46 per cent B. subtilis 23 per ce 

B. (Proteus} vulgaris 36 per cent B. janthinus 23 per ce 

'B. mes enter icus vulgatus.. 29 per cent B.fluorescens putidus 22 per ce 

Sarcina lutea 27 per cent B . fluorescens liquefaciens . 16 per ce 

Furthermore, apart from the variations from species to species, diff< 
ences have been observed by Marchal and many other investigate 
between one strain and another of any single species isolated from t 
same or different soils. It must be remembered, therefore, that in t 
study of ammonification in soils and culture solutions, due considei 
tion should be given to differences in physiological efficiency as they d 
manifested by strains and species of microorganisms. 

Apart from the ammonifying bacteria already mentioned there i: 
group of organisms studied by Miiller, Pasteur, van Tieghem, Leul 
Miquel, Beyerinck and others. These are the so-called urea bacter 
capable of intensive transformation of urea and allied compounds ir 
ammonium carbonate, by means of the enzyme urease. 

NH 2 

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

NH 2 

Morphologically these organisms include spherical and rod for , 
spore-bearing and non-spore bearing species. Most of the urea bactt i 
are particularly prominent in the transformation of animal manures 
Ammonifying Efficiency. Lipman and Burgess have found mari i 
differences in the ammonifying efficiency of fifteen organisms in p e 
cultures using peptone, bat guano, sheep and goat manure, dii 
blood, tankage, cottonseed meal and fish guano. The nature of e 
soil as well as the nature of the nitrogenous material markedly mofly 
an organism's ammonifying power. B.tumescens on the whole appear jo 
have been the most efficient organism tested. Comparing these findi ;s 



ECOMPOSITION OF ORGANIC MATTER IN THE SOIL 327 

th those of Marchal the former have obtained results in soils, while 
e latter's work was with solution cultures, the application of which 

soil conditions is not always permissible. In point of fact the am- 
nnifying efficiency of organisms is greater in sandy soil and possi- 
}y in others than in solutions, as Lipman and Burgess have obtained 

ransformation of 41.98 per cent of peptone in nitrogen and 36.06 
]r cent of bat guano nitrogen into ammonia by Sarcina lutea and 

mycoides, respectively, in twelve days at temperatures between 
and 30., while Marchal obtained similar transformations in 
irty days at 30. in albumen solutions. 

It is also of interest to note that investigations with soil fungi have 
j sealed the fact that certain species are even more efficient am- 
nnifiers than B. mycoides. McLean and Wilson, Waksman, Cole- 
nn and Kopeloff have worked with organisms like Trichoderma 
:ngi which is capable of transforming more than 50 per cent of 

nitrogenous material added in such experimentation. 

NITRIFICATION. Experimental Study. The term nitrification refers 

the oxidation either of ammonia or of nitrites to nitrates. In a 
\ 3ader sense nitrification may be defined as the production of nitrates 

m decomposing organic matter. Saltpeter or niter, the terms 
i merly applied to potassium nitrate, possessed, for a long time, a 
] culiar interest because of its relation to gunpowder. Whether it be 
tie or not that gunpowder was known to the Chinese before the be- 
ining of the present era, there is no doubt that for several centuries 
i played an important part in the political and economic history of 
hrope. The large quantities of gunpowder consumed in the almost 
i essant wars created a steady demand for saltpeter that was not 
ridily met by the saltpeter refiners of India, Hungary and Poland, 
liropean nations, particularly France, were therefore thrown on their 
en resources and were forced to develop the domestic production of 
s- tpeter. The industry came under government control and experts 
ue appointed to study the so-called saltpeter plantations and the 
nditions affecting the appearance and increase of nitrates in com- 
I$t heaps and in the soil. Much knowledge was thus gained about 

fition even though it was not suspected that living organisms 
mcerned in the process, 
h the rapid development of chemistry in the latter half of the 
nth century a nearer approach was made to the understanding 



328 MICROBIOLOGY OF SOIL 

of the true character of nitrification. The observations of Cavendis 
in 1784 that potassium nitrate is formed when electric sparks are passe 
through air confined over a solution of potassium hydrate formed tb 
starting point for various theories that attempted to account for nitrat 
formation on the basis of purely chemical reactions. The formation < 
nitric acid and of its salts was thus assumed to be due to electric di: 
charges in the atmosphere, to combustion processes in nature, or to i\ 
oxidation of organic matter and of calcium, magnesium, iron and mai 
ganese compounds in the soil. Much credence was given to the latt< 
explanation because of the almost universal occurrence of nitrates 
arable soils. 

The first indication that nitrate production in the soil and in d 
caying organic matter is due to biological activities was given I 
Pasteur in 1862. A few years later Muller expressed his belief in tl 
biological origin of nitrates and nitrites in sewage and drinking wate 
It was not, however, until 1877 that the true character of nitrificatic 
was made clear. In that year Schloesing and Mtintz demonstrate 
that dilute solutions of ammonia could be changed into nitrate by beii 
passed slowly through long tubes filled with soil. The amounts 
nitrate nitrogen found in the teachings corresponded almost exact 
to the amount of ammonia nitrogen used up. When the soil in tl 
tubes was first sterilized by heating or by means of chloroform and oth 
germicides, the ammonia passed through unchanged. But when so 
sterilized by heat or chloroform were reinfected with small quantiti 
of fresh soils nitrification again proceeded in a normal manner. 

The biological nature of nitrification having been thus establish' 
numerous investigators tried to isolate the specific organisms in pu 
culture. A large amount of work in this direction was done 1 
Schloesing and Miintz, Celli and Marino-Zuco, Munro, Warington, t 
Franklands and many others. A large number of bacteria, yeasts ai 
molds were tested with negative results. Warington, who gather 
a great mass of valuable information about nitrification, almc 
succeeded in securing pure cultures of nitrifying bacteria. Final! 
Winogradski showed in 1890 not only that nitrification is caused 1 
specific bacteria, but explained also why the others failed in securi 
pure cultures. He proved that these organisms do not develop coloni 
on the ordinary gelatin and other organic media, a fact whose recc 
nition was largely responsible for his successful solution of the problei ; 



DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 329 

tie medium subsequently employed by him consisted of silica jelly 
operly supplied with inorganic nutrient salts. After him other in- 
stigators proved that agar, deprived of its soluble organic matter, 
psum and sandstone disks, filter-paper pads, etc., could be used 
ectively as solid media. 

XilroHs and Mtric Bacteria. Winogradski's investigations led to 
K conclusion, foreshadowed by the earlier work of the Franklands and 
larington, that the oxidation of ammonia proceeds in two stages, viz., 

(1) 2 NH 3 + 3O 2 = 2 HN0 2 + 2 H 2 O 

(2) 2 HNO 2 + O 2 = 2HN0 3 

The organisms oxidizing ammonia to nitrites, and designated as 
urous or nitrite bacteria, were called by Winogradski NUrosomonas 
j'd Xitrosococcits. The former include species or varieties isolated 
m soils in Europe, Asia and Africa, and the latter those isolated from 
sils in America and Australia. The organisms oxidizing nitrites to 
i rates and known as nitric or nitrate bacteria, were included by 
'inogradski in the genus Nitrobacter. 

Apart from these bacteria there is an organism, according to Kaserer, 
fat can oxidize ammonia directly to nitrate. He named it B. nitrator. 
lie reaction is illustrated by the following equation: 

NH 3 + H 2 CO 3 -f O 2 = HNO 3 + H 2 O + CH 2 O - 41 Cal. 
CH 2 O + O 2 = H 2 CO 3 + 132 Cal. 

Enough energy for the completion of the reaction is obtained by the 
(iidation of the formaldehyde (CH 2 O). Beyond the preliminary 
snouncement of Kaserer's there are no experimental data to prove 
tie existence of this organism, even though other evidence of an 
iiiirect nature may be construed to lend support to his theory. 
4t whether it be proved or not that ammonia may be oxidized 
1 nitrate by a single species, it is evident that the number of species 
(jncerned in nitrate production is relatively small. 

Relation to Environment. The conditions that affect nitrate forma- 
ts in soils may be classified under the following heads: (a) supply of 
<|ygen; (6) range of prevailing temperatures; (c) amount and dis- 
t tuition of moisture; (d) quantity of lime and of other basic materials; 

quantity of ><>lul>le mineral salts; (/) rharactcr and amount of 






330 MICROBIOLOGY OF SOIL 

organic matter; (g) presence of toxic substances; (H) physiologic} 
efficiency of the nitrifying bacteria. 

The rapid disappearance of organic matter from sandy soils is due i 
large measure to their better aeration. On the other hand, the decon 
position of vegetable and animal substances in heavy, ill-ventilated soi 
is materially retarded by the limited supply and very gradual renewal < 
oxygen. An intimate relation exists here between air and water in thj 
the latter replaces the former to a more marked extent in heavy than i 
light soils. The influence of both aeration and the range of moisture 
illustrated by an experiment of Lipman's in which equal quantities 
soil were kept in large boxes under' different moisture conditions. / 
the end of a year the following quantities of nitrate nitrogen we: 
found : 

< 6.52 per cent 14.75 per cent 18.62 per cent 22.05 per cent 22.12 per ce 

Nitrate [ 

nitrogen \ 697 mg. 823 mg. 720 mg. Trace Trace 

found [ 

In examining the figures recorded above, we find that moisture was tl 
controlling factor in the development of the nitrifying bacteria, wht 
the proportion of water in the soil was 6.52 per cent. As the amount 
water increased to 14.75 P er cen ^ there was a marked increase in tl 
amount of nitrate produced. Beyond that, however, the further i 
crease in the amount of water began to limit the supply of oxygen, ai 
the production of nitrate nitrogen with 18.62 per cent of water in t 
soil was somewhat decreased. A still further addition of water up 
22.05 per cent led, practically, to saturation, and the encouragement 
reduction rather than oxidation processes. Hence, no nitrate was ; 
lowed to accumulate in the soil. The data in question thus help 
explain why care was taken, on salt-peter plantations, to keep t 
compost heaps moist, yet not too wet. 

The influence of temperature on nitrate formation has been observ 
by many investigators. Already Schloesing and Mlintz recorded th 
at 5 nitrification is quite feeble, at 12 marked and at 37 at its be 
Other investigators have obtained substantially the same results, exce 
that the optimum has been found to be considerably lower, often t 
tween 25 and 30. Under field conditions nitrification seems to ta 
place at relatively low temperatures, as is indicated by the raj 



DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 331 

idation of ammonium salts in the Rothamsted experiments in Eng- 
nd; and the rapid decay and nitrification of clover and of other 
gume residues in the experiments at the New Jersey Experiment 
;ation. These facts have, therefore, an important bearing on the 
trogen feeding of crops in tropical, subtropical and temperate zones. 

The influence of lime and of other basic substances including the 
.rbonates of magnesium, potassium and sodium, and of the oxides of 
on is of far-reaching importance in all nitrification processes. It is 
ell known that applications of magnesian and non-magnesian lime, 
,arl or wood ashes promote nitrification in the soil and in compost 
baps, a fact that was well recognized by the ancient niter refiners. The 
i vorable action of lime is readily explained by its ability to neutralize 
ganic and mineral acids and to render, thereby, the soil reaction 
: vorable for the rapid growth of ammonifying, as well as of nitrifying 
ncteria. Furthermore, the reserve of basic material serves to neutral- 
e the acid formed by the bacteria and prevents thus the accumulation 
an undue amount of acidity. 

The role of certain mineral salts in promoting nitrification is quite 
gnificant. Small amounts of sodium chloride have been found to favor 
Itrification in the experiments of Pichard and also those of Lipman. 
he former showed also that sulphates not only promote nitrification, 
iit that different sulphates display marked variations in this respect, 
i the same manner nitrate formation was shown to be favorably 
tfected by phosphates in bone meal, Thomas slag, and acid phos- 
lates. Generally speaking, therefore, nitrifying bacteria are stimu- 
ted in their development by a proper supply of available mineral 
utrients. 

The exact relation of organic matter in the soil to the activities of 
'.trifying bacteria is but beginning to be properly understood. Earlier 
oservations made it manifest that heavy applications of animal 
^inures, or of green manure may not only retard nitrification, but may 
:tually cause the disappearance of a part or of all of the nitrate in the 
il. Subsequent experiments by Winogradski and by Winogradski 
id Omelianski showed that in pure cultures the presence of even slight 
mounts of soluble organic matter may depress or even suppress the 
evelopment of the nitrifying bacteria. It was, therefore, concluded 
y these authors that relatively small amounts of soluble organic 

ter may inhibit nitrification. These conclusions, based on the 






332 MICROBIOLOGY OF SOIL 

study of liquid cultures only, were given a very broad application b 
many writers on agricultural topics. More recent experiments mak 
it certain, however, that in the soil itself small amounts of solubl 
organic matter, e.g., dextrose, are not only harmless, but may reall 
stimulate nitrification. It was shown, likewise, that humus an 
extracts of humus may, under suitable conditions, stimulate nitrifies 
tion to a very striking extent. 

Certain substances in the soil may exert a toxic effect on nitrifyin 
bacteria. Ferrous sulphate, sulphites and sulphides may thus act ir 
juriously, as may also calcium chloride and excessive concentrations c 
sodium carbonate, sodium bicarbonate, sodium chloride, magnesiur 
sulphate, etc. Injury by ferrous compounds, as well as by organi 
acids, is not uncommon in low-lying fields and bogs; while injury fror 
excessive concentration of soluble salts may occur in the so-calle 
alkali lands. 

Finally nitrification in the soil should be considered from the stanc 
point of the organisms themselves. There is no doubt that continue 
growth under extremely favorable conditions leads to the develoj 
ment in the soil of nitrifying bacteria, possessing a very marked ph) 
siological efficiency. On the other hand, in ill-aerated, sour soils th 
environment would depress the physiological efficiency of the nitrify 
ing bacteria. Differences are thus undoubtedly established und( 
actual field conditions, as is made probable by the variable behavk 
of soils from different sources when used as inoculating material i 
recently reclaimed or peat swamp lands. 

Accumulation and Disappearance of Nitrates. As shown above, tli 
rate of formation of nitrates in the soil is dependent upon moistun 
temperature and aeration, as well as on the presence of organic matU 
and basic substances. On the other hand, the accumulation of nitrat( 
depends, under any given conditions, largely on the character of th 
growing crop. Observations on the rain gauges at Rothamsted showe 
an average annual loss 14 kg. (31.4 pounds) of nitric nitrogen per aci 
in the drainage water from uncropped soil. In one of King's exper 
ments, land that had been fallowed contained 137 kg. (303.24 pounds 
of nitric nitrogen per acre, to a depth of 4 feet. Adjoining croppe 
land contained only 26 kg. (57.56 pounds) of nitric nitrogen per aci 
to the same depth. Stewart and Greaves found in limestone soil i 
Utah 64 kg. (142 pounds) of nitric nitrogen per acre, under corr 



\1POSITION OF ORGANIC MATTER IN TH K SOIL 333 

pounds under potatoes, and only 12 kg. (27 pounds) under alfalfa, 
ider the same conditions fallow land contained 74 kg. (165 pounds) 
nitric nitrogen per acre. The smaller amount of nitric nitrogen found 
der alfalfa bears out the observations already made by a number of 
tier investigators that the accumulation of nitrates under legumes is 
iialler than it is under non-legumes. While several explanations have 
offered to account for this fact, it is generally agreed that legumes 
similate nitrate nitrogen more rapidly than non-legumes. Unusual 
cumstances may favor, at times, the accumulation of quantities of 
;rate large enough to destroy all vegetation. It is reported, for 
stance, by Headden that he has found in limited areas in Colorado as 
ach as 90,718.5 kg. (100 tons) of nitrate per acre foot of soil. 
The amount of nitrate nitrogen in the soil is influenced by the grow- 
* crop not alone because of the nitrogen absorbed by the latter, but 
1 cause of the moisture relations as affected by growing plants. It is 
ite apparent that a large crop dries out the soil more rapidly than a 
siall crop. When the soil moisture is sufficiently depleted, nitrifica- 
n stops and the further accumulation of nitrates becomes impossible, 
die their disappearance is hastened by the constant demands of the 
( >p. The disappearance of soil nitrates is, likewise, hastened by the 
l.ching action of rain and by certain species of bacteria that transform 
i?m into other nitrogen compounds. 

DENITRIFICATION. Experimental Study. Denitrification may be 
(fined as the reduction of nitrates by bacteria, involving the evolu- 
i>n of nitrogen gas or of nitrogen oxides. In a more general way, 
(nitrification has been defined as the partial or complete reduction of 
i rates by bacteria. The term direct denitrification has been sug- 
$5ted for complete reduction, and indirect for the partial reduction 
t nitrites or ammonia. The term denitrification should not be em- 
pyed to designate losses of nitrogen gas due to the oxidation of 
nmonia, or to the disappearance of nitrates following their conversion 
i o proteins by microorganisms. 

i The reduction of nitrates in the presence of fermenting organic 
filter was noted by Kuhlmann as early as 1846. The same fact was 
r:orded many years later by Froehde and by Angus Smith. In 1868 
Sboenbein expressed the belief that nitrates may be reduced to nitrites 
1 fungi. For more than a decade after that, data were rapidly accu- 
nlating in support of Schoenbein's contention, until in 1882 Gayon 






334 MICROBIOLOGY OF SOIL 

and Dupetit made it certain that nitrate reduction with the evolutioi 
of nitrogen gas may be caused by a "ferment." Finally, in 1886, th> 
same investigators described two organisms, B . denitriilcans a, and B 
denitrificans 0, capable of completely reducing nitrates. Subsequent! 1 
the studies of Giltay and Aberson, Burri and Stutzer, Severin, vai 
Iterson, Jensen, Beyerinck and of many others not only greatly in 
creased the number of known denitrifying bacteria, but added much t 
our knowledge concerning the development and activities of thes 
organisms. It has been shown that a very large number of specie 
can reduce nitrates to nitrites and ammonia; moreover, a considerabl 
number of organisms are already known that can cause the complet 
destruction of nitrates with the evolution of nitrogen gas or nitrogei 
oxides. The following reactions illustrate diagrammatically the corc| 
plete or partial reduction of nitrates. 

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

4 HN0 2 = 2H 2 + 2 N 2 + 3 2 

In the soil, manure or other culture media the denitrifying bacteri 
which are, for the most part, aerobic develop also under anaerobi 
conditions and transfer the oxygen of nitrates and nitrites to carbo 
compounds. This is illustrated by the equations suggested by va 
Iterson: 

5C + 4KNO 3 + 2H 2 O = 4 KH CO 3 + 2N 2 + C0 2 
3 C + 4KNO 2 + H 2 O = 2 KH CO 3 + K 2 CO 3 + 2 N 2 

When nitrates are reduced to nitrites in the presence of amim 
compounds, or even of ammonium compounds, elementary nitrogei 
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 
NH 4 C1 + KNO 2 = KC1 + 2 H 2 O + N 2 

An organism has been described by van Iterson that can decompo; 
nitrates in the presence of cellulose : 

5C 6 H 10 5 + 2 4 KNO 3 = 2 4 KHCO 3 + 6CO 2 + i 2 N 2 + i 3 H 2 

Still more interesting is Thiobacillus denitrifieans described tj 
Beyerinck as capable of reducing nitrates in inorganic media. Tl 
nitrate oxygen is used to oxidize elementary sulphur: 

6KN0 3 + 58 + 2 CaCO 3 = 3K 2 SO 4 + 2CaSO 4 






MPOSITION OF ORGANIC MATTER IN THE SOIL 335 

The Actinomyces reduce nitrates to nitrites, but they do not cause 
ay loss of free nitrogen, for the nitrites are utilized by the organisms, 
ad complete denitrification does not take place. Thus these organ- 
i:is may prevent the leaching out of nitrates and nitrites in the soil, 
c the active denitrification by other organisms. 

Relation to Environment. Nitrate reduction is favored by insuffi- 
cnt aeration, as well as by an abundance of readily decomposable 
cyanic matter. In fine-grained, compact soils nitrate formation and 
rrate reduction may alternate, depending upon the more or less 
cnplete replacement of soil air by water. Similarly, in soils receiving 
e:essive amounts of animal manure denitrifying bacteria may cause 
tj reduction of nitrates. In greenhouse soils excessive moisture, as 
\11 as excessive amounts of organic matter, may be present and may 
pvent the accumulation of nitrates. It has also been shown by 
Iklevski that, contrary to opinions previously held, denitrification 
r y occur in manure heaps. In the better aerated surface portion of 
i nure heaps conditions are favorable for the oxidation of ammonia 
t nitrites and nitrates. The nitrous acid may combine with ammonia 
tform ammonium nitrite, the latter decomposing, spontaneously, into 
^ter and nitrogen gas. It is very likely, also, that the nitrites and 
r rates are reduced by the denitrifying bacteria in manure. On the 
cier hand, in manure kept moist under the feet of cattle nitrite and 
rrate formation is prevented and losses by denitrification are not 
1 ely to occur. 

The economic significance of denitrification was overestimated at 

ce time, on account, largely, of the assertion of Wagner in 1895 tnat 

ijall soils receiving applications of horse manure, the nitrates in the 

si itself as well as those added in commerical fertilizers are almost 

tain to be destroyed by denitrification. Subsequent experiments 

many investigators demonstrated that under field conditions, deni- 

cation is a factor of slight moment; however, in the greenhouse, 
i the manure heap (under certain conditions) and in market gardening 
manure is used at the rate of 45,359 kg. to 54,431 kg. (50 to 60 

) per acre, the danger of denitrification is real. 

ANALYTICAL AND SYNTHETICAL REACTIONS 

COUNT OF BACTERIAL SUBSTANCE IN THE SOIL. Various decom- 
dtion processes in the organic matter of the soil may be designated 



MICROBIOLOGY OF SOIL 

as analytical in that protein, carbohydrates and fats are split into mor 
simple compounds. At the same time, the microorganisms concerne 
in the decomposition processes multiply very rapidly and fashion th 
complex compounds of their cell-substance out of the simple cleavag 
products in their medium. In other words, analytical and synthetic; 
reactions proceed hand in hand in the soil. 

While it is not definitely known how large a proportion of the so 
humus consists of the dead and living cells of microorganisms ther 
is a mass of indirect evidence to show that these cells form a very cor 
siderable proportion of the total quantity of organic substances in th 
soil. For instance, it has been demonstrated that a large proportion c 
the dry matter of solid animal faeces may consist of bacterial cells. A 
various times and by different investigators the proportion of bacterh 
substance has been estimated at from 5 to 20 per cent or more of th 
total- dry weight of faeces. A heavy application of barnyard manui 
may introduce, therefore, several hundred pounds of bacterial cells pc 
acre of soil. Moreover, because of the extensive changes in the so 
humus itself, as is evidenced by the rapid formation of nitrates, lar 
masses of bacterial substances are constantly being formed and di; 
integrated. 

AVAILABILITY or BACTERIAL MATTER. Substances of microorgan 
origin are decomposed more or less rapidly, according to their con 
position. The extent of transformation under favorable conditions 
indicated by an experiment performed by Beyerinck and van Deldei 
in which 50 per cent of the nitrogen in Azobacter cells was transforme 
into nitrate in seven weeks. On the other hand, the humus of peat an 
muck soils, or that of worn-out soils, may contain microorganic residu< 
of so inert a character as to yield but little available nitrogen 1 
crops. 

TRANSFORMATION OF PEPTONE, AMMONIA AND NITRATE NITROGEI 
The cleavage of protein compounds into peptones, amino-acids an 
ammonia, and the oxidation of the latter into nitrites and nitrates, ma 
be properly included among analytical reactions. It should not t 
forgotten, however, that in the accompanying synthetical reactions tf 
compounds just mentioned may be transformed back into compkj 
proteins. It happens, thus, that large quantities of the availab| 
nitrogen compounds may be withdrawn from circulation by mien 
organisms that use these as building material. Under extreme coi 




(IMPOSITION OF ORGANIC MATTER IN THE SOIL 337 



icroorganisms may become serious competitors of 
I jits for available nitrogen food. 

Manure stored in heaps not infrequently deteriorates in quality, 
t en when losses by leaching are excluded. This deterioration is largely 
c e to the change of the water-soluble ammonia and amino-compounds 
i .0 insoluble protein substances. While the extent of the change into 
I Dtein compounds is variable it may range from less than a tenth of the 
vter soluble material to more than three-quarters or four-fifths of it. 
j!50 in the soil the same processes take place, but not so intensively. A 
1 ge number of species of molds and bacteria have been isolated and 
1 as to their ability to transform ammonia, amino- and nitrate 
i rogen into protein compounds. Among the more recent investi- 
^tions in this field those of Lemmermann and his associates testify that 
i three weeks 5 to 6 per cent of the nitrate added to the soil was changed 
i o protein. In the presence of barnyard manure the proportion 
t nsformed was increased to 15 per cent. In the case of ammonium 
c npounds the transformation may be even more far-reaching, amount- 
ij, at times, to more than 25 to 30 per cent of the material originally 
I :sent. Generally speaking, molds will assimilate ammonia nitrogen 
nre readily while bacteria and algae will assimilate nitrate nitrogen 
1 preference. However, the preference of molds for ammonia nitrogen 
i often more apparent than real, because of the rapid formation of 
i (1 residues in culture media rich in certain ammonium compounds. 
hrilarly, some species of bacteria will assimilate ammonia nitrogen 
i preference to nitrate nitrogen. 



CHAPTER III 
FIXATION OF ATMOSPHERIC NITROGEN 

THE SOURCE OF NITROGEN IN SOILS 



:>rogre 



EARLY THEORIES. When chemistry had made sufficient progre 
to allow the analysis of soils and plants it was recognized that nitrog 
is always present in both. It was also recognized that the soil nitrog< 
is almost wholly confined to the surface portion and is evidently 
atmospheric origin, since the unweathered, underlying rock is devo 
of this constituent. The vast accumulations of nitrogen, known 
exist in all arable soils, were ascribed, therefore, to the residues of mai 
generations of plants; and the assumption seemed to be justified th 
the atmosphere, 79 per cent of whose bulk consists of nitrogen gas, 
"the direct source of this element to plants. It was not long, howevi 
before plant physiologists demonstrated experimentally that nitrog 
gas as such could not directly serve as food for plants. There th 
arose one of the most interesting and, for a long time, one of the m( 
puzzling problems in agricultural research. Among the earlier i 
vestigators de Saussure believed, at the beginning of the nineteen 
century, that nitrogen is taken up from the soil in combined for 
Liebig in 1840 advanced his well-known " mineral theory" accord! 
to which plants secured their nitrogen from the air, in the form 
ammonia. He assumed, thus, that plants cannot use elements 
nitrogen, and that the supply of atmospheric nitrogen in the form 
ammonia was great enough to meet the needs of growing vegetatii 
The latter view was not accepted by Lawes and Gilbert of the Ro 
amsted Station in England. By a series of elaborate and carefu 
controlled experiments they demonstrated in 1858 that nitrogen in 
elementary form cannot be used by plants. They further demonstra 
that the amount of combined nitrogen brought down in the form 
ammonia, nitrites and nitrates, by atmospheric precipitation was 1 
slight when compared with the quantities annually removed by cro 
Hence the problem as to the source and maintenance of combiijl 
nitrogen in the soil seemed to be more puzzling than ever. 

338 



FIXATION OF ATMOSPHERIC NITROGEN 339 

MICAL AND BIOLOGICAL RELATIONS. The second and third 
rters of the nineteenth century saw the birth of a number of theories 
ling with this problem. It was suggested that nitrogen compounds 
be formed in the soil by the oxidation of nitrogen to nitric acid, 
pounds of iron, manganese and lime were supposed in some way 
tcmake such oxidation changes possible. It was likewise suggested 
tU nascent hydrogen may be generated in the decomposition of organic 
ntter in the soil, and reacting with elementary nitrogen, may give 
to ammonia. The various hypotheses were not supported by 
erimental proof; moreover, the situation was complicated by the 
wledge, based on empirical observations, that crops of the legume 
ily seemed to be more or less independent of the supply of combined 
ogen in the soil. Indeed, clovers and other legumes had, appar- 
ly, the ability to increase the content of combined nitrogen in the 
as was indicated by the experiments of Boussingault and of Lawes 
[ Gilbert. Finally, the mystery was solved by the investigations 
o Berthelot and Hellriegel and Wilfarth who furnished the proof that 
ebentary nitrogen may be utilized by plants when certain biological 
rations are met. These relations involve the presence and activities 
o-lnicroorganisms that by themselves, or in conjunction with higher 
pnts, make available to growing vegetation the great store of 
a lospheric nitrogen. 

NON-SYMBIOTIC FIXATION OF NITROGEN 

HISTORICAL. Non-symbiotic nitrogen fixation, or Azofication, has 
alhady been defined as the production of nitrogen compounds out of 
aiiospheric nitrogen by bacteria independently of higher plants. The 
p.'t played by bacteria in this process was not recognized until 1885, 
wj>n Berthelot published some of his data on the accumulation of com- 
bisd nitrogen in uncropped soils. His results seemed to explain a 
n nber of scattered observations, made since the middle of the century, 
oijthe apparent increase of the nitrogen content of cultivated soils. 

While Berthelot's experiments proved that the nitrogen gains 
ocjurred only in unsterilized soils and were, therefore, due to micro- 
oinnisms, it remained for Winogradski to demonstrate, in 1893, that 
ij formation of nitrogen compounds by certain types of bacteria 
nv be accomplished in culture media nearly or quite devoid of com- 




340 



MICROBIOLOGY OF SOIL 



bined nitrogen. Soon after that he succeeded in isolating his organisn 
in pure culture, and described them as anerobic bacilli allied to tho 
of the butyric group. In 1901 our knowledge of Azobacteria w; 
enriched by Beyerinck's discovery of a group of large, obligate aerob 
bacteria that he designated as Azotobacter. Since that date it has bet 
found that the ability to fix atmospheric nitrogen is possessed also 1 
certain molds and by various species of bacteria. However, this abili 
is not only extremely variable, but is also very feeble as compar 
with that of the members of the two groups described by Winograds 
and Beyerinck. These two groups may, therefore, be designated 
including the nitrogen-fixing bacteria par excellence. 

ANAEROBIC SPECIES. The species isolated by Winogradski \i 
named by him B. (Clostridium) pasteurianus (Fig. 119). It was found 




f I 




FIG. 1 19. B. (Closlridium) pasteurianus, a non-symbiotic nitrogen-fixing organi: 
(After Winogradski from Lipman.) 

grow readily under anaerobic conditions in culture solutions conta - 
ing dextrose and the necessary mineral salts, but no combined nitrog . 
The products of growth included protein, butyric and acetic ac 
carbon dioxide and hydrogen. In the presence of other bacteria 
(Clostridium) pasteurianus was found to develop also under aerc 
conditions. Subsequently studies by Winogradski and other inve 
gators showed that B. (Clostridium) pasteurianus, and varieties of 
species are very widely distributed in cultivated soils. More recei v 
Bredeman made a thorough and extended study of anaerobic ^ 
bacteria and demonstrated their almost invariable presence in a IE 
number of soil samples from Europe, Asia and America. In his opir i 
they correspond more or less closely to B. amylobacter described m 
years before by van Tieghem. 

AEROBIC SPECIES. A more or less pronounced power to fix atn 



FIXATION OF ATMOSPHERIC NITROGEN 341 

peric nitrogen is apparently possessed by a considerable number of 
aobic species. Lipman has demonstrated the fixation of small 
a ounts of nitrogen by Ps. pyocyanea and Lohnis secured similar results 
vh Bad. pneumonia, B. lactis viscosus, B. radiobacter and B. 
pidigiosus. Gottheil has detected fixation by B. ruminatus and B. 
siplex; Pillai has described a nitrogen-fixing aerobic bacillus, B. 
nlabarensis; Westermann studied a similar organism that he named B. 
diicus; while Beyerinck and van Delden observed, some years earlier, 
t-t. certain strains of B. mesentericus could fix relatively large amounts 
(nitrogen. Similarly Ps. radicicola has been found to possess a slight, 
b: nevertheless an appreciable power to fix elementary nitrogen in 
c ture solutions or in the soil. 




FIG. 1 20. Azotobacter vinelandi, a non-symbiotic nitrogen-fixing organism. 
{After Lipman.) 

'But while nitrogen fixation among aerobic soil bacteria is not as 
u ommon as was at one time supposed, this function is so feeble and 
M -.triable in most instances, as to be of negative, or, at best, of doubt- 
f i economic significance. On the other hand, the aerobic, Azotobacter, 
ti t described by Beyerinck in 1901, may be regarded not only as pos- 
*t ing a very pronounced ability to fix atmospheric nitrogen, but as 
P nng a r61e of some moment in maintaining the supply of combined 
n ogen in the soil. 

To the two species of Azotobacter, A. chroococcum and A. agilis 
d:ribed by Beyerinck and van Delden, Lipman, added A. vinelandii 



342 MICROBIOLOGY OF SOIL 

(Fig. 1 20), A. beyerincki and A . woodstownii, andLohnis and Westermani 
A . vitreum. Of these species A . chroococcum and A . beyerincki are mo: 
common and are widely distributed in cultivated soils of Europe an 
America, and probably also of the other continents. They are absei 
in acid soils deficient in humus, and most common in limestone regioi 
and in irrigated soils rich in mineral salts. Their food requirements a 
covered by solutions containing potassium phosphate, magnesiu 
sulphate, calcium chloride and ferric sulphate, and some organ 
nutrient, such as dextrose, saccharose, xylose, mannit, acetate, pr 
pionate, butyrate, malate, ethyl alcohol, etc. An alkaline or neutr 
reaction and the presence of salts of iron are essential for the vigoro 
development of Azotobacter, while humates have been shown 1 
Krzemieniewski to exert a stimulating influence on the growth of the 
organisms, even though not acting directly as a source of food ai 
energy. As shown by Lipman and others, Azotobacter may gain ; 
increased power of fixing atmospheric nitrogen in the presence of oth 
organisms. It is resistant to drying, notwithstanding the fact that 
produces no spores, and has been successfully isolated from soil samp) 
that had been kept in a dry state for several years. For some reas 
it may be detected in the soil most readily in the fall and winl 
months. 

As to the nitrogen-fixation by fungi, it has been shown elsewhc 
that the evidence is, if anything, of a negative character. Soi 
algae are able to fix atmospheric nitrogen, especially those that li 
symbiotically with azotobacter. 

ENERGY RELATIONS. In the fixation of nitrogen by bacteria t 
necessary energy for the process is furnished by the carbohydrat 
organic acids, alcohols or other organic nutrients employed in 1 
culture media. Since any given quantity of organic nutrient posses 
a definite amount of potential energy the fixation of nitrogen is nee 
sarily limited by the supply of such potential energy. This limitat: 
was already recognized by Winogradski in his experiments with 
(Clostridium} pasteurianus. For every gram of dextrose used up th 
was produced, on the average, 2 to 3 mg. of combined nitrogen. In 
experiments of Bredeman with B. amylobacter, and of Pringsheim w i 
"Clostridium americanum" the amounts fixed were, at times, c 
siderably larger. On the whole, however, it has been proved b}i 
number of investigators that Azotobacter can fix much larger quanti^ 



FIXATION OF ATMOSPHERIC NITROGEN 343 

c! nitrogen than the anaerobic bacilli. The extended investigations 
(iLipman showed that A . vinelandii has the ability to fix more nitrogen 
rjr unit of organic nutrient consumed than either A. chroococcum or 

beverincki. Under favorable conditions A. vinelandii may at times 
f 15 or even 20 mg. of nitrogen per g. of mannit used up. Krze- 
rieniewski found in experiments with A. chroococcum that additions 
( humates to the culture solutions increased the nitrogen fixed from a 
riximum of 2.4 mg. to a maximum of 14.9 mg. 

The practical bearing of the foregoing data lies in the fact that the 
fation of nitrogen in cultivated soils is limited, among other things, by 
(3 energy available, that is, by the quantity of readily decomposable 
qanic residues. An indication as to the extent of these is given by the 
nount of humus present; nevertheless, this must remain an indication 
nrely, for most of the humus is too inert to serve as a source of energy 
i Azotobacter. From the data at present available different investi- 
jtors have estimated the quantity of nitrogen fixed by Azotobacter 
i\ 6.8 kg. to 1 8 kg. (15 to 40 pounds) per acre, per annum. Assuming 
ij/orable conditions for fixation, so that 500 g. (i pound) of nitrogen 
(iuld be fixed for every 125 g.(ioo pounds) of carbohydrate consumed, 
ilwould still take an equivalent of 680 kg. to 1,814 kg. (1,500 to 4,000 
Junds) of sugar to produce this quantity of combined nitrogen. It may 

! noted in this connection that Azotobacter have been demonstrated 
live in symbiosis with algae, obtaining thereby the necessary energy 
1- their activities. This may explain, perhaps, the remarkable facts 
< served by Headden in Colorado, relating to the accumulation of such 
ormous quantities of nitrate in the soil, as to destroy all vegetation. 
me instances the nitrates were found to be present to the extent of 
8 kg. (100 tons), or more (per acre), to a depth of a few inches. If 
accumulation of combined nitrogen was due to Azotobacter, as is 
iiimed by Headden, and the bacterial residues oxidized by nitrifying 
ia to nitrates, it is difficult to account for the source of the 1,000 
tons of carbohydrates necessarily used up in the process of 
n, unless it could be proved that the energy was furnished by 

SYMBIOTIC FIXATION 






[ISTORICAL. Empirical observations extending well back into 
:ient agriculture have led to the recognition of the soil-enriching 



344 MICROBIOLOGY OF SOIL 

qualities 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 othei 
hand, he points out that luzerne (alfalfa), lupins and vetches improve 
the land and act as manure. He points out, also, that it was tht 
practice of Roman farmers to plow under lupines in order to enrich th< 
soil. In the centuries following the fall of Rome the use of legumes foii 
soil improvement persisted to some extent in Italy, France and othej 
countries; yet the practice was not followed consistently and the fer 
tility of European soils was declining for lack of available nitrogen 
and, to a large extent, also of phosphoric acid. The more general intro 
duction of clover into Germany and England in the eighteenth centun 
helped to restore the fertility of many farms, and led, ultimately, to th 
recognition of the peculiar place held by legumes in the maintenance 
of soil fertility. But while practical farmers knew of the soil-enrichinj 
power of legumes, and while they retained their belief in it even whei 
it seemed contrary to scientific authority, they did not know the secre 
of this power. It remained for Hellriegel and Wilfarth to demonstrat 
in 1886, and more fully in 1888, that this power, already hinted at b 1 
the investigations of others, is the resultant of the combined activitie 
of the plants and of bacteria that enter their roots, and produce ther 
the well-known nodules or tubercles. They showed in no uncertaii 
manner that legumes can improve the soil only in so far as they ad< 
nitrogen to it with the aid of the bacteria in the tubercles; in othe 
words, legumes were shown to enter into a symbiotic relationship wit! 
certain bacteria and to acquire, thereby, the ability to fix atmospheri 
nitrogen. 

The presence of tubercles on the roots of leguminous plants was firs 
recorded by Malpighi in 1687. He regarded them as root galls. Th 
botanists who studied them in the first half of the nineteenth centur 
classified them as modifications of normal roots or as pathologies 
processes. In 1866 the Russian botanist Woronin found that th 
tubercles were filled with minute bodies resembling bacteria and cor 
eluded that they were pathological outgrowths. Some years late 
Frank, in 1879, not on ty showed that tubercles are almost invariablj 
present on the roots of legumes, but that their formation may be prt 
vented by sterilizing the soil. Frank was thus in possession of fact 
that might have revealed to him the true nature of the root-tubercle; 



FIXATION OF ATMOSPHERIC NITROGEN 345 

Iwever, he later modified his belief in the origin of tubercles as due 
t outside infection, and accepted the interpretation of his pupil 
lunchhorst who claimed that the bacteria-like bodies in the tubercles 
vre merely reserve food materials. Because of their resemblance to 
t:teria Brunchhorst named them bacteroids. 

The studies of Marshall Ward, published in 1887, proved not merely 
ttt tubercle formation is due to outside infection, but that such infec- 
tn may be brought about at will by placing the roots of young plants 
i contact with pieces of old tubercles. Hellriegel in his preliminary 
c nmunication of 1886 also showed that outside infection is necessary 



the production of tubercles, and called attention to the true func- 



k >:, 
< V *** 





- 1. Ps, radicicola. i, From Melilotus alba; 2 and 3, from Medicago saliva. 
4, from Vicia mllosa. (After Harrison and Barlow from Lipman.) 

t T of the latter as laboratories wherein nitrogen compounds are 
r nufactured out of elementary nitrogen. The true worth of Hell- 
r rel's investigations was brought out more clearly in another paper 
t ,t he published jointly with Wilfarth in 1888. The authors showed 
t .1: in sterilized soils legumes behaved precisely like non-legumes, and 

ultimately of nitrogen hunger when not provided with nitrates or 

suitable nitrogen compounds. On the other hand, when the 

s rilized soil was later infected with a few drops of leachings from fresh 

sjl that had supported a normal growth of legumes, the starving plants 

i red and grew vigorously. Under the same conditions non- 
1'umes did not recover. The recovery of the starving legumes was 
t<> be coincident with the formation of tubercles. 



346 



MICROBIOLOGY OF SOIL 



Hellriegel and Wilfarth's studies were soon confirmed by the inve< 
tigations of others. Wigand showed in 1887 that the tubercles cor 
tained within them were true bacteria. In the following year Beyerinc 
reported the successful isolation of these bacteria on artificial medi; 
and named the organism B. radicicola (Fig. 121). Prazmowski als 
isolated pure cultures of Ps. radicicola, and followed the entrance < 
the organisms into the root hairs of young plants, their passage throug 
the cell-walls, and their transformation into bacteroids. These faci 
were all confirmed by other investigators, and it was further shown b 
Schloesing and Laurent that properly inoculated legumes not only ca 
grow in soils devoid of combined nitrogen, but that when growing i 
such soils in a confined atmosphere they decrease the quantity i 
nitrogen gas surrounding them by transforming it into nitrogen con 
pounds. It was, therefore, made clear by these investigations, and 1 




FIG. 122. Sections through root tubercles, i, Cell from tubercle of 
sativum, showing bacterial filament; 2 and 3, cells with bacterial filaments 
tubercle of Trifolium pannonicum. (After Stefan from Lipman.) 

others not mentioned here for lack of space, that the belief of practic 
farmers in the soil enriching qualities of legumes was amply justifie 
It was shown, further, that the later experiments of Boussingault, 
well as those of Lawes, Gilbert and Pugh failed to solve the proble 
because these investigators treated their soil so as to prevent t 
survival and subsequent entrance of Ps. radicicola, and deprived t 
leguminous plants of the ability to utilize atmospheric nitrogen. 

MODES or ENTRANCE AND DEVELOPMENT. Tubercle bacteria cc 
sisting of small motile rods usually enter the legumes by way of the ro< 
hairs. For this reason young tubercles, with but few exceptions, 
found pn young roots. The organisms multiply at the point of infecti 
and penetrate into adjacent plant-tissue by means of a hypha-li 



FIXATION OF ATMOSPHERIC NITROGEN 347 

ollow thread or tube that seems to consist of a gelatinous material 
? ig. 122). The tubes branch out as they pass from cell to cell and carry 
ic invading organisms with them. The bacteria which may be readily 
etected within the tubes and cells are the involution forms of Ps. 
idicicola and assume various irregular shapes. They are designated 
5 bacteroids. Stefan has suggested that bacteroids may be produced 
ithin the tubes and, possibly, from the buds or swellings that appear 
i the tubes. While still young, the bacteroids are capable of dividing, 
at as they grow they swell up and finally degenerate. 

RKSISTANCE, IMMUNITY AND PHYSIOLOGICAL EFFICIENCY. The 
vasion of legumes by Ps. radicicola and the acquisition by the plant, 
Kinks to this invasion, of the power to fix elementary nitrogen are cited 

> a case of symbiosis. However, some writers would regard the pres- 
ce of Ps. radicicola in legume roots as a case of parasitism. According 

> them symbiosis presupposes the living together of two organisms 
ith resulting benefit to both. In the present instance, however, 
mditions may arise when the host plant is injured, rather than bene- 
ted; and similarly, conditions may arise when the invading bacteria 
-e suppressed by the plants. Making due allowance for the ob- 
:ctions raised we still find, nevertheless, that in the broad relation of 
ic two groups of organisms there is an apparent benefit to both plants 
id bacteria. The former gain an adequate supply of nitrogen and 

latter a supply of carbohydrates and of mineral salts. 
A more detailed study of this relation shows that the plants resist 
ic entrance of bacteria. When an abundance of available nitrogen 
impounds is supplied tubercle formation may be largely or wholly 
ippressed. In that case the plants secure their nitrogen from the soil 
id are not only independent of the bacteria, but are strong enough to 
sist their entrance. It is further claimed by Hiltner that tubercle 
acteria differ in their virulence, and that the more virulent the organ- 
rns, the more readily will they penetrate the root tissue. Moreover, 
E believes that when a plant is invaded by brganisms of any degree of 
ftrulence, the host plant becomes immune to a large extent and can keep 
ut all but the most virulent bacteria. The use of the term virulence, 
t this connection, has been objected to, since it is borrowed from 
limal pathology and is likely to be misleading. It is better to employ 
e term physiological efficiency as implying not only a more pro- 
ced ability to enter the plant roots, but also to fix atmospheric 



MICROBIOLOGY OF SOIL 

nitrogen. It is conceivable that strains of Ps. radicicola may be de 
veloped 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 fixatior 
of nitrogen is accomplished by the bacteria within the tubercles. Th< 
claim, at one time, advanced by Stoklasa, that the fixation is accom 
plished by the plants themselves with the aid of enzymes produced bj 
the bacteria in their roots, has been disproved. It is known that th< 
period of active nitrogen assimilation by the plants coincides with th< 
appearance of the bacteroids in the tubercles, and it is supposed tha 
the microorganisms fashion nitrogen compounds out of atmospherii 
nitrogen by using the carbohydrates and organic acids in the plan 
juices as a source of energy. The plants then seem to utilize the solubli 
nitrogen compounds that pass out of the bacterial cells. It is furthe 
supposed that bacteroid formation is an attempt on the part of thi 
microorganisms to adjust themselves to the drain caused by th> 
activities of the host plant. 

VARIATIONS AND SPECIALIZATION. Apparent differences in bacteri; 
from different legumes were noted by Hellriegel. Some of his experi 
ments indicated that bacteria from clovers could not produce tubercle 
on lupines and serradella. Analogous differences were found fr 
Nobbe and his associates, nevertheless they were finally led to conclud 
that the root invasion of legumes is caused by a single species. How 
ever, continued association with any particular legume accomplishet 
in the end a certain modification, or specialization, as it were, of thi 
microorganisms, and they were then no longer able to invade the root 
of other legumes. Later, Hiltner and Stormer have been led t 
modify this view and have arranged the tubercle bacteria in tw 
groups, possessing, according to them, well-defined morphological an* 
physiological differences. One of these groups is included under th 
species " Rhizobium radicicola" and the other under " Rhizobiw 
beyerinckii" The former comprises the organisms from lupines, serra 
della and soy beans while the latter comprises all of the others. 

RELATION TO ENVIRONMENT. Nitrogen fixation by leguminou 
vegetation is readily influenced by soil conditions, particularly th 
supply of lime and of other basic substances; the supply of organi 
matter and the aeration of the soil. As to the first of these it is we 



FIXATION OF ATMOSPHERIC NITROGEN 



349 



iown that all legumes, with the exception of lupines and serradella, 
e stimulated in their growth by generous applications of lime. 




FIG. 123. These two pea plants were grown in clean quartz sand to which had 

added small quantities of all the necessary elements of plant food except 

\en. The conditions were exactly identical except that plant A was without 

)t nodules (see Fig. 124) and plant B had numerous nodules well developed (see 

- 125). (Mich. Exp. Station.) 



top dressing of lawns with lime, marl or wood ashes encourages 
appearance of white clover; an adequate supply of lime makes 



350 



MICROBIOLOGY OF SOIL 



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 direct action on the plants 
as well as on the bacteria in the soil. Similarly, the tubercle bacterig 
are favorably affected in their survival and multiplication by ar 
abundant supply of organic matter. On the other hand, acid soils 01 
those deficient in humus and inadequately aerated are but ill suitec 
to the activities of Ps. radicicola. 




FIG. 124. Roots of Plant A without nodules (Fig. 123). 

SOIL INOCULATION* 

By soil inoculation is now understood the adoption of som 
artificial method for supplying suitable quantities of nitrogen-fixinj 
organisms to soils deficient in these types. The first attempts at soj 
inoculation were made in 1886 by Hellriegel and Wilfarth during th 

* Prepared by S. F. Edwards. 



FIXATION OF ATMOSPHERIC NITROGEN 



351 



cirse of their studies on the cause of nitrogen accumulation by 
li umes. They found that when leguminous plants were grown in 
s rile sand, nodules were formed on the roots only after the addition 
oa small portion of aqueous extract of fertile soil, or an extract of 
c shed nodules, or in some cases (lupines and seradella) by soil itself 
f m a field on which these crops had been grown. The first successful 
aificial production of nodules by the aid of pure cultures was made 




FIG. 125. Roots of plant B with nodules (Fig. 123). 

1889 by Prazmowski In the course of studies on the method of 
.ranee of the organism to the root hairs of the host plant. 
The first inoculation experiments in a large way were those made in 
at the Moor Soil Experiment Station, Bremen, Germany, where 
th taken from fields that had borne luxuriant crops of various 
umes was scattered over reclaimed heath or swamp soils upon which 
1- umes had not previously grown, with the result that in every instance 
field on the inoculated portions of land was greater than on the 



35 2 MICROBIOLOGY OF SOIL 

uninoculated plots. After such favorable results, it was but a natura 
step to try the effect 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 ther 
was no increase in the vigor or amount of the crop as a result of th 
inoculation. 

METHODS or SOIL INOCULATION. From these early experiment 
results there evolved two general methods of inoculation, namely, th 
application of soil from an already inoculated field, and the applicatio 
of pure cultures of the nodule-forming bacteria to the seed befor 
sowing. 

Inoculation with Legume-earth.- The use of soil as inoculatin 
material was tried by various experiment stations of the United State 
with results not varying widely from those secured in the pione< 
experimental work at Bremen. It was found in general that tl: 
commonly grown crops, such as the common clovers, peas and bean 
made little or no increase as a result of inoculation with old legume-soi 
With new crops, however, such as alfalfa and soy beans when they we: 
first introduced, it was found impossible in many places to secure 
successful stand until the fields on which these crops were to be gro\\ 
had received a top-dressing of soil from land that had already grov 
the crop in question; and it became a common practice to inocula 
soil in this manner before seeding with these new crops. It was ear 
observed, however, that this method of soil transfer for inoculatk 
purposes was not an unmixed benefit. Aside from the expense ai 
difficulty of handling and transportation of soil, fungus and bacteri 
diseases, not only of legumes but of other crops, as well as the see 
of noxious weeds, were transmitted from one field to another and ev 
from one section of country to another. It was to avoid this difficul 
that the preparation of pure cultures was introduced. 

Inoculation with Pure Cultures. Nitragin. The first pure cultu 
method was launched in 1896 by Nobbe and Hiltner, German inves 
gators, who prepared cultures of the legume bacteria on nutrient gelat 
and arranged with a firm of manufacturing chemists to place them 
the market under the trade name of Nitragin. 

Dried Cultures. In the United States the matter of pure cultui 
was first taken up by the Department of Agriculture about 19* 
Cultures of the nodule-forming bacteria were cultivated in nitrog* 



FIXATION OF ATMOSPHERIC NITROGEN 353 

ee culture media, dried on cotton and distributed to farmers with a 
nail package of salts from which a culture solution was to be made 

the farmer and applied to the seed. This method gave poor results, 
liefly because the bacteria could not withstand the drying on cotton, 
fterward the cultures were sent in a liquid condition with somewhat 
ore satisfactory results. The dry cotton cultures were exploited 
>r a time by a commercial firm under the name of Nitro-culture, and 
imewhat similar cultures were placed on the market in England under 
Le name of Nitro-bacterine. Cultures of both kinds, however, were 
lown to be valueless, both by microbiological and by planting tests. 

Cultures on A gar. Very satisfactory results were secured from the 
te of pure cultures at the Ontario Agricultural College, Guelph, where 
arrison and Barlow, in 1905, originated the method of growing the 
|icteria on a nitrogen-poor agar medium. By this method, the farmer 
is simply to apply the bacteria to the seed just before sowing. These 
iltures, used on all the common legumes, sown in all kinds of soil, 
live favorable results in 65 per cent of cases in trials extending over a 
briod of ten years. Similar agar cultures are now prepared by com- 
ercial firms who have adopted the method of Harrison and Barlow, 
kd also by some of the U. S. Agricultural Experiment Stations. 
I Importance of Inoculation. Inoculation with pure cultures affords 
i.e farmer a rapid, easy, and cheap method of supplying the bacteria 
Isential for getting a successful stand of any legumes. Failure to secure 
^benefit from this method of inoculation may usually be attributed to 
isuitable soil conditions rather than any inherent failing in the cul- 
jres used. No method of inoculation will compensate for poor 
iiysical or chemical condition of the soil itself. The principle of using 
Uncial cultures to be applied with the seed is sound, and if the cul- 
contain large numbers of virile bacteria, there is little reason 
they should not prove of benefit when used under soil conditions 
would seem to need inoculation. 

tobacter Cultures. Some experimental work has been done in 
of cultures of Azotobacter for soil inoculation. The results are 
dictory, and more work needs to be done to prove the value 
cultures. 



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 
becomes deeper and its constituent particles smaller thanks to th( 
uninterrupted process of subdivision. Finally, lichens, algae anc 
bacteria make their appearance, the organic debris accumulates, anc 
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 entirel) 
a mechanical or chemical process. Even before the layer of weatherec j 
rock acquires any appreciable depth microscopical and macroscopica 
forms of life gain a foothold on the uneven surface. With the aid o 
sunlight they build organic compounds and make use of the combined o 
elementary nitrogen of the atmosphere. Their life activities result ii 
the production of carbon dioxide and of varying organic and inorgani 
acids which in their turn react with the constituents of the rock particles 
In this manner the biological activities become of utmost moment ii 
the transformation and migration of mineral substances in nature j 
They assume an important role in the circulation of calcium and mag 
nesium, with the accompanying phenomena that find most strikin 
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 i 
the soil, as well as in the transformation of phosphorus and its migratio 
from inorganic to organic compounds. 

LIME AND MAGNESIA 

REMOVAL AND REGENERATION OF CARBONATES. Lime and maj 
nesia are present in soils in different combinations. They may occi 

354 




CHANGES IN INORGANIC CONSTITUENTS 355 

tes, carbonates, phosphates, humates, sulphates, etc. In 
hmid climates the carbonates are being continually removed from 
vathered rock material, as is plainly shown by the composition of 
<3 linage waters. The losses become much greater in cultivated soils 
links to the humus and the microorganisms present in them. The 
a solute amounts lost from year to year will depend on the proportion 
olime and magnesia in the soil, the mechanical composition of the 
]; cr, its content of humus and the methods of tillage and fertilization, 
/cording to Hall the soils of the experiment fields at Rothamsted, 
citaining about 3 per cent of calcium carbonate, are losing lime at the 
re of 362 kg. to 453 kg. (800 to 1,000 pounds) per acre annually. In 
c tain sections of Scotland where liming has been practised for a long 
t e the farmers estimate the loss of lime from the land at 6 bushels 
p acre, annually; that is, approximately at the rate of 226 kg. to 
2 kg. (500 to 600 pounds). In New Jersey, New York, Pennsylvania 
al other eastern states farmers who use lime more or less regularly 
a )ly i ton of it at the beginning of each five-year rotation. This would 
pi vide for an annual loss of 181 kg. (400 pounds) per acre. The loss of 
li e and magnesia is increased under intensive methods of agriculture. 
Vien animal manures and green manures are employed, microbial 
a ivities are stimulated, the production of carbon dioxide is encouraged 
a I the loss of the soluble calcium bicarbonate made greater. The 
rnoval of lime is hastened even to a more striking extent when 
amonium salts are applied to the land. The resulting nitrification 
a I loss of lime are illustrated by the following equation : 

( H 4 ) 2 S0 4 + 2CaC0 3 + 40 2 = Ca(NO 3 ) 2 + CaSO 4 + 4H 2 O+ 2 CO 2 

As was already indicated, the loss of calcium and magnesium car- 
ite from the soil is effected largely through the activities of bacteria 
of other microorganisms. At the same time microorganic life is 
msible for the restoration of varying amounts of carbonates. It 
been demonstrated that, in the weathering of the complex silicates, 

ites and silicic acid may be formed in considerable quantities. 

presence of decaying organic matter and the consequent evolu- 
of carbon dioxide the formation of carbonates from silicates may 
[tensive enough to balance the losses. Similarly, calcium carbonate 
be formed in the soil from humates and from the calcium salts of 
organic acids. They may be formed, also, through the activi- 



356 MICROBIOLOGY OF SOIL 

ties of denitrifying and other reducing bacteria from the correspondin 
nitrates and sulphates. As pointed out by Nadson ammonium cai 
bonate produced in the decomposition of protein compounds may reac 
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 rea<j 
with carbon dioxide as follows: 



CaS + C0 2 + H 2 = CaC0 3 + H 2 S 



on Vi: 



Magnesium would be subject to similar reactions and Nadson 
observed the formation of a mixture of calcium and magnesium ca 
bonates (corresponding to dolomite in composition) in media inoculate 
with a pure culture of B. (Proteus) vulgaris. 

LIME AS A BASE. The carbon dioxide generated in vast amounts 
the life processes of most soil bacteria, the nitrous and nitric aci< 
formed by the nitro-bacteria, the sulphuric acid produced in tl 
oxidation of hydrogen sulphide and of sulphur by the so-called sulph 
bacteria, and the great variety of organic acids formed in the decor 
position of carbohydrates, fats and proteins all react with basic su 
stances in the soil. Of these basic substances calcium carbonate is 1 
far the most prominent. Combining with the different acids \ 
maintains a favorable reaction for microorganic life in the soil. 

The calcium salts thus formed are more or less soluble. In tl 
manner enormous amounts of lime are annually carried to the oce; 
as bicarbonates, and to an appreciable extent also as nitrate a: 
sulphate. Thus soil bacteria help to furnish shell fish and other fon 
of marine life, the material necessary for the building of their skeletoi 
In the course of ages the latter become a portion of the solid land and 
coral reefs, chalk cliffs and marl beds offer to microorganisms a n< 
opportunity to start calcium carbonate on its migrations. 

EFFECT OF CALCIUM AND MAGNESIUM COMPOUNDS ON BACTERI 
ACTIVITIES. Being basic in character calcium and magnesium c 
bonates are of great service in maintaining a suitable reaction in t 
soil. But somewhat apart from this service calcium and magnesh 
compounds seem to be particularly important for the growth of cerfc 
organisms. It has already been observed by Winogradski and Oni 
lianski that magnesium carbonate is especially useful in facilitating \\> 



CHANGES IN INORGANIC CONSTITUENTS 357 

lation and culture of nitrate bacteria. Heinze and others have 
ted the favorable action of calcium carbonate on the growth of 
-otobacter, while the beneficial influence of calcium carbonate and sul- 
ate on the development of Ps. radicicola has been repeatedly observed 
different investigators. 

PHOSPHORUS 

AVAILABILITY OF PHOSPHATES. Phosphorus exists in the soil largely 
the form of phosphates of calcium, magnesium, iron and aluminum, 
small portion of it occurs in organic combination in lecithin, phytin 
d other compounds. The soil phosphates possess a very slight degree 
solubility and often fail to become available rapidly enough to meet 
demands of the growing crop. Fortunately the presence of 

Jrbon dioxide generated from decaying organic matter hastens the 

? ution of the inert phosphates, thus: 

Ca 3 (PO 4 ) 2 + 2CO 2 + 2 H 2 O = Ca 2 H 2 (PO 4 )2 + Ca(HCO 3 ) 2 

}>r this reason a maximum supply of available phosphates may be 
<i:ured by plants in the presence of readily decomposable organic 
utter. 

I Apart from carbon dioxide as a means for making available inert 
josphates, bacteria produce organic and inorganic acids that are of 
cect 'service. The influence of nitrous, nitric and sulphuric acids, all 
< them products of bacterial activity, is undoubtedly of some im- 
] rtance. The influence of lactic, acetic and butyric acids, as well as 
( the more complex humic acids, must be of considerable moment. 
1>r instance, in the decomposition of bone meal by B. mycoides, 
loklasa found that 23 per cent of the phosphoric acid had become 
sluble, whereas in similar uninoculated portions of bone meal only 3 
jr cent of soluble phosphoric acid was found. The significance of 
ganic acids produced by microorganisms is brought out even more 
rongly in the loss of phosphates from acid soils. 

In so far as the organic phosphorus compounds are concerned bac- 
irial activities are important in that the processes of decay restore the 
nosphorus to circulation. Hence, it will be seen that microorganisms 
;e directly concerned in the migration of phosphorus from the soil to 
je plant and from the plant back to the soil. 

RELATION OF PHOSPHORUS TO DECAY AND NITROGEN FIXATION. 
as bacteria influence the transformation of phosphorus compounds 



3 5$ MICROBIOLOGY OF SOIL 

in the soil, so phosphorus itself affects the growth and activities < 
bacteria. As one of the essential constituents of living cells it reaci 
on the growth of microorganisms and influences species relationship 
There are undoubtedly species whose phosphorus requirement is great< 
than that of other species. Indeed, conditions may arise that favor tt 
rapid assimilation of soluble phosphates by bacteria. In that case tb 
microorganisms would act as competitors to the higher plants. Amon 
the species favorably affected by an abundant supply of phosphati 
Azotobacter is quite prominent. Hence nitrogen fixation is in a mea 
ure dependent upon a proper supply of phosphorus compounds. 

SULPHUR 

SULPHUR COMPOUNDS IN IHE SOIL. Sulphur occurs in the soil : 
the form of sulphates and in that of organic compounds. In il 
aerated soils the reduction products of sulphates; viz., sulphites, su 
phides and even elementary sulphur may be present in small amoun 
as a transition stage. According to Berthelot and Andre the prote: 
compounds of the soil humus are quantitatively more important the 
the sulphates. However, this is not true of arid and semi-arid soi 
in which sulphates represent a larger store of combined sulphur the 
is contained in organic substances. 

SULPHUR BACTERIA. In the decomposition of protein compoun< 
with a limited supply of air, hydrogen sulphide and mercaptans a 
evolved. The quantities of hydrogen sulphide produced may 1 
large enough to become perceptible to the sense of smell, as happens 
the putrefaction of eggs. At the bottom of seas, rivers, lakes ar 
ponds (in canals, ditches, swamps, etc.) as well as in finer-grained soi 
the production of hydrogen sulphide goes on almost uninterrupted 
owing to the activities of a great variety of bacteria. The hydroge 
sulphide thus generated serves as a source of energy to a group 
organisms known as sulphur bacteria. The oxidation of the h; 
drogen sulphide by these bacteria may be expressed by the followir 
equations: 

2H 2 S + 2 = 2H 2 O + S 2 
S 2 + 2O 2 = 2SO 2 

The sulphur dioxide produced is further changed into sulphuric ac: 
in the presence of oyxgen and water. In its turn the acid reacts wil 



CHANGES IN INORGANIC CONSTITUENTS 359 

tine base, usually calcium carbonate, resulting in the formation of 
alcium sulphate. Thus: 

SO 2 +O+H 2 O = H 2 S0 4 
H 2 SO 4 +CaCO 3 = CaSO 4 +H 2 O + CO 2 

i We owe much of our knowledge concerning the sulphur bacteria to 
unogradski. This investigator showed that in places where hydrogen 
alphide is generated in considerable quantities sulphur bacteria grow 
gorously and accumulate granules of sulphur within their cells. 
/hen the cells containing sulphur granules are removed to suitable 
icdia, in which no hydrogen sulphide is present, the sulphur seems 
) be gradually oxidized and disappears and the bacteria finally die of 
tarvation. Thanks to the sulphur bacteria, the higher plants are 
nabled to utilize again the sulphur once locked up in plant and ani- 
ml tissues, and liberated thence by decay bacteria. The circulation 
f sulphur is thus made possible and the cycle is completed when the 
ulphates are again used by plants to build protein compounds. It 
my also be noted in this connection that " Thiobacillus denitrificans ," 
escribed by Beyerinck, may also oxidize elementary sulphur. In 
lis case, however, the oxygen is derived from nitrates instead of the 
tmosphere. Thus: 



6KN0 3 + 58 + 2CaCO 3 = 3K 2 SO 4 + 2CaSO 4 + 2CO 2 + 3 N 2 

SULPHOFICATION. Lint has found that under optimum temperature 
tnd moisture conditions, sulphur applied at the rate of 600 pounds 
>er acre was almost completely oxidized within ten weeks. Boullanger 
ind Dugardin in explaining the fertilizing action of sulphur on the 
of its effect on the supply of available nitrogen found that am- 
mincation was increased by small amounts of sulphur, nitrogen- 
ition was not affected and nitrification was depressed. It has been 
)ointed out by Kossovitch, Brioux and Puerbet that the mechanism 
)f sulphur fertilization is very complex and that the oxidation of free 
sulphur occurs entirely by bacterial and not by chemical means. 
Srown and Kellogg have recently advanced evidence to prove that 
have a definite sulphofying power which is determinable in the 
laboratory by a newly devised method. They claim that the process 
sulphofication is mainly brought about by bacterial action, but 



360 MICROBIOLOGY OF SOIL 

probably there is also a small production of sulphates in soils due to 
chemical action. 

It has been observed that soils differentiated by various treatments, 
vary widely in sulphofying power, the presence of organic matter being 
responsible for an increase up to a certain point. Aeration and mois- 
ture must be optimum for favorable sulphofication while the addition 
of carbohydrates to soils depresses the process. 

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. desulphuricans and Msp. 
(Bstuatii. When grown under anaerobic conditions in culture media j 
supplied with combined nitrogen and organic nutrients these organisms 
were found capable of reducing sulphates. The oxygen withdrawn 
from the sulphates 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 organisms 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. mycoides and by B. (Proteus) i 
vulgaris. The calcium sulphide according to him may react with car- 
bon dioxide and water, giving rise to the formation of hydrogen sul- 
phide. 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. 







CHANGES IN INORGANIC CONSTITUENTS 361 

amounts occur as nitrate, carbonate and in organic compounds, 
portion present as silicates is often very large in clay-loam soils, 



ar mnting not infrequently to 22,679 kg. to 34,019 kg. (50,000 to 
7000 pounds) per acre-foot. Unfortunately for the farmer, the grow- 
in crops fail, in many cases, to secure sufficient quantities of available 
push for their rapid development, notwithstanding these enormous 
st es of potassium compounds. However, when sufficient quantities 
if adily fermentable organic matter are present and the generation of 
ca>on dioxide is rapid the silicates weather sufficiently fast to meet 
ih lemands of maximum harvests. The part played by carbon dioxide 
in ic transformation of inert potash compounds may be illustrated by 
th following reaction: 

A1) 3 K 2 6Si0 2 + CO 2 + 2H 2 O = A1 2 O 3 2SiO 2 2H 2 + K 2 CO 3 -f 4Si0 2 

,'nder actual conditions it is the aim of the farmer to stimulate 
ba erial activities (and, therefore, the production of carbon dioxide) in 
hhand by the use of animal manures or green manures and of com- 
mrial fertilizers. Apart from the influence of carbon dioxide avail- 



potash compounds may likewise be formed on account of nitric, 
huric, acetic, lactic, butyric and other acids produced by different 



ab 
su 
sojbacteria. 

OTHER MINERAL CONSTITUENTS 

RON. The investigations of Ehrenberg, Winogradski, Molisch, 
Adr, Ellis and others have accumulated a mass of data relating to the 
so- tiled iron bacteria. These organisms belong to the class of higher 
bateria and recently forms, such as rod-shaped bacteria, have been 
isoted which have a marked ability to precipitate iron oxide out of 
sol ions of iron salts. Winogradski believed that the reaction is a 
ph iological one in that the microorganisms oxidize ferrous to ferric 
conounds, and utilize for their growth the energy thus made available. 
Th investigations of Molisch, Adler and Ellis show, however, that the 
iro bacteria can exist very well without iron compounds; and that the 
prepitation of iron oxide is due to mechanical rather than chemical 
inifeiices. But whether physiological or mechanical the influence of 
tha microorganisms is felt in the formation of bog iron, and in the 
fill*; up of iron pipes; in the latter instance much annoyance is occa- 
sioilly experienced by those in charge of municipal water supplies. 




362 MICROBIOLOGY OF SOIL 

Compounds of iron are of considerable significance in the 1 
processes of many bacterial species. For instance, it was shown 
Lipman and after him by Koch, that Azotobacterwi]]. not develop in c 
ture media devoid of iron compounds. In field practice small appli< 
tions of ferrous sulphate often seem to exert a favorable effect on cr 
growth, and there is reason to suspect that soil-microbial activities i 
of some moment in bringing about the results noted. 

ALUMINUM, MANGANESE, COPPER. Weathering processes and t 
relation of carbon dioxide to these processes have already been d 
cussed in connection with calcium and potassium compounds. Tc 
great extent aluminum is affected by these reactions, for in the decomj 
sition of feldspar, kaolinite is one of the important products form 
Hence, bacteria become a factor of considerable importance intheforn 
tion of hydrated silicates of aluminum, at least, in the presence 
organic matter. Moreover, it is recognized in the ceramic industi 
that after it is dug clay must undergo ripening in order to be suita 
for certain purposes. The ripening process involves the activities 
bacteria. Unfortunately very little is known about the reactions t : 
occur in the ripening of clay. 

As to manganese and copper there is scarcely any experimental ( - 
dence available as to the part played by their compounds in the s , 
particularly in so far as they affect microorganic life. To some ext( 
it is known that where Bordeaux mixture has been employed for spr 
ing potatoes, cranberries, fruit trees, etc., plant growth is subsequer y 
stimulated to a striking extent. In view of the very slight quantitie f 
copper that are actually added to the soil by these sprays, it is poss je 
that the effects noted are caused by stimulated or changed micro jl 
activities. This view finds some support in the influence exerted iy 
copper sulphate on the growth of algae in lakes, ponds, and shalfv 
streams. 

It has also been reported that the decomposition of complex silic -s 
has been effected from powdered minerals by nitrite bacteria. 

ANTAGONISM 

A subject which bids fair to become a fertile source of investiga n 
is the application of certain biochemical laws, as established by I b 
and Osterhout in the animal and plant worlds respectively, to ie 



CHANGES IN INORGANIC CONSTITUENTS 



;t of salts on the physiological efficiency of soil bacteria in pure and 

ted cultures, as well as in the soil. C. B. Lipman has advanced in- 

>mation concerning the antagonism between anions as related to 

irogen transformations in soils, with special reference to the reclama- 

of alkali lands. Antagonism exists to a more or less marked 

at between anions of alkali salts (as for example between NaCl 

Na 2 SO 4 , Na 2 CO 3 and Na 2 SO 4 and between NaCl and Na 2 CO 3 ) 

i the ammonifying or nitrifying powers of the soil are employed 

criteria. The nitrogen-fixing flora, however, is not similarly 

:ted, apparently offering greater resistance. The practical sug- 

i.tion carried out of such data then, involves the addition of salts 

ithe toxic salts already contained in a given soil, and thereby im- 

)ving its ammonifying and nitrifying power. 



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 food 
It has a pleasant taste and aroma and is generally liked as a food < 
drink; but unless properly cared for will not long remain in its norm; 
condition. No article of human diet is more susceptible to undesi 
able changes, due to the delicate nature of the milk itself and to tl 
conditions naturally surrounding its production and handling. Tl 
injurious changes which commonly occur in milk are of two kind 

ABSORBED TAINTS AND ODORS 

Milk is very quickly affected by odors of any sort. The forei} 
odor may be absorbed before the milk leaves the udder if the cow h 
eaten strong feeds, such as cabbage, onions, etc., or it may be absorb 
after the milk is drawn from the cow. If milk is exposed to a 
strong odor, such as silage or foul air, resulting from lack of venti! 
tion in the stable at milking time, these odors will be taken up by t 
milk with surprising rapidity. If placed in an ice chest with fre 
strawberries or pineapple, or foods like cabbage or turnips, the mi 
will very quickly absorb the odor of these foods. The absorpti 
of any foreign odor gives to milk a decidedly disagreeable taste. Tl 
is true even when the odor which is absorbed is pleasant in itself 
in the case of strawberries or pineapples. When the "off" flavors '<. 
due to absorption they are strongest at the outset and become 1< 

* Prepared by W. A. Stocking with the exception of the paragraphs treating* the acid-forirj: 
bacteria, prepared by E. G. Hastings. 

364 



THE RELATION OF MICROORGANISMS TO MILK 365 

jionounced as the milk becomes older, especially if it is subjected to 
sme method of aeration. 



CHANGES DUE TO MICROORGANISMS 

While absorption of foreign odors is not uncommon, probably 
cst of the undesirable flavors, found in milk when it reaches the 
(nsumer, are caused not by absorption but by the growth of 
Jcroorganisms in the milk. In this class the changes are slight at 
4t and increase with the age of the milk. Changes of this sort 
i'lude the common phenomena of souring and curdling, the so- 
cjlled sweet curdling, ropy or slimy milk, bitter flavors, gassy milk 
i d a large variety of changes usually known as barny or cowy odors 
id flavors. If milk [could be kept free from microorganisms, it 
ight be kept for some time without showing perceptible changes in 
;pearance or taste. No other food product will undergo fermenta- 
t>n changes as rapidly as milk because it is an ideal culture medium 
r the growth of most kinds of microorganisms, especially bacteria 
i d yeasts. Not only does milk contain the needed food elements but, 
ling in liquid form, they are easily available for the use of micro- 
Nanisms. The proteins and milk sugar are most easily attacked 
id it is the breaking down of these which causes most of the changes 
i the milk. 

MICROBIAL CONTENT OF MILK 

I When we recognize the extreme ease with which milk undergoes 
tcterial changes, we are not surprised to find that ordinary milk, 
\ien delivered to the consumer, contains relatively large numbers 
c bacteria. The amount of care exercised in the production and 
Indling is a most important factor in determining the bacterial 
m lamination of milk. On this basis milk may be roughly divided 
ilo three classes. 

COMMON MILK. Age is one of the chief factors in determining 
t: germ content of milk. We are, therefore, not surprised to find 
t 1 milk in large cities having a much higher germ content than in 
s aller cities and towns. The normal germ content of ordinary 
ilk as it is found in the cities may be shown by the following 
tDles. 



366 



MICROBIOLOGY OF MILK AND MILK PRODUCTS 



BACTERIA IN BOSTON MILK* 

Average taken from 2,394 Samples 
From June to September 



Below 100,000 bacteria per c.c 

Between 100,000 and 500,000 per c.c 

Between 500,000 and 1,000,000 per c.c.. . . 
Between 1,000,000 and 5,000,000 per c.c. 

Above 5,000,000 per c.c 

Uncountable plates 



BACTERIAL COUNTS OF CHICAGO (RAW) MILK| 



Per cent 

42.0 

29.75 

9-75 

12.75 

5-o 

o-7S 






Date 


Number of 
samples 


Average 
count 


Lowest 
count 


Highest 
count 


January, 1910 
April, 1910 
Tulv IQIO 


64 

43 
181 


1,067,000 
5,948,000 
12,548 ooo 


27,000 
14,000 
8 ooo 


5,500,000 
150,000,000 













BACTERIA IN MILK OF CONNECTICUT CITIES t 



Bacterial count 

Under 50,000 

50,000-100,000. . . 
100,000-500,000. . 
500,000-1 ,000,000. 
Over 1,000,000. . . 




These figures give the results of 2,467 samples collected in seventy-five 
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 collect! 
in Rochester during the year 1909 as 446,099 per c.c. Of these samples 1.79 per ce 
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 sampl 
representing fifty-seven dairies was 53,000 per c.c. 

In Ithaca, N. Y., 148 samples were taken for the year beginning April i, igc 
and ending March 31, 1910. The average bacterial count of these samples w 
221,000. 

The immense numbers of bacteria found in milk in the large citi 
are usually the result of the rapid growth of the Bad. lactis acidi groi 
resulting from the age of the milk and the temperature at which it h 

* Data given by Hill and Slack, 
f Data given by Tonney. 
J Data given by Conn. 






THE RELATION OF MICROORGANISMS TO MILK 367 

bei kept. Such milk may also contain large numbers of those sapro- 
pl tic organisms which occur in the atmosphere and about the stables 
an milk-house. The number of this group depends largely upon the 
?a tary conditions of production and the initial contamination. In 
or nary milk organisms of the Bact. lactis acidi type will constitute a 
ye' large percentage of those present when the milk reaches the city 
evi before it shows any perceptible signs of souring. During the past 
fe years great progress has been made in the production of clean milk, 
an at present quite an important part of the general milk supply of our 
cil s 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 
Delected, Inspected, or Guaranteed. As commonly used these terms 
m<n milk which has been produced and handled with considerably 
m<e care than ordinary market milk but not with the extreme care 

ired for certified milk. Guaranteed milk is produced by herds 
wl :h have been shown by the tuberculin test to be free from tubercu- 
los). Considerable care is exercised in all the operations of handling 
th milk. The result is that these milks usually have a much lower 
gen content than the ordinary milk supply of the same city. Some- 
tiiis the germ content of such milk compares favorably with that of 
ce lied milk. These milks may contain various types of normal milk 
or nisms but they should not contain any tubercle bacteria. 

CERTIFIED MILK. Certified milk means milk which has been pro- 

du;d according to the regulations of and under the supervision of a 

miical milk commission. The stables and cows are kept extremely 

dca. No dust is allowed in the stable at milking time. The cow's 

flats and udder are washed just before milking, the milkers wear white 

su:> and wash their hands before milking each cow. Small- top pails 

an used and the milk is cooled as soon as drawn from the cow. The 

ex^me care exercised in the production and handling of this milk has 

marked effect on the number of bacteria found in it. The follow- 

ints are typical of certified milk. 



3 68 



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 


17 


5,794 


2 


13 


4,176 


3 


30 


6,825 


4 


12 


i,475 


5 


7 


2,294 



New York City, Oct., 1909 to Sept., 



Farm number 


Average count 


I 


11,132 


2 


10,516 


3 


8,504 


4 


16,193 


5 


2,863 


6 


11,246 


7 


23,705 


8 


5,370 


9 


15,062 


10 


459 



Chicago J 



Dairy number 


Number counts 


Average number bacte 


I 
2 

3 


51 
60 

43 


5,612 
4,078 
6,502 


4 


17 


2,553 



Brooklyn 

Moak gives the average of 321 counts for certified milk delivered in Broo, 
during the first six months of 1910 as 4,095 bacteria per c.c. The best average ii 
any one farm was 561 bacteria per c.c. 

* Data given by Arms, 
t Data given oy Park. 
J Data by Heinemann. 




THE RELATION OF MICROORGANISMS TO MILK 



SOURCES OF MICROORGANISMS IN MILK 



369 



The sources from which bacteria get into the milk have been the sub- 
ct of much investigation during the past few years, until now the chief 
mrces of contamination are pretty well understood. These sources 
jiay be grouped in a general way under the following heads: 





IG. 126. 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 
jcreted by the normal udder of a healthy cow is probably free from 
icteria. It is very difficult, however, to obtain milk from the udder 

24 



37O MICROBIOLOGY OF MILK AND MILK PRODUCTS 

which does not contain bacteria in greater or less numbers. This is due 
to the fact that immediately after secretion the milk becomes contami- 
nated by bacteria which exist in the interior of the udder. Early inves- 
tigators, notably de Freudenreich and Grotenfelt, believed that milk 
while in the udder was entirely free from microorganisms. Later inves- 
tigations, however, 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 se- 
creting cells. These organisms, which normally exist in the milk pas- 
sages of the udder, gain entrance through the orifice in the end of th( 
teat where they find suitable conditions for growth and, once inside 
work up through the milk cistern to the larger milk ducts and finalh 
though all parts of the udder (Fig. 126). The number of bacteria founc 
in the udder varies widely in different cows as may be seen by th< 
following figures: 

BACTERIAL CONTENT or 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. !>85o bacteria per c.c. 

If portions of milk are taken at different intervals during the proces 
of milking in such a way that all external contamination is prevented, i 
will be found that the first few streams of "fore-milk" contain man 
more organisms than the milk drawn later. After the first ten or twelv 
streams the number of organisms will decrease quite rapidly, normall 
becoming less and less until the final strippings, when there is usually 
marked increase. This condition indicates that the larger number < 
organisms exist in the milk cistern and larger milk ducts in the low< 
part of the udder and are therefore removed during the early part of tf 
milking. The increase at the end of the milking is probably due to tf 
greater manipulation, resulting in dislodging some of the organisn 
which have adhered to the walls of the milk passages. 

Not only does the number of organisms in different cows vary, bt 
there is a marked difference in the different quarters of the same udde 
as shown by the following figures. 



THE RELATION OF MICROORGANISMS TO MILK 
BACTERIA IN DIFFERENT QUARTERS OF Cow's UDDER* 



371 



Right front 
quarter of 
udder 


Left front 
quarter of 
udder 


Right back 
quarter of 
udder 


Left back 
quarter of 
udder 


No. 

sam- 
ples 


Aver- 
age per 
c.c. 


No. 
sam- 
ples 


Aver- 
age per 
c.c. 


No. 

ss.ni- 
pies 


Aver- 
age per 
c.c. 


No. 
sam- 
ples 


Aver- 
age per 
c.c. 


Ijfd of 190002 . 79 


419 
199 
161 

249 


77 
174 
46 


378 
130 
107 

191 


80 

185 
46 


653 
636 

597 
635 


80 
186 
46 


617 
698 
342 

625 


Ilrd of 1910-11 185 
ltd of A. G. L 46 

/.stages 









.'srage germ content per c.c. in 316 samples from herd of 1900-02 518 

/arage germ content per c.c. in 730 samples from herd of 1910-11 420 

/erage germ content per c.c. in 184 samples from herd of A. G. L 320 

.-'erage germ content per c.c. in 1,230 samples from 78 cows 428 

The number of organisms normally found in the udder is much 
s aller than would be expected when we consider the fact that ideal 
oditions of food and temperature are provided there for bacterial 
pwth. The relatively small number of organisms is perhaps due to 
s ne germicidal action existing in the udder. Attempts to increase the 
grm content in the udder by injecting cultures of different species of 
S3rophytic bacteria have failed to produce a continued increase, the 
i ected organisms usually decreasing very rapidly in numbers until 
t^y disappear at the end of a few days. From the standpoint of 
dinary market milk, the number of bacteria found in the healthy 
i der is so small that it is of little commercial importance. In dairies 
Here a very small germ content is desired, however, this source of in- 
f .lion must be taken into account and in certain cases individual cows, 
i lich normally have a high bacteria content in the udder, can be dis- 
crded to advantage. 

It is evident that many species do not find the conditions in the 

i der suitable for their growth, since investigations have shown that 

imparatively few species exist for any length of time in the healthy 

i der. Certain types of micrococci are the predominating forms with 

ional cultures of other species. The Bad. lactis acidi type does 

" Harding and Wilson: Technical Bui. .No. 27, N. Y. Agril. Exp. Sta.. 1913. 




372 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

not thrive in the udder. The types of organisms commonly fouiK 
there do not seem to develop rapidly in the milk when it is held at lov 
temperatures and fail to produce any appreciable changes in it durin; 
the normal life of market milk. 

Diseased Udders. If, however, the cow is suffering from diseas< 
in the udder, the bacterial condition may be quite different from tha 
described above. In this case, the milk may be filled with the specifi 
bacteria before it leaves the udder. In cases of inflammatory trouble o 
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 

EXTERIOR OF Cow's BODY. The nature of the cow's coat am 
the condition under which she is normally kept* favor the accumulatio: 
of dust and bacteria upon her body. Unless special care is taken t 
keep the cow's body free from dirt, the organisms which fall into th 
milk from this source at milking time will constitute one of the mos 
important sources of contamination. The importance of this sourc 
of contamination may be recognized when we see what large number 
of microorganisms may be carried by small particles of dust or a 
individual cow hair. 

The importance of this source of contamination depends ver 
largely upon the conditions under which the cows are kept and the car 
exercised in cleaning just previous to milking. In many of the certifie 
milk dairies this source of contamination is reduced to a minimum an 
has little effect upon the milk. 

ATMOSPHERE OF STABLE AND MILK HOUSE. The atmosphere c 
the stable is often a very important factor in determining the bacteriz 
content of fresh milk. In sanitary dairies this factor is fully reco 
nized and every effort is made to prevent the presence of dust in th 
atmosphere at the time of milking. The atmosphere is sometime 
sprayed either with the hose or with steam in order to settle ever 
particle of dust at milking time. In stables where the importanc 
of this factor is not recognized and dust is allowed to exist in th 
atmosphere at milking time, the number of bacteria in the milk wi 
be materially increased. 

THE MILKER. Not infrequently the milker himself is an importar 
source of contamination. If his clothing and hands are dirty or 
he brushes against the cow, the dust thus dislodged may carry int 
the milk large numbers of microorganisms. This is shown in the di 



THE RELATION OF MICROORGANISMS TO MILK 



373 



re nee in the germ content of milk drawn by two men milking in the 
5 ne barn under identical conditions. 

INFERENCE IN NUMBER OF BACTERIA IN MlI.K Dk.UVN BY MEN IN SAME STABLE 





Number of milkings 

I 


Xumber of bacteria 
per c.c. 


Iker No i 


10 


2 A?o 


Iker No. 2 


19 

1 


17,100 






r j 127. Colonies developing in agar plate held for ten seconds in position of 
milk pail after udder was brushed gently with the hand. (Original.) 

i THE UTENSILS. If properly cared for, the dairy utensils should 
n| add to the germ content of the milk. Not infrequently, however. 



374 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

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 ster- 
ilizing are not sufficient and bacteria multiply in large' numbers in th< 
cracks and crevices and contaminate each new lot of milk put intc 
them. Sometimes the utensils which are properly constructed ma) 




FIG. 128. Colonies developing from cow-hairs planted in agar plate. (Origina, 

contaminate the milk because they have not been properly cleanse 
and sterilized. The use of steam is the most efficient means of ste 
ilizing all dairy utensils, but boiling water may give very satisfactoi 
results if used at actual boiling temperature. If not used at the boilii 1 
temperature some of the more resistant organisms will not be kill< 
and will be left to inoculate the fresh milk. The ropy milk organisi 
B. lactis viscosus, often remains in the utensils from day to day in tf 
way. 



THE RELATION OF MICROORGANISMS TO MILK 



375 



WATER SUPPLY. Sometimes the water used for washing the dairy 
tensils is a serious source of contamination. Serious epidemics of 
isease have been traced to this source where the utensils were washed 
ith water contaminated by typhoid or other disease organisms 
nd were not sufficiently sterilized to kill those remaining in the uten- 
1s. Such dairy troubles as ropy milk and gassy milk may be caused 
y the water used for washing purposes. 




<IG. 129. Colonies developed from a bit of dust found in cow stable, 
culture. (Original.) 



Agar plate 



METHODS OF PREVENTING CONTAMINATION OF MILK 

INDIVIDUAL Cows. Normally the number of microorganisms 
3und in the udder is not sufficient to be a serious source of contami- 
ation for market milk. There are, however, certain cows which 
.ave a much higher germ content than others, and where a very low 




376 



MICROBIOLOGY OF MILK .AND MILK PRODUCTS 






count is desired in the milk, it may sometimes be advisable to elimi- 
nate such cows from the herd. 

CARE or 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: 

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 
2 


Apr. 13 
Apr. i < 




Not wiped 
Wiped 
Not wiped 


2,780 
53 
1,310 


7 


Apr 1 6 




Wiped 
Not wiped 


310 
800 




May 28 


' 


Wiped 
Not wiped 


754 
1,13 






I 


Wiped 


590 



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 i 
by reducing as far as possible the area through which dust can fall ink 
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 

No. 2 


/ Open 
\ Small top 
i Open 


15,500 
7,750 
3,700 


No 3 


\ Small top 
( Open 


1,100 
30,000 




\ Small top 


4,700 



THE RELATION OF MICROORGANISMS TO MILK 



377 




FIG. 130. Some different styles of small top milking pails which 
are practical and efficient. (Original.) 

\VOID DUST IN THE ATMOSPHERE. Many of the necessary 
oj -aliens of the cow stable stir up large quantities of dust and fill the 
microorganisms. It is astonishing to see how many bacteria 
ca 
of 

prious to milking time, the atmosphere of the stable will be filled 
wi|i organisms which may settle into the milk while it is exposed during 
th process of milking. The effect of this source of contamination 
m; be seen by the following experiments: 

( TERIAL CONTENT OF MILK AS AFFECTED BY FEEDING DRY HAY AND GRAIN 



adhere to a small piece of hay or may be found in a gram of any 
ur common dairy feeds. When these materials are fed dry just 



Experiment 



Date 



Nature of sample 




May 4 
May 17 
May 1 8 



Before feeding 
After feeding 
Before feeding 
After feeding 
Before feeding 
After feeding 



Number bacteria per 
c.c. 



350 
1,450 
2,900 
4,400 
4,100 
7,200 



)AIRY UTENSILS. All utensils which are to be used in connection 
milk should be so constructed that there are no cracks or crevices 

vhich the milk can accumulate and from which it is not easily 
wned. A milk pail with an open seam may be the cause of serious 
treble in the dairy. The dairy utensils should be simple in construc- 
Ri and so made that they can be thoroughly cleansed with ease 




378 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

and made of such material that they can be thoroughly sterilize 
either with water which is actually boiling or in steam. 

THE MILKER. No food material requires greater care and clean! 
ness on the part of those handling it than does milk. All persons havir 
to do with the handling of this delicate food product should constant. 
keep in mind that clean hands and clothing and extreme cleanliness 
every operation is very necessary if milk of good quality is to be ol 
tained. 



GROUPS OR TYPES or MICROORGANISMS FOUND IN MILK 
AND THEIR SOURCES 






In studying the types of bacteria found in milk, it is convenie 
to arrange them in groups based upon their action on the milk ai 
their effect upon persons consuming it. There are certain types 
organisms which are very troublesome to the milk dealer but which a 
not injurious to the consumer. Other species which may be of little 
no significance from their action on the milk are of greatest significan 
from the standpoint of the consumer since most of the disease organisij 
which may be carried by milk have no appreciable action upon it. St 
other forms are of but little importance to either the dealer or the cc 
sumer and others are troublesome to both. 

GENERAL SIGNIFICANCE OF ACID-FORMING BACTERIA. Of allt 
bacteria that find their way into milk, those that are able to ferment t 
milk sugar, producing from it different kinds and amounts of acids, fi 
more favorable conditions for growth at ordinary temperatures, 15 
45, than do those belonging to other groups. Because of their grea | 
rapidity of growth and because of the inhibiting effect of their by-prc 
ucts upon the other groups of bacteria, the acid types tend to predo 
inate in milk and the specific change which they produce, the sourii 
is of such common occurrence that it is often looked upon as somethi 
inherent in milk. 

GROUPS OF ACID-FORMING BACTERIA.* The acid-forming bacte 
that are constantly present in milk represent many kinds which differ 
morphology, in cultural characteristics, and in their products of fermi 
tation. They may be divided into four groups that vary greatly as 
as their importance in the handling of milk is concerned. If milk is pi 
duced under clean conditions and is kept at temperatures ranging frj 

* Prepared by E. G. Hastings. 



THE RELATION OF MICROORGANISMS TO MILK 379 

i' to 35, the acid fermentation will be almost wholly due to a group of 
ria closely allied to one of the pathogenic forms, Strept. pyogenes 
(osenbach). To representatives of this group, which is of the great- 
e importance in all phases of dairying, have been given various names 
I different investigator?. The most important organism of this group 
is ne to which the name Bact. lactis acidi is applied. The group undoubt- 
e y includes a large number of organisms, all of which produce, how- 
efcr, a similar change in milk. 

Second in importance is a group of organisms, of which the best 
klown representatives are B. coli communis and Bact. lactis aerogenes. 

large number of organisms of this group have been described and 
1. The most important characteristics of the representatives 
rationed will, however, suffice to characterize the group. A third 
(. >up is represented by Bact. bulgaricum and the rod-shaped organisms 
t it have been studied especially by de Freudenreich. A fourth group 
i'ludes many acid-forming cocci, some of which exhibit proteolytic 
[ >perties while others do not. Organisms of the third and fourth 
pups exert little or no effect in the normal acid fermentation of milk, 
c hough they are constantly present in varying numbers, as can be 
(monstrated by appropriate means, and are of importance in certain 
pases of dairy manufacturing. 

In any sample of milk the relative number of bacteria belonging to 
( h of the first two groups is dependent upon the conditions surround- 
i; production, especially with reference to cleanliness. The bacteria 
I longing to the first group come largely from the milk utensils and are 
i o found in the dust of the barn and on the coat of the animal. The 
sarce of the second group is largely the fecal matter that gains entrance 
t the milk, although they are also found in the upper layers of the soil 
i(\ on grain. They are introduced into the milk with the dirt. The 
caner the conditions of production, the smaller will be the number of 
lese two groups of organisms found in fresh milk. 

The manufacture of the leading type of butter and of all kinds of 
< eese is dependent on the action of microorganisms, hence dairy manu- 
1: taring should be classed as a true fermentation industry. In all 
ech industries one of the factors determining the quality of the product 
i the type of microorganism employed to produce the desired f ermen- 
tion, and the importance of insuring the presence of desirable organ- 
ins, and the exclusion of harmful kinds is well recognized. 






380 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

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 o 
acid-forming bacteria, the biochemistry of each group will be empha 
sized rather than the cultural and morphological characteristics of th< 
members of the group. 

Characteristics of the Bact. Lactis Acidi Group * 'The organisms o 
this group are widely distributed in nature, as is shown by the constant 
with which milk undergoes the characteristic fermentation produced b 
the members of the group. 

The cells are oval in form, about o.6ju to i/z in length, and 0.5^ i; 
diameter. The shorter cells appear nearly spherical, which, togethe 
with the fact that chains of cells often occur, has led some to classif 
them among the cocci and Kruse has applied the name Strept. lacticus t 
a member of the group. In milk the cells are usually in twos, the oute 
ends of the two cells being pointed. None of the group is motile; spore 
are not formed and capsules are often noted. The members of th- 
group are Gram-positive. 

The optimum temperature for growth lies between 30 and 35, th 
minimum growth temperature ranging from 10 to 12, while the maxi 
mum is 1 2. They are to be classed as facultative aerobes. The growtl 
on all culture media is marked by its meagerness; in the absence of a fer 
mentable carbohydrate, no growth usually occurs; peptone favors th 
growth even in milk. In the case of freshly isolated cultures, th 
growth is almost invisible, on slopes of sugar agar appearing as sma! 
discrete colonies. On sugar agar plates the colonies are small, oftei 
surrounded by a hazy zone, and always occur below the surface of th 
medium. In lactose-agar stab cultures growth occurs along the entir 
line of inoculation, but there is no surface growth. No liquefaction o 
gelatin occurs. In bouillon the medium is uniformly turbid or it re 
mains clear with a slight sediment. On potato, growth is slight or i 
absent. Milk is usually curdled within twenty-four hours at the opti 
mum temperature by members of the group, although some fail to cur 
die the milk, since the maximum amount of acid produced is notsuffi 
cient to cause this phenomenon. Still others cause curdling in the pres 
ence of small amounts of acids, in which case a rennet-like enzyme ma; 

* Prepared by E. G. Hastings. 



THE RELATION OF MICROORGANISMS TO MILK 381 

t present. No gas is produced in the fermentation of lactose, hence 
t 1 curd formed in milk is perfectly homogeneous; it shows but little 
tidency to shrink and to express whey. In litmus milk the color is 
dcharged from the entire mass of medium before curdling occurs, due 
reduction of the litmus to the colorless leuco-compound. Through 
t action of the oxygen of the air the litmus is slowly reoxidized and 
t pink layer, which immediately after curdling is but a few millimeters 
i: lepth, is slowly extended until the entire mass of curd has a uniform 

rolor. Saccharose, dextrose, maltose, and mannit are fermented. 
j The maximum amount of acid produced by organisms that are most 
tbical of the group is determined by the composition of the medium. 
I is often said that the organisms causing the normal souring of milk 
r .resent a group than can grow in a strongly acid medium. This is 
t e as far as acid salts are concerned, but free acid totally inhibits 

h. In a culture medium, which contains no substance that can 
cnbine with the acid formed and thus remove it from the sphere of 
a ion, no growth, or but very slight growth occurs. In sugar bouillon 
a 1 in milk, the amount of acid formed is determined by the amount of 

ances in these liquids that can combine with the acid. In milk 
s h compounds are the casein and some of the ash constituents, 
e ecially the phosphates. In normal milk, the maximum acidity 
aained ranges from 0.9 to 1.25 per cent calculated as lactic acid. If 
ti content of neutralizing compounds per unit volume is varied by 
cicentration, dilution, or by the addition of such substances as cal- 
c m phosphate, the maximum amount of acid produced by typical 
c lures will be changed. In sugar bouillon the maximum acidity 
p cluced rarely exceeds 0.25 per cent. 
The fermentation of lactose is usually expressed as follows: 

C 12 H 22 O n + H 2 = 4 C 3 H 6 3 . 

Tas 3^2 parts of lactose should yield 360 parts of lactic acid. The 
tloretical yield of lactic acid is never obtained, for the action of the 
o anism on the carbohydrate is much more complex than is represented 
b the equation given. In the following table are given data obtained 

a number of investigators. 

These data signify that other compounds than lactic acid are 
icned in the fermentation of lactose by these acid-forming bacteria. 

tic acid (CH 3 .COOH); formic acid (H.COOH); propionic acid 




382 


MICROBIOLOGY OF MILK AND MILK PRODUCTS 


Sugar content of 
milk, 
per cent. 


Sugar fermented, 
per cent. 


Lactic acid calcu- 
lated, 
per cent. 


Lactic acid found, 
per cent, of theo- 
retical 


4-54 
4.96 

4-94 


0.6o 
0.56 
0.65 


0.632 

O.SQO 
0.684 


89-56 
98.13 
97.89 



(C 2 H 5 .COOH); traces of alcohols, aldehydes and esters have bee 
found. The lactic acid formed is the dextro modification. It is be 
lieved that the fermentation is due to an enzyme, lactacidase, one of th 
intracellular enzymes that can be demonstrated only with difficulty. 

Milk fermented by members of this group has a mild acid taste, a 
agreeable odor, and the curd can be so finely divided by agitation as t 
produce almost as perfect an emulsion as in raw milk. The organisrr 
are to be classed as desirable from the standpoint of the dairy mam 
facturer, and the fermentation produced by them may be called a tri 
lactic fermentation. 

Characteristics of the B. Coli-aerogenes Group* 'This group includi 
a considerable variety of organisms, which differ in morphology, in cu 
tural characteristics and undoubtedly in the character and amounts j 
their by-products. They are more distinctly bacilli than the membe 
of the preceding group ; are motile or non-motile ; none produces spor 
and they are usually negative to Gram's stain. The optimum grow 
temperature, 35 to 40, is somewhat higher than for the precedii 
group, the vegetation range being 15 to 45. They are to be classed 
facultative anaerobes. 

The conditions for development are less narrow than for the Ba 
lactis acidi group, growth occurring on all the ordinary culture med 
and in the absence of carbohydrates. Indol and hydrogen sulphi 
are often formed and nitrates are reduced. The growth is usually pr 
fuse, the colonies large and surface growth occurring in stab oil tun 
Gelatin is not usually liquefied. 

Lactose, dextrose and saccharose are fermented, with the product! 
of varying amounts of gas in which have been found carbon dioxi( 
hydrogen, methane, and free nitrogen. The maximum amount of a< 
produced in any culture medium is quite similar to that formed by t 
members of the previous group. The relative proportions between t 

* Prepared by E. G. Hastings. 



THE RELATION OF MICROORGANISMS TO MILK 383 

in -volatile and volatile acids are far different, lactic acid comprising 
le< than 30 per cent of the total acid formed, while volatile acids, such 
as cetic and formic, make up the remainder. Traces of succinic acid 
(( I^COOHa)) and alcohol have also been found. The lactic acid is 
of he laevo-form. 

Milk is usually curdled, although some members of the group do 
nc produce enough acid to cause curdling. Depending on the amount 
of;as formed, the curd may be almost perfectly homogeneous or it 
m 7 be very spongy. In all cases the curd shrinks to a greater or less 
ernt and thus becomes so firm that it is difficult or impossible to 
enilsify it again. The odor of the fermented milk is offensive and the 
tae disagreeable and sharp. The organisms of this group are to be 
dised as undesirable and the fermentation produced by them cannot 
v i-ctly be called a lactic fermentation. 

Representatives of these two great groups of acid-forming 
baeria are to be found in every sample of market milk in varying 
pnortions. Both find in milk favorable conditions for growth, and 
th normal souring is produced conjointly by them, each producing 
it;3\vn specific products, the relative amounts of which are largely 
dt jndent on the number of each group that is originally introduced 
ini> the milk. The value of milk for butter and cheese is determined 
b)|the relative amounts of the products of the desirable and the 
ui esirable acid-forming bacteria. 

The difference in taste and odor between milk fermented by pure 
cv ures of Bact. lactis acidi, and that which has soured spontaneously, 
enhasizes the difference in the products of the fermentations produced 
b}:he two groups of acid-forming bacteria. 

Characteristics of the Bact. Bulgaricum Group* The organisms of 
th| group are to be classed as true lactic bacteria, since they produce 
al|ost exclusively lactic acid from the sugar fermented and only small 
qmtities of other acids as formic, acetic, and propionic. They vary 
wely in form and size; but are usually large rods, 2/i to 3/x long and 
o. i. to iju wide. There is a tendency to form long threads. They 
ai Gram-positive and when stained with methylene blue often show 
diinct granules in the cells; with Neisser's stain the appearance of 
scie cultures is similar to that of the diphtheria bacterium. They 
ai non-motile and do not form spores; capsules are seldom noted. The 

Prepared by E. G. Hastings. 



384 MICROBIOLOGY OP MILK AND MILK PRODUCTS 

optimum growth temperature is from 40 to 50 and the minimum 
asserted to be 25, although for many members of the group it must 
much lower. 

The growth on all ordinary culture media is meager or is abser 
the colonies are often microscopic in size and show radiating threac 
Free acids do not inhibit development and the term acidophilous h 
been applied to the group. They grow slowly in milk, even at t 
optimum temperature, and curdling may not occur for several daj 
the curd is homogeneous and in litmus milk reduction occurs. T 
maximum amount of acid varies from 1.25 to 4.0 per cent. Soi 
members of the group produce dextro-, others laevo-acid, and racen 
acid is formed in some cases. The curd may be easily broken by agii 
tion, and through the solvent action of the acid is partially dissolve 
The organisms do not liquefy gelatin, but the casein of milk is partia 
changed into soluble decomposition products, as was first shown by 
Freudenreich, and later confirmed by Hastings. 

It has been supposed by many that this group was confined 
and characteristic of certain of the fermented milks, especially th< 
of eastern Europe and western Asia, such as Yogurt and Matzo< 
Recent work has shown that this group is widely distributed in natu 
Representatives of this group are found constantly in milk and otl 
dairy products. Their presence in milk can be demonstrated 
placing a sample of milk in a corked bottle, and incubating at 37. 1 
acidity of the milk increases rapidly at first, due to the growth of i 
members of the two previous groups. These ordinary acid-form 
organisms are soon inhibited by the appearance of free acid, but 
acidity of the milk nevertheless continues to increase slowly, a 
with this continued increase a change in flora is noted, the she 
plump bacilli ceasing to predominate and long slender rods constan 
increasing in numbers. The source of this group is undoubte> 
the alimentary tract of the animal. 

Characteristics of the Coccus Group* This group is well represen 
by the bacteria which form the characteristic flora of the udder. Tl 
vary greatly in size and in other properties. They retain Grai 
stain; many are chromogenic, the color ranging from a white t< 
deep orange. They grow slowly on all ordinary culture media, ] 
the growth is not necessarily meager. Generally they are aerol 

* Prepared by E. G. Hastings. 



THE RELATION OF MICROORGANISMS TO MILK 385 

though many grow under anaerobic conditions. Gelatin may be 
|iietied or not. Milk may or may not be curdled, the curd often 
sembling that formed by rennet-like enzymes. They produce no 
ctic acid, but only acetic, propionic, butyric and caproic acids, 
id hence cannot be classed as lactic bacteria. 

BACTERIA HAVING No APPRECIABLE EFFECT ON MILK. This 
Joup is made up of many different forms. They produce no changes 
lich can be detected either by the eye or the taste. They do not 
ivelop very rapidly in milk, and some species gradually disappear 
pile others increase in numbers. Many of the organisms in this 
Joup are chromogenic, orange and lemon yellows being among the 
jore common forms. They are mostly cocci and do not liquefy gelatin. 
om the standpoint of the commercial milkman these organisms 
;e of little significance and this is probably also true from the stand- 
iint of the consumer. 

THE CASEIN-DIGESTING OR PEPTONTZING BACTERIA. These organ- 
ins digest the casein either with or without coagulation. Many of 
i|em coagulate the casein with an alkaline reaction. They liquefy 
I latin. Most of the organisms of this group are rods of various shapes 
; d sizes, some of them being the largest rods found in milk. Some are 
)tile and some non-motile. Some representatives of this group 
joduce little or no odor, but many of the species develop very strong 
jitref active odors. Barny or cowy odors or other off- flavors sometimes 
and in milk and dairy products may be caused by the action of this 
jpe of bacteria. They are associated with filth and their presence 
i milk indicates insanitary conditions of production or handling. 
, PATHOGENIC ORGANISMS. This group includes all those species 
lich may gain access to milk, which are capable of causing specific 
-es in human beings. They are of the greatest importance to the 
nsumer. They do not appreciably affect the physical or chemical 
joperties of the milk, or produce any changes in its appearance, 
ivor, or keeping quality which would indicate their presence. 
me of them do not even develop in milk, as is the case with the Bad. 
iterculosis. Others, as the diphtheria bacteria and typhoid fever 
Icilli, may grow in milk with great rapidity. This group also con- 
jbis certain species which produce diarrhceal disorders, especially 
j infants and young children. Some of them are probably organisms 
jiich are also included in the peptonizing group. The specific 

25 



386 



MICROBIOLOGY OF MILK AND MILK PRODUCTS 



pathogenic organisms, possibly with the exception of Bad. tubercu- 
losis, 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 bac- 
terial 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 numbei 
of organisms which it contains as a result of the conditions undei 
which it has been produced. There are differences not only in tht 
numbers of organisms but also in the species which may be found ir 
different samples of fresh milk. Both of these factors are important 
in the later changes which may take place. The effect of numer 
ical initial contamination may be seen in the following tables when! 

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 



187,000 



Bacteria 12 hours 



432,000 



Bacteria 36 hours 



633,500,000 



Hours to 



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 


149,650,000 


99 



Milk Having Small Initial Contamination 



Bacteria per c.c. in fresh 
milk 


Bacteria 12 hours 


Bacteria 36 hours 


Hours to curdling 


325 


1,712 


10,125,000 


121 



THE RELATION OF MICROORGANISMS TO MILK 387 

iilk starting out with different numbers of organisms was kept under 
imilar conditions until coagulation. Plate cultures made from these 
*iree samples show the relative development of the number of 
rganisms. 

These samples were all kept at a constant temperature of 21 and 
le difference in the numbers of bacteria and the curdling time can 
lerefore be fairly attributed to the difference in the initial contamina- 
on of the three samples. All three of the samples showed a normal 
evelopment of the lactic organisms, which constituted over 99 per 
;nt of the total organisms present at the time of curdling. While 
lis may be considered as showing the normal effect of the original 
mtamination upon the milk, it is well to bear in mind the fact that 
icre are many apparent exceptions due to some particular type of 
-ganism predominating and interfering with the normal development 
: the lactic types. 

STRAINING. The straining of milk is one of the most common 
Derations in connection with its handling and is considered by most 
lirymen as one of the most essential from the standpoint of the qual- 
y of the milk. If milk is strained through cheese cloth or wire 
mze much of the insoluble dirt can be removed. This has led to the 
meral belief that straining improves the sanitary and keeping quali- 
es of the milk. 

The effect of straining on removal of insoluble dirt is shown by 
ie following results of the 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 



.. 


47O 




u -yo 

2 CCS 


4Q- 


^/ j 
10 8 


i-5;> 
.7 C 1C 






.4 2 AC 




oi 8 




7 -JO 

3-10 


78 6 









j It may be noticed that even after straining the milk contained 
ppreciable quantities of insoluble dirt which had passed through 
e strainer_cloth. The difference in per cent of dirt removed in 




3 88 



MICROBIOLOGY OF MILK AND MILK PRODUCTS 



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 miJk and the nature of the strainer. Not infre- 
quently passing milk through a strainer not only fails to improve its 
keeping quality but actually injures it. This has been shown by a 
number of investigators. The effect of straining upon the germ con- 
tent may be seen in the following 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 


2 600 


3 600 


No. 2 


7,400 


6,900 


No t . 


12,800 


IO.C.OO 


No. 4 


8,800 


11,375 


No <;. 


8,800 


2,700 









The effect of straining upon the keeping quality is shown in th< 
following experiments where the milk was strained through the sam< 
form of strainer mentioned above and the samples kept at constan 
temperature of 21 until coagulation. 

EFFECT OF STRAINING UPON KEEPING QUALITY OF MILK 





Not strained, 
hours to coagulation 


Strained, 
hours to coagulatio 


Experiment No. i. 


42 


42 


Experiment No. 2 
Experiment No. 3. 


57 

T.S 


55 

T.C 


Experiment No. 4 


89 


54 


Experiment No. 5 


CQ 


5 









It will be seen that in no case was the keeping quality of thes^ 
samples increased by the straining process while in some cases 
was materially injured. 



Tot 



THE RELATION OF MICROORGANISMS TO MILK 389 



Cotton filters are more efficient than cheese cloth and in some 
ases the keeping quality of the milk may be improved by this process. 

AERATION. This is the process of exposing the milk to the atmos- 
here by allowing it to run over the surface of the aerator in a very 
lin film. If milk has been produced under such conditions that it 
as absorbed foreign odors, this process may be of value in getting 
[d of the absorbed odors, but from the bacterial standpoint the process 
f aerating is not desirable, since it gives one more opportunity for 
ie milk to become contaminated with organisms from the atmos- 
ihere and from the aerator itself. It is possible to aerate milk under 
Lch conditions that the germ content will not be increased, but if 
eration takes place in the cow stable or other place where the atmos- 
;here contains dust the number of organisms will be greater after 
leration than before, the amount of increase being proportional 
D the sanitary conditions under which the aeration is done. It is 
ven possible that the milk may absorb foreign odors during the proc- 
ss of aeration and be of poorer quality than it was before. It is thought 
y many that the process of aeration is necessary in order to get rid 
f the so-called animal odors commonly found in milk. These odors 
re, however, not normal to the milk but are absorbed from the foul 
ir in the stables or other sources. This is shown by the fact that some 
f the very finest quality of certified milk is bottled while still con- 
lining the animal heat with the least possible exposure to the air, 
ghtly sealed at once and plunged into ice water. Such milk contains 
o suggestion of animal odor. Aeration may be of value in removing 
ndesirable odors from milk which is not produced under good 
initary conditions, if done in an atmosphere free from all dust and 
dors, but it is not necessary for milk of good quality. The common 
elief that aeration is valuable is probably due to the fact that most 
era tors are coolers as well, and the beneficial results are due to the 
ooling and not the aeration. 

CENTRIFUGAL SEPARATION. It is a common practice in some dairies 
> pass the milk through a centrifugal separator or clarifier to 
move any dirt which it may contain. This operation is effective 
jr the removal of much of the insoluble dirt which may be in the milk, 
ut it is of undetermined value as yet from the standpoint of the 
acterial content and the keeping quality of the milk. In spite of the 
act that the separator slime is very rich in bacteria, the milk and 



390 



MICROBIOLOGY OF MILK AND MILK PRODUCTS 



cream as they come from the machine will normally show larger 
bacterial counts in agar and gelatin plates than will the milk before 
treatment, due of course to the breaking up of colonies. The usual 
effect upon the germ content of passing milk through a separator or 
clarifier may be seen in the following tables: 

INFLUENCE OF PASSING MILK THROUGH A CENTRIFUGAL SEPARATOR UPON THE 
GERM CONTENT OF THE SKIM MILK AND CREAM 





Bacteria in 
whole milk 


Bacteria in 
skim milk 


Bacteria in cream 


Sample No i 


39,000 
44,000 
56,000 

200,000 


69,000 
76,000 
75,000 
336,000 


75,000 
790,000 
820,000 
330,000 


Sample No. 2 


Sample No 3 


Sample No. 4 




EFFECT OF A CENTRIFUGAL CLARIFIER UPON THE GERM CONTENT OF MILK 


Sample Bacteria before i Bacteria after : Numerical 


Percentage 



Sample 
number 


Bacteria before 
clarifying 


Bacteria after 
clarifying 


Numerical 
increase 


Percentage 
increase 


I 


6,000 


9,000 


3,000 


SO 


2 


15,000 


22,000 


7,000 


46 


3 


60,000 


156,000 


96,000 


1 60 


4 


133,000 


197,000 


64,000 


48 


5 


370,000 


643,000 273,000 


73 



These increased counts do not mean that there is an actual 
crease in individual bacteria in these samples due to the action of| 
the separator or clarifier. What it does mean is that the small clus- 
ters or groups of organisms, as they exist in the whole milk are thrown 
apart by the centrifugal force and therefore develop individual colonies 
in the plate cultures. 

TEMPERATURE. The temperature at which milk is kept is one of 
the most important factors determining the development of its micro- 
bial content. Every one at all familiar with milk knows that it spoils j 
very quickly if allowed to stand at warm temperatures. If, how- 
ever, 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 atj 
temperatures of 10 or lower. There are, however, certain species! 






THE RELATION OF MICROORGANISMS TO MILK 



391 



hich will grow with considerable rapidity at temperatures below 10, 
pecially some of the spore-bearing non-acid forms. If the tempera- 
re of the milk is allowed to rise above 10, the growth of the common 
ecies increases rapidly. The influence of temperature upon the 
velopment of bacteria may be seen in the following experiment 
lere a given lot of milk was thoroughly mixed and divided into 
ven portions, which were then held at the temperatures indicated 
r twelve hours, at the end of which time they were plated for the 
tal germ content. 

?FECT 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 


c. 


F. 






4.5 


40 


4,000 


75 


7 


45 


9,000 


75 


10 


50 


18,000 


72 


12-5 


55 


38,000 


49 


iS-5 


60 


453,000 


43 


21 


70 


8,800,000 


32 


26.5 


80 55,300,000 


28 



The fresh milk showed a count of 5,000 per c.c. and curdled in 
ty-two hours at a temperature of 21. The curdling time of these 
mples was determined by placing them at a constant temperature 
21 at the close of the twelve-hour period and holding them at this 
mperature until coagulation took place. The difference in time of 
irdling therefore is due to the maintenance of the special tempera- 
ire for twelve hours only and not for the entire period up to the time 
curdling. 

PASTEURIZATION. The term pasteurization is used to designate 
ie process of heating milk to a temperature sufficient to destroy 
portion of the bacteria and then cooling it to a temperature which 
11 prevent the rapid development of the organisms that are left, 
temperatures commonly used for this purpose vary from 60 to 
The length of time the milk is exposed to the high temperature 
also vary from a few seconds to thirty minutes, depending upon 
method employed. The two chief purposes for the pasteuriza- 



3Q2 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

tion 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 the keeping quality of the 
milk, the method used is that known as the "flash" 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. By this method, it is possible to destroy 
a large percentage of the organisms in the raw milk, and material!) 
increase its keeping quality, but the temperature and time to which 
any particle of milk is exposed cannot be accurately controlled, anc 
this method cannot be depended upon to kill all of the disease-pro 
ducing organisms which may be in the milk. This method has beer 
largely abandoned for the pasteurization of market milk. 

Where the chief purpose of pasteurization is to render the milk free 
from disease-producing organisms, the so-called "holding" method i< 
employed. This consists in raising the temperature of the milk to aboui 
60 to 63 and holding it at this temperature for a period of twenty tc 
thirty minutes. If this method is properly done, most of the organism; 
except certain spore forms should be killed and the milk at the end o: 
the pasteurizing process contain only a small percentage of its origina 
germ content. 

Formerly it was believed that heating milk to a high temperatun 
killed all the lactic acid organisms, and favored the subsequent growth o 
other more undesirable species, but more recent studies on the bacteria 
flora of milk, pasteurized by the "holding" method, have shown tha 
some strains of the lactic acid bacteria can survive the relatively lowe 
temperatures used in this method, and that the later development o 
the different groups of bacteria is similar to that in raw milk of equa 
bacterial grade. 

Pasteurization at the temperatures used in the holding process doe: 
not seem to cause any injurious chemical changes in the milk constitu j 
ents, or affect its digestibility. 

Proper pasteurization gives a valuable means of rendering the mill 



THE RELATION OF MICROORGANISMS TO MILK 393 

si ply for our cities reasonably free from pathogenic microorganisms, 
bi, in order to insure this safety, the work must be carefully done, 
ai all later contamination avoided. Preferably, the work should be 
d<e under expert, municipal supervision. Undoubtedly the ideal 
m hod is pasteurization in the sealed bottle which is to be delivered to 
tr consumer, since this method reduces to the minimum the danger 
of ubsequent contamination. 
Pasteurization must not be regarded as a substitute for care and 



cl 



nliness or a means of renovating old or dirty milk otherwise unfit 



fo|use, but rather as an additional means of protecting the consumer 
inst disease-producing microorganisms in the milk supply. 
THE USE OF CHEMICALS. The addition of certain chemicals to milk 
retard the growth of bacteria. The chemicals most commonly used 
folthis purpose are calcium hypochlorite, borax and formalin. While 
tt< keeping quality of milk may be materially increased by the use of 
sui chemicals, their use has been opposed by health authorities and is 
cc'rary to the Pure Food Laws. If milk is handled with any degree 
of are, there should be no need for the use of chemical preservatives. 
TJy are simply a means of counteracting the unsanitary conditions of 
th production and handling. The same results can be obtained by 
chnliness in the production of the milk and the use of low temperatures 
fo preventing the contamination and subsequent growth of the 
ba:eria in the milk. The developments in the production of clean 
m ; of the past few years have illustrated very clearly that the use of 
chnical preservatives is not necessary. 

NORMAL DEVELOPMENT OF MICROORGANISMS IN MILK 

flora of any particular sample of fresh milk is determined by the 
litions under which it is produced. In stables where extreme 
iliness is practised the flora may be practically limited to those 
sp ies which occur in the udder of the cows, but under ordinary condi- 
ticjs there will be in addition to the normal udder types such others as 
occur on the cow's body and in the dust and atmosphere of the 
stales. Market milk, therefore, when first obtained from the cow 
or narily contains a mixed flora, the different types present depending 
upp the sanitary conditions under which the milk is produced. 

future development of this initial flora is largely dependent 




394 



MICROBIOLOGY OF MILK AND MILK PRODUCTS 



upon the temperature at which the milk is kept. If the milk is held t 
temperatures between 10 and 21 there will result what may be coi 
sidered as the normal development of milk fermentations. Thes 
changes may be divided for convenience into four periods or stages. 
FIRST STAGE. GERMICIDAL PERIOD. It has been shown by a nun 
ber of investigators that instead of an increase in the numbers of bacter 
in fresh milk there is normally a decrease in the number during tl 
first few hours after its production. The rapidity of this decrease ar 
the length of time over which it extends seem to be determined large 
by the temperature at which the milk is kept. The higher the temper 
ture the more rapidly the number of organisms decreases and the mo 
quickly the end of the germicidal period is reached. If the temper 
tures are kept fairly low the rate of decrease is much slower but the d 
cline will extend over a considerably longer period. This is shown 1 
the following examples given by Hunziker. 



TABLE SHOWING THE GERMICIDAL ACTION IN Cow's MILK 



Name 
of 
cow 


Milk, 
warm 
and 
fresh 


Temp. * 
of 

milk 


After 
hours 


After 
6 
hours 


After 
9 

hours 


After 

12 

hours 


After 
IS 

hours 


After 
24 
hours 


After 
32 
hours 


hoi 




( 


40 


i, 080 


1,220 


1,040 


1,020 


1,120 


i,36o 


1,040 




May. 


I 212 { 


55 


I 260 


I 400 


i 500 


1,462 


1,360 


i, 080 


3.SOO 


i", 






70 


1,000 


1,340 


1,860 


3,460 


3,46o 


64,000 


800,000 






{ 


40 


4,400 


4,260 


3,620 


3,700 


3,900 


4,000 


3,900 


, 


Ida 


5 120 } 


55 


3.900 


3,460 


2,980 


2,800 


2,920 


3,260 


3,220 


? 






70 


3,560 


2,120 


1,880 


1,880 


1,240 


4,960 


58,400 






f 


40 


1,170 


1,070 


1,120 


870 


1,120 


990 


1, 060 




Julia 


1,345 < 


55 


1,080 


99O 


980 


1,400 


1, 080 


1,080 


3,110 


68, 






70 


1,000 


1,000 


1,200 


5,600 


17,720 


1,600,000 







The exact reason for this decline is at present not well understo 
Some investigators believe that milk possesses a certain germicidal 
tion or property which results in the destruction of a portion of 
organisms found in the milk at the outset. 

The work of other investigators seems to show that the so-caljl 
germicidal action is felt by certain species and not by others as is irl- 
cated by the following sample. 



* Fahrenheit. 



THE RELATION OF MICROORGANISMS TO MILK 



395 



Age of milk 


Total 
bacteria 


Acid 
bacteria 


Per cent, acid 
bacteria 


Liquefying 
bacteria 


h 


... 12,550 


1,250 


10 


200 




12.2^0 


2,OOO 


16 


2OO 




IQ,6$O 


2,250 


23 


800 


ours 
ours . . . . 


. . 56,900 
114,250 


20,250 
68,400 


36 
60 


550 
I,9OO 













th 



T s would seem to indicate that the decrease in number is due not so 

m;h to a definite germicidal property possessed by the milk as to the 

iual dying out of certain species which for some reason do not find 



milk a suitable environment for development, while other types, 



filing the milk suitable to their needs, develop uniformly from the 
stfe 

R.osenau and McCoy found that the germicidal properties of milk 
e destroyed by boiling or by heating it above 80 and that lower 
teperatures destroyed it for certain organisms. These workers also 
fo id that there was marked agglutination of the organisms in raw milk 
ar conclude that this accounts for the decreased number of colonies 
dqeloping in plate cultures and that the germicidal action is therefore 
m e apparent than real. 

SECOND STAGE. PERIOD FROM END OF GERMICIDAL ACTION TO 
TIE OF CURDLING. The period following immediately after the ger- 
m dal action is characterized by the rapid development of the lactic 
ounisms. Under normal conditions this group develops much more 
ly than any other type. Not only do they increase rapidly in 
i\ numbers but their percentage also rises rapidly. There may 
continual increase in numbers in the other species, but their growth 
ch less rapid than that of the Bact. lactis acidi type. As this period 
nces certain of the miscellaneous types may cease to grow entirely. 
Dftng this time the gas-producing acid organisms of the B. coli and 
. lactis aerogenes type may develop more or less rapidly, but if the 
is held at temperatures not much above 20, the Bact. lactis acidi 
will develop much more rapidly, so that by the time the milk be- 
cojes sour and curdles, this type will constitute 99 per cent approxi- 
ly of the total number in the milk. From the standpoint of the 
consumer milk ceases to be of value when the end of this period is 



396 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

reached, but there are further developments which are of importance 
certain lines of dairy manufactures, notably cheese making. 

THIRD STAGE. PERIOD FROM TIME or CURDLING UNTIL ACIDI 
is NEUTRALIZED/ At the time milk curdles it contains enormous nu 
bers of the lactic bacteria. The number usually runs into the millic 
and may be even higher than one thousand million per c.c. By t 
time the coagulation takes place the acidity of the milk is so high tl 
the growth of the lactic organisms is checked and from this time 
their number decreases with more or less rapidity. 

During the period following the curdling certain other types 
organisms which have existed in the milk during the earlier stages n 
begin to grow. The organisms especially important in this stage ; 
Oidium lactis, certain species of molds, and yeasts. These organis 
are able to grow in a highly acid medium, and as a result of th 
development the acid is decreased until the milk finally presents 
neutral or alkaline condition resulting from the decomposition of 1 
proteins in the milk. 

FOURTH STAGE. FINAL DECOMPOSITION CHANGES. The reduct 
of the acidity affords favorable conditions for the growth of certain tyjls 
of organisms which have remained in the milk during the earlier sta 
tut have been practically dormant. In this fourth stage the conditi* 
are suitable for the growth of the liquefying and peptonizing bacteji 
and they now grow rapidly, causing the decomposition of the cast 
The changes resulting from this type of organisms are of special sign 
cance in cheese making and are discussed more fully in another chapl 

ABNORMAL FERMENTATIONS IN MILK 

GASSY FERMENTATION. It frequently happens that instead of 
normally rapid development of the Bad. lactis acidi type of organis s 
in the milk, other acid producers develop rapidly, with the product i 
of more or less gas. The organisms most prominent in this type of i 
mentation are the B. coli communis and the Bact. lactis aero genes tyi 
This group of organisms contains a number of varieties, some of wh 
produce little or no gas while others develop large amounts. Tl 
action in milk is usually accompanied by disagreeable odors and flav< 
They grow readily in the presence of air and therefore develop abund 
colonies on the surface of plate cultures. This distinguishes the m< 



THE RELATION OF MICROORGANISMS TO MILK 397 

of this group quite clearly from those of the true lactic group which 
gjw chiefly below the surface of the medium. The members of this 
giup do not form spores, but certain varieties are quite resistant to 
h t and will oft times survive pasteurizing temperatures which com- 
ptely destroy the Bact. lactis acidi group. They grow most rapidly 
ajhigh temperatures, between 20 and 37. 

SWEET CURDLING FERMENTATION. This phenomenon is caused by 
a ariety of organisms which cause the milk to coagulate without the 
p duction of acid. The coagulation is brought about by a rennet-like 
eyme produced by this type of bacteria. The resulting milk is 
e icr neutral or alkaline in reaction. Usually the coagulation of the 
nk is followed by the digestion of the casein as a result of another 
e:yme which is also produced by these bacteria. The coagulation 
cised by these organisms is slower than in the case of the acid 
flmers and the curd is usually soft and mushy as compared with the 
c - d formed in the normal acid fermentation. The members of this 
g>up get into the milk from and along with dust 

i dirt associated with unsanitary conditions. 

me of the species produce spores and are not 

led by the ordinary methods of pasteurization. 

is fact accounts for the occurrence of sweet 

rdling of pasteurized milk. This group of or- 

nisms is unable to develop rapidly in the pres- 

ce of the lactic bacteria and for this reason we 

not commonly get the sweet curdling of raw 

Ik. The presence of these organisms is evi- 
nce of insanitary conditions. Frequently they 

velop very disagreeable flavors in the milk. 

ROPY OR SLIMY FERMENTATION. One of the FIG. 131. Ropy 

>st common milk infections causing trouble to "?* m lifted , ith a fork - 

(After Ward.} 
milk dealer is that which causes a ropy or 

my fermentation of milk. This is sometimes spoken of as stringy 
(Fig. 131). Several species of organisms are capable of pro- 
cing this condition. These organisms grow most freely in the 
esence of an abundant supply of oxygen and for this reason the cream 
ually becomes slimy before any changes are apparent in the under- 
ng layers of milk. B. lactis viscosus is perhaps the most common 
ecies in this group. The slimy condition in the milk is supposed 




398 



MICROBIOLOGY OF MILK AND MILK PRODUCTS 



to be the result of a very viscid capsule surrounding these organisi 
(Fig. 132). Representatives of this group are quite resistant to he 
and frequently pass uninjured through the methods of cleansing a 
scalding used under ordinary dairy conditions. Because of th 
dairy utensils once infected become a constant source of infectic 




FIG. 132. Bacillus lactis viscosus from a milk culture. (After Ward.) 

This trouble can be effectively stopped by a thorough scalding of 
utensils coming in contact with the milk. 

BITTER FERMENTATION. Bitter flavors in milk may be the res I. 
of bacterial changes after the milk has been drawn, or due to certa 
feeds which the cows have consumed. If the cows are allow L 
to eat certain kinds of vegetation, such as "rag weed" and cert? 
other plants, they may impart a bitter taste to the milk, in which a 
the abnormal flavor will be apparent when the milk is fresh and usua 
becomes less pronounced as the milk becomes older, because of t 
volatile nature of the substances causing the bitterness. Most of 1 
cases of bitter milk and cream, however, are due to the growth 
certain types of bacteria in which case the bitterness increases in 
tensity with the age of the milk. Some of the species capable 
producing bitter milk grow at quite low temperatures, which accouij; 
for the fact that the most trouble with bitter flavors is found in ml 
and cream which has been held at low temperatures for some tirj. 



THE RELATION OF MICROORGANISMS TO MILK 



399 



LCOHOLIC FERMENTATION. The bacteria as a group are not 
a fe to act on the milk sugar and produce alcohol, but it sometimes 
pens that yeasts get into the milk in sufficient numbers to ferment 
milk sugar, producing appreciable amounts of alcohol. To the 
ir handler this trouble is not usually serious but the action of the 
sts is frequently of considerable importance in the cheese industry. 
OTHER FERMENTATIONS. It frequently happens that a consider- 
ab variety of disagreeable flavors and odors develop in milk. These 
ny be due to the direct absorption of odors from the foul stable 
osphere or strong-smelling feeds, such as silage; or they may be, 
no doubt frequently are, the result of the growth of certain types 
ojbacteria which have entered the milk from dirty surroundings, 
i growth of some of these organisms is frequently the cause of the 
sc ailed cowy and stable odors and flavors. 

COMMERCIAL SIGNIFICANCE OF MICROORGANISMS IN MILK 

DELATION OF DlRT CONTAMINATION TO GERM CONTENT. To the 

ccimercial milkman bacteria are of importance only as they influence 
tl: length of time the milk will keep in a salable condition. The 
ccsumers do not want milk that is sour or has unpleasant flavors 
ai odors. In order to sell his milk, therefore, the milkman must 
aiid the presence of these undesirable conditions, and in proportion 
ashe recognizes the relation between germ life and the quality of 
h: product, will he pay attention to the presence and development 
ofiiicroorganisms in his milk. In like manner, the presence or ab- 
:e of dirt contamination is important from the commercial stand- 
pat since it bears a relation to the bacterial count, and, therefore, 
cts the keeping properties of the milk. Under normal conditions 
*e is a fairly direct relation between the amount of visible or soluble 
and the number of bacteria found in any given lot of fresh milk. 
T s relation may be shown by the following samples taken from four 



erent milk producers: 



T .1 Number of 
samples 


Average mg. A ?% mber 
dry dirt per liter bacteria per 


Average hours 
to time of curdling 


A : 5 


SI-5 


115,000 


175 


B 16 

21 


58.8 
70.0 


273,600 78 
428,600 75 


D 17 


71-9 


949,400 68 



400 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

This relation does not always hold for the reason that a gram 
one kind of dirt may contain infinitely more organisms than an eqi 
amount of some other kind. The difference in the solubility of vario 
forms of dirt always causes apparent discrepancies in this norrr 
relation. In the majority of cases, however, the relation shown 
the above examples will hold reasonably true in the case of fresh mi 
There is also a general relation between the number of bacteria 
fresh milk and the length of time it will keep before souring and cuij 
ling. In this case the relation is in inverse ratio, the smaller the init 
contamination, the longer the keeping time, and vice versa. This 
lation is also shown in the table given above. There are many 
regularities, however, in this relation because of differences in 1 ' 
flora of fresh milk. It may frequently happen that a sample of m : 
containing a relatively high number of organisms will not sour 5 
quickly as another sample with a smaller original germ content. 1 ; 
associative action of the different species of organisms is an import; : 
factor here. In making comparisons of this sort, it is, of cour , 
necessary that the different samples be held at the same temperatui . 

MILK AS A CARRIER OF DISEASE-PRODUCING ORGANISMS 

It is not the purpose of this chapter to discuss in detail the disea 5 
which may be carried by milk, but a chapter on bacteriology of rr i 
would be incomplete without a brief discussion of this import; t 
subject. 

From the standpoint of their relation to the health of the c 
sumer the microorganisms in milk may be divided into three gro' 
on the basis of whether they are beneficial, inert or injurious to hea' 

Acid Forms. The preservative properties of sour milk have b a 
known since very ancient times. Its use as a preservative for rm 
eggs and other perishable food products demonstrates the value 
sour milk as a means of preventing decomposition. It has also b n 
known for a long time that sour milk has a certain therapeutic va e 
because of the action of the lactic bacteria in preventing harn 
fermentations in the digestive tract. More recently the work>f 
Metchnikoff has shown the usefulness of sour milk both for the tr< 
ment and prevention of intestinal disorders by inhibiting the devel 
ment of the putrefactive bacteria in the digestive tract. In viev f 
the value of sour milk for preventing certain forms of disease and|:s 



I HI. RELATION OF MICROORGANISMS TO MILK 401 

ibiting action on certain undesirable organisms the Had. lactis 
i type of bacteria must be regarded as beneficial organisms, and 
tlu- standpoint of the health of the consumer their presenee in the 
is to be welcomed rather than discouraged. As the value of 
milk drinks becomes better known tin- importance of this group 
milk bacteria will be more fully recogni/ed. 

// or Inert !-<>rms. In ordinary milk there is a large class of 
which, SO far as known, ha\e no appreciable effect either 
)on the composition of the milk or the- health of the persons consuming 
This group include^ a number of species, many of them being 
forms, some of them appearing in plate cultures as ehromogenic 
lonies. They grow more or less freely in milk, depending upon the 
millions, but they are usually held in (heck by the acid forming bac- 
ria and do not constitute a very important part of the tlora of normal 
ilk, They are, therefore, of little significance from the practical 
undpoint except as they indicate the conditions under which the milk 
been produced and handled. 
Injurious Organisms. The diseases which may be carried by milk 
e of two classes. 

Kf>i<:> tises. The human diseases most commonly carried 

milk are typhoid fever, diphtheria and scarlet fever and occasionally 
her diseases such as septic sore-throat, cholera and foot-and-mouth 
The first three are by far the most important of this group. 
he outbreaks of typhoid fever which are traceable to milk occur 
iust frequently. There is a large accumulation of data showing the 
; epidemics caused by infected milk. An epidemic caused 
the milk supply has certain characteristics whi h distinguish it 
urn epidemic resulting from other (aviso. A considerable number 
cases of the particular disease will appear almost simultaneously 
be distributed along some particular milk route. I'sually 
idemic stops as suddenly as it began except for a few secondary 
itracted from those first taken. The source of the disease 
nanisms is a human patient suffering from the disease, The infection 
t the milk may be direct, as when a sick person handles a milk, 
r it may be indirect as when a person caring for a patient also works 
bout the milk. In other cases it may be caused by contamination 
f the water used in washing the utensils or by cows wading in water 
<-d streams and getting the organisms on their body whence 

26 



4O2 



MICROBIOLOGY OF MILK AND MILK PRODUCTS 



they fall into the milk pail at milking time. The return of milk bottlt 
from the sick room sometimes is the means of infecting the milk suppb 




600 
SO 

60 


Jan. 


Feb. 


Mar. 


Apr. 


May 


Jan. 


My 


Auq. 


bep 


Oct. 


m 


Dec. 


600 
80 
60 
40 




































K 






















\ 












ZO 

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FIG. 133. 

Unfortunately the specific organisms of these diseases grow readi 
in milk and a small infection is all that is necessary to render tl 
milk dangerous by the time it reaches the consumer. 



THE RELATION OF MICROORGANISMS TO MILK 403 

Non-epidemic Diseases. There is another class of diseases which 
be carried by milk which are not characterized by a sudden out- 
, and for this reason are not so readily recognized as being asso- 
ted with the milk supply. One of these diseases, namely tuber- 
losis, is caused by the specific, well-known organism, Bact. tuberculo- 
, which may get into the milk from the udder of a tuberculous cow 
by the organisms which have been given off from the digestive 
ct of the animal becoming scattered about the stable and finally 
ting into the milk with particles of dust and filth. In some 
|ses the milk may become infected by persons having the disease 
ing permitted to handle the milk. Fortunately for mankind Bact. 
berculosis does not multiply in milk. 

Regarding the danger of contracting tuberculosis from the use of 
ilk there is at present some difference of opinion, but the con- 
nsus of opinion at the present time seems to be that there may 
it be very great danger for healthy adults, but that a considerable 
ircentage of the cases of tuberculosis of children may be traced from 
e milk supply. Fortunately the temperatures used in the process 
pasteurization by the "holding" method are sufficient to destroy 
iv of the disease bacteria known to be carried by milk. 

There is another class of disorders not so well defined as the above 
it which are nevertheless of great importance from the standpoint of 
ablic health, especially of young children and also to some extent of 
lults. This group includes such disorders as infantile diarrhoea, sum- 
er complaint, cholera infantum and other disorders of the digestive 
act. The organisms producing these troubles doubtless belong to the 
oup of putrefactive bacteria which come from filth. Some of the gas 
reducers and some of the peptonizers are probably responsible for 
lese troubles. Shiga isolated from a large number of cases of infant 
iarrhoea a bacterium which he named Bact. dysenteries, but in general 
:ie specific organisms responsible for these intestinal troubles are not 
ell known. Their importance, however, is shown by the relation of 
le germ content of milk to infant mortality (see Fig. 133). 

BACTERIOLOGICAL ANALYSIS OF MILK 

The development of our knowledge of the relation of bacteria to the 
rholesomeness of foods has led to a study of the bacterial content of 



404 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

milk as a means of determining its purity. The methods used for th 
purpose have followed quite closely those of the water bacteriologists. 

For many years, dairy bacteriologists have endeavored to determine the numb 
of organisms in milk by plating it into nutrient agar or gelatin. By this methc 
the number of colonies developing in the plates is assumed to represent the ger 
content of the milk. But even when the best methods are employed, the plate com 
represents only the approximate and not the exact number of bacteria in any lot 
milk. It should also be borne in mind that such counts are always underestimate 
because of the fact that not all species will develop in any given medium or incubatu 
temperature. The careful worker can recognize certain types of bacteria in pla 
media, but the addition of blue litmus solution to either agar or gelatin, great 
assists in the differentiation of types and species. 

THE DIRECT MICROSCOPIC METHOD. The plating method is expensive becau 
of the large amount of time and materials needed. It is not possible for one pers< 
to handle a large number of samples at one time. In routine work in the city labor 
tories this labor has been a serious drawback to this method. In order to decrea 
the labor and give greater possibilities to the work Stewart devised a method 1 
which the bacterial condition of milk can be studied by direct microscopic examin 
tion. His purpose was to determine only the species present, but later Slack ai 
still more recently Breed developed the method for determining the approxima 
numbers as well as the general species present in a given sample of milk. 

LEUCOCYTES. The microscopic examination of milk sediment revealed the fa 
that frequently a sample would be found which showed the presence of leucocytes 
greater or less numbers. The presence of these cells was regarded as importa 
because it was assumed that they showed the presence of inflammation and pus fc 
ma tion in the udders of the cows producing the milk. 

Several methods have been used for determining the leucocyte content of mil 
"The Smear Sediment" and "Blood Counter" are methods which more strict 
belong to laboratory practices and will not be considered in this place. 

BACTERIOLOGICAL MILK STANDARDS 

The relation of the bacterial content of milk to its wholesomene 
has led to the adoption of certain standards by the boards of health 
our cities. These standards recognize the fact that the germ content < 
milk in the large cities is greater than in the smaller ones because of ti 
greater distance from which it is shipped and its age on arrival to tl 
city. New York City in 1900 adopted a maximum limit of i,ooo,oc 
per c.c. Later Boston established a limit of 500,000 Chicago i,ooo,oc! 
from May to September, inclusive, and 500,000 from October to Apr I 
inclusive and Rochester 100,000. Other cities have made simil; 
standards. 



THE RELATION OF MICROORGANISMS TO MILtf 405 

Stokes' standard for the number of leucocytes permissible in normal 

k was 5 per field of the J^2 objective in his smeared sediment prepa- 
r;ion. Bergey found so many samples running above this number 

t he made the limit 10 cells per field and felt that no milk containing 
nre than this number should be used for food. Later Slack raised 
limit to 50 cells per field. The reason for changing the stand- 
was due partly to the larger numbers found as a result of improved 
nithods but more especially to the discovery that milk from appar- 
ejly healthy cows normally contains leucocytes in excess of the first 

ndards set. 

With the development of the dairy score card, there was a decided 

dency to place emphasis on the sanitary conditions at the farm rather 
tin on the germ content of the milk. But it was soon discovered that 

farm score did not necessarily show the true condition of the milk, 
all at present, the tendency seems to be toward placing more confidence 
rthe germ content as the best measure of the true conditions of pro- 
ciction and handling. However, the fact must be recognized that our 
nthods of bacteriological analysis are not sufficiently accurate to 
ji.tify the bacteriologist in passing judgment concerning the quality of 
sy milk supply on a single analysis. In order to secure results which 
r : at all trustworthy, a series of analyses must be considered. 
. It is held by some that a numerical standard is of little value since 
t j actual number of organisms present in a given lot of milk may not be 
correct measure of its wholesomeness. For this reason some cities 
p'y little attention to the numbers of bacteria present but base their 
s-ndards wholly on the species and the quality of the milk is judged on 
t; presence and numbers of streptococci, B. coli, leucocytes, sediment. 
)ilk is passed or condemned on the basis of any one or combination of 
lese conditions. 

In recent years there has been a tendency to combine these two 
sindards using the total germ content as a measure of the care the 
] Ik has had and the presence or absence of certain groups or species as 
; indication of the occurrence of pathological conditions in the cows 
]ioducing the milk. The practice in most city laboratories now is to 
take use of both the numbers and the species present in determin- 
i|5 the quality of the milk supply. 



406 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

VALUE OF BACTERIOLOGICAL MILK STANDARDS AND ANALYSES 

Regarding the value of bacteriological standards for milk there i 
still some difference of opinion among milk bacteriologists. The gen 
content of any lot of milk is largely dependent upon three factors: th 
number of organisms getting into the fresh milk; the temperature a 
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 an 
one of these conditions or a combination of them. A high count mear 
that there has been carelessness either in the production, resulting i 
high initial contamination, or in the subsequent handling permitting 
rapid multiplication of the organisms, or that the milk is old. 

On the other hand, milk with a low germ .content can be obtaine 
only where the original contamination is small and the milk has bee 
held at low temperatures. A low count, therefore, means care both i 
the production and later handling of the milk. 

While the germ content may be regarded as a general index to tf 
care the milk has received, it may not at all indicate its wholesomenes 
A high count may be the result of the rapid growth of the lactic bacteri; 
in which case the milk may be perfectly safe and wholesome. On tl 
other hand, the count may be quite small but contain pathogenic specie 
The bacteria count is valuable as showing the sanitary conditions 
production and handling, but much care should be used in the inte 
pretation of such results. In some ways a direct microscopic examin; 
tion of the milk sediment is much more satisfactory. The skillc 
analyst can recognize certain types which may indicate the sanitai 
quality of the milk. With sufficient experience one can recognize stre] 
tococci, certain other groups and leucocytes. The presence and abui 
dance of one or more of these groups may indicate the nature of tl 
original contamination and the existence of diseases in the udders 
cows. If rightly interpreted the information thus obtained is of miu 
value. The weakness of this method lies in the fact that it is not alwa; 
possible to recognize the above types of organisms. In a smear prep 
ration it is not possible to differentiate between pathogenic and no 
pathogenic streptococci or between B. coli and certain other type 
The presence of unusual numbers of streptococci and pus cells nic 
indicate the existence of disease in the cows and when this condition 
found in the milk it is often possible to trace it back to the farm and 1 



THE RELATION OF MICROORGANISMS TO MILK 



407 



the diseased cow and prevent her milk from being used for human 
isumption. 

j The tendency at present is to combine the quantitative and quali- 
jive analyses and the results thus obtained in the hands of the careful 
rker are of much practical value in controlling the quality of a city's 
|.k supply. 







CHAPTER II* 
THE RELATION OF MICROORGANISMS TO BUTTER 

Butter is the fat of milk that 'has been largely freed from the othe 
constituents of milk by the processes of creaming and churning 
If milk is allowed to stand, the fat, which is in the form of minut 
globules, accumulates in the upper layers of the milk because its spe 
cine gravity is much lower than that of milk serum. In modern prac 
tice the fat is concentrated in a portion of the milk by passing the mil 
through a cream separator. In the rapidly revolving bowl of the separa 
tor the centrifugal force exerted is many times greater than that of gra\ 
ity and the fat is rapidly and efficiently removed. The cream, which i 
obtained by these methods, contains varying amounts of fat whic 
is further concentrated, by subjecting it to agitation in the churnin 
process. The globules of fat cohere to form larger and larger masse 
until the entire amount of fat is brought into a single mass, the buttei 

TYPES OF BUTTER 

SWEET-CREAM BUTTER. If little or no increase in the acidity c 
the milk or cream develops, previous to churning, the butter will hav 
certain marked characteristics and is called sweet cream butter. 1 
is especially characterized by its low flavor, since it has only th 
flavor of the fat of milk which is not marked. This is usually known a 
the primary flavor of butter. Sweet-cream butter is also marked b 
the rapidity with which it undergoes decomposition changes, especial! 
when it is made from raw cream. 

SOUR-CREAM BUTTER. If the cream is allowed to undergo the aci 
fermentation, the butter will differ markedly both in degree and kin 
of flavor from that prepared from sweet cream, and as a rule itskeepin 
qualities are much better than those of sweet-cream butter. This typ 
of butter is made throughout northern Europe, England and he 

* Prepared by E. G. Hastings. 

408 



TH 



E RELATION OF MICROORGANISMS TO BUTTER 409 

. and in America. It may be said to be the standard butter 
, orld since it is the type made in all the great dairy countries. 



S^ 



et-cream butter is made especially in southern Europe, and in 



lilted amounts in other countries. 

The intensity and kind of flavor of butter is thus dependent on 
tl acid fermentation of the milk or cream. It is not believed that 
tt fat undergoes any changes during the acid fermentation of the milk 
wch could produce the flavor of sour-cream butter, but rather that 
tt increase in flavor is due to the absorption by the butter fat of certain 
of ic compounds formed in the acid fermentation. It is not essential 
tH the fat be present during the acid fermentation in order to impart 
fl.-or to the butter. If sweet cream is mixed with sour milk and 
cllrned at once, the flavoring compounds are absorbed by the fat from 
d fermented milk, and the butter will have much the same flavor, 
bi'h as to intensity and kind, as though the fat had been present 
d ing the fermentation. The churning of a mixture of sweet cream 
aJ sour milk is used commercially and is identical with the methods 
alloyed by the manufacturers of oleomargarine and renovated 
biter to impart flavor to the flavorless fats they employ. It is 
imossible to recognize these substitutes for butter by their flavor 
sii:e it is identical with and derived from the same source as the flavor 
o mtter. 

In the past many ideas have been expressed as to the source of 
ti flavor of butter; some have asserted that it is due, in part, to the 
d omposition of the proteins of milk by proteolytic bacteria. Both 
pctical experience and experimental work have demonstrated the 
c-lnection between the acid fermentation of milk and the flavor of 
b| ter, and it is certain that what is now considered the finest type of 
bj.ter can be made from cream in which only acid-forming bacteria 
(s|; Chap. I) have grown. 

FLAVOR OF BUTTER 

CONTROL OF BUTTER FLAVOR. The commercial value of any 

sjnple of butter is largely determined by its flavor. If it is lacking 

iillavor and aroma, or if it has a poor flavor, it brings a low price. The 

Dortance of being able to control the flavor, both as to degree and 

d, in the manufacture of butter has increased greatly in recent 



410 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

years, because of the introduction of the creamery system, which h; 
largely supplanted the making of butter on the farm. The financi 
success of any creamery is largely dependent upon the ability of tl 
butter maker to control the flavor of the product, so that it shall 1 
uniform from day to day. It is asserted that one of the facto 
in the remarkable invasion of Denmark into the butter markets 
the world is the uniformity of the Danish butter, not only from a sing 
creamery, but from all the creameries of the country. To the Dan 
we owe the most improved methods for the control of the flavor 
butter. 

The other points, texture, color and salt, which the judge of butt 
takes into consideration, can be easily controlled, since they are due 
mechanical operations. The flavor, on the other hand, is due to the b 
products which are formed by microorganisms in the fermentation of tl 
milk and cream, and which are absorbed and held by the fat. If any 
the products formed possesses a disagreeable taste or an offensive ode 
the flavor and aroma of the butter will be impaired. It is thus evide 
that the control of the flavor of butter is dependent on the control of t! 
acid-forming bacteria that ferment the milk and cream. This is the pro 
lem of the modern butter-maker and the modern methods seek to gi 
him this control, to enable him to eliminate the undesirable bacteria, . 
coll and Bad. aero genes, the second group,* and to insure the predon 
nance of the desirable bacteria, Bact. lactis acidi. This genei 
statement is not to be interpreted as meaning that all bacteria th 
injure the flavor of butter are to be included in the group mentione 
for many other types of bacteria, when present in milk in large numbei 
may injure the flavor of the butter prepared from it. 

The acid fermentation of the cream is most frequently called V 
ripening of cream and sour-cream butter is frequently called ripene 
cream butter. The ripening of the cream not only increases the flav 
of the product, but it enhances its keeping quality. The ripening of t 
cream also aids in the mechanical process of churning, the sour crea 
churning more easily and with less loss of fat in the butter milk. 

KINDS AND NUMBERS or BACTERIA IN CREAM. The number ai 
kinds of bacteria found in cream are dependent upon the number ai 
kind in the milk from which the cream is obtained. The cream wi 
however, contain a greater number of bacteria per unit volume than t 

See Chap. I, Div. IV, in which the groups of bacteria are considered. 



THE RELATION OF MICROORGANISMS TO BUTTER 



411 



, since the immense number of fat globules passing through the 
serum carry mechanically a considerable proportion of the bacteria 
milk into the cream. This phenomenon is to be noted in gravity 

ming, but to a much greater extent in the removal of the cream by 

of the separator. 

SPONTANEOUS RIPENING OF CREAM. By this expression is meant 

fermentation of the cream by those acid-forming bacteria that have, 
f Jtn one source and another, gained entrance to it, but which have not 

n intentionally added. Under these conditions the butter-maker can 
e| rt but little control over the fermentation. A very considerable part 

he butter made from such cream has an excellent flavor, because at 

temperature at which cream is usually kept, Bact. lactis acidi and 

ted organisms are the primary factors concerned in its fermentation 
i their by-products produce desirable flavors in butter. It has often 
m asserted that the highest type of butter can be made only from 
mtaneously ripened cream. 

As the cream from many farms was assembled at a creamery for the 
jiufacture of butter, it became evident that some means of controlling 
5 type of fermentation in the cream was needed. If the milk had been 
)duced under clean conditions, and had been received at the creamery 
'ore the acid fermentation had gone on to any extent, and if the 
am was then kept at temperatures most favorable for the lactic bac- 
ia, the product was likely to be of good quality, but such ideal condi- 
ns did not always obtain. Cream containing a large proportion of 
rmful bacteria, or in an advanced state of fermentation, or possessing 
undesirable flavor was often received, and the butter-maker could 
t control the quality of the product under such conditions. 

USE OF CULTURES IN BUTTER MAKING. As the science of micro- 
logy progressed and the role of microorganisms in all kinds of fermen- 
tion became known, it was evident that the control of the causal or- 
rdsm is an important factor in determining the quality of any product 
the fermentation industries. In the manufacture of butter, the first 
. j p in this direction was the addition of some fermented milk, cream, or 
buttermilk to the cream to be ripened. In this manner the number 
acid-forming organisms in the cream was greatly increased, and the 
mentation went on more rapidly and in a more definite direction than 
thout such additions, as the bacteria added were largely of the 
ble group, Bad. lactis acidi. The addition of fermented milk to 



412 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

accelerate the souring of cream antedates by many hundred years tfc 
science of bacteriology. 

The next logical step in the development of the process was the use ( 
the same types of bacteria from day to day. Cultures of these were ot 
tained by allowing a quantity of milk to sour, and if it had the desire 
flavor, a small amount of it was added to another quantity of milk thj 
had been heated, in order to destroy the acid-forming bacteria it coi 
tained. By the daily preparation of some heated milk, and the inocul; 
tion of it with the soured milk previously prepared, the butter-mak 
could use the same types of bacteria for an indefinite time for additio 
to the cream. 

It had been found by Hansen that, in order to control the flavor < 
beer, pure cultures of yeasts must be used for the fermentation of ti 
wort. The success of this method in the brewing industry led to tl 
introduction of pure lactic cultures for the fermentation of cream. Tl: 
use of such cultures was suggested independently by Storch, a Danis 
bacteriologist and by Weigmann, the director of the dairy experimei 
station at Kiel in Germany, in 1890. Many cultures were isolated an 
tested as to their effect on the flavor of butter. Those found to t 
desirable could be maintained in the laboratory, and could be furnisht 
to butter-makers to be used and propagated in a manner similar to tl 
method employed with the impure and less constant home-made starter 
The pure cultures of lactic bacteria are widely used at present in tl 
butter-producing countries of the world and their use is being constant] 
extended, as butter makers come to recognize the importance of coi 
trolling the ripening of cream. 

It was found that the butter made from cream ripened by pure lact 
cultures did not possess as high a flavor as did the finest butter mac 
from naturally ripened cream. This led to the search for organisn 
that could be used alone, or together, with the lactic bacteria, and whk 
should give the high flavor desired. Such cultures were found, bi 
their use did not prove practical, either because they did not maintai 
their properties on continued cultivation, or because of their effect c 
the keeping quality of the product. The difference in flavor in the cai 
of butter made from naturally ripened cream and that from cream ri] 
ened by pure lactic cultures is undoubtedly due to the products of tl 
B. coli-aero genes group. 

The acid in spontaneously soured milk is very evident to the tas 



THE RELATION OF MICROORGANISMS TO BUTTER 413 

n the acidity is 0.6 per cent and above; the volatile acids formed by 
members of the colon-aerogenes and coccus groups impart a sharp, 
pigent taste. In milk of like acidity fermented by pure cultures of 
I ;/. lactis acidi, the acid is scarcely evident to the taste and there is no 
s rpness, due to the absence of volatile acids. This same difference 
a >ears in the butter made from the two kinds of milk. 
The low flavor of the butter made from cream ripened by pure 
Lures was one of the factors that prevented the rapid introduction 
othe cultures in this country. The demands of the butter market 
e changed and the mild flavored butter, which is now considered 
t DC the finest, can be made by the use of pure cultures in the fermenta- 
ti i of pure sweet cream. 

COMMERCIAL CULTURES. In this country the preparation and 
ribution of cultures for the ripening of cream is largely in the 
bids of commercial firms; hence, the term "commercial culture" 
pplied to them. The different pure cultures are propagated in 
q laboratory of the maker; they are sent out either as liquid cultures, 
almall mass of milk or bouillon inoculated with the organism, or in 
a ryform, the latter being prepared by mixing a culture of the organism 
w|h an inert substance, such as milk sugar, milk powder, or starch, 
aji drying at a low temperature. In a liquid the organisms are 
ejosed to the effects of their own by-products, and the vitality of 
til culture is rapidly lost. Such cultures must be used when fresh 
irprder to give good results, and they cannot be kept in stock by the 
ninufacturer or dealer. The resistance of Bad. lactis acidi to desic- 
cjion is great; it thus lends itself to the preparation of the dry cultures, 
irwhich the organisms remain in a dormant condition and retain 
ir vitality for long periods. 

Most of the cultures now sold are pure, as this term is used in bac- 
ology, still others contain non-acid-forming organisms, intentionally 
led or introduced accidentally during the process of preparation. 
I:he lactic bacteria are present in such cultures in large numbers, 
impurities are usually of small practical significance. In the past 
stalled "duplex" cultures have been sold which were supposed to 
tain an acid-forming organism and a second organism that was 
enhance the flavor of the product. Such cultures are no longer sold. 
For the propagation in the creamery the contents of the container 



P 



chased are added to a small mass of milk that has been heated 



414 MICROBIOLOGY OF MILK 'AND MILK PRODUCTS 

to destroy all non-spore-forming bacteria and other microorganism 
the milk, after being inoculated, is incubated at favorable temper; 
tures and when curdled can be used for the inoculation of the secor 
and larger quantity. The process of inoculating a quantity of mi 
is carried out daily. It is impossible for the butter-maker to propaga 
the culture so as to maintain the original purity, but with care in tl 
heating of the milk the sterilization of all utensils and the maintainii 
of proper temperatures, the contamination that occurs will not inju 
the culture for practical work. The cultures propagated under su< 
conditions gradually deteriorate and recourse must be had soon 
or later to a. fresh culture. The contamination that is of the greate! 
practical significance is undoubtedly that with other acid-formii 
bacteria rather than with the forms that remain in the milk aft 
heating. 

Many of the cultures gradually lose their fermentative propertii 
and do not form acid rapidly and in sufficient amounts to insu 
exhaustive churning and to produce the desired degree of flavor 
the product. Cultures frequently become slimy or ropy on prepay 
tion. This is not necessarily due to contamination with sped 
slime-forming organisms but rather to a change in the lactic organic 
itself. Such an abnormality usually persists for only a short peri 
and the conditions that govern its appearance and disappearar 
are not known. It is asserted by practical butter makers that t 
development of too high an acidity in the cultures as they are prof 
gated in the creameries permanently impairs the value of t 
culture. 

The cultures are propagated in skim-milk. Where this is not &vi 
able, unsweetened, condensed milk or milk powder have been employi 
Efforts have been made to grow the bacteria in some other kind 
medium than milk, but without success. The starter is said to 
ripe or in the best condition for use soon after curdling, or when 1|; 
acidity is 0.5 to 0.7 per cent, as at this time it contains the maximu 
number of living cells. The practical man thus uses the curdl :; 
as an indication of the ripeness of the starter. The curdled milk shoi 
show no free whey, and the curd should be easily broken up to fo 
a creamy mass that can be uniformly incorporated with the crea 
The temperature of incubation and the amount of initial inoculat: 
determine the rapidity with which the acid fermentation will progni, 




THE RELATION OF MICROORGANISMS TO BUTTER 415 

th( naker seeking to regulate these so that the culture shall be ripe at 
thfiesired time each day. 

*SE OF PURE CULTURES IN RAW CREAM. The cream as it reaches 
mery contains a greater or less number of acid-forming bac- 
teiji that ultimately will cause it to ripen and the flavor of the butter. 
w i be due to the by-products of the mixture of bacteria. If, through 
th addition of a pure culture, the relative number of organisms that 
ar ;nown to be favorable is greatly increased, the flavor of the product 
sh ild be improved. This has been found to be true in practice and 

now believed that pure cultures are of value not only in the ripen- 
in of sweet cream, but that the addition of a relatively large amount 



of 



tarter to cream that is already fermented will enhance the value 



ofie butter. 
JSE OF PURE CULTURES IN PASTEURIZED CREAM. It is evident 



th 



the maker has but imperfect control over the fermentative proc- 



ds when raw cream is treated with a pure culture. To insure more 

p< ect control the destruction of the contained bacteria and the 

sipequent inoculation of the cream with a pure culture is indicated. 

Tl; introduction of the process of pasteurization of cream for butter 

irking was due to Storch. In Denmark this method is used almost 

eiiusively. It has been introduced into the other dairy countries 

o'the world and is constantly spreading. Pasteurization combined 

h. the use of the pure culture represents the highest type of modern 

ter making, and where the raw product can be obtained in a fresh 

dition the butter-maker has perfect control over the bacteria 

t!t cause the ripening; hence he can control the flavor of the butter, 

b h qualitatively and quantitatively. 

The intensity of flavor of butter is dependent upon the amount 
cacid that is developed in the cream or more correctly on the ratio 
t ween the amount of fat and the by-products of the acid fermenta- 
tn. If these by-products are small in amount, as in cream having a 
U acidity, the flavor of the butter will be low. If the acidity is 
uwed to reach the maximum, the flavor will be much higher. Thus 
maker can control the intensity of flavor of butter as accurately 
he can the kind of flavor. With rich cream, the acidity that can 
developed is small and the ratio between the fat and the products 
:ermentation is low; thus, the flavor of butter made from very heavy 
am is certain to be low. 



41 6 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

PURE CULTURES IN OLEOMARGARINE AND RENOVATED BUTTER. 
It was previously mentioned that the manufacturer of butter subs 
tutes employs the same methods to impart butter flavor to his prc 
ucts as does the butter-maker. The oleomargarine manufactun 
employ pure cultures of lactic bacteria for the fermenting of m 
that is mixed with the fats they employ. The same practice is follow 
by the manufacturer of renovated butter. Many of the creamer 
of the western states receive cream that is shipped long distanc, 
and is collected from the farms but once or twice a week. It is th 
in an advanced state of fermentation when it reaches the creame . 
In order to prepare from this grade of cream, which often has a im ; 
undesirable flavor, a merchantable product, various means are e- 
ployed to remove the flavoring substances and to replace them w i 
desirable flavors from the pure cultures. The acidity may be reduc'l 
by the addition of lime so that the cream can be pasteurized;^ 
cream may be aerated by passing air through it, or it may be mbjl 
with water and reseparated. After such treatment it is mixed wi 
a large proportion of milk fermented by a pure culture and churn i. 
The resulting product is constantly sold as the highest grade of cream* > 
butter. 

ABNORMAL FLAVORS OF BUTTER. Most of the abnormal flavors i 
butter are traceable to the partial replacement of the desirable ac- 
f orming bacteria with other types of microorganisms Many samp > 
of butter having abnormal flavors have been examined, and the org; 
isms believed to be the cause isolated and studied but it cannot be s I 
that any particular group of microorganisms can be associated with a 
of the abnormal flavors met. It is asserted that " oily " butter, i.e., tl 
having the taste of machine oil, is caused by bacteria and by microorg; - 
isms that decompose the fat, as Oidium lactis, yeasts, and liquefying b - 
teria. Organisms of the B. coli group that produce a turnip-like Sa- 
in butter have been described by Weigmann. The flavors of put 1 
butter, fishy butter and also many other abnormal flavors have bi 
ascribed to bacteria. 

Other abnormal flavors may be due to the presence in the milk 
certain aromatic principles contained in the feed and excreted in b 
milk. Cabbage, turnips, and other plants impart their char acted; |: 
taste to the milk and butter. 



THE RELATION OF MICROORGANISMS TO BUTTER 417 

DECOMPOSITION PROCESSES IN BUTTER 

Butter is a finished product at the time it is removed from the mod- 
churn and all subsequent changes are likely to cause more or less 
erioration. The specific causes of these changes are not well known 
it is very evident from a study of the conditions that favor or retard 
appearance of the flavors, characterizing these changes, that biolog- 



factors are concerned. When raw cream is used, sweet-cream butter 
very poor keeping qualities. As the proportion of acid-forming 
b teria in butter is increased, either by the fermentation of the cream, 
b the addition of pure cultures, and through the use of the latter in 
ajnection with pasteurization, the keeping qualities are enhanced. 
Othe butter made from ripened cream, that prepared from cream, 
hi died in a clean manner, and thoroughly pasteurized and ripened with 
a ure culture of Bad. lactis acidi, has the best keeping quality. If 
frh, sweet, clean cream is pasteurized, the butter will have better 
k^ing quality than when made from the same cream pasteurized and 
ri ned with a pure culture. This is evidence that not only the bac- 
teli other than Bact. lactis acidi are harmful, but that this organism, 



has usually been considered without influence on the keeping 
lity, must be classed as one of the factors in the decomposition of 



tH 

q 

buer. 

[t has been shown that the bacterial content of the water used for 
th washing of the butter has an influence on the keeping quality. If 
th water is of surface origin and contains the bacteria peculiar to these 
ty is of waters, its influence may be marked and some method of treat- 
m t must be followed. Filtering or heating the water has been re- 
soled to, the latter with marked success. A pure water will contain 
sopw bacteria that they will not exert any noticeable influence on the 
koing quality of the butter. 

(Storage temperature also has a marked influence on the deterioration 
obliges in butter. Modern butter-storage rooms are kept below 
the butter deteriorates slowly during storage at these tempera- 
but on removal undergoes change much more rapidly than 
w< Id have been true before storage. Another factor that is of influence 
in c keeping of butter is the amount of salt used. In salted butter, the 
co ained water is a concentrated brine; in such a medium most forms 

Bacteria are unable to grow. Small packages deteriorate more 

27 



4l8 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

rapidly than large ones, because the proportion of the mass of butt 
exposed to the air is relatively greater. Exposure to light is al 
claimed to exert a harmful influence. Antiseptic substances such 
borax and boric acid have a marked effect on the deterioration chang< 
The New Zealand and Australian butter exported to the English rm| 
kets is treated with preservatives. 

A large amount of experimental work has been done in order 
determine the effect of specific organisms on the keeping quality 
butter. The results obtained have not been definite and it is not certa 
that the organisms employed are constantly concerned in the deterioi 
tion changes. It is very probable that both bacteria and molds excj 
an influence. The chemical changes that take place in the spoiling 
butter are no better known than are the causal factors. It has be 
asserted that there is a decomposition of the glycerides with a resulti 
increase in free acids. It has been shown that this does not alwa 
occur; that a butter may be in an advanced state of decomposition ai 
its content in volatile acids not be higher than when fresh. Two typ 
of changes are usually distinguished, rancidity and the appearance oJ 
tallow-like odor. The latter may be due to purely chemical facto 
while the former is quite certainly biological. 

Moldy butter is a frequent trouble encountered by thebutter-maki 
If the butter is not salted, molds may develop just below the surfa< 
The most usual form of mold to appear is one with black hyphae; t 
slightest development of which will be evident on the butter. In t 
case of salted butter, mold on the butter itself is very rare, due app; 
ently to the concentration of the brine in the butter. The parchme 
paper in which print butter is wrapped and with which the butter ccl 
tainers are lined is an excellent substratum for mold growth. If t 
papers and containers are badly contaminated with mold spores, 
they have been kept under such conditions as to permit of a limit 
amount of growth before they are used, the development of the mold 
the paper after it is brought into contact with the butter is likely to 
rapid, even at low temperatures, and the butter is likely to reach t 
market in an objectionable condition. The paper may be render 
free from molds by placing it in water which has been heated to at le 
80. Butter tubs are scalded, steamed, or soaked in brine or treat 
with a dilute solution of formalin in order to destroy the mold spoi 
that may be present. The most efficient manner of preventing troulj 



THK RELATION OF MICROORGANISMS TO BUTTER 419 

to coat the inside of the butter tub with paraffin. This prevents 
rouble from the container but not from the paper. 

PATHOGENIC BACTERIA IN BUTTER 

If the milk contains pathogenic bacteria, they are certain to pass 
ito the cream and be incorporated in the butter. It is not believed 
Lat butter is an important agent in the distribution of the organisms 
I tuberculosis and typhoid fever, although both are able to exist in 
llted butter for over two months. Foot-and-mouth disease is said 
|: caused in humans by the use of butter made from the milk of 
fected animals, but this may still be regarded as a mooted 
lestion. 



CHAPTER III* 

THE RELATION OF MICROORGANISMS TO CHEESE 

GENERAL 

Cheese consists of the fat and casein of milk, together with thi 
insoluble salts; however, along with these constituents are carriec 
some of the moisture of milk, in which are dissolved small quantitie 
of sugar, albumin, and salts. The amount of moisture and solubL 
constituents found in cheese is determined by the amount of whe^ 
incorporated in the curd. 

In the process of making cheese, it is necessary to curdle the milk 
thus enabling the separation of the casein and fat from the milk serum 
Two methods are employed to accomplish this purpose, and, as ; 
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 may be accomplished bj 
allowing the milk to undergo acid fermentation, either spontaneous!) 
through the action of the normal flora of the milk, or through thi 
addition of pure lactic cultures. Most acid-curd cheeses are read} 
for use as soon as the whey has been removed by draining and thi 
curds salted. Acid-curd cheeses are not commercially important 
They are made for local consumption and are to be classed as a forn 
of sour milk. They owe their flavor to the products of the acid fer 
mentation, 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. Ove 

* Prepared by E. G. Hastings. 

420 



"our hun 



THE RELATION OF MICROORGANISMS TO CHEESE 421 



lour hundred kinds of rennet-curd cheeses are made, but only twelve 

fifteen are of great commercial importance. With few exceptions, 
hey are made from cow's milk. From the same raw material milk, 
ennet, and salt therefore, a wide variety of products, differing 

1 texture, taste and odor, is obtained. This fact indicates the im- 
ortance of biological factors in the changes the curd undergoes during 
he 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 

ft in the curd during the making of the cheese. The two great groups 

: rennet-curd cheeses gradually merge into each other in varieties 

lat by some are classed as hard cheese, by others as soft cheese. 

The rennet-curd cheeses, as a rule, are at first tough and rubber-like 

texture. The curd, which is not easily digested, is quite in soluble 

water and is devoid of flavor and aroma. The curd must pass 

rough a complete series of chemical and physical changes, which 

ter its texture, solubility, and digestibility, and give to it a flavor 

id aroma by which the different kinds of rennet-curd cheeses are 

pecially to be differentiated. 

In the hard cheeses the factors concerned in these changes act in a 
liform manner throughout the entire mass of the cheese, making 
possible to manufacture such cheeses in any desired size. In the 
se of the soft cheeses, the ripening changes are largely due to agents 
Mich grow only on the surface; the products of such agents by means 
< diffusion gradually affect the entire mass. In order that this may 
1 ;e place within a reasonable time, it is essential that these cheeses 
1 made in small sizes. Then, too, the soft texture of such cheeses 
i.kes it impossible to handle them commercially in large sizes. 

CONDITIONS AFFECTING THE MAKING OF CHEESE 

I QUALITY OF MILK. In the curdling of milk by rennet the solid 
jlies present in the milk are retained in the curd, thus the fat 
)ules are held, as are also the bacteria. The latter continue to 
|w as they would have done in the milk except that growth 
ilst take place in the form of colonies as in the solid culture media 
Ojthe bacteriologist. The bacteria, however, produce the same fer- 
nlitation in the curd as they would have done in the uncurdled milk. 



422 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

The butter-maker can control, through pasteurization and the u 
of pure lactic cultures, the fermentation of the cream. The pasteur 
zation may be so efficient as to destroy all non-spore-forming bacter 
since the quality of the product will not be impaired by the use 
temperatures approximating the boiling point. The cheese-mak 








* * 
' 5* 

. '4* 



FIG. 134. The type of curd obtained from milk in which the acid-forming fl( 
consists largely of organisms of the B. coli-aero genes group. Many gas holes a 
few irregular shaped, angular, mechanical holes due to imperfect "mattini 
(Original.} 

is much more dependent on the original quality of the milk, since 
has not been found possible to make most of the important varieties 
cheeses from pasteurized milk. If undesirable forms of microorganisi 
are present in the milk, they will pass into the cheese and there produ 
their harmful effects. Through the addition of pure cultures 




IflCROORG \MSMS TO CHI 



423 



;,/;' to the milk, the proportion of desirable bacteria ran 

.-ed ami a partial control of the fermentation thu^ secured. 

rur Oi'MUY OK MIIK. Methods by which the cheese 

m determine, in a rough manner, the kinds of bacteria present 




unl obtained from milk in whirh tin- .u M foriui: 
. holly of /''.:. ;. ,' | - hoK-s and nn m.ii kc.l mc Imnical 

the run! ha, "ni.ith-.l" almost ju-rfr. tly. (Original.) 



devised. The b.uleria ino^l dn.i.led and most frequently 
sent are those f the />. < <>li- <!<-r<><-ii, -\ group. 
I'he mi'tliod mo>t fre(|iientl\ usel for their detection > 

ling a simple of the milk to be tested ;( temperatures ranging 
t<> 40 for a few hour-, and noting the type of curd that U 
Milk Miitable for i'hee>e making should show the solid eurd 
;./<// group, while gtSS} Mirds 01 




424 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

and partially digested curds are indicative of bacteria that are likely 
be harmful in the cheese. 

An improvement over the fermentation test of foreign origin h 
been devised by Babcock and Russell and is known as the Wiscons 
Curd Test. It has for its basis the same principle as the simple ferme 
tation test; however, a modification is introduced; the milk is curdled 1 
the addition of rennet and the curd is cut and drained to free it from t 
whey as completely as possible. 

The undesirable organisms most likely to be present in milk a 
those of the B. coli-aerogenes group; therefore the jars containing tl 
curds should be kept at temperatures, 35 to 40, that will favor th( 
development. The great advantage of the Wisconsin Curd Test is i 
greater delicacy, since the bacteria are concentrated in a small volum 
and thus their presence is more evident than would be the case in tl 
larger mass of curd obtained when no rennet is added. The curd a 
also be removed from the jar, cut, tasted, and its texture determined, <. 
of which aid in judging the quality of the milk. The curd, should ha^ 
a clean acid odor and taste; it should be free from sliminess on the su 
face, and possess a uniform texture. Such a curd can be obtained on 
in the presence of a considerable number of lactic bacteria. Vei 
clean, fresh milk is likely to give an undesirable result, since mi 
always contains microorganisms which will grow rapidly at the hi 
temperature in the absence of the acid-forming bacteria and which w 
usually produce undesirable flavors in the curd. This fact should 1 
kept in mind in the testing of market milk. 

RIPENING OF MILK.* The methods for the determination of acidil 
in milk have very considerable limits of error. It is not possible 1 
detect any increase in acidity until the number of acid-forming bacter 
has increased to hundreds of thousands per c.c. Originally it w; 
thought that no acid was produced by the growth of the acid-formir 
bacteria during the initial stages of their development. This peric 
during which bacterial proliferation was taking place, but without a 
apparent increase in acidity, was known as the "period of incubation. 
It is now certain that this rests upon our inability to detect sma 

* In order to illustrate the r61e of microorganisms in the making and ripening of cheeses, 
somewhat detailed summary of the present knowledge concerning their action in Cheddar chee 
will be given. Many of the factors concerned in the ripening of this kind of cheese also functi< 
in the ripening of other rennet cheeses. In their description only such additional factors net 
be considered as are not active in Cheddar cheese. 



THE RELATION OF MICROORGANISMS TO CHEESE 425 

peases in acidity. The Cheddar cheese-maker desires milk that shall 
itain such a number of acid-forming bacteria that during the opera- 
is that are carried on in the first part of the cheese-making process 
re amounts of acid shall be formed in the curd. He thus wishes to 
>w, as accurately as can be determined under the conditions found 
the factory, the number of bacteria in the milk which he is to use. 
information is gained either by the titration of the milk with a 
idard alkali solution or by determining the time required for the 
ig of a definite quantity of milk at a definite temperature by a 
quantity of rennet. Very much smaller increases in acidity 
be detected by the so-called rennet test than by titrating 
milk. If the milk shows the desired acidity when it reaches the 
];ory, the making process is immediately begun. If the milk is too 
||et, or in other words, too low in its bacterial content, bacterial 
rth is favored by warming the milk to temperatures most favorable 
lactic bacteria, 30 to 32, and by the addition of pure cultures 
<3act. lactis acidi which are identical in nature and the method of 
pagation with those used in butter making. The development of a 
jit acidity is known as the "ripening" of milk, 
[n order to insure proper rennet action the maker of Cheddar cheese 
cUjres the milk to have an acidity of about 0.2 per cent. He thus 
nes milk that has passed through the period of incubation and in 
:h the acidity has begun to increase. 
"URDLING or MILK. Under the influence of a favorable tempera- 
tu and the slight acidity, the milk is quickly changed by the rennin* to 
a m, jelly-like mass that is cut, with appropriate knives, into small 
cdjs. The curd encloses over 80 per cent of the bacteria in the milk. 
Ti same factors that favor the curdling of the milk favor the shrinking 
of ie curd and the expulsion of the whey from the cubes. The develop- 
m t of acid within the curd is rapid, due to the concentration of large 
nubers of bacteria in a small volume and to the favorable environ- 
m<t. During the six to eight hours that elapse between the curdling 
of ie milk and the pressing of the curd, the increase of acidity is over 
o.] >er cent per hour. The following table gives the acidity of milk and 
th ivhey expressed from the curd at various stages in the making of a 
1 Cheddar cheese. 

."he rennet used in cheese-making is obtained by extracting the abomasum, the true diges- 
tovcomach of the calf, with a solution of sodium chloride. The extract contains two enzymes 
i cring or curdling enzyme, rennin, and a proteolytic enzyme, Pepsin. 



426 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

Acidity of milk before adding rennet o. 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. 16-0. 18 per cent. 

Acidity of whey when curd is packed o . 24-0 . 30 per ,cent. 

Acidity of whey when curd is milled 0.65-0.75 per cent. 

Acidity of whey when curd is salted 0.90-1 . 10 per cent. 

MANIPULATION OF THE CURD.* The curd particles at first sb 
little tendency to cohere; but, as the acidity increases, the nature of t 
curd changes, and, when the whey is removed, the pieces of curd so 
cohere and ultimately form a single mass in which the original cubes 
curd cannot be detected. The fusion of the curd particles is known 
" matting" and is an important step in the Cheddar process. The la 
of acid formation within the curd prevents matting while the curd is 
the vat, and may even render difficult the fusion of the particles un< 
pressure. The nature of the change which the curd undergoes at t 
stage in the manufacture is not well understood, but probably is due 
a combination between the paracasein and the lactic acid, the result 
compounds differing from the paracasein in physical properties and 
solubilities. 

RIPENING OF CHEESE. Cheese in ripening undergoes profou 
physical and chemical changes under the influence of a number 
factors, which for purposes of discussion may be divided into two grou 
those by which the content of soluble nitrogen in the cheese is increas 
and the digestibility enhanced; and those which cause the formation 
flavoring substances. During the ripening of the cheese the ma 
can do little toward the control of the factors which ultimately det 
mine its commercial value. As in butter, the flavor is the most imp 
tant characteristic of the ripened cheese and the most difficult 
control. 

Theories of Cheese Ripening. Many theories have been advam 
to explain the changes that occur during the ripening process. Ducla 
a French microbiologist, studied the bacterial flora of Cantal die 
by aid of the crude methods available before the introduction of 
gelatin-plate method. By the use of the dilution method, us 
bouillon as the nutrient medium, he isolated a number of kinds 
spore-forming bacteria. The organisms formed two enzymes, < 
a curdling enzyme related to rennin, the other a proteolytic enzy 

* Cheddar cheese. 




RELATION OF MICROORGANISMS TO CHEESE 427 

was given the name casease. A chemical study of the by- 
p ducts of the organisms, when growing in milk, revealed a number 
o rompounds that had previously been found in ripe cheese, such as 
ledn, tyrosin, and the ammonia salts of acetic, valeric and carbonic 
a is. The cultures often possessed a cheese-like odor. These facts 
1<, Duclaux to believe this class of organisms were responsible for the 
Fining of the hard cheese in question. The generic name Tyrothrix 
J; applied on account of the supposed relation to cheese. This term is 
s ,1 found in current bacteriological literature. The methods employed 
U Duclaux were such as favored the growth of the liquefying, rather 
tin the acid-forming bacteria. To the latter more recent investi- 
cr ors have devoted attention. 

iThe theory that the proteolytic bacteria function in the ripening 
ojhard cheese has been more recently emphasized by Adametz. It 
hiufficient to say that the number of spore-forming proteolytic bac- 
ti.a in cheese is not sufficiently large, nor is their presence so constant 
tit any importance can be attached to them. Any agent to be con- 
s^red as a factor in the ripening process must be present in every 
c ese in sufficient numbers to account for the change for which it is 
c .sidered responsible. Such agents should be capable of demonstra- 
1 1. It should be remembered that, by following the rules laid down 
b the practical maker, a normal cheese can invariably be made, 
hice the factors of importance in the ripening must be constantly 
psent in the milk or rennet. It is doubtful whether the liquefying 
tyteiia will satisfy this requirement. It has been shown by de 
frudenreich that such organisms, even when added to milk in large 
nnbers, exert no influence on the ripening of hard cheese, since the 
editions within the cheese are not such that growth can occur. 
|De Freudenreich, a Swiss microbiologist, by the aid of modern 
nthods, demonstrated the constant presence of certain classes of 
ai-forming bacteria in Swiss cheese, and to them ascribed an impor- 
ts t role in the ripening of this hard cheese. He was led to this con- 
C:3ion by their great numbers in the fresh cheese, and by the fact 
cheese made from milk drawn under aseptic conditions, which 
n tains no lactic bacteria, do not ripen; through the discovery, 
at certain of the lactic bacteria, predominating in Swiss cheese, 
of the Bad. bulgaricum group, exert a solvent effect on the 
of milk, although they are devoid of action on gelatin. 



428 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

Babcock and Russell demonstrated the presence of an inner t 
proteolytic enzyme in milk, to which the term galactase was appl 
This enzyme can be demonstrated by preserving a sample of fr 
milk with chloroform or other mild antiseptic. At 37 curdlg 
occurs in three to four weeks; the content of soluble nitrogen in 
milk is slowly augmented. The presence of this proteolytic enzy 
together with the fact that a normal cheese cannot be made from n t 
in which this enzyme has been destroyed by heat, led these inve - 
gators to consider this inherent enzyme of milk an important fac r 
in cheese ripening. 

Present Knowledge of Causal Factors* The r61e of certain factis 
in the ripening of Cheddar cheese has been established beyond doi t 
by the chemical and bacteriological investigations of recent ye? 
It is certain that acid-forming bacteria are essential factors in 
ripening of this kind of hard cheese, and probably all kinds of ren 
cheeses. 

As has been shown the growth of acid-forming bacteria is ra 1 
during the making of Cheddar cheese. The growth continues dur r 
the pressing and subsequent thereto; the maximum number of la( : 
bacteria is found when the cheese is one to five days old. As many 
1,500,000,000 per g. of the moist cheese have been demonstrat 

Causes of Proteolysis. The proteolytic action of rennet extract 
the paracasein of cheese was demonstrated by Babcock andRuss 
and by Jensen. This property is due to the fact that rennet extr; 
also contains the enzyme pepsin, which for its action outside the be 
requires conditions similar to those which obtain in the stoma'j; 
in other words, the presence of sufficient acid to activate it. Thehyd 
chloric acid secreted by the walls of the stomach acts as the activati 
agent in the body. The acidity resulting from the fermentation 
the sugar in the curd is sufficient to activate the pepsin. Under 
influence the paracasein is partially converted into soluble decompc 
tion products such as albumoses and peptones. In the absence 
acid-forming bacteria no acid is formed; consequently the pepsin d<> 
not become active and no proteolytic effect is produced. Under thi : 
conditions the curd remains tough and elastic and the solubility 
not increased. It is thus evident that acid-forming bacteria are esst 
tial factors in cheese ripening. The pepsin of the rennet extract a 

*Cheddar cheese. 



THE RELATION OF MICROORGANISMS TO CHEESE 



429 



tiu;alactase suffice to account for the initial proteolysis of the para- 
caan. Since neither of these enzymes forms ammonia, which is 
alvjys found in ripe cheeses, some other factor must be responsible 
for he production of this compound. It may owe its origin to micro- 

tns not yet discovered. 

retention of Putrefaction. The various stages in the decomposition 
of ilk have been outlined in a previous chapter. Briefly they are as 
fol ,vs: The first evident change is the curdling due to the acid-forming 
saoria. Succeeding this, the acid, semi-solid mass or curd is a favor- 



abl 



substratum for the characteristic mold of milk, Oidiumlactis, which 



W ^r ^ : 




A B 

G. 136. Proteolytic action of rennet extract in the absence and in the presence 
of id-forming bacteria. A, sterile milk agar; a strip of filter-paper treated with 
ren\ was allowed to remain on the medium for one hour at 37. No digestion of the 
cas. . B, milk agar inoculated with Bad. lactis acidi; incubated for twenty-four 
hot at 37, then treated as A. True digestion of the casein is indicated by the 
dec ig. (Original.) 

od forms a white, velvet-like layer over the surface of the milk. Like 
Ir molds, this form can use acids as a source of energy. The acid is 
thd oxidized to carbon dioxide and water, and thus the reaction of the 
mi is slowly changed until a point is reached which allows the putre- 
fadve bacteria, that have remained dormant during the period of un- 
f arable environment, to develop. The curd is accordingly pepton- 
and putrefaction occurs. If the acid reaction is maintained 
igh the prevention of mold growth, the milk will be preserved from 



. 

430 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

the attacks of putrefactive organisms and will remain unchanged for 
unlimited time. 

The second r61e of the acid-forming bacteria in cheese is to prot t 
it against the putrefactive organisms that are constantly present 
milk and hence in cheese. The acid reaction of the cheese, due to 
persistence of lactic acid, or to the formation of volatile acids after = 
initial fermentation, is sufficient to prevent the growth of the putrefj- 
tive bacteria within the cheese. If the cheese is made from milk wh i 
contains no acid-forming bacteria and few putrefactive ones, or if |e 
sugar is removed from the curd by washing it with water, the cheese ^iJ 
not ripen since there is no acid to activate the pepsin; the curd will - 
main in much the same condition as when it was removed from e 
press. Cheese made from milk containing no acid-forming bacteria t 
many putrefactive bacteria is likely to undergo putrefaction, since e 
latter class of organisms finds conditions for growth in the absenci if 
an acid reaction. Such a condition is rarely noted in a hard ch( e 
under normal conditions, but may be produced experimentally. ' !e 
biological acid may be replaced by other acids added to the curcjn 
appropriate amounts, since these will activate the pepsin and pro :t 
the cheese against the attacks of putrefactive bacteria; but it is not j> 
tain that the cheese will develop a normal flavor when lactic aci is 
replaced by mineral acids. 

Other Groups of Bacteria in Cheese. It has been shown at the \ ; 
consin Experiment Station that other groups of bacteria are constai 
present in Cheddar cheese. The development of certain members of 
Bad. bulgaricum group occurs somewhat later than that of th6 1 1. 
lactis acidi group. It occurs largely after the sugar has disappeaji. 
Their numbers approximate those of the Bact. lactis acidi gr<p. 
Coccus forms are also found in great numbers in the cheese. ' 
probable that these two groups may be responsible for the amm 
production, since typical cultures of both groups are able to proc :e 
small amounts of ammonia in sterile milk. 

Flavor Production in Cheese. The factors that have been discu 
are undoubtedly the most important ones concerned in the proteo 
of the curd, and are thus the factors concerned in the changes of text 
solubility and digestibility. The flavor, which develops during 
ripening process, has been regarded as due to the proteolysis of the p 
casein. A thoroughly ripened cheese contains a large amount of in 



RELATION OF MICROORGANISMS TO CHEESE 431 

and related compounds. It was thus natural to consider the flavor 
dj to these simple products of protein degradation. More recently it 
hi been discovered that the intensity of flavor does not necessarily cor- 
riond to the content of the cheese in these products; indeed a cheese 
ny have a high content of nitrogen as ammonia and yet be low in 
I'or. 

The Wisconsin Experiment Station has found that the volatile fatty 
a Is of Cheddar cheese increase as the ripening progresses. In the 
fc owing table are given the data obtained from the detailed study of a 
n mal Cheddar cheese. 

ACIDS IN ioo G. OF DRY MATTER 

c.c. of N/io alkali neutralized 



3 days 42 days 3 months SM months 10 months 



tic acid 


84. OQ 


00.28 


124 oo 


103. 70 


74- IO 


tic acid 


11-59 


29.44 


24.25 


25.86 


12 .64 


oionic acid 


0.41 


2.15 


3-42 


1.07. 


2.63 


yric acid 


0.73 


2.17 


3.50 


4.82 


5-45 


role acid 


o.oo 


0.36 


0.96 


1.25 


2.23 



It will be noted that the content of the higher volatile acids, those 
e: ecially marked in odor, continually increases. It is possible to sepa- 
ir^ other volatile compounds found in cheese from the volatile fatty 
a Is by distilling with steam, neutralizing the distillate with an alkali 
a,l redistilling; the second distillate will contain the alcohols and esters 
pi sent in the cheese. Such a distillate prepared from Cheddar cheese 
ie ound to possess the characteristic aroma of the cheese in question. 
Tie esters it contains are largely those of ethyl alcohol. The acid-form- 
ii bacteria, as stated previously, produce varying amounts of volatile 
a,is and slight amounts of alcohols and esters. It is likely that the 
part of the volatile compounds found in the ripening cheese is 
in fermentations which take place subsequent to the initial 
tation of the lactose. The flavor of Cheddar cheese, therefore, 
its origin very probably to the fermentation of the lactose, and to 
further change which the products of the Initial fermentation un- 
ro under the influence of biological factors yet unknown. That some 





432 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

biological factor is concerned in the production of flavor in Chedd; 
cheese is indicated by the fact that if changes are made in the methoc 
of manufacture, changes in flavor are likely to result. If the salt 
omitted, the typical flavor does not appear. This can be explains 
only by the action of the salt on certain types of bacteria, which, 
its absence, are able to grow and produce compounds that are n< 
found in a normal cheese. Apparently the methods of manufactu 
establish a certain equilibrium in the bacterial life which results in tl 
production of definite substances in amounts varying within certa 
limits. If any condition is varied too widely, a deviation in the micr 
bial balance is produced and the products formed in the cheeses a 
changed in kind or in amounts, either of which may result in a change 
flavor. 

ABNORMAL CHEESES 

The development of a normal texture and flavor in Cheddar chee 
is largely dependent on the presence of definite types of bacter 
If these are replaced, wholly or in part, by other kinds, the product 
likely to suffer in texture, flavor or both. As has been emphasiz 
previously, the bacterial content of the milk is of the greatest importan 
in cheese, since the organisms in the milk pass into the cheese and th 
produce the same products as they would have done in the uncurdl 
milk. All abnormalities of the cheese so far as they are occasioned 
bacteria are due to the abnormal flora of the milk. To the r 
material the maker must direct his attention if a fine product is to 
prepared. 

GASSY CHEESE. The most frequent trouble encountered and t 
one of greatest economic importance is the fermentation caused 
organisms belonging largely to the B. coli aero genes group. It has be 
seen that these produce in milk gases, such as carbon dioxide a: 
hydrogen, and offensive smelling and tasting compounds. In che( 
similar compounds are formed by these organisms; the gas causes t 
more or less abundant formation of holes which give to the cheese jud 
an indication of what may be expected with reference to flavor, i 
milk contains some of the gas-forming organisms, but it is only wh 
they are numerous that marked injury is done. 

Gassy cheese may also be due to the presence of lactose-fermenti 
yeasts which are usually found in milk in such small numbers that th 



THE RELATION OF MICROORGANISMS TO CHEESE 433 

pnot compete with the lactic bacteria in the fermentation of the sugar 
; the cheese. At times the number may be increased to such an extent 
at the major part of the sugar is fermented by them, alcohol and car- 
Im dioxide being produced. An outbreak of gassy Swiss cheese was 
und by Russell and Hastings to be due to such yeasts that had gained 
iitrance to the milk from the whey-barrels because of careless washing 
i the milk cans. The cheese makers of the country are realizing the 
iportance of the contamination of the milk from the transportation of 
taey and milk in the same can. The most practical means of prevent- 
g trouble from this practice is to heat the whey to 68 as it passes from 
e cheese vat to the storage tank. This temperature destroys the 
.rmful microorganisms, and if the storage tank is kept in a sanitary 
ndition the whey is sweet when returned to the farm in the milk can. 
has been demonstrated that such a treatment of the whey results in a 
arked improvement in the quality of the product. 

MISCELLANEOUS ABNORMALITIES OF CHEESE. Bitter cheese is 
oduced by bacteria that form a bitter principle. An outbreak of 
tter cheese investigated by Hastings was found to be due to the re- 
acement of the normal acid-forming flora by a lactic organism which 
oduced such an intense bitterness as to mask the acid taste in the 
ilk and cheese. 

Colored cheese is produced by chromogenic bacteria. In case the 
lonies are not numerous and the pigment formed is not soluble in any 
the constituents of the cheese, the color will appear as colored specks, 
ch as the rusty spot investigated by Connel and Harding, which is due 

red forms of B. rudensis. If the colonies are very numerous, or if 
e pigment is soluble, the curd may be uniformly colored. 

Putrid cheese is caused by the absence of sufficient acidity to hold 
e putrefactive bacteria in check. This trouble is rare in cheddar 
, since such cheese is made from ripened milk. Fruity flavors are 
:ed to be due to yeasts which form fruit esters. 

Moldy Cheese. In the moist air of the curing-room the cheese forms 
i excellent substratum for the growth of common molds whose pig- 
ented spores discolor the surface of the cheese and thus impair its 
ilue because of the appearance rather than by any effect in the flavor. 
heddar cheese is protected effectively from molds by dipping the 
icese, when two or three days old, in melted paraffin which excludes 
e air from the spores on the surface of the cheese. 



- r- 

3 

Mn 






434 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

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-calle< 
"foreign cheeses" in this country; however, with some "foreigi 
cheeses" the manufacture has been successful only in such localitie 
where such types originally developed, and where the climate and othe 
conditions are favorable to a normal ripening. 

CHEDDAR CHEESE. Cheddar cheese, treated in much detail in th 
foregoing considerations because it is the most important America) 
cheese, is made in England and her colonies and in the United States 




FIG. 137. Typical development of "eyes" in Swiss cheese. (Original.} 

It appears in many varieties and by the American consumer is ofte 
called American cheese in distinction from the foreign cheeses. Thi 
distinction is not wholly applicable at the present time. 

EMMENTHALER CHEESE. Swiss or Emmenthaler cheese originate 
in Switzerland, but is now made in various other countries. Alarg 
amount is made in Wisconsin, Ohio and New York (Fig. 137). It : 
characterized by its sweetish flavor and by the so-called "eyes," v whic 
are holes formed by gas, produced in a fermentation that occurs subst 
quent to the fermentation of the lactose. The number of eyes is nc 
large and they are evenly distributed throughout the mass of the chees 
except near the surface. 

The cheese is made from as fresh milk as it is possible to secun 
The rennet used is prepared by placing a piece of the dried rennet i 
whey and incubating the same for twenty-four to thirty-six hours at 30 



THE RELATION OF MICROORGANISMS TO CHEESE 435 

us is employed in place of the commercial extract used by the Cheddar 
iiker. It serves not only to curdle the milk, but adds to it a large 
i.mber of acid-forming bacteria that have grown in the rennet solution 
(jring the period of incubation. The number is not, however, suffi- 
int to cause any development of acid during the making process which 
(frers from the preparation of Cheddar cheese in the method of firming 
i|e curd. This is accomplished by heating the curd to 52 to 60, and 
] cutting it into pieces scarcely larger than grains of wheat. The salt 
iiapplied to the exterior of the cheese by immersion in brine for one to 
l|ar days and by sprinkling salt on the surface. 
I The fermentation of the lactose proceeds rapidly during the pressing 
dd subsequent thereto, so that within a few days the sugar has disap- 
I ared. The lack of the development of acid during the making probably 
juilts in a somewhat different relation between the acid and protein 
m that existing in a Cheddar cheese, which, together with the ab- 
ice of salt gives a somewhat different environment, thus making possi- 
the development of a different flora. There is no ground for believing 
tit the agents concerned in the proteoly tic changes are other than those 
Ut function in Cheddar cheese. The flavor must, however, be due to 
<|aer factors; this is indicated by the fact that if the milk is ripened 
ij in the Cheddar process, or if salt is added to the curd the flavor will 
< proximate the Cheddar flavor. The formation of the eyes is inhib- 
jd by salt, as is indicated by their relative scarcity in the outer layers 
< the cheese. Jensen has shown that the eyes are due to the f ermenta- 
t>n of lactates with the formation of propionic and acetic acids, and 
rbon dioxide. The causal organism is found in the milk and the whey 
j met. It is believed that lactic bacteria of the Bact. bulgaricum group 
important factors in the ripening of Swiss cheese. They are pre- 
in large numbers in the rennet and cheese. Mixed cultures of an 
m of this group and a mycoderma are used with success for the 
tion of the whey in which the rennet is to be soaked. The exact 
of this form of lactic organism is not known; de Freudenreich con- 
d them to be concerned in the proteolysis of the paracasein, 
he had found that the content of sterile milk in soluble nitrogen 
d when inoculated with the organism. It is probable that 
ormation of eyes and the flavoring compounds are due, in part at 
ist, to the same factors. 
In the other kinds of cheeses to be described, the r61e of the acid- 







43 6 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

forming bacteria is similar, if not identical, to their r61e in Chedda 
cheese, i.e., in activating the pepsin of the rennet and in preventin 
the growth of putrefactive bacteria. The factors concerned in flavo 
development are different. 

ROQUEFORT CHEESE. This cheese, which is prepared almos 
exclusively in the Department of Aveyron in southern France, is mad 
from sheep's milk. Its most striking characteristic is the marble 
or mottled appearance of the interior, due to the growth of a mole 
Penicillium roqueforti, Thorn. The curd is inoculated with the mole 
when it is placed in the press, by sprinkling the curd with bread crumt 
on which the mold has grown. The growth and sporulation of th 
mold in the interior of the cheese are favored by piercing it wit 
small needles, thus admitting air. The characteristic flavor is du< 
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 th 
moisture and temperature conditions of the curing room. Th 
emphasizes the importance of biological factors in the ripening proces 

GORGONZOLA CHEESE, prepared in Italy from cow's milk, an 
STILTON CHEESE, made in England are similar to Roquefort i 
appearance and contain the same mold Penicillium roqueforti. 

CAMEMBERT CHEESE. The soft cheeses are best represented b 
this important French cheese made from cow's milk by the additio 
of rennet. The milk is ripened to an acidity of 0.20 to 0.25 per cer 
before the addition of the rennet. The curd, which thus contair 
many acid-forming bacteria, is neither cut nor heated in order to n 
tain the maximum amount of whey. The curd is placed in sma ( 
hoops and allowed to drain without pressure. Salt is applied to tJt 
surface of the cheese. 

The milk sugar is rapidly fermented and the resulting acidit 
is high, for the cheese contains 60 to 70 per cent of moisture whe 
fresh and 50 per cent when ready for consumption. The high moistui 
content of the cheese and the humidity and temperature conditior 
of the curing room favor the rapid development of microorganisn 
on the surface of the cheese. Both molds and bacteria thrive und< 
the influence of these favorable conditions, changing the cheese t 
a soft, smooth and butter-like mass, while a characteristic flavor 
developed. 



THE RELATION OF MICROORGANISMS TO CHEESE 



437 



three or four days the cheese becomes covered with the growth 
Wium lactis; the characteristic mold of Camembert cheese, Peni- 
ium camemberti, appears later, within five to six days. These 
fds reduce the acidity of the curd, and through the enzymes, which 
produce and which gradually diffuse into the cheese, proteolyze the 
very completely. The appearance of the cheese when cut in- 
ites the depth to which the enzymes have penetrated; when the 
ire mass is acted upon, the cheese is ready for use. The reduction of 
acidity by the molds exposes the cheese to the attacks of putre- 
ive bacteria and it soon becomes unfit for use after it is completely 
ri|;ned. A number of different kinds of bacteria are found in the 
ly surface layer, but their r61e is not known. 

: development of the characteristic flavor and aroma is dependent 
certain relation between the various biological agents concerned 
inne ripening. This balance is dependent on very narrow conditions 
oiemperature and humidity; slight changes in these environmental 
ccditions favor or retard the individual types in varying degrees. If 
tr. equilibrium essential for the development of typical flavor is 
dtaoyed this cheese fails to ripen properly and is of low value. The 
miufacture of Camembert cheese is a delicate problem in the ecology 
ohicroorganisms, and because of this fact the manufacture is attended 
w i greater difficulties than is the case with most types of hard cheese. 



CHAPTER IV* 

RELATION OF MICROORGANISMS TO SOME SPECIAL 
DAIRY PRODUCTS 

GENERAL 

There is a number of special dairy products which do not normal) 
come into a discussion of market milk, butter or cheese, but which a] 
of considerable importance. A book of this sort would not be comple 
without a discussion of some of these products from the bacteriologic 
standpoint. Some of these special products have been developt 
as commercial enterprises and the processes of manufacture have be< 
zealously guarded as trade secrets. The result is that there is very litt 
available data on the manufacture of these products and very litt 
authoritative knowledge about their bacteriological condition. It 
therefore, difficult to give a full discussion of the microbiology of the 
products. A few of the more important ones will be discussed, howeve 

CONDENSED MILK 

There are at least three quite distinct kinds of condensed mi 
made under conditions which result in an entirely different bacteriolc 
ical condition in the finished product. These different products DHL 
therefore, be considered separately. Condensed milk means simp 
milk from which a large part of the water has been removed, th 
decreasing its bulk, the purpose being to lessen the cost of transportati 
and to increase the keeping quality of the product. Water is remov 
from milk by some process of heating, either with or without vacuu 
the heating process being more or less equivalent to pasteurizatk 

SWEETENED CONDENSED MILK. This product is made by reduci 
cow's milk at the ratio two and one-half to two and three-fourl 
parts of fresh milk to (5ne part condensed milk, by means of h( 
and the addition of cane sugar. It is then put up in sealed ca 

* Prepared by W. A. Stocking. 

438 



;LATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 439 

is not intended to be sterile. The degree of heat to which it 

subjected is not sufficient to kill all of the microorganisms 
lesent and it is also subject to infection after the condensing 

completed. Cane sugar is added to the milk, making the final 
loduct contain about 25 per cent of water, 35 per cent milk solids 
d 40 per cent cane sugar. The low percentage of moisture together 
th the added sugar tends to preserve this product against the action 
c microorganisms. There may be some bacterial growth, the 
ipidity depending upon the temperature at which the product is 
Ipt, but it is usually slow and milk prepared in this way will keep 
jr a considerable time without undergoing marked bacterial changes. 
9me gas producing bacteria exist in the milk and if cans containing 
113 organisms are allowed to remain at warm temperatures, they will 
dvelop in spite of the large percentage of sugar, producing suffi- 
i|:nt amounts of gas to cause the ends of the cans to bulge out. Such 
dnsare known commercially as "swell-heads." 

UNSWEETENED CONDENSED OR EVAPORATED MILK. In this form of 
didensed milk approximately the same amount of moisture is removed 
a in the sweetened product but no sugar is added. The decreased 
^iount of moisture tends to prevent the rapid growth of bacteria, but 
t|.s is not enough to guarantee the keeping quality of the product. 
jiter the milk is condensed it is put into the can, hermetically sealed, 
id then placed in steam sterilizers and subjected to temperatures some- 
vat above the boiling-point. In this way the milk is heated a suffi- 
(jnt length of time to insure perfect sterilization of the contents of the 
cis. If this process is properly done, the finished product contains no 
ling microorganisms and from the bacteriological standpoint the milk 
snild keep indefinitely. 

Sometimes the unsweetened product is sold in bulk in cans. In 
tjs case it is subject to more or less contamination after heating and is 
ilt sterile, but because of the small amount of moisture and the concen- 
t.tion of the milk solids, the bacteria do not develop rapidly and if 
Ipt at a cool temperature, the milk will keep several days without 
forgoing appreciable biological fermentations. 

CONCENTRATED MILK. There is now on the market a form of con- 
(lised milk prepared by a different process, which is commonly known 
^concentrated milk. By this method the water in the milk is removed 
1 means of dry air instead of by vacuum as is the case of condensed 



440 



MICROBIOLOGY OF MILK AND MILK PRODUCTS 



milk. The milk is first heated and then air under pressure is forcec 
through it. By this process the milk is heated to a temperature o: 
60 (i4oF.), and this temperature maintained for two hours, durim 
which time air is forced through the milk causing violent agitation anc 
the removal of the moisture. At the end of this time the milk is re 
duced to one-fourth its original volume.* The result of this process is < 
pasteurized milk, with a marked reduction of the original germ content 
Investigations by Conn failed to show the presence of B. coll in mill 
prepared by this process. The reduction in the bacterial content of th 
milk is similar to that secured by other methods of pasteurization. N 
additional sugar is added to this milk so the product is, therefore, a pas 
teurized milk containing a small amount of moisture. Because of th 
small amount of moisture and the concentration of the milk sugar, th 
bacteria which survive the heating process do not grow rapidly at lo\ 
temperatures. The following figures will serve to illustrate the effect o 
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,OOO 

9.950 

IO,OOO 

5,OOO 



The rate at which the bacteria develop in this milk is shown by tt 





Number of sample 


Number of bacteria per c.c. 


2 days old 


4 days old 


6 days old 


I 
2 

3 
4 


l8,000 
55,ooo 

3,500 
4,400 


39,000 
28,000 

11,000 

5,270 


46,000 
39,000 

10,000 

4,630 


The lack of moisture and concentration of milk sugar prevents tl 
rapid growth of these organisms so that bacterial changes do not tal 



* Data furnished by H. W. Conn. 



OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 441 

rapidly as in ordinarily pasteurized milk retaining its normal 
moture. 

'OWDERED MILK. This product is produced by carrying the extrac- 
tidof the water farther than in the case of the condensed milks. The 
w* ;r is removed to a point where the milk solids can be reduced to a 
podered form. This product contains the original milk solids with a 
ve small percentage of moisture usually not more than 2^ per cent. 
Tire are several forms of powdered milk now on the market produced 
b) omewhat different methods. In some cases the moisture is removed 






the milk by its being exposed to a heated surface in a thin layer. 



Sc etimes the drying is done in vacuum. The resulting product is dry 
ar can be ground to the form of flour. 

Vnother process is to remove the moisture by spraying the milk by 
m ns of an atomizer into the top of a hot chamber, the moisture being 
re aved while the fine particles of milk are falling to the floor. By 
th process the product accumulates on the floor as a very dry flour and 
d( > not require any grinding. In the first process the heat is sufficient 
to asteurize the milk while in the latter process it is pasteurized before 
b( g subjected to the drying process. The powdered milks do not 
cl ft to be sterile but are preserved against subsequent action of micro- 
or misms because of the very low percentage of moisture which they 
cc-ain. It is probable that there is no appreciable increase in the 
ni iber of bacteria in milk flour and the product will keep for along time 
w lout undergoing bacterial fermentations. 

CANNED BUTTER AND CHEESE 

Some effort has been made to put up butter and cheese in hermet- 
idly sealed cans, the purpose being to increase the keeping qualities of 
tl products and influence the flavor by controlling the development of 
tl aerobic bacteria. Only a limited amount of bacteriological work 
h been done on these canned products and the biological changes 
\v .ch take place in them are not very well known. 

S:CIAL MILK DRINKS MADE BY THE ACTION OF MICROORGANISMS 

From time immemorial fermented or sour milk has been used as an 
aide of food. We are told that Abraham* placed "curdled milk" 

j' Genesis 18:8. The Hebrew word "hemah" translated in the English authorized version 
Bible "butter" means "curdled milk." Century Bible, Vol. Judges and Ruth, p. 72. 






442 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

before his guests and that Moses told the Israelites that curdled m 
was one of the blessings which Jehovah had given to his chosen peopl 
History also tells us that the wandering tribes of Arabia used fermen 
milk as a beverage. For centuries many of the tribes of eastern Eurc 
and western and middle Asia and parts of Africa have used sour m 
for food. Each of these regions appears to have had its own particu 
milk beverage resulting from the particular bacterial flora of the regi 
The sour milk products which are now on the market unde: 
variety of names have been derived from these original sour-m 
drinks of antiquity. Fermented milk beverages have become 
popular during the last few years among all the civilized peop 
partly because they make a pleasant drink but more especially becai > 
of their supposed therapeutic value, f 

KUMYSS (KOUMISS, KUMISS, ETC.). Kumyss derives its namefrti 
the Kumanes, a Russian tribe which lived along the river Kur 
This drink was prepared from mare's milk by placing it in a leatl 
bag and adding a small amount of old kumyss as a starter. J In t ; 
country kumyss is made from cow's milk. This product is now pla( I 
upon the market by a number of companies who keep their metho , 
so far as possible, from their rivals by maintaining strict secrecy t 
regard to the methods of preparation. Dr. PifTard who has dc: 
special work on this product states that kumyss is fermented by 1 
action of yeasts and lactic bacteria. This fermentation prodm; 
approximately i per cent of alcohol and about 0.75 per cent of ac 
Kumyss is strongly effervescent. The lactic organisms used in 1 
preparation of this material appear to be a strain of the common BL 
lactis acidi. Whether or not the yeasts are the common forms us 
by bakers cannot be stated with certainty. 

Kumyss can be easily prepared in the household by the additi 
of cane sugar and baker's yeast to fresh, warm milk which should 
kept at a temperature of about 38 (iooF.) until gas begins to for 
It should then be bottled and be kept at a cool temperature. In c 
or two days a slight amount of alcohol will be formed and a suffici 
amount of carbon dioxide to cause marked effervescence. 

* Deut. 32:14. 

f Metchnikoff's Prolongation of Life. 

J Milch Zeitung, September, 1889. 

New York Medical Journal, January 4, 1908. 



RlATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 443 

KEFIR (KEFYR, KEPHIR, KEFR, ETC.). Kefir was originally made 
ar used by the inhabitants of the Caucasus Mountains. It was 
m le from the milk of goats, sheep or cows and was fermented by the 
acition of "kefir grains" to the milk. The origin of these kefir grains 
is aknown but the natives believe that they were the gift of Mahomet 
ai are carefully preserved by them. 

Kefir was prepared by the natives by placing milk in a goat-skin 
b; and shaking it at intervals until it began to ferment. The kefir 
g^ns were then removed, dried and preserved for future use. The 
fenented kefir was also used as a starter for inoculating new lots. 
Ts beverage is now commonly made by more scientific methods.* 
T principal points to be observed in the preparation of kefir are 
cl nliness and proper temperature for fermentation and the regulation 




F . 138. A large-sized kefir grain and the three species of bacteria of which it is 
composed. (From Conn, after de Freudenreich.} 

c:he fermentation so that not the acid but the alcoholic fermentation 
vl prevail. f Good kefir should be highly effervescent, should be free 
fira lumps and contain about i per cent, of acid but show no marked 
tjidency to whey off. According to Kern, kefir is fermented by a 
r xed culture of yeasts and bacteria in symbiosis. He found but one 
f ra of bacteria present in the cultures he studied. De Freuden- 
ichj made an extended study of the flora of kefir. He prepared 
ti kefir from the kefir grains and isolated the organisms present, 
Itting these organisms together in different combinations in order 

I* Milch Zeitung, 1885, p. 209. 

F. Stohman, Milch and Molkerei Products, p. 1006 to 1013. 
tr. fur Bakt. Abt. 2, Vol. 3, 1897. 




444 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

to determine which were necessary for the proper fermentation 
the kefir. He found the kefir contained four different organisn 
yeasts, streptococci, micrococci, and bacilli. The yeasts and strep 
cocci were plated in gelatin without difficulty but it was very diffic 
to grow the other two organisms present on any artificial med 
He concluded that the yeasts present in kefir are not identical w 
the species commonly used in making beer and named it Sacch 
omyces kefir. The streptococcus curdled milk in less than forty-eig 
hours at a temperature of 37 but the micrococcus did not cur 
milk at all, although it produced a considerable amount of acid. 
De Freudenreich changed the name of the bacillus from Dispora caucast 
given it by Kern to B. caucasicus, because it did not produce spores as Kern s 
posed. He also found that this organism would not grow at all on media with 
sugar, very slightly on milk, serum, agar, and best of all in milk, in which it prod 
both gas and acid without curdling the milk. This organism is 5j or 6jt in len 
by ip in width, is slightly motile and retains Gram's stain. It has a thermal dea 
point of 55 for five minutes. 

The preparation of good kefir seems to depend upon the combin 
action of the four types of organisms described. Kefir is sometim 
prepared without the use of the kefir grains* by placing milk in bottl 
to which is added a small amount of compressed yeast and sucror 
The bottles are then held at a temperature of 10 to 15 about fifte< 
hours and shaken occasionally.. Kefir prepared in this way gives ; 
effervescent mild flavored drink. 

LEBEN. For centuries the Egyptians have used a fermented mi 
drink known as leben or leben raib. This was prepared from the mi 
of cows, buffaloes, and goats. In general it resembles the other fe 
mented milk drinks in the fact that the fermentation is product 
by yeasts and a variety of other microorganisms working togethc 
At least one yeast and three species of bacteria seem to be norm 
to this product. A fermented milk drink very similar to leben 
also used in Algeria. Just the action of each microorganism concerns 
in the fermentation of this product is not certain, but it is probab 
that all of the species are essential for the production of the particul; 
flavor and consistency of the fermented product. It is claimed th; 
the fermentation that takes place in the milk renders it more diges 
ible than raw milk. For this reason it is recommended for the ui 
of invalids and persons having weak digestion. 

* Milch Zeitung, 1888, p. 393. 



OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 445 
/AHOURTH OR MATZOON (YOGURT, YAHOURD, MADZOON, ETC.). A 

fei ented milk drink known by one of the above names has been used 
byhe Bulgarian tribes for a long time. It has recently been studied 
an brought to public notice by the investigations and writings of 
Mchnikoff,* who was struck by the longevity of the tribes using this 
uct as a part of their regular diet. As a result of his investigations, 
chnikoff has advanced his theory regarding the antiseptic power 



rtain strains of lactic bacteria in the digestive tract. His theory 

lat certain species or types of bacteria which are able to resist 
th action of the stomach and can, therefore, pass through into the 
inntines have the power of checking the growth of the putrefactive 
baeria existing there and thereby prevent the production and ab- 

tion of bacterial toxins which cause autointoxication. As a result 
of is experiments, Metchnikoff came to the conclusion that the acid 
or nism (Bad. bulgaricum} f found in yahourth was able to establish 
itsf in the intestinal tract and produce enough lactic acid to hold 
in icck the putrefactive processes which otherwise exist there. 

rahourth is made by the Bulgarians in skin bags in the same way 
th the Russian tribes prepare kumyss. It is similar to the other fer- 
m< ted drinks already described in the fact that it is produced by a 
mi :d flora of microorganisms. At least one yeast is present and two 
or tore species of bacilli capable of producing lactic acid in relatively 
lai; amounts. These two organisms are known as Bad. bulgaricum 
an Bacillus paralacticus. Herter states that Bad. bulgaricum is 4^ to 
6ju i length by iju in width and grows singly or in pairs and occasionally 
in lains. It stains with ordinary aniline dyes and by Gram's method. 
It -ows with difficulty on ordinary laboratory media and is therefore 
ha i to obtain in pure cultures. These organisms produce a much 
hijier percentage of acid than the common Bad. lactis acidi and also 
gr ; at a much higher temperature. 

ifhis makes it possible to secure it in practically pure cultures by 
gr /ing it in milk at a high temperature. Grown in pure cultures, the 
Bt . bulgaricum will produce from i to 2 or more per cent of acidity. 
It rows well at temperatures between 37 and 40 and even higher. 
R<intly a number of fermented milk drinks have been put upon the 
m; ket which have evidently been derived from the yahourth. These 






EL, Metchnikoff, Prolongation of Life. 
astings has found this organism also common in cow's milk in thU country. 



446 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

are sold under such trade names as zoolak, vitalac, yogurt, fermenlact 
etc. The flora of these preparations appears to be practically the sai 
as that of the original yahourth. 

All of the fermented milk drinks thus far discussed are similar in tf 
each contains a variety of microorganisms, made up of at least c 
species of yeast with one or more species of bacteria, capable of prod 
ing greater or less amounts of acid. In some, as in the case of kenr, t 
yeast fermentation is allowed to predominate, while in others, like 
hourth, the action of the yeasts is held in check by the rapid develc 
ment of the acid by the Bad. bulgaricum. All of these drinks are co - 
monly recommended by physicians because of their beneficial effi 
upon the digestive tract. 

ARTIFICIAL BUTTERMILK, In recent years there has developed 
important industry in the manufacture of artificial buttermilk. T 
is usually made by inoculating skim-milk with a culture of lac 
bacteria, either Bact. lactis acidi, or Bact. bulgaricum or a combinati 
of these two types. In making the artificial buttermilk, yeasts are i : 
commonly added. After the milk becomes coagulated, it is th 
churned in order to give it a smooth, creamy consistency, after wh ii 
it may be bottled and kept for some time by holding at low tempe 
tures. Sometimes a small percentage of whole milk is added at the ti : 
of churning to make the finished product more closely resemble natu 1 
buttermilk. In making artificial buttermilk, the skim-milk is f 
quently pasteurized in order to get rid of the miscellaneous flora wh 
it contains. The finished product, therefore, differs from ordinary b - 
termilk in the fact that it contains nearly pure cultures of the lac : 
organisms while the natural buttermilk will contain a more or 1 5 
miscellaneous flora in which the acid organisms predominate. It 
possible to obtain a more uniform product in the artificial butterm 
than in the natural product, and this is perhaps responsible for i ; 
rapid development of this industry. All of these fermented m: 
drinks contain enormous numbers of microorganisms, usually millk 
per c.c. 

FROZEN MILK 

Some effort has been made to put upon the market milk which 1 
been frozen into cakes or bricks. This has been tried both in Eun 
and in this country. Some difficulty has been met in satisfactorily fre 



N OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 447 

ing he milk and holding it in a frozen condition. The process has 
prld to be rather expensive and not very satisfactory. One difficulty 
wit this process seems to be that the quality of the frozen milk after it 
has >een melted is not as good as it was before it was frozen. From a 
b;u -riological standpoint, this process is of some interest, but it is 
doitful whether it becomes of much importance commercially. 



ICE CREAM 

e cream is one of the important manufactured dairy products and 
i ems to be increasing steadily. Its bacterial flora varies with 
me mterials used in its manufacture and the conditions under which it 
is i -de. It may be made from fresh cream which is only a few hours 
old nd under good sanitary conditions. On the other hand, it may be 
ma from cream which has been produced and handled under unsani- 
tar conditions, kept in storage for a number of days and finally manu- 
fac red in surroundings not conducive to a low bacterial content. We 
are ot surprised, therefore, to find a very wide variation in the germ 



cor 



nt of ice cream, as it is placed upon the market. 



ji examination of 263 samples of ice cream collected in the city of 
\V< lington* showed an average germ content of over 26,600,000 per 
c.c The lowest count obtained was 37,500 and the maximum was 
36; 100,000. A similar study of commercial ice cream in Philadelphia f 
shced the average bacterial content to be very high. The lowest 
coi t found was 50,000 per c.c., while the highest count was 1 50,200,000. 
ork it was found that the bacterial content of the ice cream was 
in dte direct relation to the sanitary conditions of the establishment 
wh e the ice cream was manufactured. When ice cream is manufac- 
tur 1 in a city from materials which have been shipped in from consider- 
ances and frequently held for several days in cold storage, it is 
no surprising that the germ content of the manufactured product 
sh( Id be high. In some establishments the cream is pasteurized be- 
for manufacturing, while in others it is used in its raw condition. 

i normal cream held for sometime, the lactic bacteria should exist 
in nsiderable numbers, but when cream is held at low temperatures 
the organisms do not develop rapidly. Pennington found that cer- 

: Results of work done under the direction of G. W. Stiles. 
\":>rk done under the direction of Dr. M. E. Pennington. 




448 MICROBIOLOGY OF MILK AND MILK PRODUCTS 

tain species of streptococci developed quite rapidly in cream held t 
refrigerator temperatures. Streptococci were found in fifty-five (80 
cent) of the sixty-eight samples examined. It was found that at ref 
erator temperatures the relative growth of these organisms was grea r 
than at higher temperatures, a fact which may account, in part at la 
for the frequency with which these organisms occur in ice cream. 

Frequently ice cream is held for a considerable time in a frozen c 
dition before it is sold. It has generally been supposed that there is 3 
bacterial growth in material which is held below the freezing tempt 
ture. This, however, did not seem to be the case in samples exarnii i 
by the investigators already mentioned. They found in samples h 1 
about a month that there was normally a decrease in the bacterial co 
and also in the amount of gas production for a number of days, a)(r 
which there was frequently a marked increase in the bacterial coin ,. 
These results would seem to indicate that even in the frozen condit i 
there may be some increase in the number of bacteria present. r . e 
number of these experiments, however, is not sufficient to justify v y 
general conclusions. The work of Conn and Esten* in holding milk t 
i may throw some light upon this question. 

If the cream from which the ice cream is made has been produ 1 
and handled under sanitary conditions, the bacterial content shod 
consist chiefly of organisms of the Bad. lactis acidi type, in which c e 
the high count in the ice cream might not be objectionable. If, n 
the other hand, the cream has been held in cold storage for some t e 
under conditions which inhibit the growth of the lactic organisms <i 
permit the development of putrefactive types, bacterial poisons r y 
be developed in the cream, which will be highly objectionable. Tr e 
seems to be little doubt that this is the cause of the cases of ptoirjn 
poisoning, resulting from the use of ice cream. It is known that cert a 
types of bacteria, especially those belonging to the so-called pu 
factive group, are capable of developing at very low temperatures ; 
can, therefore, produce considerable quantities of toxic products 
the cream. Whether or not these products are developed bef 
the cream is manufactured or whether they may develop in the fro n 
product cannot at present be stated. In general it can be said that 
total bacterial count does not indicate the wholesomeness of theje 
cream any more than does a similar count in buttermilk or in the c<;i- 

* Annual Report, Storr's Experiment Station, 1901. 



ATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 449 



;n cial fermented-milk drinks. The kinds of organisms present is a 
fa: nore important question from the standpoint of the wholesomeness 
of ie ice cream. However, the results obtained by many ice-cream 
mj ufacturers has demonstrated the fact that the germ content of this 
prluct can be quite definitely controlled by the same methods of care 
an sanitation as are required in the handling of other forms of dairy 
prlucts. 



DIVISION V 
MICROBIOLOGY OF SPECIAL INDUSTRIES 



CHAPTER I* 

DESICCATION, EVAPORATION, AND DRYING OF FOOD 
FACTORS THAT BRING ABOUT CHANGES IN DRIED FOODS 

The factors that bring about changes in dried foods may be considei 
under two general heads, chemical and microbial. Enzymes, althou 
the product of living cells, may represent the chemical changes, and 1 
activity of bacteria, yeasts and molds, the microbial changes. Enzyn 
are normally present in food stuffs derived from animals or plants wh: j 
have not been subjected to heating. All living cells contain enzym , 
and these may remain active for a considerable time after the dea 
of the cell. Some of these enzymes attack carbohydrates, some fa , 
some proteins, and some other compounds. Enzymes are responsi : 
for the stiffening of the muscles after death (rigor mortis], others la : 
break down the tissues and bring about a ripening of meat whereb} ; 
becomes more tender. Autolytic enzymes may in some instan ; 
produce rancidity in food products by a splitting of the fats. Bactei I 
enzymes are known that duplicate the action of practically all tin 
produced by higher animals and plants. Some of the changes p 
duced are desirable, others undesirable, particularly if action is allcw I 
to continue for too long a time. In foods dried for preservation, i 
therefore important that sufficient heat be used to destroy the enzyn 
or that enough water be removed to inhibit their activity. Ordinal 
the activity of enzymes will be inhibited by the removal of wa 
sufficient to prevent the growth of microorganisms. The action 
enzymes is characterized as reversible, that is, after a certain cono 
tration of enzymic products has been reached, the transformation cea 

* Prepared by R. E. Buchanan. 

450 



DESICCATION, EVAPORATION, AND DRYING FOODS 451 

Jtil a part of the accumulation has been removed by diffusion or other- 
de. Since many of these actions are hydrolytic in nature, water for 
Kh diffusion and hydrolysis must be present before the enzyme 
cji act. 

Bacteria are introduced in large numbers when food is handled and 
p )bably constitute the most important factor in its destruction. If 
r'isture and temperature conditions are favorable, they bring about 
i desirable changes. The amount of water present in foods may be 
I'd as a basis for their classification into four groups: first, those in 
Jich moisture is present in appreciable quantities in the interstices, 
tit is, those which seem wet. Under these conditions bacteria not 
( ly multiply but spread rapidly through the medium by actual space 
pwth, by diffusion currents, and by their own motion. Second, 
s ne foods may contain sufficient moisture for the 'abundant growth of 
t:teria, but not free water for diffusion and distribution. In these 
t ' spread of infection must be largely by direct growth of the organism 
c r cl will necessarily be slower than in the preceding. Third, the sub- 
s atum may be so dry that little or no growth of organisms may take 
I ice, yet there is sufficient moisture so that they remain viable. 
] urth, the food may be so dry that only those organisms that can 
Uhstand relatively complete desiccation will survive. These groups 
cinot be differentiated entirely upon the basis of the percentage of 
vter present, for the character of the food itself and of the material 
i solution are also important. 
1 Yeasts usually require sugars for their best development and are 

>re commonly present in foods containing this substance. They 
:; of importance therefore in fewer foods than bacteria. In nature, 
;y are frequently found upon fruits, particularly those which contain 
ble quantities of sugar in the sap. They will be found also 

the cut ends of twigs or grass culms where sugary sap has oozed 

Colonies of considerable size may sometimes be seen upon corn 
ble during damp weather. They are commonly distributed by 

id other insects which feed upon the sugary plant juices. They 

t motile, hence the spread of infection in any food must be by 

growth. 

olds, like bacteria, are ubiquitous and under proper conditions 
estroy almost any food. They grow readily in solutions and on 

ted substrata, but ordinarily are prevented by the bacteria which 



452 MICROBIOLOGY OF SPECIAL INDUSTRIES 

find the optimum condition for their growth in such conditions. Fc 
example, 'it is commonly observed that wet silage rots when expose 
to air supports a luxuriant growth of bacteria, while drier silage become 
moldy. Unlike bacteria, the molds extend through and over food whe 
there is no visible water film. The spores are much better adapted t 
air dispersal than are bacterial cells, and the hyphae penetrate mor 
rapidly than will the bacterial colony. In certain foods, therefore, a 
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 b' 
the absence of sufficient moisture in the medium to support growtl: 
This is not nearly so common as might appear at first thought. ] 
occurs in some foods as olive oil, starches, meals, cane sugar, etc., tha 
have little or no free water. Frequently the drying results in a cor 
centration of the solutes, beyond the point to which microorganism 
can adapt themselves to the osmotic pressure. When it is remembere 
that a 50 per cent solution of cane sugar is capable of exerting a pressur 
of about 226 kg. (500 pounds) per square inch, it will be realize 
that considerable readjustment is necessary in the cell of a yeast plan 
that can grow in such a medium. Drying also sometimes changes th 
former relationship of cells and tissue constituents so that protectiv 
layers may be formed. For example, in curing pork, the fat which i 
structurally isolated in distinct cells for the most part becomes diffuse 
throughout the outer layers of the tissues and forms a water-free an 
water-proof exterior. Foods are sometimes subjected during th 
process of drying to sufficient heat to destroy the microorganisms cor 
tained. At other times they are exposed to the germicidal action c 
the direct rays of the sun or to the fumes of some disinfectant c 
bleaching agent as sulphur dioxid or smoke. 

METHODS OF DRYING 

The reduction of the water in foods below the minimum require 
for the growth of microorganisms is accomplished in a variety of way; 
Most commonly heat is employed, either the sun's ray or some artificiz 






DESICCATION, EVAPORATION, AND DRYING FOODS 453 



urce. In localities where the humidity of the air is low, as in many 
the irrigated fruit districts of the western United States, exposure 
the rays of the sun results in rapid drying. With other types of 
ods and in more humid regions, artificial heat is used to reduce this 
lative humidity. Some foods cannot be dried at high temperatures 
cause of their instability. In most cases such foods must be dried 
lickly for they are readily attacked by microorganisms. These are 
.ually dried at a low temperature and in a partial vacuum. Other 
ods are dried without recourse to evaporation by the use of the 
. draulic press or by centrifugal action, the latter in the manufacture 
cane sugar. The water available for the growth of microorganisms 
ay be reduced by the addition of some crystalline substance such as 
irar or salt. The usefulness of the latter depends largely upon their 
bility to create a concentration of solutes too great for the growth of 
icteria. At the same time a considerable proportion of the water 
om that part of the food into which the solutes will not penetrate, is 
Dstracted by osmosis. 

j Many food products do not require any additional drying, as they 
iturally contain little moisture. Such are the grains and the products 
anufactured from them, as flour. The drying in this instance has 
:curred during the ripening process of the grain. When for any 
ason this does not occur, the grain will mold. It has been found 
xessary in many instances to kiln-dry corn. Grain, nuts, etc., are by 
icir nature adapted to keep under normal conditions for considerable 
^riods. Other foods require artificial drying. In these we have the 
ttergrading classes, which have been discussed above, those which 
mtain a very small percentage of water and those which have con- 
derable water but a high concentration of solutes. The absolute 
nt of water in the food is by no means an index to the amount 
is available for the growth of microorganisms. Many foods are 
opic. Foods having the same water content and percentage 
solutes will behave very differently with reference to delivering up 
ic water to any organism present. 
The effect of the concentration of solutes by drying is perhaps the 

f important factor in the preservation of food. These substances 
ved in the water may be actually antiseptic when concentrated, 
e acids of the juices of certain fruits. More often the sugars 
a concentration so great as to prevent growth by plasmolyzing 



454 MICROBIOLOGY OF SPECIAL INDUSTRIES 

the cell contents of the organisms. For every organism there is { 
maximum concentration reached sooner or later, beyond which growtl 
is impossible. 

Dried foods may be divided into three groups, using the relativ< 
abundance of carbohydrates, fats, and proteins as a basis for classification 

Carbohydrate foods are usually preserved by drying. Many, sue! 
as grains and nuts and the flours and meals prepared from them, d< 
not require artificial heating. They are, however, somewhat hygro 
scopic and in damp climates enough moisture is taken up to allow tfo 
growth of injurious molds and bacteria. Moldy corn has long beei 
regarded as the probable cause of the disease, called pellagra, in man 
Still other carbohydrate food stuffs require more or less care in the dry 
ing or curing, such as hay and fodder in general. This is usually drie<j 
by simple exposure to the air and sun until most of the water has beei 
evaporated. Fodder that has become moldy through the presence o 
too much moisture is a prolific cause of trouble in horses and less fre 
quently in cattle. The many deaths due to the so-called cerebrospina 
meningitis are suspected many times to be due to the consumption o 
moldy hay. In localities where the air is too moist or it rains so fre 
quently as to make it difficult to dry hay, curing is effected by a proces 
of self fermentation. The hay is piled in a mass while still green an< 
undergoes a process of heating. The temperature rises usually to abou 
70. The causes of this rise are somewhat uncertain, but it is prob 
ably due to the combined action of enzymes and microorganisms 
Just how much of this keeping quality is due to the heating, how mucl 
to the loss of water, and how much to the accumulation of products cj 
fermentation is uncertain. In other cases, the heated hay is spread ou 
and quickly dries sufficiently so that it may be stored. A certai 
small percentage of the nutriment of the hay is necessarily lost i 
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 moistur 
is usually removed to prevent organisms from growing, but in man 
other cases, as in the preparation of currants and raisins, the concer 
tration of the sugar and other solutes is the controlling factor. Fn 
quently as much as 30 per cent of the dried fruits is water. Fruit dry 
ing is often accomplished by the heat of the sun's rays, in other case 
artificial heat or even hydraulic presses are used. 



DESICCATION, EVAPORATION, AND DRYING FOODS 455 

Many manufactured products, particularly baker's goods, as 
cjckers, biscuits, dried yeast cakes, etc., are preserved by the 
e nination of water. 

\Macaroni and vermicelli are prepared by forcing a thick paste of especially pre- 
pLd flour and water through openings of different sizes. The product is then 
dj-d 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 
I ian Oceans, is prepared by cutting the meat of the cocoanut into pieces and 
ding them in the sun. From this copra, much of our desiccated and powdered 
cjoanut is prepared. 

Syrups, molasses, jellies, jams, and many other carbohydrate foods 
J: preserved through the concentration of the solutes. Many of these 
J: partially sterilized by the heat used in the process of manufacture, 
Jt there is usually plenty of opportunity for subsequent infection, 
ley are more frequently attacked by molds and yeasts than by 
Hcteria. An exception may be noted in Streptococcus mesenterioides 
iiich sometimes causes considerable trouble by a gelatinous fermenta- 
jm in syrups from which sugars are manufactured commercially. 
I Foods with considerable quantities of fat usually contain little water. 
j)ttonseed, olive and other vegetable oils, the plant and animal fats, 
'I lard, tallow, and butter, are quite resistant to change by bacteria 
iJess water is present and considerable traces of nitrogenous im- 
plies remain in them. With these foods the water is necessary for 
ie growth of the organism and also for the activity of the lipolytic 
zymes, which might hydrolyze fats and aid in the development of 
ncidity. Butter forms an exception to the rule that fat foods contain 
| tie water, as it usually has from 12 to 16 per cent. Where it is 
ry to keep butter-fat for long periods or under unfavorable 
itions, it is melted, the water and the nitrogenous impurities 
ved, and the clear fat preserved. Bacteria, enzymes, and a few 
s have been described that attack fats. In the process of prepara- 
or manufacture of any fat foods, sufficient heat is used to sterilize 
material and infection thereafter spreads to the interior very 
ly. The heat destroys the enzymes as well as the bacteria. 
The third class of foods preserved by drying includes those that 
in a high percentage of protein, in large part flesh foods and flesh 
atives. 




456 MICROBIOLOGY OF SPECIAL INDUSTRIES 

Desiccation, however, is only one of the agencies acting to presen 
the flesh. 

Jerked meat is sometimes prepared in localities with a hot dry climate. 
meat is cut into thin slices and exposed to the direct rays of the sun until dry. Tl 
bactericidal action of the sunlight and the rapid extraction of moisture preven 
microSrganisms from producing undesirable changes during the curing process. 

Dried beef is lean meat which usually has been treated with certain condiment 
smoked and salted and then dried. 

Dried fish such as cod, mackerel, and herring, is prepared by the use of cone 
ments, salt, and smoke in addition to the drying. 

Pemmican is prepared by drying lean meat, grinding it, and mixing it with sug; 
and fat, dried fruits, spices, etc. It is highly nutritious, not unpalatable, and cor 
pact, and will keep for a long period. It is frequently used as a concentrated for 
of food by Arctic explorers, etc. 

Beef extract is prepared by cooking minced beef and water in a receptacle und 
a slight steam pressure. The digestion is continued for several hours. The liquid 
filtered off and concentrated in a partial vacuum to the desired consistency. 

Gelatin is prepared by boiling bones and tendons, sometimes also horn and hi( 
scraps and concentrating the gelatin which dissolves from these. 

Somatose, sarco-peptone and related so-called predigested protein foods are mi 
tures of albumoses and peptones prepared by the artificial digestion and drying 
proteins, usually flesh. The product is marketed as a powder. 

Milk, either with or without its butter fat, is dried by being sprayed into a war 
compartment from which the air is partly exhausted. It dries immediately, in ti 
form of a very fine powder. This powder, if thoroughly dry, will keep well and 
finding an extensive use. The high sugar content of this powder is instrumental i 
preventing the development of microorganisms. 

Eggs are dried in much the same manner as milk and the product is being use 
extensively at the present time by bakers. 

Meats are frequently preserved by a combination of drying and th, 
action of certain antiseptics or preservatives. The salting of meat owf 
its effectiveness in part to the abstraction of water. In most case; 
the surface of the meat and probably even the other portions ar 
protected in large measure by the diffusion of the fat and the saturs 
tion of tissues and by the formation of water-proof fat films. Th 
autolytic enzymes are active in the fresh meat and soon becom 
inert upon the removal of water. The organisms responsible fc 
decay of preserved meats and flesh foods are usually bacteria. Som 
of these break down the protein into simpler chemical compounds, c 
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 

s d to have had its origin in the experiments of Spallanzani, who in 

5 boiled meat extract for an hour and hermetically sealed the flasks, 

asr which treatment no change occurred in the material. An applica- 

i of this principle was suggested as early as 1782 by the Swedish 

mist, Scheele, who advised the exposure of vinegar in bottles to the 

iperature of boiling water in order to effect its preservation. Some 

is later the principle was applied to the conservation of food by a 

Fnch confectioner, Nicholas Appert, who in 1811 published an 

austive treatise on "The Art of Preserving Animal and Vegetable 

stances." His method was to enclose the food in a glass jar which 

-> then corked tightly, and placed in boiling water, the length of 

t e of heating varying with the article treated. 

Jn 1810 Peter Durand secured a patent from the English govern- 

irit for the preservation of fruits, vegetables and fish in hermetically 

stled tin and glass cans. He did not claim to be the discoverer of the 



cess, but said it had been communicated to him by a "foreigner 



nding abroad." Although the secret of the process was jealously 
ded, the employees of different establishments became familiar 



wih its essentials, and in this manner the industry found its way to 
A erica. One of the first to introduce the process was Ezra Daggett, 
w), with his son-in-law Thomas Kensett, in 1819 engaged in the manu- 
fa.ure of hermetically sealed goods, the principal foods packed being 
senon, lobsters, and oysters. In 1820, William Underwood and 
C tries Mitchell, emigrant employees from a canning factory in England, 
ojned a factory in Boston where they canned plums, quinces, cran- 
bjries and currants. 
In the earliest days of canning, glass jars were used exclusively, but 

Prepared by S. P. Edwards. 

457 



45$ MICROBIOLOGY OF SPECIAL INDUSTRIES 

were gradually abandoned as it was found that they could not readi 
withstand the extremes of temperature, and were expensive, bulk 
and costly in transportation. In 1825, Thomas Kensett secured 
patent on the use of tin cans in preserving food, and in the same ye; 
began using the process in his factory. The early manufacture of t 
cans was by hand and crude, the bodies being cut with shears and t 
side seams made with a plumb joint (that is meeting but not ove 
lapping), and then soldered together. Heads were made to set in 
the body and were soldered in place in a very crude manner. T 
making of 100 cans was considered a good day's work for one ma 
Improvements were gradually made, however, in their manufactui 
until finally can making became a distinct industry and now all t 
parts are made and put together by mechanical devices. 

In the original Appert process, the goods were cooked in open kettl 
the highest temperature obtainable by this method being the boili 
point. A little later common salt was used to aid in securing a higt 
temperature, and this was followed later by the use of calcium chlori 
which made possible a temperature of 115. In 1874, a closed ket 
was invented for superheating water with steam, and this was imn 
diately followed by another improved kettle in which dry steam 
used, the principle employed being that of the modern autoclav, 
which method any desired temperature may be obtained and modif 
to suit the requirements of different classes of food. 

ECONOMIC IMPORTANCE 

FROM STANDPOINT OF HEALTH AND DIETETICS. The value o 
variety of foods, especially fruits and vegetables, is recognized 
dieticians. Unfortunately, however, the season for fresh fruits ,' 
vegetables is comparatively short. Moreover, many foods grca 
exclusively in one section of country will not withstand shipping i a 



fresh condition to other sections. In spite of improved method; 
refrigeration, it is not practicable to ship fresh sea foods to far inl 
towns, or to send some perishable products of warm climates to c d 
countries. The canning and perserving industry overcomes tl 
difficulties by supplying pure, clean, wholesome fruits, vegetal: 
meats, and fish to any region the year round, and at prices c 
paratively low. 



IN THE PRESERVATION OF FOOD PRODUCTS 459 

)M STANDPOINT OF COMMERCE. In its commercial aspect, the 
jortance of the industry can scarcely be estimated. Canned 
lucts make possible the carrying of larger stores of provisions by 
and navies and expeditions for exploration than would otherwise 
sible. In fact, the stimulus which prompted the investigation of 
>rt was a prize offered by the French Navy Department for a 
mlhod of preserving foods for provisioning ships more satisfactory 
th|i by pickling, drying, or other methods in use up to that time. 

I 'Although the preserving industry was established in three great 
comercial centers in the United States as early as 1825, it did not 
borne of much importance until the last decades of the nineteenth 
ce ury. There were many hindrances to the progress of the industry, 
sui as the secrecy observed in the process, skepticism of the public 
re rding the healthfulness of canned foods, the general prejudice 
agnst them, and the high cost of production. These obstacles have 






ually been surmounted, and at the present time the several 



niches of the industry have collectively assumed large proportions. 

Kn idea of the magnitude and importance of the industry in the 
lilted States may be gained from statistics for 1914 compiled by the 



ional Canners' Association, and here reproduced by permission, 
pack of tomatoes was 15,222,000 cases; of corn, 9,789,000 cases; 
of peas 8,847,000 cases. This does not include fruits, the pack of 
:h in California alone in 1912 was 4,833,900 cases. Nor do these 
res include the great variety of other vegetables, fruits from other 
as than California, preserves, oysters, meats, or fish. ' The average 
holds two dozen cans, and sells at an approximate average price 
2.40. It is apparent from these data that the canning and preserv- 



st; 

ca 

of 

in; industry is one of immense value, and that it constitutes a large 

fa 3r in the feeding of the race." 

ALTERATION OF FOOD 

>ICAL CHANGES. Appearance. Some physical changes attend 
:onservation of foods by heat, approaching more or less closely the 
iges incident to the ordinary preparation of fresh foods for the 

In the preserving of some vegetables, notably peas and 
[ragus, the canner subjects them to a blanching process which con- 
in submitting the vegetables to the action of hot water for a short 



460 MICROBIOLOGY OF SPECIAL INDUSTRIES 

time, the object being, first, to remove the mucous substance from 1 
outside and a part of the green coloring matter so as to have a cL 
liquor in the can, and second, to drive water into the vegetables so tl 
all will be tender. The blanching process also improves the color [ 
these vegetables. 

Mechanical Disintegration. In the case of very soft fruits or ve 
tables, the high temperature essential for sterilization causes a slij 
amount of mechanical disintegration, which is not objectionable, h( 
ever, unless excessive, as there is little deterioration in appearance, r 1 
none in food value. In the case of meats, practically the only physi 
change is the shrinkage during the parboiling previous to placing 
the cans. 

CHEMICAL CHANGES. Appearance. The chemical changes in fo 
preserved by heat may be considered under two heads: first, thosci 
which the appearance is modified; and second, those in which the f 
itself is altered. Some change of color sometimes occurs and res' 
from various causes. In colored vegetables, such as peas, string be; 
and asparagus, a part at least of the loss of color is due to the oxidat 
of chlorophyll. With a few foods, iron sulphides are occasion; jy 
formed by a combination of sulphur of the foods with the iron of |e 
container. This seldom occurs however, and is not of great imj :- 
tance. Some fruits packed in glass gradually lose their color by ox: L- 
tion on exposure to the light. 

Chemical Change. So far as chemical alteration of the food it If 
is concerned, there is little change and none other than would occu n 
the ordinary preparation of the food for the table. The albumins -e 
coagulated. The fats probably remain unchanged. Of the can- 
hydrates, the chief action is on the sugars. The cane sugar is wh 
or partly inverted by the combined action of the heat and the frui 
vegetable acids. The starch undergoes little if any cleavage, inasm 
as this change occurs only in the presence of acids, and in foods wi 
high acid content, the proportion of starch is relatively low. The o 3 
amyloses probably undergo little if any change. 

Palatability and Digestibility. It is often contended that car 
foods are less palatable than fresh foods of the same kind. This fl 
of agreeableness to the taste is, however, more seeming than real, jid 
arises largely from the prejudice of the consumer against food conseipd 
in tin cans, rather than from any actual change. When the preser jig 



AT IN THE PRESERVATION OF FOOD PRODUCTS 461 

is operly done the product should be no less attractive to the eye, no 

le< pleasing to the palate, and of no less value from the standpoint of 

iiility than the same food when served in the fresh condition. 

BIOLOGICAL CHANGES. Vital Disorganization. The entire industry 
of mservation of foods by means of heat is based on a microbiological 
It is a universally recognized fact that the ordinary spoilage of 
fo. is a microbiological change, hence the individual desirous of 
pr ecting food from spoilage must give consideration to the microbial 
agits responsible for the change. 

formal Flora and Fauna. Aside from a few types of organisms 
ca ing disease or specific poisonous conditions, we are unable to desig- 
na definite species as those causing canned goods to spoil. Consider- 
in; he great variety of foods preserved by heat, and the different con- 
diims under which they are grown and secured, it naturally follows 
th the normal flora and fauna of food to be preserved in this manner 
we Id embrace a wide variety of species, including some higher fungi, 
mils, yeasts, bacteria, and low animal forms. Generally speaking, 
th microbial flora of fruits consists mostly of molds and yeasts, 
altjugh bacterial forms may also be present. In the case of vege- 
tal 2s, and of fruits coming in contact with the earth, more species of 
haeria are apt to be present, many of them spore formers able to 
wi stand a high temperature. Finally, in meats and fish the living 
ions may include not only molds, yeasts, and bacteria, but animal 
foiis as well, such as the organisms of tseniasis (tapeworm) and 
tritinosis. In the preserving industry, therefore, consideration must 
be iven to all these forms, not only from the standpoint of the suc- 
celul preservation of the various foods, but also from that of pro- 
tec ng the health of the consumer from possible poisonous substances 
prjent or produced in the conserved food, and from possible infection 
\vi pathogenic organisms present in the food before it is packed. 

PASTEURIZATION 

)NOMIC CONSIDERATIONS. In the preservation of food by heat, 
:esses are applicable, pasteurization, and sterilization. In 
jurization, the aim is not to effect the permanent preservation of 
fo<js or drinks by destroying all life present, but rather to destroy 
cejiin species of organisms, thus checking the natural fermentation, 
Fecting a temporary preservation. 



462 MICROBIOLOGY OF SPECIAL INDUSTRIES 

The principle of pasteurization may be said to have originated 
the early work of Spallanzani and Scheele, already mentioned, al 
was employed by Appert in his later investigations. The operat 
as carried out by Appert does not, however, appear to have foil 
general application until Pasteur revived the method, and as a res t 
of his activities in attempting to secure a general adoption of 
practice to prevent the spoiling of wine, the process was named fr 
him. 

SPECIFIC APPLICATION. Beer. Pasteurization is of economic 
portance particularly in the dairy and fermentation industries, 
brewing, "The process of pasteurization is in use with even the smal t 
brewers in the United States, beer being pasteurized even for k .1 
consumption." The beer is pasteurized in bottles by being subjec i 
to a temperature of 58 to 63 for one-half hour. The entire pro< 
as practised in the large breweries, requires less than an hour, and 
eludes the warming of the cold bottles to pasteurizing temperature, 
pasteurizing proper, and the cooling to a little above room tempt 
ture. The process is a continuous one, the bottles being put into 
machine at one end and taken out at the other. Experiments hie 
been made in pasteurizing beer in large containers, steel, copij:, 
aluminum, and tin having been tried, but without complete suoiis 
as yet. 

Fruit Juices. The essentials in the pasteurization of wine and f it 
juices are similar to those for beer. There is, however, no univejil 
rule of application. Details of the process must be arranged to it 
the character of the different liquids under treatment. 

Cream and Milk. Pasteurization as employed in the dairy indu; y 
varies in its method of application according to the purpose for wl 
it is used. In factory butter making, it must be employed to sec 'e 
the best results. Milk as ordinarily received at creameries contis 
a widely variant microbial flora, many of the species exerting a gre 
or lesser influence in determining the flavor of the finished prod 
By pasteurization of the cream, the butter-maker destroys most of 
organisms present; and by the use of a culture starter of lactic ; 
bacteria, he is able to control the fermentation, and is assured 
uniform quality of product from day to day throughout a season, 
added value of pasteurization is that all pathogenic organisms 
destroyed, thus aiding in the prevention of such diseases as mighpe 



JAT IN THE PRESERVATION OF FOOD PRODUCTS 463 

through this product. In creameries, the usual process of 
urization is what is known as the continuous or flash process, in 
the milk is subjected to a momentary heating to about 85, 
through the pasteurizing machine being so regulated as to 
all the milk up to the desired temperature, the heating being 
diately followed by rapid cooling, and subsequent addition of the 
ic starter. 

In the pasteurization of milk for infant feeding, a lower temperature 
i employed. A temperature sufficiently high to kill the organism of 
tberculosis (the standard for pasteurization) by momentary heating, 
parts to the milk a cooked flavor, making it less palatable, and coagu- 
es some of the protein constituents making it less digestible. The 
d$ired end may be reached however, by using a lower temperature for 
donger period of time, and the method generally recommended is to 
Hat the milk to 60 to 65 for thirty minutes. This heating is suffi- 
cint to render harmless any pathogenic organisms likely to be present 
i the milk, without the objectionable features attendant on heating 
t a higher degree. 

I Condensed Milk. It is commonly stated that Gail Borden invented 
t-2 process for preparing condensed milk, in 1856. Previous to this 
Ijwever, milk had been condensed in France, Germany, and England 
c early as 1825 to 1835. While he cannot, therefore, be called the 
ventor of condensed milk, to Borden belongs the credit of having 
tat prepared it by a rational process, and in a practicable form. 
i In the manufacture of condensed milk, good fresh milk is evaporated 
i a vacuum pan similar to those used in sugar factories, at a tempera- 
Ire of 40 to 50 until the volume is reduced a little more than half, 
dne sugar being added so that the finished condensed milk usually 
(jntains 40 per cent, cane sugar. The evaporation must be conducted 
^|th great care, otherwise the lactose crystallizes out, and this causes 
3 product to feel "sandy" to the tongue. When the evaporation 
milk is complete, the yellowish white syrup is sealed up in tins 
hold about 450 g., and this quantity is equivalent to about ij^ 1. 
al milk. The addition of cane sugar acts as a preservative, 
although the finished product may contain some living organisms, 
id to keep indefinitely if unopened, and will even keep for a 
r of days after opening. Occasional losses do occur by spoilage 
finished product, either from the growth of occasional types 



464 MICROBIOLOGY OF SPECIAL INDUSTRIES 

of bacteria tolerant of the high percentage of cane sugar, or frc 
yeasts. 

STERILIZATION 

ECONOMIC CONSIDERATIONS. For certain classes of food produc 
pasteurization is widely applicable, and is of immense value from 
economic standpoint. Preservation by pasteurization is at best, ho 
ever, temporary. Spores of spore-forming bacteria are certain to 
present on many kinds of foods, and these, unharmed by pasteurizi 
temperatures, develop vegetative cells, and spoilage occurs. 

For permanent preservation, therefore, sterilization must be adopt 
and it is upon the principle of sterilization, coupled with prevention 
future contamination by hermetically sealing the container, that t 
whole canning and preserving industry is based. The method 
applicable to nearly every class of food, and with less alteration in t 
food than any other method of conservation. The principle employ 
to-day is essentially the same as that used by Appert over 100 ye; 
ago. Although he knew nothing of microorganisms or their relation 
the spoilage of food, Appert's experiments taught him that not 01 
must the food to be conserved be heated thoroughly, but that it mi 
be so sealed as not to allow air to enter the container. In the light 
microbiological science, it is clear that the success of the process depen 
not upon keeping out the air, but upon keeping out organisms which : 
carried in the air. 

SPECIFIC APPLICATION. The process of preservation by ste - 
ization is not so extensively practised for fruit juices and fermenil 
liquids as that of pasteurization. If too high temperature is emplo) 
for fruit juices, certain compounds of agreeable taste and aroma ; 
destroyed, with a consequent deterioration in the flavor of the prodii 
Fruit juices may, however, be sterilized by heating at a low temperat 
for several successive days. 

The method of Appert has its widest application in the conservation of fruits ; 
vegetables, meats and fish. Whatever modifications are made in the handling of 3 
different classes of foods the essentials are the same. The raw material a 
thorough cleaning and removal of waste if any, is filled into the cans and submit 1 
to the sterilizing process, the degree of heat and time of processing varying v 
different products. From the character of the flora, fruits as a rule require a o 
paratively low temperature for sterilization, while some vegetables and meats 



HEAT IN THE PRESERVATION OF FOOD PRODUCTS 465 

qije a very high temperature to destroy the bacterial spores sure to be present. 
Bjfly, the methods employed in canning some foods follow: 

\[ ea l. In the meat-canning industry, lean meat is largely selected for two reasons. 
Fd well-finished carcasses bring a better price when offered for sale in the fresh con- 
diin; and in the second place, lean meat has a better appearance in the canned 
ste than fat meat. The selected meat is cut into pieces of approximately from 
i 14 pounds in weight, according to the size of the tins in which it is to be preserved. 
TJ pieces are cut as nearly as practicable the same size, not only for purposes of 
arlarance in the cans when opened, but also that the process of sterilization may 
bttiore uniformly carried out. If the pieces were of different sizes, the smaller 
orl would become thoroughly cooked and disintegrated before the larger ones were 
st lized. 

Vfter the pieces have been selected and dressed they are parboiled before being 
piled in the containers, the time ranging from eight to twenty minutes, according to 
ttsize of the pieces of meat. The object of parboiling is to secure the shrinkage 
w :h always takes place on heating. Meats put into tins in the fresh state and 
st lized shrink to about two-thirds of their original volume. When the meat is 
pi directly into boiling water, there is less loss of protein than when the meat is 
pled in cold water and heated gradually. During parboiling, the meat loses 
aht i per cent of the protein content, about one-third of the total meat bases, 
ai 50 per cent of the mineral matter. 

This shrinkage by parboiling tends to make a more concentrated article, thus 
fairing transportation, and, pound for pound, the nutritive value is not lowered. 
P :tically, the nutritive value of a pound of properly canned beef is about one-third 
gi.ter than that of i pound of fresh beef of the same kind. After parboiling, the 
m] t is placed in tins, by hand or by machinery, and to each can is added a small 
q ntity of "soup liquor," the manner of preparation of which is not disclosed by the 
p;cers. It may be regarded as a thin soup, the object of which is to fill up the 
sjj-.es between the meat, and to add condimental substance to render the meat more 
pitable. 

After the cans are filled, they are closed and processed in suitable retorts by steam 
uer 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 
aJDmatically sealing them in the exhausted state, thus removing all the air and other 
g; :s, after which they are placed on an endless conveyor and dipped into an oil 
bn at a temperature of 115, the speed of the conveyor being so regulated that 
tl cans remain in the bath a sufficient length of time to complete sterilization 
bore they are carried out at the opposite end. They are next carried automatically 
ii a solution of carbonate of soda, and finally into pure water, after which they 
a dried, painted with a shellac or lacquer and labelled. 

Fresh meats other than beef or pork are canned in a fresh state. When game 
a wild fowl, as well as domesticated chickens, ducks, geese, turkeys, and pigeons are 
Uii, the general process is as already described. Horse meat is used more or less 
c unonly in some European countries, but probably rarely in the United States. 

F ish. The process of fish canning does not differ materially from that of other 
30 






466 MICROBIOLOGY OF SPECIAL INDUSTRIES 

meats. On account of its proneness to rapid decomposition, especial care must 
observed that the fish are in a perfectly fresh state before canning, and that 
sterilization be most thorough. Salmon is the principal fish for the preservatioi 
which dependence is placed on sterilization alone, most fish being preserved 
other methods. 

Vegetables and Fruits. Corn. The young tender ears of sweet corn are pic 
from the stalk, preferably in the early morning, keeping the husks on, and 
taken in this condition to the factory. They are husked and the silks removed 
passed through machines with sets of knives which cut the grains evenly from 
cob, care being observed not to cut the corn so closely as to cut off particles 
the cob with the corn. After the corn is cut off the cob, some canners add a "syri 
of water, salt and sugar, and cook the corn for a few minutes at 80, after whic 
is filled into the cans, sealed, and sterilized. Another method is to fill the uncoo 
corn directly into the cans, fill them with "syrup," hermetically seal and steril 
The temperature employed varies somewhat, but usually lies between 115 
121 for thirty minutes for No. 2 cans holding 20 ounces. Proportionately Ion 
time is required for larger cans. Most of the operations are carried on by machin 
The sterilization is sometimes done in the ordinary canners' retort, or the cans E 
be placed on an endless conveyor, dipping into water or brine of a proper t 
perature, the speed of the conveyor being so regulated that the cans are sufficier 
heated to sterilize them during the passage. 

Peas. In the pea-canning industry the vines are cut with a mower, loa 
onto racks like hay, and hauled to the vining machines. The viner is a mad 
consisting of an outer and an inner cylinder revolving in opposite directions, 
inner one bearing paddles or beaters so arranged that as the vines pass through 
machine the paddles break open the pods. As the peas are thrown out, t 
pass through perforations in the outer cylinder, while the vines are discharged 
the opposite end. The shelled peas are next washed to remove all dirt and i 
the mucous substance from the surface, thus insuring a clearer liquor in the c 
Grading for size is done by passing the peas over sieves, or into a revolving cylin 
having four sections with perforations of different sizes. The peas are next bland I 
in hot water to remove the mucous covering and to drive water into the peas 
that all will be tender. The time of blanching varies from one-half to five or m 
minutes, large mature peas requiring more time for the blanching than smaller 01 
After blanching, the peas are filled into the cans by special machines, the cans 
filled with "liquor" consisting of water, salt, and sugar, sealed, and steriliz 
The time of processing varies, the average being 115 for thirty to thirty- five minu 
for the ordinary sized can. 

Fruits. The essentials in the canning of fruits do not differ from those 
vegetables. Stone fruits may be canned either with or without the pits. In 
case of such fruits as cherries, or other acid fruits, the tin can is coated on the ins 
with a laquer or enamel which protects the tin from erosion by the action of 
acid juices. The time and temperature of processing of fruits is usually less tl 
that required for vegetables, for the reason that in the presence of the fruit aci 
the organisms are more easily destroyed than in foods in which acids are not prese 




HEAT IN THE PRESERVATION OF FOOD PRODUCTS 467 

CONTROLLING FACTORS IN SUCCESSFUL CANNING 

j CLEANLINESS. Too much emphasis could hardly be placed upon 
tie importance of cleanliness throughout the whole preserving process, 
hd especially in the preparation of the product for preserving. Vege- 
Lbles that have come into contact with the soil are pretty certain to 
(arbor many spores of bacteria, and if as many of these are removed 
B possible .by a thorough preliminary cleansing, sterilization may be 
ifected with greater ease and certainty. The necessity of cleanliness 
i the part of factory employees is needful only of mention, not only 
om the esthetic standpoint, but also from that of good health. 

THE SOUNDNESS OF RAW MATERIAL. The necessity of sound and 
jholesome raw material is fully as great as that of cle