Hit Aid | i it if i iH a tt ety ae RHE bree CORNELL UNIVERSITY. THE Koswell P. Flower Library THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE. 1897 icrobio TT Q MICROBIOLOGY MARSHALL pees Are MICROBIOLOGY A TEXT-BOOK OF MICROORGANISMS GENERAL AND APPLIED CONTRIBUTORS . T. Bioletti, Berkeley, California. W. J. MacNeal, New York, New York. . E. Buchanan, Ames, Jowa. E. F. McCampbell, Columbus, Ohio. . Dorset, Washington, D.C. ° Z. Northrup, East Lansing, Michigan. . F. Edwards, Lansing, Michigan. E. B. Phelps, Washington, D. C. Fidlar, London, Ontario. oO. Rahn, Elbing, Germany. .D. Frost, Madison, Wisconsin. L. F. Rettger, New Haven, Connecticut. . Guilliermond, Lyons, France. M. H. Reynolds, University Farm, St. Paul, . MacL. Harris, Chicago, Illinois. Minnesota. . C. Harrison, Macdonald College, Que., Canada. W. G. Sackett, Fort Collins, Colorado. . G. Hastings, Madison, Wisconsin. W. A. Stocking, /thaca,, New York. WwW. Hill, London, Ontario. ee Thom, Washington, D.C. . E. King, Detroit, Michigan. J. L. Todd, Montreal, Quebec. . G. Lipman, New Brunswick, New Jersey. E. E. Tyzzer, Boston, Massachusetts: EDITED BY CHARLES E. MARSHALL Amherst, Massachusetts PROFESSOR OF MICROBIOLOGY AND DIRECTOR OF GRADUATE SCHOOL MASSACHUSETTS AGRICULTURAL COLLEGE “SECOND EDITION REVISED AND ENLARGED ss WITH 186 ILLUSTRATIONS e PHILADELPHIA e of P. BLAKISTON’S SON & CO. 1012 WALNUT STREET QR fj 96 eee ; No. S000 CopyRIGHT, I911, BY P. Biaxiston’s Son & Co. Reprinted with Corrections, June, 1912 CoPYRIGHT, 1917, BY P. BLaxisTon’s Son & Co. THE MAPLE PRESS YORK PA CONTRIBUTORS BIoLETrI, FREDERIC T., M. S. Professor of Viticulture and Enology, Viticulturist of Experiment Station, University of California, Berkeley. Bucuanan, R. E., B. S., M. S., Pu. D. Professor at Hacteriolosy, 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. Epwarps, S. F., B. §., M. S. .Formerly Professor of Bacteriology, Ontario Agricultural College, Guelph, Canada. Director of The Edwards Laboratories, Lansing, Michigan. Fiptar, Epwarp, B, A., M. B. Chief of Division of Pathology; Pathologist of London Asylum and of Victoria Hospital; Professor of Pathology,-W. U. Medical Faculty; Pacteriologist of London Board of Health, London, Ontario. Frost, W. D., Px. D., D. P. H. Professor of Agricultural Bacteriology, University of Wisconsin, Madison. Guittrermonp, A. DocTEuR is 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. Hastines, E. G., M. S. : Professor af Agricultural Bacteriology, Bacteriologist of Experiment Station, University of Wisconsin, Madison. Hitt, H. W., M. B., M. D., D. P. H. Diveetor of Institute at Public Health; Chief of Division of Epidemiology; Professor of Public Health, W. U. Medical Faculty; Medical Officer of Health of London, London, Ontario. . Kinc, WattER E., M. A., M. D. Formerly Protessne af Bacteriology and Bacteriologist of Experiment Station, Kansas Agricultural College, Manhattan. Assistant Director of Research Laboratory, Parke, Davis & Co., Detroit, Michigan. ! Vv vi CONTRIBUTORS Lipman, Jacos G.,. Px. D. Dean of Agriculture, Rutgers College; Director of Experiment Station, New Brunswick, New Jersey. MacNEAL, Warp J., Pu. D., M. D. Professor of Bacteriology and Director of the Laboratories, New York Post- Graduate Medical School and Hospital, New York. McCampBeELt, Eucene F., Pa. D., M. D. Professor of Preventive Medicine, Dean of the Medical College, Ohio State University. Norturvp, Z., M. S. Assistant Professor of Bacteriology and Hygiene, Michigan Agricultural College; Assistant Bacteriologist and Hygienist of Experiment Station. PHELps, Earte B., B. S. Professor of Chemistry, Hygienic Laboratory, U. S. Public Health Service, ' _ Washington, D. C. Raun, Otto, Px. D. Formerly Assistant Professor of Bacteriology, Illinois University, Urbana. Now Elbing, Germany. Retrcer, L. F., Px. D. Assistant Professor of Bacteriology and Hygiene (in Sheffield Scientific School), Yale University, New Haven, Connecticut. Reynotps, 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. SackEeTT, WALTER G., B. S. Bacteriologist, Colorado Experiment Station, Colorado Agricultural College, Fort Collins. Steckine, W. A., M. S. A. Professor of Dairy Industry, Cornell University, Ithaca, New York; Dairy Bacteriologist of the Experiment Station. Tuom, Cartes, Pu. D. ‘Mycologist, Bureau of Chemistry, U. S. Department of Agriculture, Wash- ington, D. C. Tonvp, J. L., B. A.. M. D., D. Sc. i 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 f inseets, a growing subject, in a distinct chapter. The study of microérganisms 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. \ AMHERST, MASSACHUSETTS. vil 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 microdrganisms 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. CHartes E. MarsHatt, Epitor. AMHERST, MASSACHUSETTS. CONTENTS TITLE Pace . . ‘ ‘ dh UN Cae tat 4 . ii CONTRIBUTORS . . ‘ . “ 3 v INTRODUCTION (Editor) $3 , ee oR ee vii ContTENTS (Editor) . . . c el eee 5 xiii HISTORICAL REVIEW (Harrison) de F ' I PART I.—MORPHOLOGY AND CULTURE OF MICROORGANISMS GENERAL (Editor). OUTLINE oF PLANT Groups (Thom) OvuTLINE oF PRotozoaL Groups (Todd) (Tyzzer) CHAPTER I.—ELEMENTS oF MicroBiAL CyToLocy (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.—Motps (Thom)... . 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). a Sp - 59 Morphology of certain types,—Definition and bases of ‘classification. — “Cy boloey, = 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). a Form,—Fundamental form eps, gradations, anivoliition: fein, —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, minute 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.—INVISIBLE MICROORGANISMS (Dorset) . RCE a aa ae es TEC A brief general discussion of the available knowledge of invisible microdrganisms. CHAPTER VI.—Protozoa (Todd, revised by Tyzzer.). ..... eee te 120 Introduction.—Structure of protozoa.—Activities of protozoa, —Tocomotion: and: re- production, developmental cycle, encystment.—Parasitism.—Discussion of classifi- cation.—Technic. xiii xiv CONTENTS PART II.—PHYSIOLOGY OF MICROORGANISMS (RAHN) DIVISION I.—Noutrition AND METABOLISM. (A Few Paragraphs on Protozoal Nutrition by Todd, revised by Tyzzer.) INTRODUCTION—Principles of nutrition and metabolism, energy supply of micro- Organisms 2 4 + a * ee w ® 4 ‘ =) aa * CHAPTER I.—Foop oF MICROORGANISMS . , The composition of the cell,— Moisture, cell wall, “cell contents: Amount of food Te- quired.—Food for growth (sources of carbon, nitrogen, hydrogen, oxygen, minerals). —Food for energy.— Oxygen relations. CHAPTER II.—Propucts oF METABOLISM. .. . 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 wee General theory of metabolism,—Metabolism, katabolism and suabolism—Tntras 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.—Puysicat INFLUENCES CHAPTER I.—MolstureE. ¢ Osmotic pressure (Plasmolysis), ‘Salt ‘and sugar nolitions: colloidal solutions. —Des- iccation. CHAPTER. II.—INFLUENCE OF TEMPERATURE. Optimum temperature.—Minimum température: —Magionm jemibenstares Bie. 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 . . enue Phototaxis.—X-rays.—Radium rays. CHAPTER IV.—INFLUENCE oF ELECTRICITY . . ‘CHAPTER V.—INFLUENCE OF MECHANICAL EFFECTS . Pressure.—Gravity.— Agitation. DIVISION III.—CuHemicaL INFLUENCES CHAPTER I.—STIMULATION oF GROWTH, ...... ta be & Bee WOR a Chemotropism and chemotaxis. . 141 148 158 + 189 207 213 222 . 226 . 227 » 230 CONTENTS XV CHAPTER II.—INuHIBITION or GROWTH... . see « 232 Poisons, germicides, disinfectants, antiseptics, preservatives: Mode of scion Factors influencing disinfection.—Classification of disinfectants. DIVISION IV.—Moutuat INFLUENCES SyMBIOSIS.— METABIOSIS.—ANTIBIOSIS. ‘ . ‘ 241 PART III—APPLIED MICROBIOLOGY ‘ DIVISION I.—MicrosioLocy oF Arr (Buchanan) CHAPTER I.—TuHE MIcROORGANISMS OF THE AIR AND THEIR DISTRIBUTION. . . . 247 Microérganisms present in the air.—Occurrence in the air—How microérganisms 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 J1.—MicrosiaL 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.—MIcrRoBIOLOGY OF WATER AND SEWAGE CHAPTER I.—Microdrcansism 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.—MtcroBioLoGy oF SEWAGE (Pheips). . 274 Bacterial flora of sewage.—Types of sewage bacteria, _Putrefactive and anastobic 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.—MicrosioLtocy oF Soi (Lipman) CHAPTER I.—MIcro6RGANISMS 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, alge, 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.—Decomposit1ion oF ORGANIC MATTER IN THE SOIL. Carbohydrates,—Origin, decomposition of cellulose, the production of methane ital 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.—FrxaTion or ATMOSPHERIC NITROGEN. (Methods of Soil Inoculation, by Edwards.) . a The source of nitrogen in agilg arly theories, chemical aid Biological fetations! — 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. 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,—A vail- 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.—MiIcrosioLocy oF MILK AND MILK PropuctTs CHAPTER I—TuHE RELATION oF MICROORGANISMS TO MitK. (Stocking.) (The Acid- forming Bacteria, by Hastings.) . Importance of milk as a food. — Absorbed taints and odors. Changes iiue to micro- 6rganisms.— Microbial, content of milk,—Common milk, special milk, certified milk. —Sources of microérganisms 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 microérganisms found in milk, and their sources,—Gen- eral significance of acid-forming bacteria, groups of acid-forming bacteria (character- istics of the Bact. lactis acidi group, characteristics of the B. coli-aerogenes group, characteristics of the Bact. bulgaricus group, characteristics of the coccus group) (Hastings), bacteria having no perceptible effect upon milk, the digesting or pepton- izing, pathogenic organisms.—Factors influencing the developing of microérganisms in milk,—Initial contamination, straining, aeration, centrifugal separation, tempera- ture, pasteurization, the use of chemicals——The normal development of micro- érganisms 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- 315 338 354 . 364 CONTENTS xvii mentation, other fermentations.—The commercial ‘significance of microérganisms in milk,—Relation of dirt contamination to germ content.— Milk as a carrier of disease organisms,—Those microérganisms 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.—TueE RELATIONS OF MICROORGANISMS TO BUTTER (Hastings). . . . 403 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 IJI.—RELAaTION OF MICROORGANISMS TO CHEESE (Hastings) . e 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) 4 22 854448 see Be ww oe Se eR Se ee AaB General.—Condensed milk,—Sweetened condensed milk, trisweetened 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.—MrcroBioLocy OF SPECIAL INDUSTRIES CHAPTER I.—DesiccatTion, EvAPORATION, AND DryiInG oF Foops (Buchanan) . . 450 Factors that bring about changes in dried foods.—Inhibition of growth of micro- érganisms 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 Foops (Edwards) . he oe 2 G57 Historical résumé.—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.—TueE PRESERVATION OF Foop By CoLtp (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. XVili CONTENTS CHAPTER IV.—PRESERVATION OF Foop By CHEMICALS (MacNeal). ...... 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.—MicrosiaL Foon Poisontinc (MacNeal) General considerations.—Infections of food-producing animals transmissible toi man. —Human infections transmitted in food—Food poisoning due to the growth of saprophytic bacteria in the food,—Poisorious 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). Introduction.— Microérganisms of certain portions of the alimentary canal '—Miero- érganisms of the mouth, microérganisms of the stomach, microdrganisms of the » 479 488 496 intestines, ‘microérganisms of the feces.—General method of study,—Collection of material. CHAPTER VII.—THE MIcroBIOLoGy oF ALCOHOL AND ALCOHOLIC PropuctTs (Bioletti) . Wine: Grape juice, and wine as culture media.—The microérganisms found on grapes (molds, yeasts, pseudo-yeasts, bacteria)—Microérganisms found in wine,— Aerobic organisms (mycoderme, acetic bacteria), anaerobic organisms (slime-form- ing bacteria, propionic and lactic bacteria, mannitic bacteria, butyric bacteria).— Control of the microédrganisms,—Before fermentation, during fermentation, after fermentation.— Beer: The raw materials and microérganisms 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.—Méiscellaneous 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.—MANvUFACTURE OF VINEGAR (Bioletti). 5 S 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.—MANUvuFAcTURE OF OTHER FERMENTED Propucts (Bioletti). Preparation and conservation of food material—Compressed yeast, bread, yeast as as food, vegetables, starch, sugar, tobacco.—Preservation and conservation of miscel- laneous products,—Indigo, retting, tanning. CHAPTER X.—MANUuFACTURE oF VaccINES (KING) 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. - 538 560 CHAPTER XI.—THE MANUFACTURE OF ANTISERA, AND OTHER BIOLOGICAL PRODUCTS, RELATED To SpeciFIc INFECTIOUS DISEASES (King) é eh Antitoxins——Diphtheria antitoxin, tetanus antitoxin. —Antimicrobial serume== 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. + 575 CONTENTS xix DIVISION VI.—Microsiat Diseases or Prants (Sackett) INTRODUCTION ‘ Gi 588 CHAPTER I.—Buicurs. woe «+ 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.—Gatts anp Tumors. - 605 Crown gall.—Olive knot.—Fingers and toes. of ‘eabbages (Toda, revised ‘by Tyzzer).—-Tuberculosis of sugar beets. CHAPTER III.—Lear Spots... - 612 Citrous canker.—Angular Jeaf-spot of cucumbers. —Leaf-spot ‘of the larkspur. — Bacterial spot of plum and peach.—Leaf spot of sugar beet. CHAPTER IV—Rorts 617 Black rot of cabbage. —Wakker's Ss 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 tomate’ egg plant, Trish potato, and tobacco.— Additional bacterial diseases. DIVISION VII.—MicropiaL DiskAsEs oF INsEctTs (Northrup) . 632 INTRODUCTION.— Miscellaneous insect diseases,—Saprolegniaceez, Entomophthor- acez, and Entomogenous fungi (Thom).—Bacterial disease of June Beetle larve, Lachnosterna spp.—Flacherie (silk worm).—Bacterial disease of locusts.—Bacillary septicemia of caterpillars, Arctia caja.—Graphitosis——American foul brood.—Sep- ticemia of the cockchafer, Melolontha vulgaris—European foul brood.—Bacterial septicemia of larve of the Lamellicorne.—Bacterial disease of the gut-epithelium of the lug-worm, Arenicola. ecaudata.—Sacbrood of bees.— Wilt disease or flacherie of the gipsy moth caterpillar, Porthetria dispar.—Pebrine. x DIVISION VIII.—MiIcrRoBIOLOGY OF THE DISEASES OF MAN AND DoMmESTIC ANIMALS CHAPTER I.—MErHops AND CHANNELS OF INFECTION (McCampbell) a oe 659 Infection defined—Microérganisms of diseases considered and classified, '—Patho- genic bacteria, pathogenic protozoa, ultra-microscopic microdrganisms 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 microérganisms 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 microérganisms are disseminated.—The methods by which in- fectious microérganisms are eliminated from the body.—The effect of infectious microérganisms upon the body,—The period of incubation, local reactions, general reactions (metabolism, blood-forming organs, parenchymatous tissues, epithelial and endothelial tissues, erythrocytes and leucocytes, antibody formation). XX CHAPTER II.—Immunity AND SUSCEPTIBILITY (McCampbell) . se & _ CONTENTS General,—Definition, hypersusceptibility or anaphylaxis, predisposition ‘and: non- inheritance of infectious diseases.—_Immunity,— Natural immunity and susceptibility (racial immunity and susceptibility, familial immunity and susceptibility, individual immunity and susceptibility), factors of natural immunity (the protection afforded the body by the surfaces, skin and cutaneous orifices, subcutaneous tissue, the 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.—MicrosiaL Diseases OF MAN AND DomEsTIC ANIMALS ‘ Diseases caused by molds and yeasts (various authors),—Pneumomycosis (Thom), thrush'(Thom), dermatomycoses, barbers itch, etc. (Thom), favus (Thom), actino- mycosis (Reynolds), mycetoma (Fidlar), mycotic lymphangitis (Reynolds).—Dis- eases caused by bacteria,—Botryomycosis (Reynolds), gonorrhcea (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, hemorrhagic septicemia (Reynolds), leprosy (Fidlar), plague (Fidlar), swine erysipelas (Dorset), tuberculosis (Reynolds), foot rot of sheep (Dorset), malignant cedema (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) —Ameebic dysentery, entero-hepatitis of turkeys, kala-azar, Delhi boil, sleeping sickness, human trypanosomiases of Sout’: 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 spirochetal diseases, syphilis. CHAPTER IV.—Conrrot oF INFEcTious DisEasEs (Hill) . Principles—Practice—Disinfection—Carriage of infection by biological duents: INDEX OF CONTRIBUTORS . eee 5G % é toe BS INDEX OF SUBJECTS . - 684 724 850 863 865 POI AASY Po LIST OF ILLUSTRATIONS Frontispiece . Jansen’s Microscope .. . ‘ boa eX . . Cells of Saccharomyces cerevisiae. Cells made up of energids Diffuse nuclei of bacteria . Nuclei in Cyanophycee . Chromidia in protozoa . Micro- and macro-nucleus in an infusorian. . Division of micro-nucleus and chondriosomes. . Formation of chloroplasts . . . Mitochondria developing into amyloplasts . . Chloroplasts of different forms . ae . Metachromatic corpuscles . . Illustrating cyst and thread membranous walls. . Organs of locomotion in bacteria . . Division of Spongomonas uvella and Monas termo . Transverse section illustrating trichocysts and cilia attachments . Schizogony in Ame@ba polypodea - j Sporogony i in Saccharomyces cerevisiae, B. mycoides and Leucocytozoon lovatt. . . . . Karyokinesis in Acanthocystis aculeata and Coleosporium senecionis . . Protomitosis in Ameba mucicola, Ameba froschi, Euglena splendens, and Ameba diplomitotica . . Mesomitosis in Pelomyxa palustris, ‘U1 rospora lagidis, and Galactima succosa . . Conjugation in "Schizosaccharomyces octosporus ‘ Nuclei in mycelium of Thamnidium elegans and Mucor circinelloides. . Fragments of mycelia of molds with dividing nuclei. Filaments of molds showing chondrium . Nucleus of Mucor in various stages of division . . Metachromatic corpuscles in Dematium . Metachromatic corpuscles in asci . . Metachromatic corpuscles in conidia . . Metachromatic corpuscles in cell of perithecium of Pestularia: vesi- culosa. . . Mucor, general . . Mucor, zygospore. . Penicillium expansum . . Aspergillus glaucus . . 4 . Aspergillus fumigatus, A. nidulans . Cladosporium herbarum . . Spores of Alfernaria. . Fusarium . . . Oidium lactis. . Monilia candida . . Yeast cell . 2 xxl XXli LIST OF ILLUSTRATIONS 42. Spore-bearing yeast cells. . 2... ee ee ee ee 61 43. Saccharomyces cerevisie showing vacuoles and metachromatic cor- puscles stained. . . 62 44. Saccharomyces cerevisia showing cells with nuclei, nuclear division and glycogenic vacuoles with grains. . . 62 45. Saccharomyces cerevisia showing cells stained by a special method re- vealing a chondrium consisting of granular- and rod-mitochondria. 62 46. Saccharomyces cerevisie with both nucleus and metachromatic granules. . are ae . ; 63 47. Saccharomyces ellipsoideus cells with nucleus . . . a « « 104 48. Copulation and sporulation in Schizosaccharomyces octosporus. ac 66 49. Various stages of nuclear division during Sarena in Schizosac- charomyces octosporus . . Sols : ‘ . 66 50. Cellular fusion in Schizosaccharomyces ‘pombe. - ae 67 51. Heterogamous copulation in Zygosaccharomyces chevalieri. 68 52. Sporulation in Saccharomyces ludwigit. . . . 69 .53. Germination of ascospores in SECM ludwigit : ‘ : . 70 54. Wine and beer yeasts. . 3 5 s 72 55. Wild and pseudo-yeasts . : Bie noms . 75 56. Types of micrococci. é : 77 57. Types of bacilli. . : ; : ; 77 58. Types of spirilla . . . ch e2 & A i ‘ . 78 59. Involution forms. . be RE 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 Bact. anthracis. . . we aog ns 86 67. Plasmolytic changes. . . . 4 ek oy 87 68. Karyokinetic appearances in Bact. gammari : ee . 89 69. B. megatherium in process of division . . : 90 70. Diffuse nucleus in Chromatium okenii and Beggiatoa ‘alba. . gI 71. B. butschlit in division. 93 72. B. sporonema in spore formation with vestiges of ancestral sexuality 94 73. B. radicosus with nuclear appearances. . . ae e 2 O04 74. B. flexilis in division of cell and formation of. spores. a 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. . . . ; ae ihe te 8 99 77. Structure of bacterial membrane in section. . 4 , 101 78. Capsules (Bact. pneumonia) . . : . . 102 79. Distribution of nuclear substance and various flagella s » « 103 80. Monotrichous bacteria (Msp. comma)... . is! 9 ate 103 81. Monotrichous bacteria (Ps. pyocyanea) . ; ; ee a @ 2103 82. Lophotrichous bacteria (Ps. syncyanea) al 3 ‘ b> fe oe. TOS 83. Lophotrichous bacteria (Sp. Debra) Oe ee ae : . 103 84. Peritrichous bacteria (B. meets nate wet og eo 103 85. Crenothrix polyspora... . . BO see ahs) he ne » . 107 86. Chlamydothrix hyalina. . fee Bren eee le dine ss» ¢ IO 87. Cladothrix dichotoma.. . 2. 6 we ee ee ee ee we TID 88. Beggiatoa alba . hy Smee Pee ve. og SRLS * 89. Pasteur-Chamberland ‘or Berkefeld filtering apparatus. o's ey ae AL7 LIST OF ILLUSTRATIONS . Ameba ves pertilio. : . Paramecium caudatum dividing ‘without mitosis. . Stages in division of Ameba polypodia. . . . . . Multiplication of Coccidium schubergi . 5 . Herpetomonas musce-domestice. . Trypanosoma tince and aad perce. . Trichomonas ebertht . ‘ . Lamblia intestinalis . : . Development of sporozoits in Laverania malaria Ameba proteus . . Influence of oxygen on microorganisms. . Crystals of bacteriopurpurin . . Carbon cycle. . Nitrogen cycle . Sulphur cycle . 4 . Action of light on bacteria. . Action of light on molds. . Action of light on mold colonies . Chemotaxis . sash is 2 he S . Curve of disinfection. 2 2)... . Influence of filtered water on typhoid fever and Asiatic cholera. . Section of sand filter . . eae ‘ . Unglazed porcelain filters . 114, 115. Location of wells on farm . . Construction of model well. . Trickling filter, sand filter, dosing tank, septic tank . 3 ODES CAME i ga see ate. Gee ces dk sate ee . Non-symbiotic nitrogen-fixing organism (B. pasteurianus) . . Non-symbiotic nitrogen-fixing organism (Azotobacter vinelandi) . Ps. radicicola. .. 1... - Aue don . Section through root tubercle. . 124, 125. Influence of Ps. radicicola . Section of cow’s udder. . . Bacterial colonies in dust from udder }. Bacterial colonies from cow’s hair. . Bacterial colonies from dust.of stable . . Small-top milk pails. ea . Ropy cream . : . Ropy cream organisms B . Chart of Rochester milk supply. . Gassy cheese. . . : : Cheese from lactic starter . . Influence of lactic organisms on casein degradation . . Swiss ag : a Kepfir gr 138a Tatsss foe Teces examination 139. 140. 141. 142. 143. 144. 145. ‘ 146. 147. 148. Bacteria of slimy wine. Bacteria of wine diseases. . Vinegar bacteria Vinegar barrel . Rapid process vinegar ‘apparatus . Ps. medicaginis. Pear blight. . . Walnuts affected by bacteriosis. Crown gall. Roots of cabbage | plant affected with ‘ ‘stump-root” ss 349, 350, 351 ar - 369 . 373 Xxili 4 T21 124 : ; 126 . 131 132 133 134 135 144 . 157 177 184 . 185 186 . 222 . 223 224 230 ae 233. . 259 267 269 271 272 285 286 340 341 345 374 » 375 : 377 397 » 398 402 . 422 423 . 429 . 434 - 443 . 505 . 513 514 540 » + 544 - 547 . 591 - 597 - 603 . 605 . 609 Xxiv : LIST OF ILLUSTRATIONS 149, Plasmodiophora brassice@. . . . 1. we ee es Jo ud ah ere GLO 150. Oidium albicans. . e Bie dee Sows a 25 151. Oidium albicans. (Kohle and Wassermann.). fe. EG . 725 152. Trichophyton tonsurans A io ws e foe gM aw Be 720 153. 154. Actinomyces bovis. #4 eae 8 be oe ae wee : 728, 729 155. Gonococct . Shah & oD oe stageets Se 735 156. Bact. anthracis, ‘thread formation ‘ a eee eee 751 157. Bact. anthracis, spores. . . : 5 Gee aaa ees 4 751 158. Organisms of anthrax in capillaries r 4 Mey wit 8 eae 3 af - 753 159. Bact. diphtheria ; : : eee ie . 761 160. Westbrook’s VypEs @ of Bact. ‘diphtheria . eeu i ee on de » » 762 161. Bact. mallet . z j ee . 769 162. Bact. pestis. . . 6 Bs ee el ok 777 163. Bact. tuberculosis, ‘branching forms . ‘ iS cae ils 28 . 782 164. Bact. tuberculosis, from sputum. z noe 782 165. Bact. tuberculosis,in culture... . . es aS 2 8 . 783 166. B. tetani, with spores : ; See ‘ ‘ ch ZOE 167. B. typhosus Recta: ce : ar oes ia AOS 168. Msp. comma. . Bo ie, a ae wea ots . 799 169. Msp, comma colonies in gelatin. oe . . 800 170. Kidneys in hog eee pene points Bede Be she . . . 808 171. Negri bodies... . “ ‘ ye Ska ate gh i 818 172. Amebacoli.. . . bl eae hte ee . 823 173. Leishmania donovani... .. ed 2. fie . 2. 825 174. Structure of trypanosome a fe 827 175. Trypanosoma gambiense. . .. . j 828 176. Glossina palpalis . . ‘ ‘ 5 j j . 829 177. Colonization in Trypanosoma lewisé . Ie a Se RSS soe . 832 178. Malarial parasite in human and mosquito cycles . . 835 179. Longitudinal section of Anopheles. Bee sad sasor ig gsr adeh ders Saas . 838 180. Babesia bigemina. .. . ‘ . ‘ es . 840 181. Ornithodoros moubata : Bienes casey aS i Rae B45 182. Spirocheta duttont.. . . . S5 wh fae any ge. A Sg : hy . 846 183. Treponema pallidum. . ei ‘ ‘ ‘ : . 848 Colored Plate The Malarial parasites... . 1... . ee ee . 8357836 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 microérganisms. : In the year 1683 Antonius van Leeuwenhoek, a Dutch naturalist and a maker of lenses, communicated to the English 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. XN 2 HISTORY OF MICROBIOLOGY that microdrganisms were capable of reproduction outside of the body, and that they might be conveyed from place to place by the air. The important réle 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 oo 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. 1. From that date the microscope gradually improved. In 1844 the immersion lens was introduced by Dolland. In 1870 Abbé 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. Y c ee c 5 a bb Q2, dA t Fr Fic. 1.—Longitudinal section of a compound microscope made by Zaccharias Jansen (1590). a@, Microscope tube; b, objective tube; c, ocular. In 1786 O. F. Miiller (a Dane) first attempted to classify, according to the Linnean system, the various organisms previously discovered, and characterized four or five genera—among them, the genus Vibrio, in which, under the terms bacillus, lineola, and spirillum, we recognize forms that correspond with our “bacteria.” From the middle of the eighteenth century until well on into the 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 preéxisting 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 preéxisting 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 Abbé 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 Franz Schulze, and the discovery by Schroeder and Dusch in 1853, that putrescible fluids will not decay after boiling, if protected from the bacteria of the air by means of a cotton-wool filter or plug; and the epoch-making experiments of Pasteur in 1860, with the now well-known Pasteur flask, showed conclusively that the hypothesis of spontaneous generation, or abiogenesis, could not be proved. - Liebig, the celebrated German chemist, strenuously opposed the theories of Pasteur; his authority and the brilliancy of his expositions influenced the scientific world during the period 1840-60. 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 microdrganisms,” 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 “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 1860 the 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 flourishes without free oxygen. He devised an improved method of making vinegar, and demonstrated the presence of the acetic organism which he named Mycoderma aceti. Later he studied the diseases of wine, and 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 microérganisms. 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 Pébrine. 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 réle 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 animal in a sterile condition, thus proving that the souring of milk was caused by organisms from some external source. In 1872, F. Cohn’s System of Classification, based on morphological characters, appeared. He distinguished six genera—micrococcus, bac- terium, bacillus, vibrio, spirillum, and spirochete; 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 microdérganisms 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 réles 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 protozoén of malaria; in 1881 Robert Koch introduced the poured gelatin and agar plate, which made it possible to obtain pure cultures without difficulty. Investigators were quick to take advantage of this method; and notable results followed. Eberth and Gaffky discovered the bacillus of typhoid fever, and succeeded in growing it in culture media. In 1882, Loeffler and 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, Loeffler grew the organism in pure culture. ~ In 1884, Koch published his results on the etiology of tuberculosis, in a paper which will remain as a classical 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 1885, Nicolaier observed the tetanus bacillus in pus produced by inoculating mice and rabbits with soil; later, in 1889, Kitasato isolated this organism, and showed that the cause of the failure in earlier attempts to isolate it were due to the fact that it could grow only in the absence of free oxygen. The specific infecting agents in pneumonia were discovered by Friedlander and Fraenkel about this time, as were also several 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. *All 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 been engrossed with his prophylactic studies. In 1880, he dis- covered a method of vaccination against fowl cholera; and in 1881 he published his method of vaccination against anthrax. On a farm at 'Pouilly le Fort, sixty sheep were placed at Pasteur’s disposal; ten of these received no treatment, and twenty-five were vaccinated. Some days afterward the latter 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 that 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 Yersin and Kitasato independently discovered the bacterium of bubonic plague. The now well-known serum diagnosis of typhoid fever, whereby living and motile typhoid bacilli are clumped and lose their motility when placed in the diluted serum of a patient suffering from the fever, was due to the work of Gruber and Durham, and the exploitation of the method by Widal dates from 1896. Tn 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 syphi- lis, a spirochete-shaped organism, which he named Treponema pallidum, and which had escaped earlier discovery on account of its being refractory to the ordinary staining methods. No one can deny that the progress of microbiology in the last forty years has been extraordinary; but much still remains unknown. The 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 zodlogist 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. TS I2 MORPHOLOGY AND. CULTURE OF MICROORGANISMS OvTLINE oF PLrant Groups* The following is a diagram of plant groups, showing one scheme of placing the bacteria, yeasts, and molds in relation to othergroups. Onlya few of the sub-groups can be shown in such a scheme. Schizophyta | Schizomycetes (fission-fungi), bacteria. (fission- plants) Schizophycee (fission-algz), blue-green alge. Chlorophyceze—green alge. Alge Pheophyceze—brown alge. Rhodophycee—red alge. Characee. Myxomycetes. Chytridinez. Zygomycetes (Mucors). Saprolegniacez Phycomycetes deter find. Oomycetes | Peronosporaceze (downy mildews). Hemiasci (Monascus). Protoascinez (Sac- Thallophyta charomyces, Yeasts). 5 Protodiscinez. Plaats Hung Cece Euasci } Discomycetes. Plectascinez °(Asper- gillus). Pyrenomycetinez. Penicillium, Fusarium, Alier- Imperfect Fungi, } aria, Conidia only Oidium, Cladosporium, and others. Rusts Basidiomycetes Smuts Mushrooms. Bryophyta (mosses and liverworts) Pteridophyta (ferns, etc.) Spermatophyta (seed plants). *Charles Thom. OUTLINE OF PROTOZOAL GROUPS 13° OUTLINE oF ProtozoAL GRrouPs* “AN OUTLINE CLASSIFICATION OF THE PRotozoa,” embracing only parasitic and more especially the pathogenic forms. For discussion of classification see p. 130. Protozoa Rhizopoda Flagellata Sporozoa Infusoria Entameba buccalis Entameba coli Entameba | Entameba tetragena (histolytica) Entameba meleagridis Plasmodiophora { Plasmodiophora brassice. ; 5 Leishmania donovani Leishmania : fs . Leishmania tropica Crithidia Leishmania infantum Trypanosoma gambiense Trypanosoma cruzi Trypanosoma brucez Trypanosoma Trypanosoma evansi Trypanosoma equinum Trypanosoma dimor phon Trypanosoma lewisi Trypanosoma equiperdum Trypanoplasma = eee | Trichomonas intestinalis richomonas ; ae Minis Trichomonas vaginalis Plagiomonas Lamblia {Lamblia intestinalis ae Coccidium cuniculi (Eimeria stieda@) occidium say'e ‘ Coccidium avium Plasmodium vivax Plasmodium ; Plasmodium malarie Plasmodium falciparum Proteosoma Hemosporidia Heeteoproteus Lankesterella (and other Hezemogre- garines) Hepatozoén Babesia Babesia bovis (bigemina) Babesia canis Sarcosporidia { Sarcocystis{ Sarcocystis miescheriana Haplosporidia { Rhinosporidium { Rhinosporidium kinealyi Myxosporidia { Myxobolus { Myxobolus pfeifferi Microsporidia { Nosema { Nosema bombycis {Balantidium{ Balantidium coli * J. L. Todd. Revised by Ernst E. Tyzzer. 14 MORPHOLOGY AND CULTURE OF MICROORGANISMS Toxoplasma Chlamydozoa Ultramicroscopic viruses Spirocheta obermeieri Parasites of uncertain Spirocheta duttoni position Spirocheta { Spirocheta vincenti | Spirocheta theileri Spirochata gallinarum Treponema pallidum Treponema | Treponema pertenue CHAPTER I* ELEMENTS OF MICROBIAL CYTOLOGY CELLS AND ENERGIDS' The microérganisms are confined to cells, such as alge, 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.3,4). There are also some cells which have a special structure. In each cell are enclosed several nuclei. If certain amoebe 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 withthe cytoplasm which surrounds it, equivalent to a _cell, may be called specifically an energid. Some alge and fungi are made up of threads of cells enclosing several nuclei; each cell in- cluded in @ thread consequently represents a group of organized ele- ments, the union of several energids in the same anatomical unit (Fig. 3, A). 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 microérganisms 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. 15 16 MORPHOLOGY AND CULTURE OF MICROORGANISMS fluid are found: the mucleolus, a spherical corpuscle made up of pyrinin 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 isasmall chromophilic grain which is often surrounded by a clear zone of protoplasm called archoplasm. as er” Fic. 2. Fic. 3. Fic. 2.—Cells of Saccharomyces cerevisiae. Frc. 3.—Cells made up of several energids. A, A portion of the mycelium of a mold, Aspergillus ochraceus. (After Dangeard.) B, Cell of an amceba, Pelomyxa palustris (After Doflein). i 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 réle 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 17 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- 5) terial of nuclei, and their evolution during the forma- Si tion of spores, force the observer to admit that these ¢ E cs represent grains of nuclear substance, and that bac- as - teria have a kind of diffuse nucleus, which is scattered Fie) pape in the form of small grains (Fig. 4) in the cytoplasm fuse nuclei of of the cell. bacteria. A, B. ae d : mycoides. (After Forms of Nuclei in Microérganisms—The nucleus Guillier mond.) of primitive microérganisms is far simpler than in ee the higher forms, where it becomes fairly complex. Swellengrebel.) Consequently in the Cyanophycee or blue alge, the ; lowest of all alge, 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 microérganisms the nucleus is Se & much more complex. Yet frequently this ; G nucleus is found in a primitive state quite different from typical nuclei of higher i 2 organisms. In some amcebe, the nucleus it is formed simply of a poorly defined mem- e ES) brane filled with nuclear fluid, and a large 5 B body of chromatin resembling a nucleolus A called the karyosome or centriole-nucleolus Fic. s—Nuclei of Cyano- (Fig. 21), because it acts both as a cen- phycee. A, Thread of Rivu- triole and as a nucleolus. In the center of laria bullata with nuclei in Heo fan 1 process of division. B,-D, the karyosome is frequently seen a more Fragments of threads of Calo- jntensely chromophilic corpuscle corre- thrix pulvinata showing nuclear - , i Eieane sponding to the centriole (Fig. 20, B, a). Many protozoa and some alge 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 microérganisms (protozoa and fungi) the nucleus 2 ) Woes 18 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. 109, 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 macronucleus 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 Tap 6 Chromidia anor that all cells have two nuclei. Recent tozoa. A, The cycle of the mi- evidence reveals that there are in the (After SOLAN cytoplasm of most protozoa small chro- meba hystolytica. (After Hart- mophilic granules, like the chromatin meee t, 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.—A ppearance and Properties of Cytoplasm.—Cytoplasm may be defined 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 fi IQ 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 microérganisms, 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 réle in the cell. These structures, visible in the living organism, but stained AA a fe ! f 1 Ke 4 L "gn ‘ V4 < ss Aa i i 3 Ait oF Ree, bf Sn i) Ne se Kis “AY ch =~ hie 0 Woe ‘A -B . yy" A Fic. 7.—Glaucoma piriformis, Fic. 8.—Division of micronu- infusorian with (NV) macronu- cleus and of the chondriosomes cleus, (7) micronucleus (ch) in Carchesium polypinum, infu- mitochondria, (vp) pulsating sorian. (After Fauré-Frémiet.) vacuole. (After Fauré-Fré- miet.) 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. 7, 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. All the mitochondria of one cell are called the chondrium. ‘These structures seem to be made up of lipoidal substance and phosphates of albumin. The mitochondria cannot generate themselves directly from the cytoplasm, but are formed always from preéxisting 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, whateyer 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 chloroplastids, make the chlorophyl RYR LT oS k a4 \, Za NY x) . | Fae | +f | | & aAl Sa al feated a > oh paar) > Sy Sd * », Cal Bn fa ioe Sve OS an ie { Netge eee \Q yey) bat 2 emma A Fic. 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 amyloplastids, confine themselves to forming starch from the excess sugars found in the cells; still others, the chromoplastids, 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 alge 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. rr), etc.—but all appear to be made up of a mitochondrial substance. Their physiological réle 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- Ge \ a7" cAl iw f__-S\) ee / Fic. Io. Fic. 11. Fic. 10.—A cell from the root of a bean in which the rod-mitochondria (ch) form in the course of their development amyloplasts from (p) which spring grains of starch (a). Fic. 11.—A, Euglena viridis with its star-like chloroplasts (chl.) at the center of the organism, the pyrénoid body (Py) surrounded by grains of paramylum (Par), eye-spot (0), contractile vacuole (v), flagellum (f), nucleus (x). (After Dangeard.) B, Microglena punctifera, with two elongated chromatophores arranged longitudinally. (After Stein.) quently the complex chloroplastids of the alge 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 Cyanophycee are the only microdrganisms in which the chon- drium has not beenfound. In the Cyanophycee the chlorophyl and the blue pigment (phycocyanin) associated with it are diffused throughout the cytoplasmic area surrounding the riucleus. The very primitive structure of the alge 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, 11). 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 microdrganisms are the granules called metachromatic corpuscles (Fig. 11, '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 alge and bacteria, and in many protozoa. Glycogen and paraglycogen are equally well distributed in micro- drganisms (fungi, protozoa). Among alge, glycogen is found only in the Cyanophycee, 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 Mucorine, 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. MEmBrRANE.—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 microérganisms is closely associated with the mem- brane, for the movement of cytoplasm and the flexibility of the mem- Fic. 12.—Metachromatic corpuscles (cm) in Sarcosporidia, Sarcocystis tenella. (After Erdmann.) Fat globules (g) in Trypanosoma rotatorium. (After Doflein.) brane are essential factors. Cells as a rule have a membrane of different 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 (alge, 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 Cyanophycee, in bacteria surrounded by capsules, and in zoéglea. The membrane then becomes extremely thick (Fig. 13, B). LocomotivE STRUCTURE.—Most alge and fungi cannot move. Many bacteria and all protozoa have more or less perfected locomotive structure. The Cyanophycee 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 amceba. The naked cell of the amceba pushes out pseudopods, simple expansions of the ecto- plasm arising’ at any part of the body, which take various shapes, and reénter the body without leaving the least trace of their existence. It is a result of motility of the cytoplasm, one of its essential properties, _ shown here exteriorly because of the absence Fic. 13.—A, Cyst of Amebalimax. (After Dan- Of @ Cellular membrane. geard.) B, Thread of nostoc The locomotive structure is more com- surrounded by a thick muci- : laginous case. plex in other protozoa; the pseudopod is replaced by contractile appendages— flagella, or vibratile 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 body, 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- Ree eth like pseudopods are found with a rapid \‘ oN (i rhythmic movement which may serve as eK j ) % intermediate forms. Be that as it may, the 4 _ Aon oe Biv method of forming these organs is of special -— interest. Apparently they are formed under the influence and at the expense of the cen- Frc. 14.—Organs of loco- triole motion in bacteria. A, B. d P subtilis. _ (After Fischer) In the Flagellata the flagellum is always B, Microspira comma. inserted in the centriole or in a similar organ (After Fischer and Migula.) ; : 5 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 in general in 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 blepharoplast or 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 ise sry . , end DOV ANE a AWAY Fic. 15. Fic. 16. Fic. 15.—A, Spongomonas uvella. 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 (Z) exists the final nuclear division. (After Martin.) Fic. 16.—Fragments of the peripheral portion of Prorodon teres (infusorian) with vibratile cilia and their basal corpuscles. (ect) Ectoplasm; (end) endoplasm; (tr) 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 27 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- tion (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 Coy es) cea 5 oe A met is ! Se) be Be Fic. 17.—Schizogony in Ameba Fic. 18.—Sporogony. A, Formation polypodia with amitotic division of spores in Saccharomyces cerevisie. B, of the nucleus. (After Schulze Formation of spores in B. mycoides. (After and Lange.) Guilliermond.) C. Formation of spores in Leucocytozoon 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. Nuciear Divisron.—Nuclear division may occur in one of two ways, one very complex, (1) 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 Heliozoin, 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, A, 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, 0). Following this, the karyosome dissolves in the nucleoplasm, supposedly conveying material to the chromatic network which enriches itself noticeably in chromatin. Thechromaticnetwork then relaxes, thickens and transforms itself into a more or less spiral cluster, the spireme (Fig.19;A,c). Atthesametime 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 20 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 éelophase or phase of reconstitution of the two nuclei which terminates the process. In this phase, the chromosomes “9 9 d e fF g Fic. 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; (f) anaphase; (g) telophase. (After Schaudinn.) B, In Coleosporium senecionis (Uredinee). (a) Nucleus at rest with its centriole extranuclear; (b) formation of chromosomes; (c) equatorial plate; (d) metaphase; (¢) anaphase; (f) (g) (z) telophase. (After Madame Moreau.) € 4 + eo ® ) ° / é y y (o form a spiral chromatic cluster making a spireme at each of the poles (dispireme stage, Fig.19, A,g); 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 Uredinee (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 adefinite 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 réle 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 microdérganisms. 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 Cyanophycee (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 31 Pata fs i” r | r ors Ana, / \ ay tae ¢ ik [Sigest / HA ae eS ‘ A, By) { 4 NWS ox t vy ay Mt (Ney f, Never, peat Ds NB @) <@ wi-” et 1 4 { x Se ae / ' ) eta: 9 b rth seen Fic. 20.—Protomitosis. A, In Ama@ba mucicola. (a) Nucleus at rest; (b) beginning of prophase; (c) division karyosome; (d) division of centriole; (e) (f) equatorial plate; (g) metaphase; (z) (k) telophase. (After Chatton.) B,In Ameba froschi. (After Négler.) C, In Euglena splendens. (After Dangeard.) D, In Ameba 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. s, 0). We find in the Ameba 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, f), 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, 7, k). The nuclear membrane exists during the entire phenomenon. In other microérganisms (Amebe, Flagellata, Euglene) 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, c), and the two resulting centriole-threads pass to the extremities of the achromatic spindle, while the karyosome codperates in the formation of the chromosomes (Fig. 21, A, d, ¢). In other cases (various fungi, Gregarine, etc.), the centriole be- comes extranuclear, and the karyosome acts as a true nucleolus (Fig. Fic. 21.—Mesomitosis. A, In Pelomyxa palustris. (a) Nucleus at rest; (b) —(e) division of centriole; (f) (g) equatorial plate; (z) anaphase. (After Bott.) 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; (b) anaphase; (c) telophase. 2t). 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 3 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 Amebe and Mycetozoa the karyosome enclosing all the chromatin divides itself into two equal r Fic. 22.—Conjugation in Schizosaccharomyces octosporus. (a) Two gametes in the process of fusion; (0) (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 microérganisms 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 (isogamy) 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 alge, 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 hyphe, singular, hyphae). These hyphe usually branch in more or less complex manner forming networks or webs, collectively called mycelium. Hyphz may be one-celled or composed of many cells placed end to end as shown by the cross walls, called sepia, 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 hyphe. 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 Thom. 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 hyphe 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 gemme is sometimes applied to similar structures, preferably to such as grow at once. The same end is reached in still other groups by the formation of sclerotia which are hard masses or balls of thick-walled cells filled with concentrated food materials. These sclerotia are 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 avery 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 (phylogeny) of the fungi, if known, would show several or many lines of descent rather than one. Certain of these groups may be presented briefly. BactEr1A.—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 as 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 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. Asa 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 ascus, a sac containing, when ripe, typically eight spores, some- times a less number by the failure of some to develop, sometimes a larger number, usually some multiple of eight. The ascus 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 hyphe 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 heen 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 hyphe 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. / ImpEerFECT Funci.—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- perfect fungi and their grouping regarded only as temporary, a con- venience for the identification of materials. These include many forms of 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. CytToLocy oF Mo.ps* GENERAL STRUCTURE OF Motps.—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 whén 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, 1 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 Mucorinee, 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 coenocyies (Fig. 24,1). 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. Guilliermond. MOLDS 4I There are, moreover, molds which show both these last two struc- tural types, with transitional forms between the two. For instance, in Endomyces magnusit, the mycelium, ordinarily consisting of areas, each containing many energids, can in some parts progress to a uninuclear cellular structure. The conidia or spores of many molds may have either one or many nuclei, according to the Fic. 23. Fic. 24. Fic. 23.—1, 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 Léger.) Fic. 24.—1, Mycelial filament of Endomyces magnusti. 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 Mucorinee 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 42 MORPHOLOGY AND CULTURE OF MICROORGANISMS several nuclei. The yeast-forms of the Mucorinee, 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. Cyroprasm.—The cytoplasm is a semi-fluid mass, somewhat dense, Fic. 25.—Various molds fixed and stained by a special technic, showing thelr chondrium. 1, 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 magnusit. 8 and 9g, 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. The division of the nucleus is not always easy to observe. it, one must examine the growing tips of the mycelium. 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. Nucte1t.—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). To study 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 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 Mucorinee, true métamitoses have been found. In the Mucorinee 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 Fic. 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 PRopucts.—The vacuoles always contain a great many shining granules, showing Brownian motion and capable of being stained in the living state by ‘neutral red and methylene blue. These bodies have staining qualities which permit them to be easily characterized. They are stained a violet-red by most of the basic dyes, aniline blue or violet. They also take on a very pronounced reddish tinge with hematoxylin (Fig. 27). By reason of this property of metachromatism, they have been called metachromatic corpusices. These bodies, which are very common in the Protista, have been found in yeasts, bacteria, alge and protozoa. The chemical nature of the substance constituting them is still unknown, but the name metachromatin is often used for it.1_ Some authors, among 1Because of the priority and more exact signification, the names metachromatic corpuscles and. melachromatin are preferable to the terms grains of volutin and volutin 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 réle 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, 1 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 Sy és> see” 3 x 9 TN “eae pp O®, Fic. 27. Fic. 28. Fic. 27.—Dematium species Stained by a method permitting the differentiation of the metachromatic corpuscles. 1, 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). m. Nucleus. v.c. Vacuole with metachromatic corpuscles. Fic. 28.—Various stages of the development of the ascus in Aleuria cerea. rt 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 coéxistent 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 ft 207 Conia organ of Aspe grain on one side; then disappears corpuscles. 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, undergo the same evolution as above. Thus the metachromatic corpuscles, like grains of starch in the higher 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 46 MORPHOLOGY AND CULTURE OF 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. Py». Ax 92 ie 4 yw a Vee Hey : Fic. 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. CreLt-Wati.—tThe 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 Motps* 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 Thom. MOLDS 47 constantly found upon-every kind of putrescible matter, especially as the causes of fermentation or decay infood. ‘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. Mops 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. GENERIC CONSIDERATION OF GROUPS* THE Mucors or BLtack Motps.—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 hyphe, 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 hyphe 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 rouxti (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. Fic. 31.—Mucorinee. Mucor. From Tabule Botanice, 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 oryze, 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. 4 50 MORPHOLOGY AND CULTURE OF MICROORGANISMS THamnipium.—Of related genera, Thammnidium 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. Fic. 32.—Mucorinee. Mucor, Rhizopus. A, B, C, D, Formation of the zygo- spores from conjugating branches; £, section of D; F, mature zygospores in section; G, germination of zygospores; H, diagram of fruiting stolons of Rhizopus nigricans; K, section of sporangium during spore formation, highly magnified (From Tabule Botanice.) (Reduced one-half.) (By permission of A. F. Blaskeslee.) Thamnidium elegans, Link, produces primary and secondary spor- angia on different hyphe, together making white colonies. The fertile side branches are produced in whorls and bear whorls of branchlets from their centers which in turn produce sporangioles from the tips of short straight twigs or branchlets. PENICILLIUM.—The extremely abundant green molds most fre- quently belong to the genus Penicillium, although some members of other groups may be confused with them at times. Characters—Colonies are composed of loosely woven hyphae, branched, septate, colorless, or bright colored. The fertilehyphze MOLDS 51 (conidiophores) are mostly erect, arising either from submerged hyphe, or as branches of aerial hyphz, septate, usually branched only in the fruiting portion. Conidial fructifications consist of more or less com- Voy 7 t a gn rd Se ee ATL S Fic. 33.—Penicillium expansum, Link. a, b, f, Branching and arrangement of branches of conidial fructification (goo); c, d, e, conidiiferous cells and conidial chains (Xg00); g, h, j, k, l, sketches of fructifications (X140); m, n, 0, germination of conidia (Xg00); r, s, sketches from photographs showing in s loose aggregations - of conidiphores beginning to develop into zonately arranged coremia, in‘? a coremium x mm. in height. (From Bul. 118, Bureau of Animal Industry, U.S. Dept. 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 of nutrient media for members of the group. The larger number of the species grows best at temperatures from 15° to 30°; a very few of them reach their optimum at 37°, but many species are entirely inhibited and some killed at blood-heat. Vegetative mycelium begins to be produced at temperatures very close to freezing, but colored conidia are produced slowly or not at all at low temperatures. The species of Penicillium thrive through a wide range of concentration of culture media, though perhaps the most characteristic growths are produced in media high in water content. The common species of each genus will grow in all the standard bacteriological media. With few exceptions the species grow well in synthetic media composed of assimilable car- bohydrates and inorganic salts. A few species require the presence of 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, Thom, 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, Thom, 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 spores which has been used physiologically to detect the preserice of arsenic by its ability to set free arsine from such substrata. Except species associated with particular processes or substrata, the identifica- tion of the green species of Penicillium requires special methods and greater care than is possible aside from special study of the group. ASPERGILLUS (AND STERIGMATOCYSTIS).—The genus Aspergillus in- cludes numerous species which develop under widely different condi- tions. Many of these forms reach their typical development under drier conditions than Penicillium and Mucor, such as stored grain, her- barium specimens, dried flesh, or foods containing concentrated sugars, such as jams, jellies, etc. Some excite processes of fermentation, and a few are associated with diseases. Characters —The vegetative hyphe are creeping, submerged in the substratum or sometimes aerial also, loose, floccose, branched, septate, usually colorless, and sometimes bright colored. Conidiophores or fertile hyphe are erect, unseptate, or few-septate, usually much larger in diameter than the vegetative hyphe, and gradually enlarged upward, ending in a more or less abrupt dilation or head which bears closely packed columnar sterigmata or conidiiferous cells over the whole or a large part of its surface (Fig. 34,6). Each of these cells bears, in one group of species, a single chain of conidia, in other species (called by some authorities Sterigmatocystis) three or four secondary sterigmata which bear the conidial chains. Part of the species produce also thin- walled perithecia as yellow or brown spherical bodies upon the surface of the substrata. These perithecia are filled with eight-spored asci (Fig. 34, e). A few species produce sclerotia instead of perithecia, but many species are not known to produce either perithecia or sclerotia. Important Species—Among the species constantly met with, “Aspergillus niger is recognizable by its black or very dark brown spores and in some strains by black sclerotia. Several black-spored forms are described, but their separation is usually impossible from the data given. Aspergillus niger ferments sugar solutions with the production of oxalic acid in considerable quantity. Of green forms, Aspergillus* glaucus, Link (Aspergillus herbariorum, Wiggers), and Aspergillus repens, De Bary, both produce abundant yellow perithecia. These abound upon herbarium specimens, hay, *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 by 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 3u* in diameter. This species produces a destructive disease of birds known as aspergillosis. The same species is sometimes reported as pathogenic to man. % Fic. 34. Fic. 35. Fic. 34.— Aspergillus glaucus. a, Conidiophore showing increased diameter over the vegetative cells at its base (128); b, sterigmata (450); ¢, conidia, smooth thick walled in this variety, other varieties are spiny (450); d, perithecium( 128); e, ascus containing ascospores (X450). (Original ) Fic. 35.—Aspergillus. (1) A. fumigatus, Fres; (2) A. nidulans. 1 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. ‘io Aspergillus oryze has been used to produce “Taka-diastase” from rice in Japan. Other species produce amylase also, but in different degrees. Aspergillus wentit, Wehmer, characterized by its long conidiophores and coffee-colored heads of conidia, is found in the soja preparatiorf in Java. Of other forms constantly met, Aspergillus candidus has white or *The unit of measurement is the micron (u) or micro-millimeter (oor mm.or ¥soo0 in.) MOLDS : 55 pale cream fruiting surfaces, Aspergillus flavus produces several shades of yellow and green, Aspergillus ochraceus, ocher or tan. Much confusion is still found in the literature of this genus, so that ‘frequent references to the activities of particular species are difficult or impossible to verify. : CLADOSPORIUM (AND HorMODENDRON).—The species of Clado- sporium occur frequently in cultures of decaying vegetable matter, of milk and cream, or butter. The colonies liquefy gelatin. Both 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.* Wy fed Fic. 36. _Fic. 37. Fic. 38. Fic. 36.—Cladosporium herbarum, showing the forms of conidiophores and conidia which are very common upon laboratory culture media. (Original.) Fic. 37.—Spores of Alternaria sp. (Original.) « Fic. 38.—Fusarium from decaying potato. a, Spores showing curvature and septa; 6, germination of spores; c, development of spores in petri-dish culture; d, mass of spores as found in culture. (Original.) y 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 Fusartum.—The frequent occurrence of species of’ Alternaria 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 Alternaria are brown, Indian-club form, muriform (divided into several cells by longitudinal as well as cross-walls), and are con- nected together into chains (Fig. 37). The spores of Fusarium are *This species has been shown to be a conidial form of Spherella tulasnei Janczewski, but the bacteriological student will meet only the conidial stage. 56 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. Fic. 39.—Oidium lactis. a,b, Dichotomous branching of growing hyphe; 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; #, branching chain of oidia; k, 1, m,n, 0, p, s, types of germination of oidia under varying conditions; ¢, diagram of a portion of a colony showing habit of Oidium aa as seen in culture media. (From Bull. 82, Bur. Animal Industry, U. S. Dept. Agr. Orp1um.—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 hyphe 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. Ozdium 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. Monitt1a.—Monilia candida (Bonorden), Hansen. The line be- tween the Mycoderma group of yeasts, Oidium and Monilia, and the Fic. 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, Dematium pullulans, 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 hyphe submerged in the substratum. At first both myce- lium and conidia are colorless, later some or all of the cells develop ° 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. SAPROLEGNIACEE AND ENTOMOPHTHORACEH.—These are two groups of Phycomycetes which differ from the mucors in habit and in their prominent development of sexual reproduction. ENTOMOGENOUS FuNcI.—Numerous species have been identified as 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 microérganisms. 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. cerevisie of beer). The true yeasts occur in the form of spherical or more or less elon- gated cells varying in normal width from 2.54 to 124. 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 Gymnoascee. 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 such 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. Fic. 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 1° 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. ludwigit). 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. Fic. 42.—Spore-bearing cells. A. S. pasteurianus. (After Bioletti.) B. Sch. octosporus. (After Schiinning.) 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 isseemingly more complex and consequently more difficult to interpret on account of the abundance of the stainable granulations which sometimes accumulate in the cells and 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 cerevisie which, because of the is relatively large size of its cells, lends itself better than (@) (#) any other yeast to a cytological study. Examined in \ os) _\ the living state, highly magnified, the cells of this (=>) yeast show a dense and homogeneous cytoplasm with Vd a group of small vacuoles or a single large vacuole at Fic. 43—Sac- the center. In the vacuoles and also in the perivacu- EOE fe olar cytoplasm, we can clearly distinguish a great cells examined in many small shining granules, of varying sizes, which the living state manifest Brownian motion. It is easy to stain them in a solution of | se : : ; : neutralred. The in the living state (Fig. 43) with a very dilute solu- vacuoles, stained tion of neutral red or methylene blue. These are pale red, contain . metachromatic only metachromatic corpuscles. corpuscles _col- _ ci i — i Beiaark sed: In fixed and stained preparations (Fig. 44, 1-10) 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- Fic. 44. Fic. 45. | Fic. 44.—Saccharomyces cerevisig. 1-10, 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. Fic. 45.—Saccharomyces cerevisie. 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 63 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 ofa 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). Asin molds, these corpuscles appear not only in-the vacuole, but also in the perivacuolar cytoplasm; theré 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. Fic. 46.—Saccharomyces cere- In the cytoplasm of yeasts, also, have eras ane Py mead ee been noted granulations, which can be the metachromatic corpuscles. stained with ferric hematoxylin, which 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 membrane of a nature not yet clearly defined. CyroLocicaL PHENOMENA DURING MuttipLicatTion.—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 be in the mother cell before the appearance of oe the bud. Only when the bud is quite large does the nucleus begin to divide. It is elon- bs & 3 gated so that one end penetrates the bud; the nucleus then resembles an elongated dumb- bell with the larger head remaining in the Fic. Fe ana mother cell and the other, smaller head, in the ellipsoideus. Young cells 2 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- 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 i 65 of the S. cerevisie. 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, 11). 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 zodgloea (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 Ascosporrs.—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 5 66 MORPHOLOGY AND CULTURE OF MICROORGANISMS described, with one nucleus and one or more metachromatic vacuoles containing corpuscles (Fig. 48, @). 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 OD ED € IO O-f) cells (Fig. 48, b, c, d). The septum @ separating the two gametes in the a ch A oD OD middle of the channel is quickly sv U7 New i‘ em q. absorbed, and the two cells then + CO OY) have free communication. The'cyto- aie = plasm of the two cells draws together and mingles in the channel; there the Fic. | 48.—Successive stages of two nuclei draw near to each other copulation and sporulation in Schizo- ‘5 : cj saccharomyces octosporus.| (Fig. 48, ¢) and fuse into a single nucleus (Fig. 48, f, g, 2). 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, 7). Occa- sionally, however, it retains a vestige of the individuality of the two gametes, showing two swellings joined by a semewhat narrower middle portion (Fig. 48, 7). 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- matic corpuscles. The nucleus which occupies the center of the zygote begins to divide. The ascus, containing sometimes four, sometimes eight ascospores (Fig. 48, 7), 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 _, F!6._ 49-—Schizosac- 5 charomyces octosporus. shows a very clear structure with a nucleolus Various stages of the and a chromatic reticulum (Fig. 49, a). It ao during 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. 49,5 andc). The YEASTS 67 nucleolus always persists on one side of the spindle. At a subsequent stage the chromatic granulations or chromosomes are divided between the two poles of the spindle, the nucleoplasm is mixed with cytoplasm, then the spindle elongates, while the chromatic granulations form a homogeneous mass at the two poles (Fig. 49 d, e,gandh). The nucleolus is quickly absorbed, then the two nuclei are formed at the expense of the two chromatic masses (Fig. 49, f). 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- brane is absorbed only at the end of the phenomenon. They show the characteristics of the mesomitoses which have been described in the asci of the higher Ascomycetes. i a. mo a x _ / \ Zn Nee <<) Iq 1 3 VA om fA Pry 4 | \ ’ 4 7 Oy ew i] Og @ 18 26 7 8 Fic. 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, 7); 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. S600 20 Go Ga 6 T ies CAL eg Ge oa @ 3b 90 Se AD 8 ® oe W S oS \7 18 A @ .- @ 10 ag Red &, Yo &- “e i902 pS eR Fic. 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 réle 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 690 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- he ( 4 be \ (Y AN /*\ , \ ‘ Fic. 52.—Sporulation in Saccharomyces ludwigtit. Figs. 1 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 79 MORPHOLOGY AND CULTURE OF 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 ro). 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, 11 and 12), then gradually dis- solve. The ascospores constantly increase in size at the expense of 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 of 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). : Fic. 53.—Germination of ascospores in Saccharomyces ludwigit. 1, 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. /udwigii, copulation, suppressed at the beginning of sporula- YEASTS co 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. cerevisie. 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. 4 \ 42 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. - f Fic. 54.—Wine and beer yeasts. A, S. ellipsoideus, young and vigorous; B, S. ellipsoideus, (1) old, (2) dead; C, o nn bottom yeast; D, S. cerevisiw, 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 arrack. It fer- ments the sugar produced from rice by the molds, Mucor oryzeand Rhi- ZOPUS Or yZe. : 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 Deveee 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 sometimes producing a branching mycelium. Spores are formed easily and abundantly. The apiculate yeast, S. apiculatus, is very abundant on grapes and most acid fruits. Itis 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 produced in the fermenting liquid. The fermentative power is high and a large percentage of alcohol may be formed. Several other species of this gents have been isolated from grapes and from Jamaica rum. PsEupDo 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 Toruia. 74 MORPHOLOGY AND CULTURE OF MICROORGANISMS They are usually small, spherical or slightly elongated. Some species produce a little alcohol and some none. They seldom occur in suf- ficient quantities to be harmful and one form is accredited with pro- ducing the special flavor of some English beers. The forms included under Mycoderma resemble yeast in shape 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 of the latter is Laurent’s medium: : Ammonium sulphate, 4-71 & Potassium phosphate, 0.75 g. Magnesium sulphate, o.Iog. Water, : iL. To this is to be added any carbohydrate to be studied. Media may be made solid by the addition of gelatin or agar. Pure cultures can be made, rarely, by inoculation from a naturally pure source such as the sporangium of a Mucor. Physiological Separation.—The first attempts at purifying mixed cultures were by means of physiological differences. Pasteur freed yeast from bacteria by growing it in a medium containing 2 per cent. of tartaric acid. Effront used fluorides in the same way. These methods may be made more effective by repeated transfers of the culture. Each transfer will contain a larger proportion of the form most suited to the 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 serene ~ show only a few, some of which may be pure cultures. : Fic. 55.—Wild and pseudo yeasts. A, S. pombe. (After Lindner). B. Torule. (After Pasteur.) C, Mucor, (t) spores; (2) germinating spores and mycelium. D, S. apiculatus. E, Mycoderma vint. (After Bioletti.) ‘ ' 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 methoda 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. membranefaciens) 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 microérganisms quite distinct and characteristic, while the higher bacteria form links, as it were, between the lower bacteria and other closely related microérganisms. The morphology of the two groups will need to be discussed separately. * Forms or Lower Bacteria* J FUNDAMENTAL Form Typres.—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 micrococct (sing. coccus or micrococcus). The straight rods are bacilli (sing. bacillus) and the bent rods are spirilla (sing. spirillum). att i a0fe 3 3% eg et coon B Fic. . 56.—Types of micrococci. (After ees 5 oY 2 aK. (go ° tH 2 6 oe. (U _ Fic. 57.—Types of bacilli. (After ks *Prepared by W. D. Frost, with cytology by A. Guilliermond. 77 78 MORPHOLOGY AND CULTURE OF MICROORGANISMS e ev S Fic. 58.—Types of spirilla. (After Williams.) GrapaTions.—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- 4 : 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. Ven. VA LO Ee J AY | Fic. 59.—Involution forms. Were are illustrated unusual forms ‘of B. subtilis, de water bacteria, Bact. aceti, Bact. pasteurianum, bacteroids in root nodules, Bact. tuberculosis, Bact. diphtheria. (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.) S1zE* } 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 (4), or micro- millimeter. This is .oor 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 14. 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 0.24 and a length of o.5u, and sometimes very large as, for example} the Bact. anthracis with a width of 1.24 and a length of 5.20u. The spirilla average about 1.op in diameter but may be as long as 30u-40y. Moritity* 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. 80 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 movement which is possessed by some bacteria. ViraL Movement.—As already indicated, bacteria have the power of independent movement due to inherent vital power. Only a few of the micrococci are motile, while many of the bacilli and spirilla are. This movement is a change of position and is caused by certain protoplasmic processes which these bacteria possess, known as cilia (sing. cilium) or flagella (sing. flagellum). The fact of motility or non-motility of an organism is of considerable value to the systematist. It is determined by examination in a hanging drop. At times, however, it varies so little from the brownian movement that it is difficult to tell whether a par- ticular organism or culture does or does not possess vital movement. An opinion can be more definitely formed at times if some chemical producing an anesthetizing effect on the bacteria is introduced into ‘the examining medium. In case the organism is actually motile its’ movement will be altered by the anesthetic but in case it is merely a brownian movement there will be no change. OrcaNs oF Locomotion.—The protoplasmic threads referred to as the organs of locomotion are known as flagella, or cilia. The difference between the cilium and flagellum is the fact that a cilium has a simple curve while a flagellum has a compound curve, like a whip lash. Most of the bacteria possess flagella rather than cilia. The size, 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 MovemEent.—Different bacteria exhibit different kinds of movement. Some dart forward with great rapidity, others Y BACTERIA ~ 81 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 QWODOGES Fic. 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 in 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 bacilli 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 endospores. Endospores are formed by the bacilli and the spirilla, but not by the micrococci. Their chief value to the cell is their _ ability to resist unusual conditions, and to enable the individuals of a ° species to pass through unfavorable conditions which to the ordinary vegetative form of the cell would prove disastrous. At the maturity of the cell, spore formation may begin. It is an open question whether spore formation occurs as a regular stage in the life history of an organism, or is produced only under the stimulus of unfavorable 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 clostridium. 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 endospores possess remarkable powers of resistance due to the concentrated character of the protoplasm, or to the character of the f Fic. 61. Fic. 62. Fic. 61.—The formation of spores. (After Fischer from Frost and McCampbell.) Fic. 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. e2en © -00C (= (= 0¢ o& oaoal 4 é 6 Fic. 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. Ina streptococcus filament, certain cells suggest to the observer the joint Spores of the alge 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 Groupinc* 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 Micrococct.—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 oT te a 6 c + @ Fic. 64.—Division forms of micrococci. @, 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. known as fetracocct, or merismopedia. Still again, the planes of division may proceed at right angles to each other in three dimensions of space. In this case packets are formed which are known as packet cocci, or sarcing. Another group of the micrococci occurs, known as the staphy- lococci, so called because they are arranged in irregular bunches, like a “bunch of grapes. This arrangement may be due to the fact that these micrococci divide in many different planes, or because during the course of their growth their arrangement is changed. CELL AGGREGATES AMONG THE Bactru1.—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 4 \-— YY -- eeeone”m & e Fic. 65.—Division forms of bacilli. a, Single; 6, pairs;¢, in threads. (After Novy.) 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. CeLtL AGGREGATES AMONG THE SPIRILLA.—Thée same kind of arrangement is maintained among the spitilla. fe of i! o % % * ae i ~ i ipo wh Ny 7 Zi aX W wilt UE “N i Nn He | at a es YM i hit Tatu a ~ Ww Wy a a fe SYN We ti imi 9 NT wn ia KAA | yt Mi; \ \ OW y fi! ! th it i x S wh it My tog Ht mea \ LN him a SL ay tl a) , a=, 8 v ait al fl Bray SN Yu Wy in Wily) ger Sse Sy YY WS | eo cpm wie Ny SONY WAY [Zon a0 DSS mal Uy 94 TSR NS mina fe “a. “aset jhe! i SRS SS TASS w y Fic. 66.—Threads of Bact. anthracis. (After Migula.) Zooci@aA.—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 zoogleic 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. Tue CytToLocy 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 the case of bacterial cells, we find most of these structures present, such as cell wall, cytoplasm, and appendages. GENERAL CONSIDERATION OF CYTOPLASM AND Nucreus.*—The cytoplasm of the bacterial cell is similar to the cytoplasm of other cells except that chemical analyses seem to show that it contains a higher / a Ag oS A” Fic. 67.—Plasmolytic changes. (After A. Fischer.) @, Cholera vibrio; b, typhoid / bacillus; c, Spirillum undula. (From Novy.) percentage 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- formly 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 aresult 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 bacteriopurpurin 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 cytologic 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 89 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 inacell. These granules he would consider nuclei. It seems to be established, however, that the ma- jority of the elements noted by Meyer Fic. 68.—Bacterium gammari are not nuclei, but reserve products and a filamentous bacterium from 5 the intestine of Bryodrilus. (After common among the Profista and known = éjdgwsky.) as metachromatic corpuscles. Véjdowsky’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, Véjdowsky 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, 0, 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, @). go 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 Véjdowski seems to resemble a filamentous mold. However this may be, one of Véjdowsky’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. mycoides, 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 Clizwrp @ |) incipient transverse septum which fixes el _-——~ 2 Stains well at the beginning of its forma- em!) SED tion and in some ways resembles a nucleus. 3 4 The studies of Penau, who also endea- Fic. 69.—Bacillus megathe- vored to prove the existence of a typical rium. (After Penau.) : : 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, 1), 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 trabecule, 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. Were, after a stage of undifferentiated structure which BACTERIA gI characterizes the youngest cells, follows a phase where the cytoplasm 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. This nucleus, however, has only an ephemeral existence and quickly undergoes a cytolysis during which it disintegrates. The disintegra- tion products then impregnate the trabecule 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 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 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 such an organ in well-defined bacteria. We must conclude, therefore, that with the exception of the results obtained by Véjdowsky, 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 Fic. 70.—1. Chromatium okenii. 2. Beggiatoa alba. These two bacteria have a central body containing chromatic grains and considered by Biitschli as the eaualens of anucleus. (After Biutschli.) nearly the whole volume of the cell and consisting of an alveolar ae plasm of highly stainable web, containing within its knots numerous chromatic granulations (Fig. “a The remainder of the cell consists 92 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 Cyanophycee, 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 okenit by Dangeard. The Sulpho-bacteria, however, are organisms morphologically entirely distinct from ordinary bacteria, and are apparently directly related to the Cyanophycee. 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. biitschliit. It is the largest bacillus known at present (4u 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, 11 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 joinéd the two rudiments of spores, gradually disappeared as well ds the cytoplasm, while the spores increased in size. Then the sporangium ended by breaking and setting free the two spores. Germination con- BACTERIA 93 sists 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 as a morphologic entity; it is scattered in the cytoplasm in the form of little grains. ) 3 10 Fic. 71.—Bacillus biitschlii. 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 Schaudinn.) In another bacillus smaller in size (B. sporonema), Schaudinn has found an analogous structure only at the time of sporulation; he does 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 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, asterosporus, alvei). Upon examination at the very outset of their development, these bacteria present a homogeneous appearance and are uniformly 4 () Fic. 72.—Bacillus sporonema. . Cell about to sporulate. 2, This cell grows narrow at the center, as if it were Boing to be divided (Schaudinn regards this pinch- ing together which afterward disappears (5), as the vestige of an ancestral sexuality like that of B. biitschliit). 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, 1). These soon FZ POT 0O DE OH 2) = = eS pe io il Fic. 73.—1-10, Bacillus radicosus. 1, Beginning of development. 2-3, Cells at the end of eight hours; 4-6, spo rulation. 9-10, Cells in which the chromatic grains are located in the middle in a mass slightly resembling a nucleus. 11-12, Spirillum volutans. 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 1 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 cases (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 cell 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 changes 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 not enable us to differentiate? That has seemed tous scarcely probable, for if this nucleus existed, it would certainly be visible in a species as large as B. biitschlii 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 consist principally of chromatin. The cells of bacteria would accordingly have a very primitive structure. Granted the clearly demonstrated existence of this particular struc- ture in the Cyanophycee, there is no reason for not admitting that the nucleus, very rudimentary in the Cyanophycee, might be even more so in 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, Ruzicka, 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 (2u wide) almost as large as B. biitschlii, and named it, B. flexilis. This species shows exactly the same cytological charac- teristics as B. biitschlii (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 micrococci 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, Fic. 74.—Bacillus flexilis. 1, 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.) Fic. 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 in 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 of B. 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 diffiise nucleus, that is, the structure described by Schaudinn and by Guilliermond. In the group of spirilla, Dobell notices these tities 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 Sulfo-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. Lire Cycre or BactErta.—The Editor feels justified in adding to the foregoing review the very recent work of Léhnis and Smith, Journal of Agricultural Research, Vol. VI, No. 18, July 31, 1916, because, both for its suggestiveness and presentation of experimental 7 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 normalshape. 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. Dirdct 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- formity and accuracy when connected with morphological investigations along these new lines. Several problems in general biology are brought under more promising aspects. Agricultural bacteri- ology and medical also will derive much benefit.” RESERVE Propucts.*—Besides the grains of chromatin which we have just been considering in bacteria are found other granulations which do not show the characteristics of chromatin and which act as products of nutrition. These Fic. 76.—Various bacteria | stained by a method which differentiates only the meta- chromatic corpuscles. 1-4, Baci'lus radicosus. 5-6, Bacil- lus asterosporus. 7, ‘The same. The cells have formed their spore and the metachromatic corpuscles outside of the spores have not yet been absorbed by granulations are characterized by the jt 89° Spirillum volutans. reddish color which they assume with 10-11, Bacillus alves. 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 eells, 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 diphtherig). 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, 1 to 4, 6, 10, 11). In some species (B. alvet, asterospdrus, Bact. tuberculosis and diphtherie), 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, 11). 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 zoogloea. 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.t—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. { Prepared by A. Guilliermond. BACTERIA IOI 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 immer 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. biitschlii. According to him, the membrane seen in profile would appear to consist of a series of disks alternately clear and cloudy (Fig. 77, A 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 Fic. 77.—A and B, (Fig. 71, 15 and 16). Structure of the.mem- The chemical composition of the membrane brane and of the ecto- eas fl derm in’ Bacillus is little known. According to some authors, pytschii. C, Membrane this membrane consists of cellulose; according of the same bacillus, : ; pee: front view. (After to others, it contains a lipoid substance; finally, — schanudinn.) _ 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 toshow 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- Fic. 78.—Capsules. Bact. pneumonie (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 Fic. 79. Fic. 80. Fic. 81. Fic. 79.—Chromatium okenii; 2, Bacterium lineola; 3, 4 and 5, sulpho-bacteria; 7, Ophidomonas jenensis; 8, and 9,'S pirillum undula; 10, Cladothrix dichotoma. (After Biltschli from Guilliermond review, Bull. Inst. Past.) Fic. 80.—Microspira comma. Monotrichous bacteria. (After Migula from Schmidt and Weiss.) Fic. 81.—Pseudomonas pyocyanea. Monotrichous bacteria. (After Migula from Schmidt and Weiss.) them and sometimes many times that length. B. symptomatici anthracis found in the soil has a flagellum sixty times its own length. The arrangement of the flagella on the bacteria is quite constant and WV whe i oo Ly ein z Fic. 82. Fic. 83. Fic. 84. Fic. 82.—Pseudomonas syncyanea. Lophotrichous bacteria. (After Migula from Schmidt and Weiss.) Fic. 83.—Spirillum rubrum. Lophotrichous bacteria. (After Migula from Schmidt and Weiss.) Fic. 84.—Bacillus typhosus. Peritrichous bacteria. (After Migula from Schmidt and Weiss, and Frost and McCampbell.) is used by some authors to differentiate genera. Very few of the micrococci are provided with flagella, as was indicated above, and in the bacilli and spirilla they may be arranged at the poles singly or in I04 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, 80and 81). Whenthey . 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 réle 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 volutans) 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 relation to a. basal chromatic grain (blegharoplast). Tur HicHer Bacterta* ' The so-called higher bacteria include some of the spiral forms, at least the larger spirochetes, the thread or ¢richobacteria, and the sulphur or thiobacteria. The spirochetes and ¢trichobacteria contain so many forms of interest that their form and structure needs special consideration. Tue LARGER SPIROCHETES.—Spirochetes differ so much among themselves that it seems necessary to divide them into two groups. The members of one of these groups, the small spirochetes, are prac- tically identical with the true bacteria, and naturally fall in the family of the Spirilliacee. Members of this group, however, so gradually approach the other group, the large spirochetes, that it is difficult to draw a line of separation between the two, yet the large spirochetes resemble in so many essential details the trypanosomes that they are usually placed as a codrdinate genus with them under the flagellates—a sub-class of the Profozoa. ‘The larger spirochetes are described as follows: Form and Size—In form the spirochetes 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. It seems probable that some of them are flattened and hence in form are more like a spirally bent ribbon than rod. Motility——These organisms move very rapidly under normal con- ditions. The character of the movement may be of three kinds: (1) Lashing, eel or snake like; (2) undulatory, compared to the flapping of a sail in the wind; (3) rotation, similar to a cork-screw when pushed into 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 spirochetes. 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 spirochetes 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 ¢richobacteria 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. 8s 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 alge. 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 er BACTERIA 107 ' resting stage may produce new threads of cells. Sometimes spores germinate while still in the old thread (Fig. 85), giving a tangled mass of cells or whorls of new threads at intervals on the old. -The conidia may be either motile or non-motile. The motility of these conidia when it exists is due to flagella. Sheath.—The threads of cells are sometimes surrounded by sheaths of varying thickness. This sheath is a thickened and hardened mem- Fic. 85,—Crenothrix polyspora Cohn, Brunnenfaden. (After Migula from Schmidt and Weiss.) brane, and forms a tube in which the different cells of the bacteria are contained. This sheath is homologous to a capsule. In it are fre- quently deposited characteristic by-products of the cell. In Creno- thrix (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 bovis (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. Tue SutpHur BactEerta.—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 spirochetes or Oscillaria among thealge. 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. é ' 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 of 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, and nowadays we are finding it as difficult to decide upon a system of clas- sification. A great many systems have been proposed, but many of them are untenable because those who proposéd them were ignorant of or unconcerned by the rules adopted by systematists in other lines. The only system that seems worthy of continued life is that of Migula, * Prepared by W. D. Frost, BACTERIA 109 who. is a trained botanist. This system, with sight modifications, is given below. In this system, the characters which separate the genera are morphological; while physiological characters, including cultural, are used for the differentiation of species and smaller groups. - One of the rules adopted by systematists in other lines is the binomial _ rule. In the violation of this rule, bacteriologists have been great sinners, and some-of the names proposed by Migula and others follow- ing his system are quite different from those by which well-known forms have been christened by their discoverers. CLASSIFICATION OF MIGULA (MODIFIED) The bacteria are phycochrome-free schizomycetous plants which divide in one,, two, or three planes. Reproduction takes place by vegetative multiplication (fission).. Resting stages in the form of endospores are produced by many species. Motility is. noted in some genera, and this is due to flagella. In Beggiatoa and Spirocheta the: organs of locomotion are not definitely known. I. Order: Eubacteria (true bacteria). The cells are devoid of any nucleus (Zentralkérper) and free from sulphur and bacteriopurpurin, colorless or faintly colored. I. Suborder: Haplobacterine (lower bacteria). I. Family: Coccacee (Zopr) Mic. The cells are globular when in a free state, but in the various stages of division appear somewhat elliptical. A few species in this family are motile. Cell division takes place in several directions of space. Frequently the cells remain attached to- gether, and under these conditions usually show some flattening of the cell at the point of junction with the cell next to it. Genus: Streptococcus BILLRorTH. : The cells are globular and do not possess any organs of locomotion. Cell division takes place in only one plane. Usually the cells remain united together after division, producing chains or diplococcus forms. No endospores have been noted. Genus: Micrococcus (HALLIER) CoHN. The cells are globular and do not possess ‘any organs of locomotion. Cell division takes place in two planes at right angles. If the cells remain attached together after cell division, merismopedia plates are formed. The plates give the appearance of a regular flat mass of cells. No endospores have been noted in this genus. Genus: Sarcina Goopsir. , : The cells are globular-and do not possess any organs of locomotion. Cell division takes place in three planes, all perpendicular to each other. Its cells remain attached after division; cube-like packets are formed. The composition of the medium zome- times prevents this typical cube formation. ' Genus: Planococcus Micura. : The cells are globular. Cell divizion takes place in two planes at right angles IIo 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 MIcULA. The cells are globular. Cell division takes place in three planes as inSarcina. . 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: Bacteriacee Micuta. 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 Fic. 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 remain attached together, forming threads, while others separate from each other soon after fission, Genus: Bacterium EHRENBERG. The cells are cylindrical, of longer or shorter length. Threads are frequently formed. The cells do not possess any organs of locomotion. Endospores are pre- duced in some few species, but in the majority no such formation oecurs. It is’ possible that endospore formation occurs only under certain envirenmental con- ditions. BACTERIA III Genus: Bacillus Conn. | The cells are cylindrical, of longer or shorter length. The rods are sometimes oval in shape. Cells are motile and possess flagella which are distributed over the entire surface. Endospore formation occurs with marked regularity. The bacteria in this genus are motile only during certain periods of their life. This period varies greatly in length and occurs only in the weariative stage. ‘ Genus: Pseudomonas Micuta. The cells are cylindrical, of longer or igen length. The cells are motile and possess polar flagella. These flagella may vary from one to twelve in number. The formation of endospores in this species is claimed by some. If they occur, it is ex- tremely rare. Occasionally certain species in this genus form themselves into threads -or chains. III. Family: Spirillacee Micuta. The cells are wound in the form of a spiral or representing the portion of a turn of a spiral. In the latter case, if the cells remain attached together in the form of a thread, a full spiral of several turns is produced. Cell division takes place in only one direction of space, and this is transverse to the long axis of the cell. Genus: Spirosoma Micuna. The cells are rigid and bent in the form of spirals. The members ofthis 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 ScHROTER. The cells are rigid, short, and bent similar toacomma. When the cells are united. together, S-shaped threads are formed. The cells are motile, possessing usually one flagellum and rarely two or three flagella. These flagella are about the same length as the cell. No endospores are formed. Some writers make no distinction between Microspira and Spirillum. The name Vibrio has also been applied by some writers to this genus... Genus: Spirillum EnRENBERG. 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 tuff 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: Spirocheta EHRENBERG. The cells are flexible spirals, very thin and long. No flagella are present. These bacteria move by rotation similar to a screw, and also by lateral motion similar to a snake. The locomotive organs, if present, are not known. No endospores are produced. 3 II. Suborder: ee (higher bacteria). Family: Chlamydobacteriacee Micuta. 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. , ‘ II2 MORPHOLOGY AND CULTURE OF MICROORGANISMS Genus: Chlamydothrix Micuta. 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 Genus: Crenothrix Coun. species can be placed in this genus. place by means of gonidia, which are round and arise di- rectly from the vegetative cell. This genus is called Lep- tothrix by Ktr1zine and Streptothrix by Coun. 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 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 Fic. 87.—Clado- acquiring sheaths. The sheath in this genus is quite thin thrix dichotoma and can scarcely be seen. Cohn. (After Fis- Genus: Sphzrotilus Kitzinc cher from Schmidt ‘ 2 and Weiss.) 1833 (Cladothrix Conn). 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: Beggiatoacee TREVISAN. Jas cast Be BGO tae Vaue ese Fic. 88.—Beggiatoa .' alba. Vaucher, Trevi- san. (After Winogradsky , from Schmidt and Weiss.) 8 Filamentous bacteria which do not contain bacteriopurpurin. The cells contain sulphur granules. Reproduction takes place in one direction of space. 4 BACTERIA 113 Genus: Thiothrix WinocRapsky. 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: Rhodobacteriacee (WiINocRADSKY’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. Z The cells are united into colonies. Cell division takes placein 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 Lamprocystacee. 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 Thiopediacez. 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 fouts and capable of motility. IV. Subfamily Amcebobacteriacee. The cells are united into colonies. Cell division takes place in one direction of space. IT4 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 WinocRADsKY. The colonies are solid, non-motile, and consist of small cells which are pressed together. V. Subfamily Chromatiacez. 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 alge than to any other forms of life. They resemble these organismsinform, ° method of reproduction, and absence of definite nucleus. It is quite impossible to decide, furthermore, whether someforms, such as Bact. viride and Bact. chlorinum, are blue-green alge 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 Spirochete, which are usually , *Prepared by W. D. Frost. BACTERIA ; 115 believed to be bacteria, have been classed as flagellates by eminent protozodlogists. Physiologically the bacteria are quite closely related to the fungi, and are frequently classed with them under the term. Schizomycetes. ARTIFICIAL CULTIVATION OF BACTERIA™ The introduction of methods of artificial cultivation marks the beginning of the science of microbiology. These methods were developed by Pasteur and Koch and are depended upon by the microbiologist of to-day as the foundation for most of his work. It has been the aim of investigation to discover a more general culture medium. So far it has been impossible to do this, but beef broth, made after a formula suggested by Loeffler many years ago, forms the basis of nearly all of our culture media. This beef broth, or nutrient bouillon, is made by extracting meat free from fat in water, adding a small per cent of peptone, correcting the chemical reaction, clarifying and sterilizing. To this broth various substances are added for special purposes; gelatin and agar, in order to solidify the media, and various sugars and other chemical substances for the purpose of determining the physiological — characteristics of various bacteria. One of the difficulties with the present methods of the artificial cultivation of bacteria is the inconstancy of the composition of the media, due to the fact that the extract of beef, the peptone, and other ingredients, cannot be obtained chemically pure. If it should prove possible to use synthetic substances, such as the polypeptids, it would mark a great step in advance, but it is probably quite impossible to devise a single medium upon which all bacteria will terete grow. Some bacteria, such as those which produce nitrification, refuse to grow on ordinary media containing organic material. The cultivation of bacteria in pure culture is dependent upon isolation, and the method of isolation suggested by Robert - Koch in 1880, and known as the plate culture method, has given eminent satis- faction. This method is dependent upon the use of liquefiable solid media, such as gelatin or agar. *Prepared by W. D. Frost. CHAPTER V a. INVISIBLE MICROORGANISMS* The term “invisible microdrganism” is used interchangeably with such expressions as “ultra-microscopic organism,” “invisible virus” and “‘filterable virus” to designate a group of microérganisms which, for the most part, cannot be discerned with the most powerful lenses. Besides being invisible, these microérganisms 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 microérganisms and in addition the usual culture tests will give negative results. Notwithstanding this apparent sterility, however, the filtrate will produce disease in cattle in the same manner as the unfiltered lymph. It is known that the symptoms produced by the filtrate are caused by a living organism and not by a toxin, because by successive filtrations and inoculations the disease can be transmitted through a long series of animals, thus indicating clearly ‘ that there exists in the filtered lymph a living organism which is capable of reproduction. Another proof that the virulence of the filtered lymph is caused by the presence of living corpuscular elements, and that it is not a mere solution of a toxin, is found in the failure of the virus to pass through filters of finer grain than the Berkefeld as, for example, ’ the Kitasato filter. The more important of the diseases which may be caused by invisible. microdrganisms 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 of Noguchi. 116 ¢ ¢ * INVISIBLE MICROORGANISMS II7 ' Fic. 89.—Apparatus for fractional filtration, designed for use with Pasteur- Chamberland or Berkefeld filters. a, Glass mantle surrounding filter; 6, Chamber- land filter; c, paraffin joint; d and e¢, rubber stoppers; f, double side-arm suction flask; _, pinchcock controlling outlet from suction flask; 4, outlet tube surrounded by: glass shield and attached to lower end of suction flask by means of short rubber tubing; i, glass shield fused to and surrounding outlet tube as a protection against contamina- tion when the filtrates are drawn off; j, glass inlet tube plugged with cotton, for ad- mitting air into suction flask; &, pinchcock governing the admission of air into flask; d,.vacuum gauge;'m,fstopcock connected with vacuum pump. (U.S. Depi. of Agri- culture, Bureau of Animal Industry, Bull. 11 32) % 118 MORPHOLOGY AND CULTURE OF MICROORGANISMS pleuropneumonia, cattle plague, swamp fever or infectious anemia of horses, chicken pest, sheep pox, and horse sickness. _ The invisibility of this group of microérganisms 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.1y4 in diameter. We know of bacteria which in size approach this limit quite closely (M. progrediens, 0.15 in diameter) and there is no reason for believing that the size of organisms is limited -by our ability to see them. As already stated, invisibility may also ‘result from a peculiarity of structure, such as complete transparency and failure to stain with the reagents ordinarily used for this purpose. - The ability of microdrganisms to pass through filters is dependent upon a variety of factors. The size and plasticity of the organism, the fineness of the pores, and the thickness of the walls of the filter as well as the conditions under which the filtration is performed, will all influence the result. The failure of the invisible microérganisms 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 exist in the animal body. While the invisible microérganisms possess certain qualities in com- mon, in some respects they differ widely from one another. Some will pass only through the coarsest of bacteria-proof filters, while others pass readily through the densest filters, thus indicating wide differences in size or in structure. Some are very susceptible to the action of germici- dal agents, whereas others are more resistant than the ordinary bacteria. Some produce disease in only one species of animal, while others show little or no limitation in this respect. The diseases produced by these microérganisms likewise differ markedly, some being comparatively benign and local in character, whereas others appear as the most pro- found septicemias. Some are extremely contagious, while others can be transferred from one animal to another only by means of an inter- mediate host. In fact these invisible microdrganisms seem to differ among themselves quite as widely as do those which are visible to us. The existence of an invisible microérganism is determined as follows: The infectious agent must pass through a bacteria-proof filter, which is free from imperfections as shown by tests with visible organisms of small size. Pressure exceeding one atmosphere should not be employed INVISIBLE MICROORGANISMS IIgQ , during filtration. The time of filtration should not exceed one hour. The filtrate should remain free from all visible bacteria as shown by , microscopic examination and cultural tests. The filtrate should possess the specific disease-producing qualities of the unfiltered material. Animals infected with the filtrate should yield material which, after filtration, will in its turn possess the attributes of the original unfiltered material. 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 rg9z2, ‘published in the Journal of Experimental Medicine; he is also requested to read the article by Léhnis 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 zodlogical point of view, the protozoa constitute an impor- tant sub-kingdom. It is sometimes difficult to say whether a minute organism isa plant or ananimal. 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 alge 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 I2I hosts 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 which 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 eyeas individuals, — Fic. 90.—Amaba vespertilio. (After Doflein.) or as 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 of Mycetozoa, which are sometimes seen on rotting wood or in tan pits, may measure many centimeters in breadth. Like all living things, the protozoa are composed of protoplasm (page 18) 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 protozoédn 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 endoplasm. 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- entiated from the remainder of the cell by being more refractile and | by being colored more deeply in specimens which have been stained by dyes. It stains deeply because it contains a substance called chro-- matin. The chromatin usually occurs in granules which may vary ' PROTOZOA 123 considerably in size and which are supported upon a Jinin 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 15) 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 réle 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 Mefazoa are composed of a great number of cells, a protozoén 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 protozoén 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 organelle 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. Fic. 91.—Paramecium caudatum: division showing the macronucleus (N) divid- ing without mitosis, the mi- cronucleus () dividing mi- totically. ¢.v1., Old,.and c.v?., new, contractile vacuoles. (Minchin, after Biitschli and Schewiakof, in Leuchart and Nitsche’s Zoologische Wandtafien, No. LXV.) 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. Cuilia 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. ’ “ ae 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 Fic. 92.—Stages in the division of Amaba polypodia. (After F. E. Schulze and Lange from Doftein.) 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- tion of this process is gemmulation, in which a small protozoén 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 protozoén disappears in a swarm of daughter cells. When a protozoén divides at a single division to pro- duce a large number of daughter cells simultaneously, the process is Fic. 93.—Coccidium schubergi. A-C, asexual multiplication; D-K, sexual multi- plication; D, microgametes; E, macrogamete; F, G, fertilization; H, 7, K, division and spore production. (After Schaudinn, from Doflein.) called schizogony and the young parasites are called merozoites, .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 ‘327 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 usual number. In dividing, cells may go through a process called mitosis during which the chromatin of the nucleus is grouped into more -or less rod-shaped masses which are called chromosomes. Thenumber of chromosomes which are formed during mitosis is constant and char- acteristic 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 of the reproductive areas of the same animals. The process by which the 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, thatis, 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 microgametes, 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. Init, 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 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 chromatin. 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 protozoén consists of the changes through which it passes in the period intervening between each fertilizing act. In many of the pathogenic protozoa, an alternation of generations occurs; that is, cycles of development in which an asexual method of re- 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, 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, the parasite which produces malaria (Fig. 177). While it is in the body , PROTOZOA 129 of its mam:masian 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 the parasite while it is in other stages are called intermediate hosts. ENCysTMENT.—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 or 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 protozodn is spread, and, as in the coccidia, the presence of such a stage is often absolutely essential in the life history 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, .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 unernployed 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- 3 S 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 ape 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 spirochetes 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 zodlogical 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 inrusoria; 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 protozoén 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- zoén 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 «131 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 rwizopopa include the simplest forms of animal life. A rhizopod, such’ as an ameeba, 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 Entameba. Different species of parasitic amcoebe may occur A in the alimentary canals of various ani- Fxg. 94 Her petomonas mals. Certain of these produce serious musce-domestice (Burnett). A, diseases (page 822) motile individual with two flag- page 622). lye ; ella; B, cyst; a, nucleus; 6/, The FLAGELLATA are distinguished kineotonucleus. (After Pro- ‘ wazek from Minchin.) y 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 Binucleata by certain systematists. Most flagellates are free-living. Comparatively few 132 ‘MORPHOLOGY AND CULTURE OF MICROORGANISMS species are parasitic, but some of these cause very serious diseases (page 824). The Her petomonad is an elongated organism whieh possesses tro pho- nucleus 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, arudimentary undulating membrane, Trypanosoma is an elongated parasite which has a trophonucleus, a kinetonucleus usually situated near its aflagellar extremity and an Fic. 95.—A, Trypanosoma tince of the tench; note the very broad and undulat- ing membrane in this species; B., C., T. perce 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 Trypanoplasma 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. Fic. 96.—Trichmonas eberthi, from the intestine of the common fowl; fll., ‘anterior flagella, three in number; P.f.., posterior flagellum, forming the edge of the undulating membrane; chr. l., ‘“chromatinic line,” forming the base of the undulating membrane; chr.b., “‘chromatinic blocks;’’ 6/., blepharoplast from which all four flagella arise; m., mouth opening; N., nucleus; ax., 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. Fic. 97.—Lamblia intestinalis. A, Ventral view; N., one of the two nuclei; ax., axostyles; fi.1, 1.2, 71.3, l.4, the four pairs of flagella; s., sucker-like depressed area on the vile 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 Gregarine 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 attachesitself toitshost. None of the gregarines are parasitic on mammals; worms are the hosts for some ofthem. The Coccidia are usually parasitic within certain cells of their PROTOZOA 135 host, for example, Eimeria stiede@ (Coccidium cunicult) (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 Fic. 98.—Sporozoits in the oocyst of Laverania malarie. 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; EH, a portion of one enlarged. (After Grassi, from Doflein.) intestines of the birds which it infects (page 832). The Hemosporidia 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 Hemoproteus are malarial parasites of birds (page 832). The Hemogre- garing 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. Hepatozoin perniciosum is the best known of a group of hemogregarine-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, Rhinosporidium 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 pébrine 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 Uliramicroscopic viruses (pages 116, 842) are all associated with important diseases in men and | in animals. The sPIROCHAT (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 spirochetes 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, a single species is taken as the type of each genus and that species, with the disease it produces, is described; if the remaining species of the genus are mentioned, they are spoken of only to indicate how they differ from the description of the type species. Tecenic* 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 between slide and cover-glass which is readily accomplished by touching paraffin with a hot needle and transferring it thus melted to several points on the slide before the preparation is made. When very minute forms are to be studied it is necessary to utilize what is known as the dark field illumination. This brings out very minute organisms and particles which being transparent are invisible to ordinary transmitted light. The dark field apparatus consists of a strong source of light such as a small arc lamp, a special condenser which deflects the light so that objects in the micro- scopic field are illuminated by light directed from the sides causing them to appear bright on a dark background. Another method of obtaining a dark field is to mix on a slide a small drop of the material to be examined with an equal-sized drop of India ink or better of saturated aqueous solution of nigrosin and then to smear this mixture across the surface of the slide when it may then be dried and examined at once by the oil immersion’lens. Only ordinary daylight is required for this method but,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 dissect 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, Lehrbuch der Protozoenkunde, Jena, Gustav Fischer; Prowazek, Der mikrochopischen Technik der Protistenuntersuchung, Leipzig; and Stitt, Practical Bacteriology, Bloodwork and Para- sitology, Blakiston, Philadelphia. 138 MORPHOLOGY AND CULTURE OF MICROORGANISMS properties of various portions of their bodies and it is Biss 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 1 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 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 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 yery readily with the platinum loop ordinarily used in bacteriology. A very practical method for making blood smears is to gather a minute drop of freshly drawn blood from a small incision or prick in the skin on one edge of the end of aslide. The latter is placed in contact with the surface of another slide and being held at an angle of 45 degrees is pushed steadily lengthwise across its surface. By increasing or decreasing this angle a thicker or thinner film may be made. Certain investigators prefer to use what is termed the wet method for the fixation of smears. In this case the smear is dropped face down immediately and before drying into a fixative composed of two parts of a solution of saturated HgCle in distilled water and one part of absolute alcohol. The technic employed in the staining of sections is then followed and the smear is not allowed to dry at any step in the procedure. , The preparation of stained sections requires a considerable amount of technical skill.. Tissue is first fixed to render its structure permanent. It is then dehydrated in alcohol of increasing strengths, next placed in chloroform or some other clearing ~ reagent when it is then imbedded in paraffin after which it may be sectioned. For the details of sectioning and the staining of sections the reader is referred to Mallory and Wright’s Pathological Technic, W. B. Saunders and Co., and Lee’s Vade mecum. The cultivation of free-living protozoa is usually accomplished by keeping a supply of the medium in which they live on hand. Hay infusion prepared by boiling * Barbour: University of Kansas, Science Bulletin 1907-4-3; also Journal of Infectious , Diseases, ro11, 8, 248, and 1911, 9, 117. PROTOZOA 139 a quantity of chopped hay in water is an easy and valuable method of preparing culture media. For the cultivation of amcebz, the following media is widely em- ployed. It should be noted, however, that the amcebe which have been cultivated are regarded as free-living forms and the attempts to cultivate parasitic amoebe have thus far been unsuccessful. MeEpiIvuM oF ‘MUSGRAVE AND CLEGG PAD AT nse savadbceanisce, da genase sua ee ae Rae epee ante Sateen sedetaisNe 20 to 30 g. Liebig’s extract of beef... 2... 2c ccc cee eee .3 to .5 g. Common isa tcc econczancaraln ag tecnd crews quccamssaratgeieeeniiaxetunees .3 to .5 g. Waterss cvictansionucaammugionnnasayanuita se aceuvelesmemure 1,000 C.C. This medium is designed to provide for slow bacterial growth in order to provide food for amcebe. On a richer medium the latter are overwhelmed by the rapid growth of bacteria. For the cultivation of trypanosomes, leishmania and other flagellates the so- called triple N media is employed. This is prepared as follows: Nicotte, Novy, MacNeat Meprum WA CEES is dssagrvcurunuayacecuaraim haven ovednaraela cavoneh ermine lanai goo C.c Dal Oh a scidiin dears mehmaaniecene baat Adela shed Raremnainaea mee 6g AR EE essen see Stn salle a cst Compara Ree eee 16 g. Dissolve, distribute in tubes, sterilize and add to the medium in each tube after liquefying and cooling to 40°-50°C. one-third its volume of rabbit blood obtained by cardiac puncture. Slope the tubes for twelve hours, incubate at 37°. for five days to test the sterility of the medium and then keep them at the ordinary temperature of the laboratory for a few days before sowing them. (The tubes should be sealed to prevent evaporation.) ; The malaria organisms have been made to continue development outside the body by the following method devised by Bass. Bass’s Method.—The blood in 10- to 20-c.c. quantities is taken from the patient’s vein and received in a centrifuge tube which contains 1/9 c.c. of 50 per cent. glucose solution. A glass rod, or piece of tubing, extending to the bottom of the centrifuge tube is used to defibrinate the blood. After centrifugalizing there should be at least 1 inch of serum above the cell sediment. The parasites develop in the upper cell layer about 149 to 49 inch from the top. All of the parasites contained in deeper lying red cells die. To observe the development, red cells from this upper 14 9-inch portion are drawn up with a capillary bulb pipette. Should the cultivation of more than one generation be desired, the leucocyte upper layer must be carefully pipetted off, as the leucocytes immediately destroy the merozoites. Only the parasites within red cells escape phagocytosis. Sexual parasites are much more resistant, and the authors think they observed partheno- genesis. The temperature should be from 4o to 41°. and strict anaerobic conditions observed. Aistivo-autumnal organisms are more resistant than benign tertian ones. Dextrose 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 MeETABOLISM.—The nutrition and metabolism of microdrganisms are based on the same principles that regulate animal and plant metabolism; in a general way microérganisms are more closely related to animals than to plants, if viewed from the standpoint of their food, their mode of digestion, and their general physiological nature. Only in a few in-, stances, i.e., in the case of life without oxygen (anaerobiosis) and in the ability of some species to use free nitrogen gas, are there processes unparalleled in the more highly developed organisms. Since it will be necessary frequently to refer to plant and animal nutrition in the course of this discussion, these principles, therefore, are briefly discussed in the following paragraph. Green plants feed only on inorganic substances. They assimilate carbon dioxide, (COz) 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 compounds constituting the plant cells are produced from these simple inorganic substances. This formation of organic compounds from inorganic compounds requires a certain amount of energy. If a certain quantity of sugar is burned to carbon dioxide (CO) and to water (H20), a 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. I4I I42 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 1 g. molecule (180 g.) of sugar. The complete equation of sugar combustion is therefore written CeHi20¢5 + 120 = 6CO2 + 6Hz:0 + 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; CO: and H.0O are absorbed by the plant resulting in the formation of sugar. But it is evident from the equation that CO: and H,0 are not sufficient 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. Chlorophyl is the green coloring substance of plants, and only green plants can use the energy of sunlight for their growth. The growth of green plants is a storing of the energy of light in the form of organic matter; their metabolism is largely synthetic, 7.e., building up. Plants without chlorophyl, however, like mushrooms, molds, yeasts and bacteria, have to provide for thett energy by some other means. Animals construct their bodies mainly of organic matter. Their body substances as protein, fat, etc., are derived from the protein, © fat, cellulose, etc., of plants or of animals. .Nevertheless, a certain _ amount of energy is required in this assimilation process, since the animal protein and fat are somewhat different from the plant protein and fat. Consequently, complex chemical changes and rearrangements, which require some energy, are necessary for growth. Energy is also lost by radiation of heat and by locomotion. Animals, being entirely unable to use the sunlight as a source of energy, obtain their energy from the digestion of organic food. The larger part of this food is oxidized completely; this part provides for the energy. The smaller part of the food is used for building the tissues of the body; it becomes part of the animal itself. Animal metabolism is largely analytic, i.e., INTRODUCTION , 143 destructive although a limited amount of energy is‘required for the © chemical changes and molecular rearrangements which are essential ; to animal tissue formation—a synthetic process. Accordingly more organic matter is decomposed than is formed. Often the same sub- stance can serve both purposes; the meat eaten by a dog furnishes to it energy as well as material for growth. In other cases, certain food compounds execute only one function and not the other. This dis- tinction between food for energy and food for growth must also enter into 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, microdérganisms have been divided into plants andvanimals, 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 microdrganisms. Since none of the plants discussed in this text-book possesses chlorophyl, none of them can use light as a source of energy, therefore they depend entirely upon chemical energy obtained by the digestion of food. This means that they require organic food almost entirely, since inorganic food furnishes energy only in exceptional cases. In this respect they resemble the animals very much. The metabolism of protozoa which in some respects calls for dif- ferential and special treatment is furnished by Todd and Tyzzer as follows: “The ingestion of food is accomplished in. some protozoa by pseudopodia; the protozodn simply flows around and so encloses a food particle (Fig. 99). In the same way, these protozoa flow away from waste particles which are to be eliminated. Other protozoa have defi- nite mouth areas for the ingestion of food, and definite anal areas for the 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 amcebe and ciliata, pos- sess contractile vacuoles. A contractile vacuole is a clear cavity which Fic. 99.—A, Ameba proteus; Na, a food particle; Cv, contractile vacuole; V, 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. The fluid which it ejects contains the soluble waste products resulting from the metabolism of the protozoén. One function of the contractile vac- uoles is, therefore, excretion; in some protozoa, they are probably also concerned with respiration. Contractile vacuoles are frequently absent in protozoa which are parasitic within other animals. “Organisms which feed upon solid food, for example the bodies of other organisms, are said to be holozoic in their mode of life. Others INTRODUCTION 145 which by reason of their generic relationship are included with the protozoa are capable under the action of sunlight of manufacturing starch. Such are said to be holophytic. Other protozoa live upon organic material in solution, the saprozoic mode of life, and finally some are especially adapted to live at the expense of other animals and are parasitic in nature. In the latter instance food may be obtained from the fluids in which they live by absorption as is the case with trypanosomes living in blood plasma or cells may be ingested as in the case of dysentery amcebe. : j “The process of respiration in the protozoa is in general similar to that of higher animals. Most of them require oxygen and eliminate carbon dioxide. The contractile vacuole which is found in certain forms is believed to have a respiratory function. Respiration may consist of the liberation of energy through oxidation or through the breaking down of complex molecules. In organisms of an anaerobic habit the respiration is probably through internal molecular changes affecting material stored in the cytoplasm. “In addition to the expulsion of solid undigested material from the cytoplasm there is evidence that waste products other than COz are excreted by contractile vacuoles. Many organisms also secrete material either of the nature of chitinous membranes on their surface or metabolic products in the form of globules, etc., within their bodies. “The most obvious evidence of liberation of energy in the physiology of protozoa is seen in their movement. Certain protozoa, Nocticula for example, however, emit light and produce the phosphorescence often observed in sea water. From analogy with higher animals it is to be supposed that heat and electrical changes are also produced. “Certain chemical substances which attract protozoa are said to be positively chemotactic, others which repel negatively chemotactic. Vari- ous forms react in a definite manner to light—phototaxis, heat— thermotoxis, gravity—geotaxis, etc. “Derangement of function may be produced associated with which are visible degenerative changes. It has also been found that certain protozoa have the ability to recover from injury and to regenerate lost parts.” ENERGY SUPPLY oF MicroORGANISMS.—The source of energy in microbial life is always of chemical origin. The simplest processes are 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 sihigh oxidize ammonia to nitrates. This process, according to Winogradski, is divided distinctly into two phases: the Nitrosomonas oxidizes the ammonia to nitrous acid, NH; + 30 = HNO: + HO + 78.8 Cal. and the Nitromonas oxidizes the nitrous acid to nitric acid, HNO, + O = HNO; + 18.3 Cal. These oxidation processes yield a certain amount of energy which enables the bacteria to build their cells from carbon dioxide, ammonia, and certain mineral salts. Without ammonia or without nitrous acid, respectively, these bacteria cannot grow for lack of energy; they would be like a plant without light. It is evident in this case that the food for energy is also used to some extent as food for growth. The nitrogen necessary to the bacteria is supplied by the ammonia or the nitrous acid. As an example distinguishing strictly between the food for growth - and the food for energy may be mentioned the hyposulphite bacterium studied by Nathanson. This organism oxidizes hyposulphites to sul- phates and sulphur, largely following the formula Na.S203 + O= NasSO4 + S + x Cal. Hyposulphite Sulphate Sulphur Besides, some more complex compounds, like sodium tetrathionate (Naz2S.O¢), are formed. The bacterium builds its cells exclusively from nitrates, carbon dioxide, and mineral salts; organic food is rejected. The hyposulphite can hardly be used for the construction of the cell, and must be considered entirely a food for energy. This distinction is not confined to mineral decomposition only. The urea bacteria get their energy from the decomposition of urea into ammonium carbonate which is hydrolysis. (NH2)2CO + 2H.O = (NH4)2COs + 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 isadded. Séhngen 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. ‘ Microérganisms 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, CO2 and H,0, 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- ion 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 compounds which are in no way essential to life manifestations; they are in the medium in which the cell grows, and thus pass into the cell without taking part in its functions. Sodium and silicon are probably elements of no use to bacteria though commonly present in the cells. Most of the compounds of. the cell are, however, essential to normal develop- ment. Some idea of the needs of the cell may be obtained by studying its composition. ' MoisturE.—The amount of water in the cells of microdrganisms 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 WaLLt.—The membrane of microérganisms does not generally consist of true cellulose (CgHi905);, though it is found in some cases. Other compounds, related to cellulose, are more common; chitin* (CisH39N2012), or another very similar nitrogenous compound is also found. The slime surrounding some bacteria, and the capsules, consist largely of carbohydrates, but often contain some protein. *Chitin when hydrolized yields glucosamine and acetic acid. CigH3oN201. + 4H20 = 2CH:OH'CHOH:‘CHOH:CHOH ‘CHNH:2°CHO + 3CH;*COOH. 148 4 FOOD OF , MICROORGANISMS 149 CELL ‘ConteNnts.—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 réle 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- érganisms may be mentioned glycogen (CgH100s), 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 Foop REQUIRED The amount of food that is ordinarily decomposed by microérgan- isms and the amount that is absolutely necessary, differ widely. The quantity of organic and inorganic matter just sufficient to support a very weak growth is certainly very small, since a few species will multiply to some extent in ordinary distilled water. Such water, after having stood for some time, is found to contain several thousand bacteria per c.c. It may seem to the layman that in such water it + 150 NUTRITION AND METABOLISM would be possible to detect easily the organic and inorganic matter of the microérganisms 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 1 square micron and whose height is 2 microns (which is a high estimaté) the volume of such a cell would be 1 X1X2 cubic microns = 0.001 X 0.001 X 0.002. mm. = 0.000,- 000,002 cu.mm. The specific gravity of bacteria being very nearly 1, 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 o.2 mg. Of this total weight, at least four-fifths is water and only one-fifth is solid matter. The total solid matter in 1 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 (CO2), ammonia (NH;), hydrogen (H), and perhaps methane (CH,). 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). Foop ror 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 0.1 mg. This corre- sponds to about 250,000,000 bacteria. MacNeal and associates found ~~ FOOD OF MICROORGANISMS I51 that the dry matter of 550,000,000 cells of B. coli weigh o.1 mg. The amount of food that is used as the building material for the cell is probably larger than the weight of the cell itself, since there will always be present waste products, but it is of the same order of magnitude, 7.e., very small and often hardly measurable. The example of the urea fer- mentation (page 146) illustrates this point very well. SOURCES OF CARBON.—The compounds which can serve as building stones for the cell vary greatly with the species. The source of carbon for all green plants is carbon dioxide, COz. Animals cannot use this,. for they all requiré complex compounds, such as carbohydrates, fats or amino-acids. Bacteria exist between the plants and animals’ in this respect. Some bacteria have already been mentioned (page 147) as being able to use carbon dioxide (COz), as the only source of carbon; they are the mineral-oxidizing species. Such bacteria are called autotrophic in their-relation to carbon, since they use it in the inorganic form. A bacterium feeding on carbon, as such, would be called prototrophic; bacteria of this class are said to exist. The vast majority | of microérganisms 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 to be equally satisfactory as construction material. Sources oF NiTROGEN.—The sources of nitrogen are equally varied; the green plants use nitrates; animals must have a number of different amino-acids; the microérganisms again are found between them. We know autotrophic bacteria, and especially molds and yeasts which can grow with nitrates or ammonium salts as the only source of nitrogen. There are three groups of prototrophic bacteria in their relation to nitrogen—the B. amylobacter group, the Ps. radicicola group and the Azotobacter 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 own 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. III, 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 OF HYDROGEN AND OxycEN.—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,O) and with carbon in organic substances (CH,) 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 todistinguish 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, K2.HPO., 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. Foop 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 as food makes us realize that oxygen is not such an extraordinary compound as animal physiology seems to indicate, but that it should be 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 microdérganisms 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 microérganisms to provide for their energy without using free oxygen. One such example has already been given in urea fermentation. (NHe)2 CO, + 2H.O = (NH,4)2CO3 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.eH12O0¢ = 2C,H;OH + 2CO2 + 22 Cal. Sugar Alcohol C.Hi20¢ = 2C3H,.O3 + 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 HA O 00000 | Dextrose, H—C—C—C—C—C—C HH HHAHHH H H O Oo || Alcohol, HC—CH C Carbon dioxide, HH | O H H H OO Lactic acid, HC—C—C HH | 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 155 intramolecular changes require a special structure. Carbohydrates © are the most excellent substances for such intramolecular decomposi- tions. S. cerevisie and B. coli can live in sugar-free broth only if ex- posed to the air. They provide for all their needs by oxidation of the protein. If oxygen is excluded, growth depends upon sugar, or a similar fermentable compound. We test for the absence of sugar in a given solution by pouring it in a fermentation tube and inoculating with B. coli: if the liquid in the closed arm remains clear, i.e., if B. colt does not grow without oxygen, it is a good indication that no sugaris present. ; It is usually assumed that in fermentations of this nature, the oxygen atoms are shifted within the same molecule. In other cases, oxygen is taken from one molecule and used for the oxidation of another. This results in one of the molecules being reduced. Nitrates are reduced in this way to nitrites, or ammonia, or nitrogen gas; sul- phates to hydrogen sulphide, and litmus or methylene blue to the colorless leuco-compounds. Such removal of oxygen from a molecule requires energy, and is possible only when the bacterium by using the oxygen for oxidation of organic matter can obtain a larger amount of energy. The following example shows such a possibility: 2KNO; + 36.6 Cal. = 2KNO, + O2 C.H;OH + O2 = CH;CO.H + H.O0 + II5 Cal. This process leaves an energy balance of 115 — 36.6 = 78.4 Cal. for the needs of the bacterium. Such decompositions are sometimes referred to as “reducing fermen- tations” but this term is not correct, as the reduction must always be accompanied by a simultaneous oxidation process. The amount of energy liberated by a fermentation without oxygen is much smaller than that furnished by complete oxidation; the intra- molecular change always leaves organic compounds which contain a considerable amount of the total energy. Yeast, in presence of very much oxygen, oxidizes sugar completely to water and carbon dioxide. Ce6Hi206 + 120 = 6CO2 + 6H20 + 674 Cal. while in: the absence of oxygen it will change the sugar to alcohol and carbon dioxide. C.Hi20¢ = 2C,.H;OH + 2CO2 + 22 Cal. The energy gained in the first process is about thirty times as large I 56 NUTRITION AND METABOLISM as that gained in the second process. This was demonstrated as early as 1861 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..... ie aris 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 microdrganisms, 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 really 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 will kill bacteria in very low concentrations. Ordinarily it is regarded as constituting over 20 per cent of our atmosphere. But if a study is made of its effect upon bacteria, it is necessary to measure it in the same way food is measured, and consider the concentration in which it is offered to the cell. Microédrganisms obtain their oxygen not as gas, but as dissolved oxygen. The solubility of oxygen is very small, about 0.0009 per cent at 20°. Practically all bacteria die readily if the FOOD OF MICROORGANISMS 157 oxygen concentration is raised to thirty times the atmospheric pressure. This would mean a concentration of 0.027 per cent. It shows that oxygen is about as poisonous as formaldehyde or bichloride of mercury. Some bacteria are extremely sensitive to oxygen, and will die if’ exposed to ordinary atmospheric oxygen. They grow only if oxygen is almost completely removed. These organisms are called the strictly anaerobic or obligate anaerobic bacteria. They are contrasted with the facultative anaerobic bacteria which thrive with oxygen as well as without, and the sérictly. aerobic bacteria which have to have oxygen for their normal life processes. No strict limits can be drawn between aerobic and anaerobic bacteria. Even the most sensitive of organisms will be able to tolerate traces of oxygen, while the strictly aerobic bacteria can multiply also if the oxygen concentration is below that of a saturated solution. The limits of growth for the anaerobic bacteria are the limit of tolerance of the poisoning oxygen; the lower limit of growth for the aerobic bacteria is a question of'too scanty food supply. The relation between bacteria and oxygen is graphically represented in the following diagram, after Kruse: 0 02 04 06 Of 10 : 20 | 30 ht ny | ——. —— Saw & G FU Fic. roo.—Influence of oxygen upon microérganisms. The lines indicate the, oxygen concentrations where growth is possible. Line t is a strict anaerobe; 2 is not quite so strict; 3 is still less sensitive though it cannot 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; | 6 can grow only with oxygen but can get along with very little: it might be one of the, urea bacteria; 8 is more dependent upon oxygen and the line would corre- spond to average molds; 7 is a peculiar type needing oxygen and yet being very sensitive toit. The sulphur bacteria, ¢.g., the Beggiatoacee, belong to type 7. Type 9 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 essentially on the same foods, is the incomplete metabolism of most bacteria, contrasting sharply against the very complete oxidation of food in the 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 final decomposition product, ammonium carbonate. Microdrganisms, on the contrary, are characterized by incomplete metabolism. They do not commonly oxidize their food to the end products but many of them ‘produce organic compounds which are not farther decomposed by them. It is this partial decomposition of organic matter which makes bacteria play such an important réle in life and industries. Our modern bacteriology is dated from the time when Pasteur showed that the alcohol in the beer fermentation, the lactic acid in the souring of milk, the acetic acid in the vinegar fermentation are products of microbial activity. The existence of bacteria had been known for nearly 200 years, but they were considered largely as a curiosity; as soon as they were recognized as the cause of fermentations, and of toxins, they received at once the greatest attention. Not all 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 many 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 chemical process which follows in all respects the laws of chemistry. That we are not familiar with all the changes taking place in the cell is not 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 microdrganisms. ‘This formulation is not always simple, because the greater number of microérganisms 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 CeH12O0¢ = 2C,H;OH + 2CO. 180 92 88 According to this formula, roo parts of dextrose should give 51.11 parts of alcohol and 48.89 parts of carbon dioxide. The actual yield comes very close to these numbers, but does not reach them; the largest amounts found were 46-47.5 per cent of carbon dioxide and 47.5-48.67 per cent of alcohol. Under the most favorable conditions, the total yield of the products of fermentation was only 95 per cent of the theoretical yield. Other products are formed besides the alcohol and‘ carbon dioxide. The amount of glycerin found in fermented liquids varies very much with the conditions of fermentation; it reaches from 1.6 to 13.8 per cent: 1 160 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 CieH22011 + HO = 4C3H.O; 342 18 360 Lactose Lactic acid the actual yield of acid is found to be between go 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: 56C6Hi206 + 42H2O = 116H2 + 114CO2 + 30CH3;COOH + Dextrose Acetic acid 36CH3CH,CH2COOH. Butyric acid Second stage: 46C6H120¢ + 18H20 = 112H2 + 94CO2 + 15CH3;COOH + 38CH;CH.CH,COOH. , PRODUCTS OF METABOLISM : 161 Third stage: CsH1205 = 2H, + 2CO, + CH3;CH.CH.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: CeH.O¢ = 2H. + 2CO, + CH3;CH.CH.CO.H CeHi206 = 3CH;CO.H C.Hi20¢ + 6H.O = 6CO, + 12H, The first fermentation continues when the others have already ceased, and thus the last stage of Perdrix’s equations is very simple. Brede- mann 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. coli. Other complications occur when an organism is able to use its own roducts 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 microérganisms 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. Microérganisms 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 microdrganisms 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 microérganisms these experiences are gained almost always from millions of cells. A simple illustration is the 11 162 NUTRITION AND METABOLISM | development of bacteria in salt solutions. If a broth culture of B. colt 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 microérganisms. The best illustration for the variability of cultivated microérganisms. is the enormous number of varieties of Saccharomyces cerevisie. Nearly every large brewery has a yeast type of its own which differs from others by the amount of alcohol and aromatic substances produced, by time and optimum temperature of spore-production, by the appearance of the budding yeast in the hanging drop, and also in other respects. The cultivated organisms are not alone in showing this tendency toward variation. The transferring of a soil or water bacterium into the ordi- 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 well in the first generations, but it gradually becomes able to grow on ' this medium. By this treatment, it loses gradually the power of pro- ducing acid and does not thrive as well in milk. The attenuation of pathogenic bacteria by cultivation on media, as potato, very different from the blood and muscle upon which they grow most naturally, or by growing them at low temperature, or above the maximum, furnishes 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 to ‘produce toxin. As by certain diet the metabolism can be changed, so certain physiological properties of bacteria can, by proper cultivation, be increased. By the frequent transferring of an organism on gelatin, its liquefying qualities can be increased, provided it had some at the start. By continued passing of a bacterium through an animal, its virulence can be increased. Strains of bacteria which will produce a very high acidity can be bred; this is illustrated by the quick-vinegar process gOS. TREN PE aha hag, 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 microdrganisms are used in alcoholic and lactic fermentation, in patho- genic bacteriology and in the mnogTaaoN of leguminous plants with nitrogen-fixing bacteria. Propucts FROM NITROGEN-FREE COMPOUNDS SucArs.—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 microdrganisms. 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 microérganisms many distinguish. somewhat crudely six common types: Complete oxidation. 5 ‘ Partial oxidation. Alcoholic fermentation. Lactic fermentation. Acid gas fermentation. Butyric fermentation. Most of these types have been mentioned previously. Complete oxidation of carbchydrates is observed most commonly among molds and mycodermas, arid also in a few bacteria, ¢.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 microérganisms 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: ' CeHi205 + 90 = 3(CO2H)2 + 3H20 Oxalic acid (CO.H). + O = 2 CO. + H.O 164. 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: CeH2O06 + 30 = CeHs07 + 2H20 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 microérganisms,. 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. Ce6Hi20¢6 = 2€C3H603 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. Manv streptococci will produce the true lactic fermentation. PRODUCTS OF METABOLISM 165 ih The last two groups of organisms, alcoholic and lactic, represent complex fermentations. There are several products formed, and as has already been pointed out in the paragraph on the equation of fermen- tations, the entire fermentation cannot be described accurately by one equation, for different fermentations operate independently and simul- taneously in the same cell. Under slightly different experimental conditions the one or other of these simultaneous fermentations may be . favored, accordingly a varying proportion of the products are formed. The typical representative of the acid-gas forming group of micro- érganisms which cause acid-gas fermentation are B. coli, and its near relative, Bact. aerogenes. Many of the gas-formers in nature belong in this group; the bacteria of the fermentations of pickles, sauerkraut, salt-rising bread, the gassy fermentation of milk are some of the many representatives. They are distinct rods, with good surface growth, and do not liquefy gelatin. They are commonly spoken of as the coli- ‘aerogenes group. Some of them have peritrichiate flagella, while others are not motile. The fermentation of dextrose brought about by these organisms has been described originally by Harden in the equation: 2C;H1.0¢ + H.O = 2C3H,O3 + CH;CO.H + C.H;OH + 2CO, + 2H. Dextrose Lactic acid Acetic acid Alcohol Harden 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. equation. Recently Kamm has shown that a good mineral food (probably phosphates are the essential agent) favors a formation of gas and of volatile acids, while a scant supply of minerals causes the bacteria to produce mainly lactic acid. We must assume, therefore, at least two simultaneous independent fermentations: C6Hi206 = 2C3H6O3 \ and CeHi2.0, + H2O = CH3CO.H + CH;CH.OH + 2CQO:2 + 2He . The first equation is already known to us; it is the true lactic fermenta- tion. The second equation may be divided still further into several simpler equations. ’ B. typhosus, causing typhoid fever, is closely related to B. coli, but 166 NUTRITION AND METABOLISM does not form gas. It forms, however, formic acid, HCO2H, which, if decomposed, would give Hz + COs. 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. Asalready 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. CeH12.0¢6 = 2H. + 2COre + C.H302 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, 7.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 Mucors, and Aspergilli. 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 réle 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. 7 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. Acips anp Atconors.—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. C3H,O3 + 60 = 3COz + 3H.0 168 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;CO.H + 40 = 2CO, + 2H.0 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;CH.0OH + 20 = CH;CO.H + H:0 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- érganisms, they are first split into glycerin and free acid. H.C —-O—CO-—C;;H3, HOH oe HO.C-Ci;Hs1 | ’ HC = O — CO cs C,5H31 + HOH i HCOH + HO.C:Ci;Ha1 | | H,cC-O-CO-CisH: HOH 4H,COH HO0.C-Ci;Ha Fat Water Glycerin Acid PRODUCTS OF METABOLISM 169 This brings about the liberation of three molecules of free acid from a neutral fat molecule. It is customary to test for the splitting of fat by determining its acidity. The glycerin is readily used up by the micro- érganisms, while the fatty acids are oxidized but very slowly. The number of organisms which can attack fat is quite small. Most molds can destroy it; one torula has been found in butter which attacks it, and perhaps a dozen species of bacteria will do the same, among them B. fluorescens and B. prodigiosus, which cause occasionally the rancidity of butter. 1 PRopucts FROM NITROGENOUS COMPOUNDS t On account of the complexity of the protein molecule, the products of protein decomposition by microérganisms 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 formula 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 most 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 .and horn are slowly attacked by a very few organisms. Gelatin, casein and serum albumin are more readily decomposed, though their solubility is quite limited. Peptones which are readily soluble are used by the vast majority of microdrganisms. Of interest in this con- nection 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 have also germicidal properties which are lost by heating or upen - standing. PROTEIN BODIES are aS numerous as plants and animals. Each species of organism seems to have its particular protein which differs from that of other species. With the more highly developed organisms, there are several distinctly different proteins found in the same individ- ual in different parts of the body. The constituents, carbon, oxygen, hydrogen, nitrogen, and sometimes sulphur and phosphorus can be determined in their relative amounts without, however, furnishing any knowledge of the structure of the molecule. The molecular weight of 17° 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 ctystalline 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 ‘terms “water-soluble nitrogen,” “acid-soluble nitrogen” and others 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 that of the protein bodies. We speak of peptonisation of proteins, é.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: radical. Some of them are simple compounds, as the amino-acetic acid NH,CH2COOH and also the amino-capronic acid usually called leucin (CH;)2CH CH, CH(NH,) COOH. Others are of a more complex nature, such as the tyrosin or hydroxy-phenyl- aminopropionic acid, CsH,(OH) CH2CH (NH2) COOH, and the tryptophan or indol-amino-propionic acid, CsHsN CH2CH(NH») COOH. Of other nitrogenous products which are not amino-acids, a few are of striking significance. The very disagreeable odor of putrefying proteins and of excreta is due to indol (CsH;N) and methyl-indol or skatol (CsHgN-CH3). Indol gives a rose color with nitrites in acid solution, and this convenient reagent is used in the identification of PRODUCTS OF METABOLISM 171 bacteria. Another group are the amins. The simplest amins are the methyl-amins, of which the tri-methylamin (CH;)3N 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 promains. The ptomains (page 491) are alkaloid-like bodies of basic character and of more or less well-known structure. Some of them are notorious for being very strong poisons, while others are quite harmless. These ‘bodies are 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.(CH:2),NH2] and the cadaverin or penta-methylen-diamin [NH2(CH2);NH:], which can scarcely be considered poisonous. The methyl-guanidin NH, ~ HN =C a NHCH; may be mentioned as an example ofavery poisonousptomain. Another poisonous ptomain is the neurin CH, = CH — N(CH3),0H 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, ¢.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; or (NH,)2CO; 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;), 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;PO,). 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 (H2S). Some microdrganisms are able to form mercaptan (CH;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 defined. 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 COs, H.O, NH; and H.SO,. The nitrogen is never changed to any oxidation product, but is found as NH, 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. 4 PRODUCTS OF METABOLISM , 173 As a third group, B. coli.may be mentioned which forms indol, but no ammonia from peptone, and whose proteolytic powers are very weak as 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- hydrates 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(NHe). + 2H20 = (NH,4)2CO3. 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 have recently been found to hydrolyze urea. All urea bacteria can live without urea, feeding on organic matter like other bacteria, but ‘most of them require an alkaline medium. Hippuric acid is split by certain bacteria to benzoic acid and amino-acetic acid which can be oxidized completely. Uric acid can be changed in several ways. In some of these changes, urea is found as an intermediary product. PRODUCTS FROM MINERAL COMPOUNDS Minerals are used by microérganisms 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- 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. Asa 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: KNO; — O = KNO, KNO; = KNO. + O ' KNO; + 2H = KNO, + H,O. The result in all three cases is the same. Many bacteria can reduce nitrates to nitrites or toammonia. A few can reduce them to nitrogen. These “true denitrifiers” are found in soil and in old manure. Their reducing process is as follows: Ca(NO,)2 — 50 = CaO + 2N. Nitrates are reduced through the efforts of the organism to secure a supply of oxygen. The denitrifying bacteria have strong oxidizing 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 H2SO4 = 40 = HS. 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 '’ sulphur to hydrogen sulphide. The potassium and sodium salts of selenic and telluric acid (H2SeO, and HTeO,) are reduced by certain organisms and not by others. The reduction results in a colored precipitate; this reaction has been suggested as a diagnostic means to distinguish different species. The reduction of arsenious oxide to arsin (AsH;) is used as a very delicate test for arsenic; it is applied in the detection of arsenical poisoning. The material to be tested is sterilized and inoculated with Penicillium brevicaule (page 52, the “arsenic mold”). This will reduce most arsenious compounds to arsin (As Hs) or to diethyl arsin, As H(C2H;)2, both of which are easily _ recognized by their very pronounced garlic odor. UNKNOWN PrRopuctTs 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. PicmEnTs 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 microérganisms 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 Oidiuwm 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 pigment as a useless excretion, either as a color-body or as a leuco-body which becomes colored through the action of atmospheric oxygen. The individuals themselves are colorless and may under certain conditions cease to form pigments. To this class belong B. prodigiosus, B. cyano- genes, Ps. pyocyanea, and others. 3. Parachrome bacteria, which form the pigment as an excretory prod- uct but retain it within their bodies, as B. janthinus and B. violaceus. 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 to obtain non-pigmentation with many species by propagating them through many generations at high temperatures. Oxygen also -is necessary for the chromogenesis of many bacteria. Some need a short exposure to daylight in order to produce their pigment, while cultures grown in absolute darkness may remain colorless. Strong sunlight, however, will check pigment production in the same degree as do’ antiseptics and other harmful influences. t PRODUCTS OF METABOLISM 177 The chemical nature of microbial pigments is little known. They are distinguished according to the solubility in various liquids, water, alcohol, ether, chloroform, benzol, and other solvents, and according to the change of color caused by acid and alkali. A group of carotin bodies, named: because of their. similarity to the pigment of carrots, the prodigiosin bodies, named after B. prodigiosus, the Fic. ror.—Bacteriopurpin, from a Rhodospirillum, crystallized from a chloroform solution.. (After Molisch.) fluorescent pigments and perhaps a few other groups are distinguished, but their chemical nature is rather vague as yet. The absorption of distinct lines of the spectrum by solutions of these pigments is claimed to be a very reliable means of distinguishing the pigments of different species. : AROMATIC SUBSTANCES constitute another group of metabolic prod- ucts. The chemical analysis accomplishes more with these com- pounds than with pigments, since they are frequently well-known compounds. The main difficulty arising in their identification is in the very minute quantities of the products available. Some substances with strong, mostly very disagreeable odors have already been men- tioned: indol, skatol, hydrogen sulphide, mercaptan, the amins and ammonia, butyric acid, and some of the higher alcohols. There re- main to be mentioned certain oils and esters giving rise largely to pleasant aromas. The formation of aromatic oils has been established although their nature is entirely unknown. The same is true with the esters. The substance causing the fishy flavor in butter is volatile with steam and is neither of an alkaline nor acid nature. The strong odor of freshly plowed earth is caused by an Actinomyces; the odor 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 least understood products of microbial action are the enzymes and the toxins. These two groups are related in many respects. The enzymes will be discussed extensively in the following chapter and toxins are treated more extensively on pages 575,676. Toxins and enzymes are formed by the cells in such small quantities that they would never have been discovered by ordinary chemical means were it not for the unusual effects which they produce, the enzymes acting upon food substances, and the toxins acting physiologically upon organisms. Toxins and enzymes are chemically unknown. It is assumed that they are chemical bodies, but even this has not been proved. A pure toxin has never been obtained and we have no criterion for its purity. The presence of a toxin is recognized only by an animal test and in this way the com- parative concentration can be determined approximately. Such standardization of toxin solutions is only comparative, however, and gives no clue as to the actual amount of toxin present. Not all ani- mals are sensitive to all toxins. It is quite possible that all bacteria produce compounds with chemical qualities similar to toxins, and only a few of them happen to react upon men or animals. Toxins are not always the product of microbial action. Vegetable toxins or phytotoxins are known, among which the ricin of the ‘castor- oil bean is perhaps the most studied representative. The best-known zodtoxin is the rattlesnake poison. These non-microbial compounds have the same quality as the microbial toxins—they are extremely poisonous. Toxins are the cause of disease in diphtheria, tetanus and botulism. Ifa culture of these organisms is filtered through a porcelain filter which removes all bacterial cells, the filtrate injected into an animal will cause the disease with all its accompanying symptoms though there are no microérganisms introduced into the animal body. PRODUCTS OF METABOLISM 179 If the filtrate is heated, however, no effect will take place after the in- jection, because heat destroys the toxin. The amount of toxin that will kill an animal is extremely small. .oocoo5 mg. of the purest tetanus toxin will kill a mouse, .ooo7 mg. of ricin will killa rabbit, less than -23 mg. of tetanus toxin will killan adult man. The body of an animal or man forms an anti-body against: the toxin which neutralizes its poisonous action. Anti-bodies are also formed against: enzymes injected into an animal. Toxins are very sensitive to heat. A short exposure to temperatures between 80° and 100° will inactivate them. They are also very sensi- tive tolight. While some toxins are secreted, others are retained within the cells of microérganisms, and never leave them until the cells die or, disintegrate. Ptomains, which are also metabolic products of micro- organisms and sometimes cause poisoning, differ from the toxins in their resistance to heat and. light (page 171). Ptomains differ in no way essentially from ordinary organic compounds; the animal or human body produces no anti-ptomains to counteract their poisonous effects. There is no chemical relation whatever between toxins and ptomains, and the physiological effects are also quite different, though they both cause poisoning. Toxins are not essential products of the metabolism of pathogens. Strains of pathogenic bacteria can be bred which do not produce toxins as chromogens can be bred without pigment, or lactic bacteria which — do not produce acid. The strains which lose their pathogenicity grow better on artificial media but are less able to produce disease in the animal. They may regain the power of producing toxin if passed through the body of the animal. The real object of toxin production by microdrganisms is not known; the microdrganisms derive no ap- parent benefit. Factors INFLUENCING THE TYPE OF DECOMPOSITION In the chapter on products of metabolism, it has been shown that the same compound can be decomposed in many different ways, and the question may well be asked what decides the type of decomposi- tion. Since bacteria are widely distributed, it must be expected that there are certain conditions which are most favorable to a given type of fermentation, while under changed conditions, other types are more 180 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 decomposition are oxidation, complete or incomplete, and alcoholic fermentation. The oxidation is brought about by molds or organisms closely allied to ‘yeasts. The latter are very common on all sour foods, especially on 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 Penicillium and 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 through a large number of experiments by Kendall and Walker, that practically all bacteria will decompose sugar in preference to proteins. If a leaf contains sugar and protein (cabbage) the sugar decomposition will be conspicuous, and the protein is not attached very readily. Putrefac- tion in the presence of sugar or of acid does not take place. Meat will not putrefy if mixed with sugar, while milk putrefies readily if the sugar is removed by dialysis. The three types of sugar decomposition which come into consideration in neutral media, are the lactic, the acid-gas and the butyric fermentations. The latter is a strictly anaerobic fermenta- tion, and thus limited to special conditions. Of the other two, the acid- ae PRODUCTS OF METABOLISM 181 gas fermentation is the most common, and the souring of vegetables of all kinds is due to this type of fermentation (pickles, sauerkraut, ensilage, salt-rising bread). Sometimes the acid-gas fermentation is followed by a butyric fermentation. The true lactic fermentation is not common, and is limited almost entirely to milk. This is ex- plained by the circumstance that the organisms causing this decom- position are parasitic in their habits, causing disease or living in the intestine of animals. In the absence of acid and sugars, putrefaction is the most common type of decomposition. Many factors aside from the chemical composition of the medium are essential. Oxygen has already been mentioned as preventing buty- ric fermentation. It will also prevent the acid-gas fermentation if too abundant. Ensilage is trampled and pressed down to avoid air spaces as much as possible, for molds will outgrow the acid-forming bacteria . - if air has free access. Absence of oxygen will prevent mold growth, and for this reason, jelly is paraffined, and butter wrapped tightly into impermeable paper. The influence of oxygen upon the type of proteix and of cellulose decomposition has been pointed out previously. The moisture content is of great importance. As will be showtt later, not all organisms have an equal need of moisture; some molds will 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 | substance is immersed in the medium. Their thread formation enables them to search a dry medium, such as flour, for moisture, the extreme of adaptation being Rhizopus, and the construction of the fruiting bodies shows that they are destined by nature to be spread by air and wind. It is no wonder that damp organic matter, if it can be de- composed at all, will show molds, and nothing else, regardless of the chemical composition, for there is no competition. Flour, moist seeds, incompletely dried fruit, damp milk powder will always become moldy. The same holds true with very concentrated sugar solutions such as syrups, jellies and jams, while in concentrated salt solutions, molds cannot thrive, and the torula yeasts are best adapted to such conditions. _ Avery important part is also the structure of the material. Micro- érganisms act mainly upon organic. matter, and since this comes from living organisms, it has usually definite structure, exceptions being milk and blood, The structure of all living organisms is such 182 . NUTRITION AND METABOLISM as to prevent the intruding of microérganisms. 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. Microdrganisms 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 microdrganisms 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 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 to ~ - the other. Molds possess this means; they can puncture cell walls, and forcing their way from one cell’to the other, they will soon 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 in the soil and surrounded by billions of microérganisms. They are 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, and thus cause a softening of the root tissue. The bacteria causing the various rots of vegetables belong to this type. “There is, then, a great variety of factors deciding the type of decomposition of organic matter in nature, and by knowing the chemical composition as well as the structure and other physical conditions, it is possible to foretell which group of organisms is most likely to 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. Or- ganisms grow and decay. The same carbon and nitrogen atoms which constitute the organic world of to-day constituted it thousands of PRODUCTS OF METABOLISM 183 years ago. The amount of carbon, nitrogen, hydrogen and of all , Other elements of life on earth is limited, and the same atoms will . be used for the future generations of life that constitute the present. There must be continuous destruction to enable new construction. Construction is mainly the task of green plants, enabled by the chloro- phyl to use the energy of sunlight in building up organic substances from minerals, water and carbon dioxide. Destruction is caused mainly by animals and other organisms which have to break down organic matter in order to exist. These two factors keep the atoms of the organic world in perpetual rotation. In this circulation of the elements it is necessary that all compounds of organic nature be decomposed finally to a form available for plant food. If this were not the case, the indestructible compound would sooner or later accumulate in such ‘enormous quantities that the elements constituting this body would be removed entirely from general circulation. Let us suppose, as an illustration, that for some unknown reason, all urea bacteria on earth would die. Urea could be decomposed no more, and the plants, unable to use urea as a source of nitrogen in place of nitrates, would get but little benefit out of stable manure. All urea would pass gradually undecomposed into rivers, lakes, and finally into the ocean where it would accumulate 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. CaRBoNn 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 chlorophy] 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 microdrganisms; 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- Lhad Organisms Respiration Ma rbondioxtde Protein Carbohydrates fat, Folin Fic. 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 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, microdrganisms have been present, in order to render the dead organic matter again available for plant and animal life. Fig. 102 gives a schematic illustration of the carbon cycle; the microbial activity is marked by heavy lines. PRODUCTS OF METABOLISM 185 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 microérganisms 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, Fic. 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 186 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 itis 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. /ydroger - Sulphide Dead TOASTS Fic. 104.—Sulphur cycle. SutpHuR 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 digested by higher animals or broken down by putrefaction to the final product, hydrogen sulphide, which is oxidized by the sulphur bacteria first to sulphur, then later to sulphates. PuospHorus Cycite.—The cycle of phosphorus has not been worked out completely, but from the discussion in the last pages, it is plainly seen that a simple cycle very much like the ones above must exist. It PRODUCTS OF METABOLISM 187 is probably much simpler because phosphorus does not enter as easily into organic compounds as nitrogen. PuysicaAL Propucts oF METABOLISM Propuction 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 1°, 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 ro° 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 microdrganisms, but it has been shown recently that even sterile hay will show a rise of temperature under certain 188 NUTRITION AND METABOLISM conditions. This does not exclude the formation of heat in hay by microdrganisms 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. PRopuction oF Licut.—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 microdérganisms 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 Jlight-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, KaTaBouisM, METABOLISM.—In the introduction to the Physiology of Microérganisms, it was stated that microdrganisms 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 microérganisms. 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 Foop.—It has been stated before that many microérganisms feed upon cellulose, starch, fat, gelatin, keratin and other insoluble compounds. It has also been previously stated that microdrganisms, with the exception of some protozoa, 189 Igo 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, invertase 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 microérganisms 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 decomposed further. The food-preparing processes are all supposed to be simple hydrolytic processes. For some of these changes the chemical equations are well known. The hydrolyzation of starch to maltose by means of diastase is represented by the equation 2(CeH1005)n + DH20 = nCiHe201. MECHANISM OF METABOLISM Ig!I The splitting up of a fat molecule into glycerin and fatty acid is also a well-known process C3Hs(CisH3502)s + 3H:0 = C;H;(OH)s + 3CisH36O2. 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. Invertase decomposes saccharose into dextrose and levulose: CisH22011 + H.O = CeHi20¢6 + CeHi20e. 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 192 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 microérganisms 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 microdrganisms 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 toa 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 triedto 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 tinued dialyzing weakens the activity which can be restored only by | adding salts. ENZYMES OF FERMENTATION.—It has been demonstrated in the above paragraph that food is prepared for digestion or fermentation by enzymes. The final decomposition, the process which yields the energy for cell life, must take place within the cell. The difference in importance of food preparation and fermenta- tion may be illustrated by the example of Rhizopus oryze. This mold attacks starch, changes it, by means of diastase, to maltose, the 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 source of its life; the others are incidental processes, not absolutely necessary 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; it 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 microérganisms, 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. 3 CLASSIFICATION OF ENZYMES Since the chemical nature of enzymes and of their action is largely unknown, they can be classified only according to the compounds they act upon. It is possible, however, to distinguish between the following four groups: Hydrolyzing, zymatic, oxidizing, reducing 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 (intra-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). (b) coagulating (coagulases): thrombase, rennet (chymosin). II. Zymases. 1. of carbohydrates: alcoholase, lactacidase. z. of other nitrogen-free bodies: vinegar-oxidase. 3. of proteins: endo-tryptase, autolytic enzymes, amidase, urease. Ill. 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. {4 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 195 Hyproiytic ENZYMES EnzyMES oF CARBOHYDRATES.—Enzymes which decompose carbo- hydrates are very commonly found in nature, because carbohydrates constitute a very extensive and common group of organic matter. By far the largest part of the dry plant consists of cellulose, starch and sugar. To decompose them, enzymes are necessary. The chem- ical reaction of these enzymes is hydrolytic; in other words, the larger molecule is broken into smaller ones by the simple addition of water. Thus, the cellulose-destroying enzyme, called cellulase or cytase, de- composes the cellulose into soluble sugars after the following formula: C6H i005 + H,O = CeH120¢ or, considering that the cellulose molecule is really many times Ce6Hi00;, the formula will be more accurately written (CoH1005)n + nH,O = nCsH12.0¢ which indicates at the same time that one cellulose molecule gives many sugar molecules. Cellulase is an enzyme which is quite difficult to obtain. Though it must be produced by all the cellulose destroying molds and bacteria, experiments “have failed in some instances to prove its presence. It is found in some wood destroying fungi and in some of the bacteria causing the rot of vegetables. The organisms of certain plant diseases force their way into the cell by dissolving the cellulose membrane by an enzyme, while certain molds are able to puncture the cell wall mechanically. Diastase, or amylase, is the starch-dissolving enzyme which is one of the most common enzymes in nature. It is found in all green plants,’ and it forms during the sprouting- of starchy seeds. Many molds and a few bacteria produce this enzyme, while yeasts generally cannot decompose starch for lack of diastase. Starch has the same formula as cellulose, and it is broken up into soluble sugars in the same way. Much * “attention has been paid to this process by the chemists, and it is found that the process is a gradual one, giving first dextrins, and finally maltose (Ci2H220i1). The hydrolysis of starch expressed in chemical symbols may be presented as follows: 2(CeH100s5), + nH2O = nCywH220i1. Starch Maltose 196 NUTRITION AND METABOLISM The disaccharides or double sugars, having the chemical formula Ci2H»20i1 are broken up into single sugars, monosaccharides, by the following process: Ci2H201 + H20= CeH1206 + CoeH1206. The two molecules of CsHi20¢ 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 Jactase 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 CsH120¢ 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 microérganisms. 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 MECHANISM OF METABOLISM 197 called fermentation products; these may result from alcoholic, lactic, butyric fermentations or some other. Emulsin is an enzyme which is able to hydrolyze glucosides. Gluco- sides occurring in plants are complex bodies which contain a sugar- radical. Emulsin splits glucosides liberating the sugar, usually dex- trose. The typical example for emulsin action is the hydrolysis of amygdalin to hydrocyanic acid, benzaldehyde and dextrose. CooHe70uN + 2H,0 =) C.H;COH + 2CgH120¢ + HCN. Amygdalin Benzaldehyde Dextrose Hydrocyanic acia Emulsin is found in many molds and bacteria, and recently has been found in yeasts. Glucoside-splitting enzymes play an important réle in the fermentations of coffee-beans, cocoa, mustard and indigo. In most of these fermentations, however, the emulsin is probably not formed by microérganisms, but by the plant, from which the ferment- ing material is derived. Enzymes or 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, but the result of the cleavage process is always the same. The name formerly assigned to enzymes of fat is steapsin, but this term is now almost 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 acentury. 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 frypsins, which act best in slightly alkaline media. Thenamesare derived from pepsin (peptase) 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 microérganisms 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 microdrganisms 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 + sign means proteolysis, a — sign means no action. MECHANISM OF METABOLISM 199 _ Milk Organism Gelatin| Serum ae Fibrin Coag. | Digest. Bact. anthracis..... i i: Sr ek eo eet + + + = + + Microspira comma... 1.6.0.0 eee + + + + + + M. pyogenes var. aureus............665 + + + _ = = Pseudomonas pyocyaned.............4- ap + + + + ics Be ViOlaceusss ence caidas agegas owes - - + = = es Be MY COULESE snirerdt 9 voineaie. wie rasa tees a ines + + + — + = B. prodigiosus..... 0.600. c cc cece _ + + + + + Aspergillus niger....... 0.00 cece eee + + me wh cy ay Aspergillus Oryz@.. 0... cece ccc ee _ + + ae 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. Coacutatinc Enzymres.—The blood-clotting enzyme (throm- base) does not occur in microdrganisms. 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 “Jab.” 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 1 g. of substance. ENERGY LIBERATED FROM I G. OF SUBSTANCE 1 Soluble Enzymes Endo-enzymes Pepsin, trypsin........... o calories Lactacidase............ 80 calories Lipase.s¢sveske9s25 xeaxes 4 calories Alcoholase............. 120 calories Maltase, invertase........ to calories UTEAS Orsi cic tecsey Gdns 230 calories TLattase. sc iiaccis aces adang dbs 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, Jlactaci- 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. MECHANISM OF METABOLISM 201 The vinegar oxidase is an enzyme which remains in the cell of the acetic bacterium, oxidizing alcohol to acetic acid. Its independence of the living cell has been demonstrated by killing the cells with acetone. The PROTEOLYTIC ENDO-ENZYMES of yeasts, only, have been studied extensively. That such enzymes exist is recognized by the observa- tion that certain microédrganisms do not liquefy the gelatin until after they are dead and the proteolytic enzymes diffuse out through the deteriorating cell membranes. That yeast in the absence of sugar loses in weight, and that leucin and other cleavage-products of protein are formed, was the first indication of a proteolytic process in the yeast cells. By pressing the juice out of the ground yeast cells, a liquid is obtained which liquefies gelatin, digests casein, albumin and fibrin. The living yeast cell does not attack these compounds, be- cause they cannot diffuse into the cell and the enzyme cannot diffuse out. The proteolytic endo-enzyme of yeast is called endo-tryptase. Its object is apparently the regulation of the protein-content of the cell and perhaps it has some bearing on the formation of cell plasma. The possible relation between enzymes and growth is discussed in a following sub-chapter. If yeast is mixed with a weak antiseptic (chloroform, toluol) the proteolytic process takes place quite rapidly. This process is called autolysis (self-digestion). Similar autolytic enzymes are found in other microdrganisms. Autolysis is a well-known process in the higher animals. To this is due the ripening of meat. Proteolytic endo-enzymes must be expected in all microérganisms which depend upon protein as food material only. These organisms will secrete certain enzymes which decompose the insoluble protein into bodies which diffuse easily into the cell. Here, proteolytic endo- enzymes further decompose these products. Such an endo-enzyme is the amidase discovered by Shibata in the mycelium of Aspergillus niger which forms ammonia from urea, acetamid, oxamid, biuret. Endo-erepsin and amidase were also found in Penicillium camembertt by Dox. Similar to these proteolytic enzymes is the urease which is formed in large quantities in the so-called urea bacteria, but it is also present in the mycelium of some molds. An endo-enzyme, splitting hippuric acid into benzoic acid and glycocoll, is found in the mycelium of a few molds. 202 NUTRITION AND METABOLISM OxIDIZING ENZYMES The most typical example of an oxidizing enzyme is the vinegar- oxidase, because its chemical action is well known. Most of the oxt- 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 amold. Many other oxidations are supposed to be of enzymic nature, but their independence of the living cell has not been proved. Many higher organisms are known to contain oxidases, the best studied are those of certain mushrooms which change the white mush- room meat into a bluish or brownish color as soon as it is exposed to the air. Oxidases are very common in most of the tissues of higher animals. RepucING 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. HO. + katalase = H,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 the alcoholase of this yeast cannot tolerate more than 8.5 per cent of alcohol. The inability of the cell to regulate enzymic action may account for the fact that often a culture produces an amount of fermentation products sufficient to kill all cells. This is observed in the lactic, acetic and alcoholic fer- mentations, and, perhaps, occurs in many others. Probably, all cells produce several enzymes. Microérganisms feeding upon various foods must form various enzymes. Frequently several enzymes are necessary for the decomposition of one com- pound. Rhizopus oryze@ 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 microdrganisms is surprising. Asper- gillus niger has the reputation of forming almost all enzymes which have ever been found in microdrganisms. Penicillium camemberti produces (after Dox) erepgin, nuclease, amidase, lipase, emulsin, amylase, inulase, raffinase, invertase, maltase and lactase. It has been believed for a long time that certain enzymes are regular products of the cell while others are formed only if the substance upon which they act is present. According to Dox’s investigations with Peni- cillium camemberti, there is no evidence that enzymes not normally formed by the organism in demonstrable quantities can be developed by special methods of nutrition. The addition of a particular food compound does not develop an entirely new enzyme, but stimu- i 204 NUTRITION AND METABOLISM ; lates the production of the corresponding enzyme which is normally formed, although in small amounts, under all conditions. THEORY OF KATABOLISM Regarding katabolism as the sum of all destructive processes of the living cell substance, 7.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- tionedin theaboveparagraph. Unquestionably, the katabolic processes are similar to enzyme processes, since katabolism is checked by heat or poison just like enzyme processes. THEORY OF 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 that in such a small unit as a microbial cell, there are contained the powers of making protoplasm, enzymes, nuclear bodies, chromatin bodies, the substance of the cell wall and probably many other unknown MECHANISM OF METABOLISM 205 compounds. All these complex substances are generally made from simple 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 usually fulfilled in the living cell, due to the fermenting processes going on continuously. There is a strange interaction between anabolism and intra-cellular fermentation proceeding in the pro- toplasm and this linking together of destructive and constructive reaction is the basis of life processes. The life processes decompose certain substances, the energy liberated allows the formation of proto- plasm, which again liberates energy. Thus a continuous formation of protoplasm is secured. An explanation of anabolism based upon chemical experiments is not possible at the present time. In the study of intra-cellular destruc- tion it is possible to trace most processes back to enzymic action. There our knowledge ceases because the nature and mode of action of enzymes is unknown. In the study of anabolism our knowledge has not even progressed so far. The most promising explanation at present is based upon the reversibility of enzymic action. REVERSIBILITY OF Enzymic Action.—Chemical reactions be- tween organic compounds proceed quite rapidly at first, then become slower and slower until the reaction stops entirely. The reaction is not 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 proceed either way, depending upon the relative concentrations of the reacting substances. The standard example is esterification. As- cetic acid plus alcohol gives ester plus water, CH;COOH + CH;CH:,OH@CH;COOCH.CH; + H.0. Acetic acid Alcohol Ester The process goes to a certain equilibrium and stops. If ester is mixed with water, it gives acid plus alcohol, until the same equilibrium is reached. If acid and alcohol are added to a system in equilibrium, more ester will be formed. If ester is added, more alcohol and acetic acid will be formed. The same is true with enzymes, at least with some enzymes. Maltase will decompose maltose into two molecules of dextrose. In a concentrated solution of dextrose, however, maltase 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 amino-acids by the reversed action of proteolytic enzymes. In the same way, cellulose may be formed from dextrose, fat from glycerin and fatty acids. Nearly all phases of growth can be accounted for in this way.. This is nothing but theoretical 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 I PHYSICAL INFLUENCES | CHAPTER I MOISTURE Moisture may be called the most important factor of life. Not only bacteria, but every microscopic and macroscopic being requires a considerable amount of moisture. Living organisms contain on the average between 70 per cent and go per cent of water, and only ro per cent to 30 per cent of solid matter. Microédrganisms which live entirely submerged in liquids need water not only within but without the cells. Bacteria, yeasts, molds, and some protozoa obtain their food by diffusion through the cell-membrane; their food-substances must ' be soluble and dissolved. No other liquid can take the place of water. The amount of water required by microérganisms cannot be stated briefly. Several factors have to be taken into consideration, as the osmotic pressure, the insoluble and the colloidal substances, the species of organisms, temperature, and perhaps others. Osmotic PRESSURE.—In the organic world we find very commonly membranes which will allow water to pass through but retain some compounds dissolved in the water. Such so-called ‘semi-permeable membranes are found surrounding the protoplasm of cells. They are not the cell wall, but separate the protoplasm from the cell wall. Similar properties are found in parchment paper, pig’s bladder, and other organic membranes. If a salt solution is poured in water, the two liquids will mix in a short time and soon every smallest portion of the mixture will have the same concentration. If a salt solution and water are separated by a membrane which does not allow the salt to pass, the water will go through the membrane toward the salt with a certain amount of pressure. This pressure depends upon the nature of the dissolved substance as well as upon its concentration. 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.* Prasmotysis.—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, microdrganisms do not show it so readily. In fact, many bac- teria, like B. subtilis, Bact. anthracis, cannot be plasmolyzed by any concentration of salt in solution. Others, as B. coli, B. fluorescens, react promptly. But even though many are killed, the rest recover from plasmolysis after a few hours, and appear normal. This indicates that the salt passes slowly through the plasma-membrane and thus increases the pressure inside the cell until finally the inside and outside pressure are the same again. The fact that many microérganisms show no plasmolysis whatever is explained in the same way. These organisms probably have plasma- membranes so constructed that the salts diffuse through nearly as fast as the water. An absolute exclusion of all soluble substances by the mem- brane is impossible since the food can get into the cell only by diffusion through the membrane. The resistance of various microérganisms against concentrated solutions depends upon the organism as well as upon the dissolved sub- stance. The sodium and potassium salts of the common mineral acids act upon a culture nearly in proportion to their osmotic pressure, but the potassium salts always retard growth a little less than the sodium salts. The effect of salts upon microérganisms is therefore not due to the osmotic pressure only; the chemical constitution of the salts also plays an important réle. *One atmosphere equals the pressure of 1 kg. per square centimeter or about 15 pounds per square inch. MOISTURE 209 The different functions of life are influenced in different degrees by concentrated solutions. Some bacteria will multiply but not form spores in salt solutions. Molds will sometimes show a good growth in concentrated sugar solutions but fail to produce spores. Bact. anthracis loses its virulence in sea water. Often, the form of microdrganisms is affected by concentrated solutions. Some bacteria grow more spherical, others become elongated or distorted. The deforming influence is not due to the osmotic pressure only, but depends mainly upon the chemical character of the salt; magnesium salts especially have a tendency to produce such involution forms. Salt and Sugar Solutions—Most experiments on the influence of concentrated solutions have been carried on with sodium chloride, be- cause of its wide application in the preservation of foods. Most micro- drganisms, especially the rod-shaped bacteria, are suppressed by a salt concentration of 8toz1opercent. At 15 per cent only few cocci develop slowly, while some species of Torule grow without a very noticeable re- tardation. Above 20 per cent the Torule are practically the only organisms which can develop. They are, therefore, found in all food products which are preserved by salt, as salted pork, beef, fish, butter, and pickles, often in nearly a pure culture. It seems that they are easily overpowered by other organisms in the absence of salt, but in salted 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 brine pickles, where the desirable bacteria can grow in the presence of salt while the undesirable ones are kept away. Possibly the salting of butter has the same effects. Another compound of great practical importance is cane sugar, which is the standard preservative for fruits and condensed milk. Its action has been studied mainly upon molds. Theoretically, dextrose should be expected to have twice as strong a preserving action as saccha- rose because it has only half the molecular weight and consequently produces twice as strong an osmotic pressure in the same percentage of concentration. 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- “4 210 PHYSICAL INFLUENCES \ centrated solutions while bacteria in general do not tolerate solutions’ higher than 15 to 4o 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. IfB. prodigio- sus does not develop in a ro 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 microérganisms. 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 4o per cent does not necessarily indicate the conclusion that any substance with less than 30 per cent water cannot be decom- 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 microdrganisms in the droplets of buttermilk MOISTURE \ 211 distributed all through the butter. The concentration in these small droplets is the deciding factor. If the growth of microdrganisms in butter is to be prevented by salt, it is unnecessary to give any attention to the fat; the bacteria live only in the water and not in the fat globules. In adding 3 per cent of salt to a butter with 15 per cert of moisture, a brine of 3 parts of salt in 15 parts of water is produced; in other words, a 20 per cent brine, because salt does not dissolve in the fat. Similar considerations will come up in the preservation of fruit, vegetables, meat, milk, and other food substances by drying or condensation. , DESICCATION.—Microérganisms do not die immediately after the removal of the water, and they do not die all at once after a given time. Death through drying is a slow and regular process. Paul and his associates founded that the number of bacteria dying in the unit of time 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 go 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 1 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 too cells per gram. After 5 days 1o cells per gram. After 6 days 1 cell per gram. After 7 days o.1 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 o on the seventh, but to one cell in 10 g. and on the eighth day one cell in 100g. The total number dying in the first day is much larger than that dying on the sixth day, but the rate is constant, go 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 is caused by an oxidation process; in purée oxygen bacteria died much faster. The poisonous effect of oxygen upon moist bacteria has already been 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. carotarum and Ps. radicicola, are readily killed. The resistance of microdrganisms 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 microérganisms 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- tors of life. It is so important that the most highly developed animals protect 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 cohstant 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 microérganisms 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 all 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 microdér- ganisms. Temperatures Species = Minimum Optimum Maximum Penicillium glaucum............... 1.35° 25°=27° 31°-36° Aspergillus niger.............--.45 7°-10° 33°-37° 40°-43° Saccharomyces cerevisie I.... . .... 1°-3° 28°—30° 40° Saccharomyces pasteurianus I....... 0.5° 25°-30° 34° Bacterium phosphoreum............ below 0° 16°-18° 28° Bacillus subtilis .......... cas oe 6° 30° 50° Bacterium anthracis................ 10° 30°-37° 43° Bacterium ludwigtt................ 50° 55°-57° 80° THE MINIMUM TEMPERATURE or the lowest limit of growth is usually farther from the optimum than the maximum temperature. It will vary with the organisms just as do the other cardinal points. But there is a natural limit drawn by the freezing-point of the nutrient liquid. Not all organisms can grow at such low temperatures, in fact the greater number does not develop below 6° to 10°. Those that can grow 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 0°. 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 215 The number of microérganisms that developed at the freezing- point was found to be: In 1 c.c. of market milk, up to 1,000 germs. In 1 c.c. of sewage, up to 2,000 germs. In 1g. of garden soil, up to 14,000 germs. THE MAXIMUM TEMPERATURE is usually about 10° to 15° higher than the optimum. The development of microdrganisms above the optimum temperature is not quite normal; there is a great tendency toward 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. Bact. 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 for 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. 216 PHYSICAL INFLUENCES . BIOLOGICAL SIGNIFICANCE OF THE CARDINAL POINTS OF TEMPERA- TURE.—The importance of the temperature requirements of certain organisms to the réle 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 Mucorinee. Pathogenic microédrganisms 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 microérganisms; 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 perc.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 Bact. aerogenes and B. coli. The odor is disagreeable and later butyric acid is produced; eventu- ally the lactic acid increases further by the action of Bact. 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 217° This indicates the importance of temperature regulation in the fer- mentation industries. Even pure cultures may give different products if working at different temperatures. Cream ripened with a pure culture starter at too high a temperature will have a sharp acid flavor. The cold curing of cheese has become a very common practice because of the much improved flavor. Bioletti claims that the value of the dry California wines would be doubled if the fermentation were carried on generally at a lower temperature. END-POINT OF FERMENTATION.—Another question is the relation between the end-point of fermentation and the temperature. Of the few data existing, many indicate that at a lower temperature the final fermentation goes farther than at a higher temperature. Miiller- Thurgau found that under exactly the same conditions with the tem- perature as the only varying factor the following final amounts of alcohol were produced by a pure culture of yeast: AtH6 mews cade 3.8 per cent alcohol. At oF essay e253 valine’ 7.5 per cent alcohol. At 18°..... eae g aden 8.8 per cent alcohol. At, O%ssay escuns “2... 9.5 per cent alcohol. Concerning the lactic fermentation some investigators find no differ- ence in the end-point, while others obtained results similar to the re- sults with alcohol. With three strains of Bact. lactis acidi were ob- tained after thirty-four days, by C. W. Brown: A B Cc At 37° 0.89 per cent 0.87 per cent 0.60 per cent of lactic acid. “At 30° 1.00 per cent 0.96 per cent o.81 per cent of lactic acid. ‘At 18° 'r.08 per cent 1.06 per cent 0.88 per cent of lactic acid. At 6° 0.70 percent 0.73 per cent 0.62 per cent of lactic acid. These results are quite logical and perhaps can be explained by the recognized experience that all products of fermentation tend to check the process of fermentation, and that any chemical product or substance acts the more vigorously upon any life process the higher the temperature. The same amount of alcohol that will still allow a slow fermentation at-10° may check the fermentation entirely at 20°. Naturally the rate of fermentation in the beginning will be higher at the higher ‘temperature but the end-point is lower. The end-point of the lactic cultures A; B, and C-at 6° is probably not final, because 218 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 rapidly with the rising temperature and the end-point will also be lower. FREEZING.—The discussion of the relation of temperature to microdrganisms has so far considered only the temperatures within the limits of growth. However, the temperatures below the minimum and above the maximum are also of greatest importance. If bacteria are cooled below their minimum temperature they do not die immedi- ately. They remain alive in a dormant condition ready to multiply as soon as the temperature rises. Even the freezing of a liquid will not kill them immediately. Of course, they cannot multiply in ice, because they have no water, consequently no food, and they cannot thaw the ice to get their water and food for lack of body temperature -of their own. As long as liquids are frozen solid the bacteria in them will remain dormant much like dried organisms, and like them their ‘number will decrease very slowly. An example is given in the follow- ing table relevant to the number of bacteria in frozen milk (after Bischoff). The decrease in numbers is not very uniform, since there are 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... ....... 000s cece eevee eee 200,000 bacteria per c.c. After Tudaye coca doen space tae Be gRaG h 105,500 bacteria per c.c. ATER BGA YSi. 5.0 6.8 dvadedcleie Gia + auecee Bae we 72,300 bacteria per c.c. Aiter SidaySict2c6 weaneads saeleins booaas 62,000 bacteria per c.c. After qidaysinacaisossepecawaneserene x 46,400 bacteria per c.c. After 7 days... ins cams endenadena wees 44,000 bacteria per c.c. After 14 :dayse.cccvdae vs sega geen gees 40,500 bacteria per c.c. After 21 days........ pears heen peer Seas +++ 30,300 bacteria per c.c. After 35 Gayss.. uve goss. tees cu eeaee ee 22,500 bacteria per c.c. TEE 40 da YS 93552 wn seiccean See aire mene Sans ao 14,200 bacteria per c.c. The table shows plainly that it is impossible to sterilize milk by freezing, but as long as it is frozen it will keep; there in no possibility of any microdrganisms 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 to changes other than microbial or the material was not completely frozen as is probably the case with salted butter. INFLUENCE OF TEMPERATURE 219 After bacteria are once frozen, they do not seem to be affected by | any lower temperature. Macfadyen and Rowland found that they tolerate very low temperatures remarkably well. Many bacteria were not killed by a twenty hours’ exposure to the temperature of » liquid hydrogen (—252°). Yeasts are not quite so resistant and the mycelium of most molds is easily destroyed by freezing, while the spores are hardier. THERMAL DEATH-POINT.—Heating above the maximum tempera- ture is quite harmful to bacteria, and the amount of injury increases with the temperature. Recent experiments have shown that heat does not kill bacteria instantaneously, but that we have an orderly process as in the case of death by drying. This can be observed only in a very narrow range of temperature, however, since the death rate rises very rapidly with the increase of temperature. 10° increase may make _ the death rate ten to one hundred times as great, and death is almost instantaneous. For most practical purposes, it is sufficient to state the time and temperature neccessary to bring about complete sterili- zation. It has become customary to define, as the thermal death- - point, the lowest temperature at which a culture will be killed in ten: minutes. As most bacteriologists will use very nearly the same tech- nic, they will have fairly uniform numbers of cells to start with, and therefore obtain ‘fairly uniform results. The thermal death-point does not depend upon the species and ‘the temperature only. It varies with the age of the culture since older cells are less resistant than younger ones especially if heated in their own products. The medium in which the organisms are heated is also of great significance. The fact that acid liquids, as fruit juices, are more easily sterilized than neutral meat or vegetables is largely due to a chemical (poisonous) action of the acids upon the bacteria. But the greater resistance of tubercle bacteria in the sputum compared with those suspended in salt solution cannot be so readily accounted for. A necessary factor for the prompt destruction of organisms by heat is the presence of moisture. The resistance of dry organisms is remarkably higher than that of the same organisms in a liquid cul- ture. The following table shows the death-point of yeast cells and spores in a dry and moist state. 220 PHYSICAL INFLUENCES , THERMAL DEATH-POINT OF Dry AND Molst YEAST Cells Spores Variety of yeast : Moist Dry Moist Dry Pale ale yeast............ 65° 95°-105° 65°-70° I15°-125° Hofbrau yeast............ 55° 85°— 90° 65° II§°-120° Saccharomyces pasteurianus | 50°-55° 100°—-105° 60° ris” RESISTANCE OF SPORES.—The organisms most resistant to heat are the spores of certain bacteria. In the chapter on moisture require- ments attention has been called to the great resistance of spores to drying. We find the same exceptional resistance to high temperatures. Boiling heat will not kill spores readily. Some bacterial spores can stand the temperature of 100° for several hours. In order to kill spores in one heating the temperature must rise to about 110° for fifteen to thirty minutes; this can be accomplished only by heating under 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 materially 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 few spores of bacteria. After cooling, these spores will germinate under suitable conditions and the vegetative cells thus appearing instead of the resistant spores are easily killed in a second heating. A third heating is necessary in order to kill any vegetative cells which may have devel- oped from spores not yet germinated before the second heating. It is essential to have the time between two heatings long enough to allow the germination of spores, and not too long to permit formation of new spores. It is customary to heat on three successive days for fifteen minutes each time. In this case, sterilization is usually complete, while a forty-five minutes’ heating at once is not sufficient to guarantee sterilization. Among the substances that are very easily sterilized are cider and other fruit juices, while milk and soil are the most difficult materials to sterilize. INFLUENCE OF TEMPERATURE 221 Dry spores will resist still higher temperatures than moist spores. Some dry spores survive an. exposure to 140° or 150° for ten minutes. It requires a very high temperature to sterilize glass, cotton, gauze, and instruments with dry heat. A discontinuous sterilization of dry mate- rial is useless, since the spores will not germinate without moisture, therefore their resistance remains unaltered. The spores of molds are more resistant than the mycelium, but if moist, they all die at 100°. The dry mold spores can tolerate a some- what higher temperature, but not as high as the spores of many bacteria. Yeast spores and yeast cells are very much alike in their resistance to heat. The table on page 220 shows hardly any difference between their resistance. 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. Fic. 105.—These plates were heavily inoculated with B. coli and B. prodigiosus respectively and then were exposed, bottom side up, to the direct rays of the sun, for four hours. On the instant of exposure, a figure O cut from black paper was pasted to the plate shading the bacteria underneath. After one, two and three hours the corresponding figures were pasted to the plates. The above picture was taken 24 hours after exposure, proving that three or four hours of direct sunlight weaken and and may even kill bacteria. B. prodigiosus proved more sensitive than B. coll. (Original.) The influence of light varies with its intensity. Direct sunlight has a very harmful effect upon microdrganisms. Most bacteria are killed by direct sunlight in a few hours; the time depends upon the organism as well as upon the intensity of light; this again varies with 22:2 INFLUENCE OF LIGHT AND OTHER RAYS 223 the amount of moisture and dust in the atmosphere, with the time of the day and with the season; an absolute measure for the action of light cannot be fixed, therefore, as easily as with the action of heat in the ther- mal death-point. The different colors of the spectrum do not act alike; the part of the spectrum from red to green is practically without influence upon microdrganisms, while the blue light acts strongest and the intensity decreases in the violet and ultra-violet. In carrying on experiments with the influence of light, it must be remembered that glass absorbs ultra-violet rays, and further that the heating of the medium by direct radiation must be avoided (Fig. 105). Fic. 106.—Phototropsim of Rhizopus nigricans. The mold is grown on gelatin with diffused light coming from right side. (Original.) Yeasts, molds, and bacteria and probably Protozoa are equally sensi- tive to light. Even the spores of most bacteria do not show a greater resistance to light, while the mold spores are an exception. The col- ored spores of the Penicillium, Aspergillus and Mucor species can be exposed to light for a long time without being killed, but the colorless spores of Ozdiwm and Chalara show no increased resistance. It is sup- posed that the pigment in mold spores is a protection against light. This is not true with the pigment of bacteria. The colored and colorless strains of pigmented bacteria show no difference in their resistance to light. The only exceptions are the so-called purplebacteria. These peculiar 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 microérganisms 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 Fic. 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 microérganisms as direct sunlight. Long exposures to diffuse light will kill most bacteria, while molds are not at all sensitive. They rather like a very dim light, and many molds grown in a dark room with light only from one side will grow toward the light. This property, which is characteristic for all green plants, is called heliotropism or phototropism (Fig. 107). It has INFLUENCE OF LIGHT AND OTHER RAYS 225 been found that molds produce mycelium mostly in the dark, while in daylight sporangia are produced mainly. This difference in the devel- opment during the day and during the night accounts for the concentric tings which are quite commonly found in older mold colonies, and which indicate the age of the culture (Fig. 107). Similar rings are . occasionally found with yeast and bacterial colonies, and are possibly due to the same influence of light. X-RaAys.—Of other rays, the invisible X-rays and the radium rays have attracted the attention of bacteriologists and physiologists. It is known that the X-rays will destroy living tissue by long exposures; microérganisms cannot be considered less resistant. X-rays are used in the treatment of microbial diseases of the scalp and skin. RADIUM RAYS are not-so well known, and their bactericidal action is doubtful. The treatment of certain bacterial diseases has been attempted, but it has not been applied as generally as yet as the X-ray method. The sterilization of milk and possibly other foods by this method has been suggested, but the practical application is at present quite improbable because of the cost and the uncertainty of the results. \ 15 CHAPTER IV INFLUENCE OF ELECTRICITY The influence of elecrticity upon microérganisms is much less than one might perhaps expect, if the electriticy as such is considered. A direct electric current passing through a nutrient medium will, of course, cause electrolysis which is usually manifested by the formation of acid on the positive pole and of alkali on the negative pole. The acid and alkali will kill microérganisms, as is discussed in the chapter on chemical influences. In this case, it is not the electricity itself that destroys the bacteria. It is also possible to kill bacterial cultures by passing an alternating current through the medium for some time. No electrolysis takes place in this case, still it is not the direct action of the current that acts upon the organisms, but rather the heat produced by the current passing through a medium of high resistance. If the culture is cooled properly the influence of the current is insignificant if at all noticeable. Whenever electricity is applied against microérganisms the effect is con-’ sidered electrochemical. The electrical current is used in a very small way in the purification of sewage. The sewage passes between two iron plates which represent the two poles of a strong current. The electrical sterilization of milk has been patented. Wines are improved by electricity. The steriliza- tion of drinking water by ozone is also an application of electricity, though of course the ozone once formed by the current acts as a chem- ical compound independently of its source, and the same effect would be produced if the ozone were manufactured chemically. 226 CHAPTER V INFLUENCE OF MECHANICAL EFFECTS PRESSURE.—The resistance of microérganisms to mechanical pres- sures is very great. Pressures of 3,000 atmospheres* will not kill the majority of bacteria in four hours. They are, however, weakened and some species will die. A specific difference between the molds, yeasts, and bacteria in this particular does not seem to exist. Of the organisms exposed to 2,000 atmospheres for ninety-six hours, Bact. anthracis, Bact. pseudodiphtheria, M. pyogenes var. aureus, Oidium lactis and Saccharo- myces cerevisie survived, while seven other organisms lost the power of multiplication. Some of these were not dead, however, since they retained their motility for several days. It is noteworthy that high pressure will destroy one quality (multiplication) and not effect another (motility). Pigment-production and virulence of pathogenic bacteria were either diminished or lost completely. The resistance against high pressure is necessary for the organisms which cause the decay of organic matter at the bottom of the oceans. Vertebrates breathe oxygen in the form of gas or have at least an organ filled with gas (fish bladder); the volume of gas is changed considerably by slight changes of pressure; this will affect organisms depending on gas. Microérgan- isms do not require gas as such. They can absorb gases only in solution. A change of pressure therefore will not cause a change of volume, since liquids have a very small coefficient of compression. The situation is entirely different if the liquid is not exposed to the pressure directly, but to compressed air. In this case, the chemical effect of the gas is the deciding agent. The higher the pressure, the more gas will be dissolved in the culture medium. The fatal pressure under these conditions will vary as much as the fatal dose of an antisep- tic; it depends upon the chemical qualities of the gas, upon the pressure (concentration), upon the temperature, and upon the organism. *One atmosphere is 1 kg. pressure per square centimeter (or about 15 pounds per square inch). 227 228 PHYSICAL INFLUENCES Some data have been given already in the chapter on oxygen require- ments. It was mentioned in that connection that Bact. butyricum can- not tolerate more than 0.65 per cent of the total oxygen content in air (o.2atmosphere); in other words, an oxygen pressure higher thano.oo13 atmosphere will kill the organism. The maximum pressure for B. prodigiosus was found to be about 5.4 to 6.3 atmospheres. Very few experiments have been made with other gases. Carbon dioxide at a pressure of 50 atmospheres retards the growth of bacteria in water and will sterilize it in twenty-four hours. Suspensions of pure cultures of B. typhosus and Msp. comma are killed by 50 atmospheres carbon dioxide pressure in three hours. Milk cannot be sterilized by his pressure but bacteria do not multiply. Carbonated milk has been recommended as a refreshing drink by several investigators. The ordinary market milk will keep about two days longer under the pressure of 10 atmospheres (150 pounds) than without pressure. If pasteurized it is said to keep for a week. Graviry.—Gravity would have a great influence upon the growth of microdrganisms in liquids if their specific gravity were much greater than that of water. This does not seem 'to be the case however. It has been estimated by accurate weighing to vary between 1.038 and 1.065. Very much higher results (1.3 to 1.5) have been obtained by centrifuging bacteria in salt solutions of varying specific gravity, but these data are 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 the culture medium, it is plainly seen that gravity has’ but little influence. The microdrganisms will live suspended in the liquid and sediment out: very slowly. The slightest current in the liquid will carry them around and distribute them through the medium. The motility is of minor importance; the actual distance covered by motile bacteria has been measured, and under the most careful exclusion of currents in the liquid has been found to be about a millimeter in a minute for B. subtilis. This is very slow compared with the speed of the circulating water moved by changes of temperature or other incidental agents. Yeast cells and other gas producers use the carbon dioxide as a ve- hicle. The gas bubbling up in the fermenting liquid keeps it constantly in motion and moves the yeast cells against gravity toward the surface where the gas escapes and lets the cells fall back to the bottom. The production of scums and pellicles on the surface by organisms INFLUENCE OF MECHANICAL EFFECTS 229 which are heavier than the liquid they float on, is often accomplished by small gas bubbles between the cells (M-ycoderme). In other instances, it may be just the floating of cells having oily surfaces. The growth is influenced by gravity very little. The sporangia of molds are the only exceptions, growing decidedly away from the center of gravity (negative geotropism). Aciration.—For the majority of microérganisms, the quiet, undis- turbed growth of the laboratory culture is the normal or the ideal one. Such cultures, if shaken for a considerable time, show a decrease of liv- ing organisms, and it is possible to sterilize cultures by continued shak- ing. The effect is not a simple mechanical breaking or tearing of the cells. The bacteria break up into the finest particles. This is also the case if cultures are exposed for several days to the trembling motion caused by the working of very heavy machines. There is no grinding or tearing effect but the cells break to pieces just the same. A slight and slow agitation seems to be advantageous for many cul- tures, only continuous heavy motion proves harmful. Different organ- isms show wide variations in their resistance to agitation. DIVISION II CHEMICAL INFLUENCES CHAPTER I STIMULATION OF GROWTH The influence of chemical substances upon microérganisms 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- tion can be stimulated by metallic salts. It is be- lieved by some physiologists that, as a law of nature, Fic. 108.—Chem- every substance that is injurious in a certain con- otaxis. (After centration is a stimulant in a lower concentration. Fischer.) ic 5 ‘ A similar action of certain chemical compounds upon enzymes has been noticed, retarding in high concentrations, stimulating in weaker solution. CHEMOTROPISM AND CHEMOTAXIS.—Microérganisms 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 microérganisms may be accounted for largely by chemical or osmotic 230 STIMULATION OF GROWTH 231 forces. In a solid medium the hyphe of molds will grow toward the best source of food supply. This growth on account of chemical stimulation is called chemotropism, analogous to the phototropism or growth toward light. If some injurious compound is offered, the hyphe will grow away from it. Thus we have to distinguish between positive and negative chemotropism. The motile organisms, bacteria as well as protozoa, demonstrate their preference for certain food compounds by swimming toward them. This is called chemotaxis (Fig. 108). Here also a positive and negative chemotaxis must be distinguished, the latter taking place if injurious substances are present. CHAPTER II INHIBITION OF GROWTH Poisons, GERMICIDES, DISINFECTANTS, ANTISEPTICS, PRESERVA-, TIVES.—A great number of inorganic and organic bodies will destroy life in comparatively weak solutions. These substances are called poisons if they are considered in their effect upon man and animals. In their application to microdrganisms they are generally called germicides (germ-killers), or disinfectants if the emphasis is laid upon the prevention of infection rather than upon the actual killing of the microdrganisms. Analogous to the general term germicides, the terms bactericide and fungicide are used occasionally. The term antiseptic means a prevention of sepsis which may be accomplished by checking the growth without necessarily killing all microérganisms. The meaning of the word pre- servative is practically the same, only the latter is used more commonly in relation to foods, feeding stuffs and preparations of similar origin while the word antiseptic is largely used in relation to microbial diseases. A strict line cannot be drawn between any of these definitions. A dis- infectant, if diluted, becomes an antiseptic. A strong salt solution is an antiseptic for some organisms and a disinfectant for others. Of the above expressions, germicideis the most definite, but is not so commonly used as the others. Mope or Action.—The action of a poison upon the cell is generally considered an action upon the protoplasm. The poison is supposed to combine chemically with the cell plasma producing compounds which interfere with the continuation of the life processes and thus cause death. If the cell has been subjected to the action of the poison only a short time, it can be saved by removing the poison. Bacteria can be treated with mercuric chloride (HgCl.) so that they will no longer de- 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 239 INHIBITION OF GROWTH 233 heat or drying. The number of cells dying in a given time interval is proportional to the number of cells surviving. In the last five years, this has been tested and found true with practically all disinfectants. Fig. 109 shows the curves plotted from data obtained with Bact. anthracis, the full-drawn line representing the number of live spores in .21 per 4000 3500) 3000) 250 Cells per c.c. 2000 1500 . #9000 x ‘ s x oN 500 2 7 a». e Pn --p- - 10 20 30 4Q 50 60 70 $O min. Fic. 109.—Curve of disinfection. Spores of Bact. anthracis in mercuric chloride solution. (After Chick.) : cent of mercuric bichloride, the dotted line the same in .11 per cent solution. The (apparent) resistance of the few remaining cells is of great im- portance in those applications of disinfection where a thorough kill- ing of all bacteria is intended, ¢.g., in the treatment of drinking water. Our 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 microérganisms. 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 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 diff- cult to poison bacteria in blood, pus, or similar material. The sensi- bility of the microdrganisms in pure water is remarkable. Very small doses which would not be considered efficient under any other condition, will destroy microérganisms 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 Recent investigations have shown the rather unexpected fact that the efficiency of a poison is not proportional to its concentration. If a certain poisonous solution is diluted with an equal volume of water, we might expect it to be half as poisonous as before, but depending upon the chemical nature of the poison, it may be more poisonous than expected, or considerably less. It follows from this that two dif- ferent poisons of the same intensity, if diluted in the same proportion, may not have the same intensity any more. Microérganisms will gradually become accustomed to certain poi-, sons, and become more resistant. This principle has been utilized in the manufacture of distilled alcohol; yeasts have been cultivated which can tolerate a high concentration of acid; the acitl serves to suppress bacteria producing undesirable fermentations. The age of the culture and the stage of development will naturally change the resistance of a species materially. The old cultures which are past the culmination of growth will be much more sensitive to any poison unless a spore-producing organism is under test. In this case, we find a greatly increased resistance, similar to the increased resistance of spores against drying and heat. THE CLASSIFICATION OF DISINFECTANTS is very difficult as long as we cannot explain completely the process of poisoning. It is im- possible to arrange them according to the intensity of action, because the intensity of influence depends not only upon the disinfectant, but also upon the species of organisms. Some yeasts can resist ten times as much alcohol as certain, bacteria. Formaldehyde is not nearly as strong an agent with molds as it is with bacteria. The dis- infectant concentration of a poisonous substance is not absolute. The simplest method of grouping is by chemical structure and qualities. Of the following natural groups can be distinguished acids (inorganic and organic), metallic salts, hydrocarbons (aliphatic and cyclic), alcohols (aliphatic and cyclic), aldehydes, anesthetics, essential oils, oxidizing agents and reducing agents. The first three groups, acids, alkalies and salts, are distinguished from the rest as electrolytes; the strength of acids and alkalies (chemic- ally speaking) is measured by the degree of electrolytic dissociation. The disinfectant value follows largely the same law. The strongest acids in the chemical sense are also the strongest disinfectants. There are exceptions, however, where, besides the poisonous effect due to 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 (HgCle) and decrease also its disinfectant power. Mer- curic chloride dissolved in absolute alcohol is not dissociated. In 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 réle 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 preference for acid media. The acid in the stomach of man and animals plays an important réle as a sterilizing agent for the food. Many microérgan- isms are killed in the stomach. In the household, the natural. acidity of fruit helps in keeping canned fruit, preserves and jellies. Especially in heating, the acid together with the high temperature has a very strong germicidal effect. Vinegar is often used to pre- serve fruit and vegetables; in some parts of the country, meat is kept in buttermilk. Benzoic and salicylic acids are often used in the pres- ervation of fruit and vegetables. Their poisonous influence is not so much due to the acid reaction but to the specific chemical character of these compounds. Of the alkalies, only one is used extensively, namely, lime; quick- lime (CaO) is considered a valuable disinfectant for excreta in privy vaults; it is universally applied as a whitewash in stables, barns, poultry houses and similar buildings. Quite commonly, it is used as ‘milk of lime” (one part of slaked lime with four parts of water). It should be kept in mind that the calcium oxide unites with the carbon dioxide of the air and thus gradually loses its disinfecting power. Of the metallic salts, many are well-known germicides. The most powerful disinfectant is mercuric chloride (HgCle) which is one of the standard disinfectants. It is generally used in a dilution 1: 1000 which is sufficient to kill all vegetative cells as well as spores in a few minutes. Quite commonly, hydrochloric acid or salt is added, to- prevent coagulation or precipitation of slimy or albuminous matter which would protect the enclosed bacteria from immediate contact with the poison. The addition of hydrochloric acid or any chloride INHIBITION OF GROWTH : 237 decreases somewhat the disinfectant value for bacteria suspended in distilled water because it decreases the electrolytic dissociation. Another disinfectant of remarkable strength is silver nitrate; it is not used commonly because of its high price. It also decomposes easily and leaves dark spots on the skin and clothes. Of the other metallic salts, copper and iron sulphate are not used extensively, though recommended for the disinfection of feces. Zinc sulphate may be applied to mucous membrane the same as silver nitrate. Many other salts may be used occasionally for disinfecting purposes, though the expense or undesirable qualities prevent their common. application. The alcohols are well known for their poisonous effects, but the value of ethyl alcohol as a disinfectant is usually overestimated. It takes quite strong alcoholic solutions, more than 20 per cent, to kill certain yeasts and the spores of some bacteria in less than a day, and a complete sterilization by alcohol in a few minutes cannot al- ways be guaranteed even with 50 to 60 per cent solution. It has already been mentioned that desiccated organisms are very resistant to concentrated alcohol, more so than to a so per cent mixture. Methyl] alcohol is weaker, the higher alcohols, especially amyl alcohol, are stronger disinfectants than ethyl alcohol. They all give good results in. the presence of water while the absolute alcohols have scarcely any effect upon desiccated bacteria. None of these alcohols in whatever concentration they may be used, can be relied upon to kill bacterial spores. Stronger germicidal effects can be obtained by the alcohols of the’ benzol group, of which phenol or so-called carbolic acid (CsH;OH) is the simplest representative. Phenol, like ethyl alcohol, is not as effective as is commonly believed. It is applied in solutions from .5 per cent to.5 per cent ordinarily, but it usually takes.a long time even for the 5 per cent solution to kill vegetative cells as Bact. tuberculosis or B. coli; it is inefficient against anthrax spores. More powerful are the higher cyclic alcohols, of which the cresols are examples. They are used extensively as disinfectants and antiseptics. They are, together with phenol, coal-tar constituents and are sold commercially under many different names, either pure or mixed with soap or other disinfectants which make them emulsify readily in water. The cresols are almost insoluble in water, and not as effective in solutions as they are in 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 anesthetics, 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 of 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 microdrganisms 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 and mustard is very small. The most important disinfectant has not been mentioned, because it does not belong to any of the above groups. This is formaldehyde. Formaldehyde (HCOH) is a gas, soluble in water to the amount of 4o per cent at room temperature; it does not attack metal, clothing, wood- work, and is, therefore, preferable to many other disinfectants for steril- izing rooms. It kills spores of bacteria in a short time in a 1 : 1000 di- lution. Its greatest importance lies, however, in its’ gaseous nature, because it can be applied to rooms and buildings by simply evaporating it. The saturated 4o per cent solution can be evaporated directly or by generating steam which passes through the formaldehyde solution; this latter method has the advantage of saturating the air with moisture, which increases the power of the formaldehyde gas. Formaldehyde can also be obtained in a dry form; it polymerizes to a white crystalline substance, paraformaldehyde ((HCOH);) which can be changed back to 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 quite inoffensive in its solid form, while the formaldehyde solution has a very penetrating odor and is exceedingly harmful to the mucous mem- brane of the respiratory organs. Of the oxidizing agents, oxygen itself has already been mentioned. Though it is able to destroy certain anaerobic bacteria, it cannot be called adisinfectant. For this purpose, oxygen must be activated; such oxygen can be obtained in the form of ozone (Oz). 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 (H2O:2) resembles ozone in its chemical reactions; it changes readily to H,O + O, and this oxygen atom in the nascent state is quite effective as an oxidizing agent. For an antiseptic, it must be used in at least a 1 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 hypochlorite 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 sewage; 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 (SO2) was for a long time a standard disinfectant and is still used occasionally for fumigating rooms, stables, barns and out-buildings though it is substituted more and more by formaldehyde which can be applied almost as easily. The burning of sulphur is an extremely simple process, but it requires a moist air to disinfect properly, and under these circumstances it will attack metal, dyes 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 microdrganisms 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 be mentioned ethoxyl, given against the protozodn 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 microérganisms are of the greatest im- portance in nature. Pure cultures in nature are very rare and of excep- tional occurrence; they are hardly ever found except in certain diseases of man, animals and plants. Generally, nature works with mixed cul- tures. All natural fermentations, decompositions and putrefractions are accomplished by a number of different species among which perhaps one dominates, but is influenced by the rest. The study of the mutual relations of microdérganisms is in the very first stage as yet; practically: all laboratory work is done with purecultures. The experiences obtained with pure cultures are not sufficient to explain all microbial activity in nature. There are many. possibilities of mutual influence between different organisms. Generally three main cases are distinguished: symbiosis, where two organisms profit by the combination; metabiosis, where one profits by the other’s action without benefiting the other in return, and antibiosis, where one organism injures the other. These cases cannot be separated strictly. The relations are not always constant through the entire development of the cultures; an originally beneficial influence may change to an injurious one in a few days. Many terms have been coined to designate all these various possibilities, but in order to avoid this multiplicity of more or less indefinite names for the various relations, the general term ‘‘association”’ has come into use, especially when the relationship is not well understood. _ SYMBIOSIS. Symbiosis is not very common among microdrganisms, and it is difficult to find examples where true symbiosis exists through the entire 16 241 1 242 ‘ # MUTUAL INFLUENCES development of both organisms. The association of lactic bacteria and Oidium lactis in milk is, for a certain period at least, a symbiosis. The bacterium will produce only a certain amount of acid, and then it can grow no more because the acid is too strong; the mold will destroy the acid and thus gives the bacterium a chance for continued activity. The bacterium produces the acid which the mold likes; the mold in turn removes the excess acid which otherwise would check the bacterial activity. True symbiosis is more common in the relation of microérganisms 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 from the air which the plant cannot assimilate directly. The typical exam- ple in the animal kingdom is B. coli in the intestine of animals, being nourished by the food of the animal and rendering the food more easily digestible. METABIOSIS Metabiosis may be considered a one-sided symbiosis; two organisms live together, but only one is benefited, the other remains uninfluenced or later may be injured by the association; the latter case is the most common. In this relation, one usually prepares the food for the other. It has previously been mentioned that the metabolic products of one species serve as food for another species, thus breaking up the various organic compounds step by step to smaller and simpler molecules. Quite commonly, each step is accomplished by a differént species of microdrganism. Consequently, metabiosis is a very common occurrence among microérganisms. 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 the MICROBIAL ASSOCIATIONS 243 association of lactic bacteria with certain protein destroying organisms. The lactic bacteria’ often develop much better if the protein-bacteria grow together with them or have grown previously in milk. Meta- biosis does not require the growth of the two associated organisms at the same time. The effect will be the same if first the one and later the . other develops, and even after the first organism is killed or removed, its effect upon the pure culture of the second will still be noticed. This does not occur in the case of symbiosis. . One species can favor the development of another by other means than food provision or preparation. Certain bacteria cannot live in acid media, and molds or 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- isms. 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, Bact. lactis acidi combats all other organisms by a rapid production of lactic acid. Thoughitis 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 Bact. 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 isunknown. Bact. 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 prodticts 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. colt 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 Il APPLIED MICROBIOLOGY DIVISION I* MICROBIOLOGY OF AIR CHAPTER I THE MICROORGANISMS OF THE AIR AND THEIR DISTRI- BUTION The atmosphere is not the normal habitat of bacteria, for growth and mulUphication cannot take place in at under ordinary conditions. The ‘phrase “microérganisms of the air” is therefore somewhat ambiguous. The small size of microdrganisms enables them to remain suspended for considerable periods when physical forces have separated them from the substrata on which they have developed. MIcROORGANISMS PRESENT IN THE Arr.—Molds, bacteria, and yeasts are all found in the air under certain conditions. The first two are usu- ally relatively abundant, the latter are less common. The common molds have adapted themselves for the most part to wind distribution. They bear spores that are smalli in size and possess a surface that is not readily moistened. These ‘spores are resistant to desiccation and light and remain viable for a considerable tine even under unfavorable conditions. Furthermore, the fruiting bodies of many, though not all molds, show a distinct negative hydrotropism, i.e., the mycelium remains in contact with the moist substratum while the threads which bear the spores rise at right angles toit. These latter are so sensitive that they can detect slight differences in the moisture con- ~ tent 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 Dayoeloetally are as well adapted for air distribution as are the molds, OcCURRENCE IN THE ArrR.—Microérganisms 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. Microérganisms 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 surface of fermenting liquids.on which bubbles are.bursting. When the drop is small enough, the air currents keep it in suspension and the water soon evaporates and frees the organism. This brings about the condition first discussed, free bacteria in the air. The decredse in weight and size incident to this loss of water probably accounts for the fact that the so-called “infectious droplets” are sometimes carried for considerable distances. How Microércanisms ENTER THE Arr.—In comparatively few in- stances do microérganisms possess mechanical devices for projecting the spores or other cells into the air for wind distribution. Usually the organism is passive and is freed only by air currents or by mechanical agitation. Some molds, as has been stated, release their spores even in the presence of moisture, so that complete desiccation is unnecessary for their dispersal. Bacteria and yeasts, on the other hand, are not usually given off from moist surfaces. Only when dry and pulverized can the bacterial medium be readily blown about. Hansen found that in the immediate vicinity of a heap of decaying malt, the air was comparatively free from bacteria. Winslow has shown that sewer air is frequently_ practically free from bacteria although the surface with which it comes * in contact teems with bacterial life. Mechanical agitation often throws large numbers of organisms into the air. Moving hay and straw, MICROORGANISMS OF THE AIR 249 grooming animals, sweeping a floor or carpet will multiply the dust and bacterial content of the air many times. In a similar-manner, tiny, germ-holding droplets may be scattered by the splashing of sewage or of fermenting or putrefying liquids, and in speaking, sneezing or coughing. . CONDITIONS FOR SUBSIDENCE OF BactERIA.—The length of time during which an organism may remain suspended in the air is dependent upon several factors. Small particles settle out more slowly than large for the reason that as the size of an object is decreased, the surface area decreases less rapidly proportionately than the volume. The lifting effect of air currents depends upon the ratio of surface area to volume and specific gravity. The smaller the object, therefore, the greater is the resistance to subsidence. Consequently, bacteria usually settle out of air. very slowly if free in a quiet atmosphere. The time of sus- pension is determined also by the velocity of the air currents. While considerable velocity may be necessary to dislodge microérganisms and bring them into suspension, a very slight air current will sustain them. Winslow has ‘found that a current of 17 inches per minute is sufficient to sustain B. prodigiosus. The relative humidity of the air is also an important factor. In a supersaturated air solid particles, such as bacteria, become foci of condensation for water and quickly settle out. When dust is present in considerable quantities, and certain elec- trical or moisture conditions exist, flocculation occurs and the larger bodies so formed subside rapidly. The character and abundance of - surfaces with which the suspended particles may come in contact also . play an important part. . Moist surfaces are much more effective in retaining. particles than those which are dry. . DETERMINATION OF THE NUMBER OF BACTERIA IN THE Arr.—The number of bacteria in the air is frequently determined by exposing open petri dishes of gelatin or agar in different places for definite periods. This is a comparative quantitative method only. The number of colo- nies developing upon these plates will give the number of dust particles having living spores or cells upon them that fall in the given area ynder the conditions of the experiment. Evidently this is of value only for rough comparative work as constantly shifting currents of air usually introduce great errors. A somewhat more accurate method is to draw measured volumes of air into a flask, the bottom of which is covered with a layer of gelatin or agar. The colonies which develop represent the number of organisms which settle out from the given volume. More 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 isso little ordinarily to be learned by a study of the air flora that a comparison of plates exposed directly will usually suffice. Where more accurate results are desired, one must resort to one of the filtration methods discussed above. Qualitative determinations of the species of air organisms are not often made. When necessary it may be done by simple examination of the colonies developed on the plates or by animal inoculations made VYAA from the water used in the air filter. It is sometimes necessary to vary ~~ the composition of the medium used in order to favor the development of certain types of organisms desired, for example, a higher precentage of molds will be found and a more luxuriant development will take place if wort agar or acid gelatin is used. NuMBER OF BACTERIA IN THE AIR.—The number of bacteria in the air is determined by a variety of conditions. The velocity of air cur- rents and the nature of the surface with which these currents will come into contact, are probably most important. Bacteria are usually more abundant on quiet days in the air of buildings than out of doors, but on windy days the reverse is true. They are often more abundant in cities than in the country. Fewer are found at high altitudes and over large bodies of water. Frankland found that there are fewer in winter than in summer. They are washed from the air during rains. Bright sun- light destroys many. The nature of the soil and the vegetation cover- ing it has a marked influence. The following figures from various . MICROORGANISMS OF THE AIR 251 authors are appended to serve as an index to what may be expected in the air content of bacteria. Locality ype le Observer Outdoor air, Boston................ 100-150 bacteria. Sedgwick and Tucker. 50- 75 molds. Open alti segeue ves vaaeepneen ss 100-150 bacteria. | Fischer. Open field... cssnig cceenia de nmames es 250 Uffelman. Seacoast: ..,cauredveavcgasaanes des 100 Uffelman. Mountain altitude, 200 meters...... ° Pasteur. Mont Blanc..................00005 4-11 Ellis. Spitzbergen (Arctic Regions)........ ° Levin. Middle of Paris.................... 4,000 Ellis. Paris Streets scciuigee 4 o:sdons Stereo’ 3,500 Fischer. Tailor’s Room in Whitechapel....... 17,000 Ellis. Boot Workshop................... _ 25,000 Ellis. SPECIES OF ORGANISMS IN THE ArR.—Penicillium is the most com: mon mold isolated from the air. Next in importance are Mucor, Rhizopus, and Aspergillus in the order given. In addition to these a considerable number of species of hyphomycetous molds are occasion- ally found. Torule, but not true yeasts, are usually common. Bac- teria are either spore-bearing soil bacilli or cocci. Of the former, B. sub- tilis, B. mycoides, and related forms are ubiquitous. Sarcina lutea and Sarcina aurantiaca and certain other chromogenic cocci are to be found in almost every plate exposed. Since the air does not have a true flora, the species as well as the number of bacteria present must depend en- tirely upon the character of the environment. CHAPTER II MICROBIAL AIR INFLUENCE INF ERMENTATION, DISEASES, ETC. Arr AS A CARRIER OF ConTAcIon.—There are many popular mis- conceptions of the influence of air upon health. Experience early taught that exposure to the night air in certain localities or to swamp air during certain seasons was generally followed by disease. Natur- ally, the air itself was held responsible. We know now that certain fevers, malaria, etc., are caused in every instance by infection with specific microdrganisms and that these organisms are not usually. car- ried by the air but by insects, such as the mosquito, in water and food. Nor can the emanations from decaying organic matter or sewer gas itself be held to produce disease directly. Before the establishment of the germ theory of disease, leading sanitarians held that sickness was induced by the gases from the decaying organic matter, by the effluvia from cesspools and by sewer gas. However important the places named may be in harboring disease microdrganisms, we have learned that the air itself rarely acts as a carrier. Sewer gas has been shown to be un- usually free from bacteria. Hazen says, “‘After many years of exper- ience and long-continued investigation, there is not the slightest reason to believe that infectious diseases are carried by the air of sewers.” Undoubtedly the air does play some part in the carrying of disease germs. In certain diseases, as the exanthemata (smallpox, measles, etc.), the infecting agent may be present on the dry skin and may be blown about and inhaled. This means, however, is not established. In certain nasal, tracheal, and pulmonary infections, the organisms may be spread through speaking, sneezing, and coughing, for the infec- tious droplets, as has been seen, remain suspended for a time in the air. Pyogenic cocci are present in the mouth and care must be used in surgical operations that the mouth is so protected that none of these organisms’ gain entrance to wounds. Rarely, if ever, are intestinal infections, as typhoid or cholera, spread through the air. We may there- 252 MICROBIAL AIR INFLUENCE IN FERMENTATION, DISEASES, ETC. 253 fore conclude that air is of secondary importance asa carrier of infection. It may be of importance in a crowded workroom, but even under these conditions it is probable that transmission of infection comes about more frequently through actual contact or through food and drink. ORGANISMS OF THE AIR AND FERMENTATIONS.—A uniform inocula- tion with soil bacteria such as produce the nodules on the roots of leg- umes is obtained over considerable areas through the action of the wind in blowing dust particles. The bacterial flora of milk is to some extent dependent upon air currents as is also the development of the molds mecessary to the proper ripening of cheese, such as the Camembert! Acetic, butyric, and other organisms are likewise distributed in this manner. The organisms responsible for putrefaction and decay, the molding and spoiling of foods are wind-borne. Freemnc Air FRoM BAcTERIA.—Air is most commonly freed from bacteria by sedimentation, for this is the ultimate fate of most dust par- ticles. We have seen that they gradually subside in a quiet atmos- phere. When large quantities of pure air are required, dust and bac- teria may be removed by passage through a spray of water or through various types of filters, such as cotton, glass, wool, etc. A familiar example of this type of filtration is the laboratory use of cotton plugs in test-tubes. It is sometimes necessary to resort to fumigation to destroy the organisms of the air when an undesirable species is present. DIVISION 1 MICROBIOLOGY OF WATER AND SEWAGE CHAPTER I* MICROORGANISMS IN WATERT Water is necessary in the life of man. Besides its.use as a beverage, for cooking, and all domestic purposes, it is largely used in many manu- facturing industries; therefore, the study of its chemical and biological content is one of the most important features of modern hygiene. All natural waters contain microdrganisms, which gain entrance from many sources. Under the influence of the sun, sea water evaporates and forms a water vapor, which we call clouds; and these, driven by the wind over the land, are precipitated as rain and in the form of snow or hail. Most of this water collects from vast areas into brooks, creeks, rivers, lakes, or in subterranean streams, and finally reaches the sea whence it came. The water vapor arising from the sea or land contains no organisms; but as soon as the vapor is precipitated microdrganisms find their way intoit. These come from the air and from the soil. Some of them find in water sufficient nutriment for their life and growth; and, because of their constant presence and evident ability to thrive in water, they are sometimes spoken of as belonging to the “‘water flora.” Others, such as *Prepared by F. C. Harrison. + For specific details regarding methods of analysis and a fuller presentation of the subject, readers may consult any of the following excellent books: 1. Savage, W. G.: The Bacteriological Examination of Water Supplies, London, H. K. Lewis, 1906. z. Horrocks, W. H.: An Introduction to the Bacteriological Examination of Water, London, J. and H. Churchill, roor. 3. Prescott and Winslow: Elements of Water Bacteriology, 2d Ed., New York, Wiley & Sons, 1913. \ 254 MICROORGANISMS IN WATER 255 the soil bacteria, are found only at certain seasons, as after rain or dur- ing flood-time, and flourish only for a time; while some few, such as intestinal organisms that find their way into water, survive for only a short period. CLASSES OF BACTERIA FOUND IN WATER The bacteria found in water are here roughly divided into: (a) natu- ral water bacteria; (8) soil bacteria from surface washings; (c) intes- tinal bacteria, usually of sewage origin. But there is no strict divid- ing line’ between these three groups; for some organisms belonging to the water flora are found in the soil, and vice versa. Water draining from manured land frequently contains intestinal organisms. The division, however, is sufficient for all practical puropses. NaturaL WATER BacreriA.—The natural water bacteria are gen- erally regarded as harmless to man. These organisms are frequently numerous in river, lake, and all surface waters; certain species predomi- nate at one season, and disappear at another. Some of the best known are mentioned below. Several investigators have grouped the bacteria found in water into classes according to their biochemical properties. Where groups are subsequently referred to, the classification is that used by Jordan and followed by many other workers. B. fluorescens liquefaciens, Group V, together with some closely allied varieteis, is probably more frequently found in water than any other form, and is easily recognized by the green fluorescence and liquéfaction it produces in gelatin. B. fluorescens non-liquefaciens, Group VI, as the name implies does not liquefy gelatin, but produces characteristic colonies with a fluores- cent shimmer, is often very abundant in river waters, and is representa- tive of a group comprising B. f. longus, B. f. tenuis. B. f. aureus, and B. f. crassus. Certain organisms which liquefy gelatin and acidify milk—classed by Jordan in his Group VIII—are quite common at certain seasons. Some of these are soil organisms and are closely related to the proteus group; and some of them are B. liquefaciens, B. punctatus, B. circulans. Chromogenic bacilli and cocci (Groups XIII, and XIV) are often present in water. Of those producing red coloring matter, the well- known B. prodigiosus is the type of the group; others are B. ruber, B. \ 256 MICROBIOLOGY OF WATER AND SEWAGE indicus, B. rubescens and B. rubefaciens. Several yellow and orange organisms are commonly found, such as B. aquatilis, B. ochraceus, B. aurantiacus, B. fulvus, etc.. At certain times, 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. coeruleus. . 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. Sot BACTERIA FROM SURFACE WASHINGS.—During times of flood, high water, and after rains, numerous soil organisms are found in natural waters; and occasionally certain species persist for a consider- able time. Among the commonest species is B. mycoides, with its characteristic rhizoid colony; also B. subtilis, B. mengatherium, and B. mesentericus vulgatus, with its allied varieties; likewise B. m. fuscus and B. m. ruber— all belonging to Jordan’s Group VII, and having many characters in common, such as characteristic ‘colonies, followed by liquefaction when growing in gelatin, production of spores, etc. Cladothrix dichotoma, one of the thread bacteria, easily recognized on gelatin plates by the brown halo that surrounds the colony, is often found in fresh and stagnant water, and in most soils. It seems to flourish wherever there is much ‘organic matter. These are the soil organisms most often found when beef peptone gelatin is used for isolating purposes; but if other media are used, a different flora appears, and we find nitrifying organisms, yellow chromogens, etc. INTESTINAL BACTERIA, USuALLY oF SEWAGE OrIGIN.—Proteus Group.—There are several groups of sewage organisms found in impure water; some of these are very abundant in crude sewage, but are not found in such relatively large numbers in contaminated water. Jor- dan’s Group III contains the organisms belonging to the large proteus group, the principal species being B. vulgaris, B. zenkeri, B. mirabilis, B. zopfii, the sewage proteus of Houston, and B. cloace._ All these are frequently found in impure water, and in sewage. In the latter Hous- MICROORGANISMS IN WATER 257 ton has found as many as 100,000 per c.c. All these organisms are mo- tile, liquefy gelatin, and produce gas in dextrose and saccharose broth, and little or none in lactose; reduce nitrates, curdle milk, produce indol, and give a fecal, disagreeable odor in broth or other media. Sewage streptococci.—The streptococci found in sewage are probably similar to those found elsewhere; but their appearance in contaminated water may be regarded as indicative of recent sewage contamination, because the bulk of the evidence available seems to show that they are delicate organisms, which rapidly die outside of the body. While it is easy to ascertain their presence in polluted water, it is almost impossible to enumerate them; and they do not furnish such good evidence of sew- age pollution as the colon bacillus. They may be said to furnish valu- able confirmatory evidence of sewage contamination. B. enteritidis sporogenes.—This resistant, spore-bearing organism is usually present in the intestinal tract of man; is found in sewage, milk, and dust; and occurs in foodstuffs, such as wheat, oatmeal, rice, etc. On account of its ubiquity and the resistance of its spores, it cannot be considered a good indicator of excretal pollution. B. coli.—The presence of this organism in potable water is gener- ally accepted as the best bacterial indicator of sewage pollution. It must be remembered, however, that there are many varieties of this organism, to which certain investigators have given specific names, even when the differences from the type organism have been very slight. It may be well to mention some of these, to avoid confusion in the mind of the reader. The true colon bacillus, B. coli, or B. coli communis, or B. coli communis verus, is a short bacillus with rounded ends, motile, forms no spores and is Gram negative, does not liquefy gelatin, produces acidity and coagulation in litmus milk, gives rise to acid and gas in glucose and lactose media, causes canary-yellow fluorescence in neutral red media, and produces indol when grown in peptone water. The term “Excretal B. coli”’ has been suggested as a convenient designation of an organism which possesses the above characteristics. A saccharose fermenting variety of B. coli has been named B. com- munior; and we have a whole series of organisms which differ more or ess in various biochemical reactions, or lack some of their positive reactions. To some of these the name “para-colon”’ has been given; and the name “‘para typhoid” has been applied to those which more closely approxi- mate to the cultural peculiarities of the typhoid bacillus. 17 258 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 in water, it is well understood at the present time that the purification of the water supply of a town.or city produces a marked decrease in the number of cases and in the mortality from typhoid fever, as the following table shows: (See also Fig. r1o.) DEATHS FROM TYPHOID FEVER PER 100,000 PER YEAR 3 . . Five years < Percentage Purification Date of Five years Place “ by change ee after change | ees an Hamburg............ Filtration | 1892-3 47 7 85 Ziitich ss <2 2ssiisaxe4 Filtration | 1885 76 Io 87 Lawrence, Mass...... Filtration | 1893 121 26 79 Albany, N. Y........ Filtration | 1899 104 28 73 Not only has such a marked improvement followed the purification of public water supplies in the case of typhoid féver, but it has been. shown by statistics that ‘where one death from typhoid fever has been avoided by the use of better water, a certain number of deaths, probably two or three, from other causes have been avoided.” In the routine examination of water, no particular effort is made to isolate this organism, owing to the difficulty of the task. The tests that the present-day investigator has to satisfy are extremely thorough; and unless the suspected organism conforms to the whole of miese necessary tests it cannot be accepted as true B. typhosus. . Msp. comma.—tThe spirillum, or vibrio, of Asiatic cholera is an intestinal organism; and the disease it produces is spread largely by water. Epidemics of cholera are more easily traced to their MICROORGANISMS IN WATER 250 AVERAGE ANNUAL DEATH RATE FROM TYPHOID FEVER PER 100,000 OF THE POPULATION. MUNICH VIENNA HAMBURG PARIS PA’ 1039 1.6 125 (27 178 18. ALBANY, NY. 18. 2016 VA. 2473 32.82 37.27 56.01 69. An instructive contrast between Altona and Hamburg before the latter filtered its water, having learnt its lesson from a sharp outbreak of cholera. A FEW = ‘ SCATTERED CASES OF CHOLERA. HAMBURG. POPULATION: 600,000 CHOLERA CASES: 17.000 - DEATHS: 8,600 ALTONA: WATER FILTER! HAMBURG: WATER UNFILTERED Fic. 110.—(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 filters which were used to purify the water (Fig. 110). In times of epidemic, the organism has been isolated from rivers, wells, and reservoirs in India, a country in which the disease is endemic. Tue NuMBER OF BACTERIA IN Rain, Snow, Hatt, Erc., AND IN WATER FROM WELLS, UPLAND SURFACE WATERS, RIVERS, AND LAKES Rarn.—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 sera steaming Month ee January. ........... 8,000 Julyiecdsniteescaas 5,600 February. .......... 1,320 August. .......... 8,300 March............. 2,920 September........ 5,770 Aprils: ocaun geen 2,140 October.......... 3,220 MAY 0% cuisine goa ei 2,440 November........ 3,250 June sas eeneysesess 5,600 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. MICROORGANISMS IN WATER 261 SnNow.—The results obtained from snow are similar to those ob- tained from rain; but as a rule the numbers are larger, a result doubtless due to the larger particles of the snow flakes. One investigator has found from 334 to 463 bacteria per c.c. of snow water. On the sum- mit of high mountains snow is practically sterile, Binot not finding a single organism in 8 c.c. of water from mountain-top snow. Water issuing from glaciers is of remarkable purity, containing only from three to eight organisms per c.c.; but the numbers are larger as the distance from the glacier increases. Hait.—Hail stones usually contain large numbers of bacteria, varying from 628 to 21,000 per c.c. of water obtained from the melt- ing hail. Fluorescing bacteria have been found in some samples; _ and the presence of these microdérganisms suggests that surface water is sometimes carried up by storms and congealed. The presence of many molds in hail is due to contamination from the air. DEEP WELLS.—Deep well water and spring water contain as a rule but few organisms, usually less than 50 per c.c. on gelatin at 20°, and less than 5 per c.c. on agar plates at blood heat. In a series of tests of water taken direct from forty-three artesian wells, 152.4 M. (soo feet) deep or more, the writer has found an average of 27 per c.c. for the gelatin and 1.5 per c.c. for the agar counts. These tests have extended over a period of several years; and water from deep springs has given similar results. SHaLLow WELIs.—The bacterial content of shallow wells depends greatly on their location and construction. Even in those well loca- ted and constructed, the number varies with the amount of rainfall, and is often large. In polluted wells, very high numbers of organisms are found. Sedgwick and Prescott found from 190 to 8,640 bacteria per c.c. in unpolluted wells. In the same class of wells, Savage found from 10 to 100 per c.c. by the blood-heat count, and 100 to 20,000 or more by the gelatin count. Sixty polluted wells examined by the writer gave an average gelatin count of 740 bacteria per c.c.; and thirty-eight wells which were free of contamination gave an average count of 4oo 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 1 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 débris, etc., influence the microbial popu- lation. A few figures may be given for illustration. BACTERIOLOGICAL EXAMINATION OF RIVERS AT AND BELOW LARGE SOURCES OF Poxttution (Boyce AND CO-WORKERS) Distance Direction pane oe Above 305 4,786 About 0.6 mile.............. Below 9,387 About 2.7 miles............. Below 13,503 About 6.0 miles../.......... Below 8,764 30,432 About 12.0 miles............. Below 4,796 12,460 About 15.0 miles....... eee Below : 3,602 9,595 About 26.0 miles............. Below | cence 7,869 In the Chicago drainage canal, Jordan found 1,245,000 bacteria per c.c. at Bridgeport; 650,000 at Lockport, twenty-nine miles below; and 3,660 at Averyville, 159 miles below. Below where the sewage of Peoria enters, the number rises to 758,000 at Wesley City, and decrease to 4,800 at Kampsville, 123 miles from Peoria. The River Rhéne 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. Laxres.—The water of lakes is generally much purer than river water. Near .the shore, the bacterial content is higher than farther out, showing the contaminating influence of habitation. Thus Lake Geneva contains as many as 150,000 bacteria per c.c. near the shore, and further out only 38 per c.c. Other figures are as follows: Loch Katrine, 74 per c.c., Lake Lucerne, 8 to 51 per c.c., Lake Champlain, 82 per C.c. SEA WATER.—There are few bacteria in sea water remote from the coast; but near the shore and in the neighborhood of seaports there may be large numbers. MICROORGANISMS IN WATER ° 268 _ Examples: 350 M. from Naples, sea water contained 26,000 bac- teria per c.c. At a distance of 3 KM., only 10. Samples taken from depths of 75 to 800 M. at distances from 4 to 15 KM. from shore were found to contain from 6 to 78 bacteria per c.c. in surface water, and from 3 to 260 at various depths below. Causes AFFECTING THE INCREASE AND DECREASE OF THE NuMBER OF BACTERIA IN WATER There is a number of causes which influence the multiplication or diminution of microdrganisms in natural waters; and while it is necessary to discuss each of these causes in detail, it must be remem- bered that a number of them may be simultaneously influencing the increase or decrease. TEMPERATURE.—In natural waters, a low temperature probably acts injuriously on parasitic bacteria, reducing their numbers; but the bacterial content of water during the hot summer months is gener- ally not so large as during the cooler seasons. Water collected for examination should be analyzed at once; otherwise, contradictory results as to numbers will be found. Usually, in most waters, there is a reduction in numbers for a few hours, followed by a large increase. Very much polluted waters, however, show a marked decrease of intestinal organisms, if the samples are kept cool. Licut.—Although the germicidal effect of sunlight is well known, yet it has not such powerful effects on the bacteria in water. Much depends, no doubt, on the turbidity and speed of the cur- rent, the maxmium killing effect being produced in shallow, clear and slow-moving water. It has been found by experiment that the germ-killing power of light extends to a depth of 3 M (about 9.84 feet). . As a means of purifying water, direct light produces very little effect. Foop Suppry.—The amount of organic matter in water directly influences the growth of bacteria. Where a large amount of this is present, the number of microdrganisms is also large. Rivers containing considerable organic matter derived from vegetable débris, etc., contain, as a rule, more organisms than rivers in which there is but little of such material. Thus the Ottawa River, which drains a large area of forest lands and is characterized as an upland peaty water carrying a rather high percentage of organic and volatile matter, contains through- 264 ‘ MICROBIOLOGY OF WATER AND SEWAGE out the year a larger number of organisms to the cubic centimeter than the water of the river St. Lawrence, which is much clearer and contains much less organic matter. Sewage water is rich in organic matter, and 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 connected with the insufficiency or unsuitability of the food supply is to be found » the main reason for the bacterial self-purification of streams.” OxitpaTion.—On the surface of waters, in rapids, falls, and tidal 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 alge, river plants, and the numerous protozoan forms, bring about a reduction of organic matter in water, and thus reduce the amount of food available for bacteria. There is also the antagonism between these forms and bacteria. The chemical products of the higher forms are considered by some authorities to be injurious to bacterial life; and many bacteria are ingested by predatory protozoa. Diturion.—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. (roo 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 of the self-purification of the Chicago drainage canal illustrates Jordan’s contention. Oruer Causes.—There is a number of other causes, not well known nor of sufficient practical importance for more detailed com- ment, which may increase or decrease the number of bacteria in water, such as the inhibiting action of microdrganisms and their products on one another, the effects of pressure, etc. A peculiar fact, which has never been satisfactorily explained, is the quick death (in three to five hours) of the cholera vibrio in the waters . of the Ganges and Jumna. When one remembers that these rivers are grossly contaminated by sewage, by numerous corpses of natives (often dead of cholera), and by the bathing of thousands of natives, it seems remarkable that the belief of the Hindoos, the water of these rivers is pure and cannot be defiled, and they can safely drink it and bathe in it, should be confirmed by means of modern bacteriological research. It is also a curious fact that the bactericidal power of Jumna water is lost when it is boiled; and that the cholera vibrio propagates at once, if placed in water taken from wells in the vicinity of the rivers. soe INTERPRETATION OF THE BACTERIOLOGICAL ANALYSIS OF WATER In making any analysis of water, all data, such as the kind of water and the particulars regarding collection, transmission, sampling, rainfall, etc., should be given, as these are a great help in interpreting the results. One analysis is rarely sufficient; examinations should be regularly and systematically made. QUANTITATIVE STANDARDS.—No absolute guide can be given to determine the potable quality of water from the number of micro- organisms in it. It may, however, be safely assumed that high bacte- rial counts indicate a large amount of organic matter. The number of organisms growing in beef peptone gelatin at 20° to 22°, and termed the ‘gelatin count,” should be given. For deep wells and springs, this should not exceed 50 per c.c.; and for shallow wells and rivers, not over 500 per c.c. After rains or floods, these figures might be exceeded, and would not necessarily indicate dangerous pollution. The number of organisms which develop on beef peptone agar incubated 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 of | specific’ disease organisms, such as typhoid and cholera microbes from water, is sufficient to condemn such a sample as unfit for use; but, on account of many technical difficulties, it is practically impossible to make such an examination. Apart from a few special cases, when it may be necessary to attempt the isolation of these pathogenic bacteria, the presence of the colon bacillus (B. coli) in small amounts of water, is generally looked upon as significant and indicative of sew- age pollution. The technical methods used in this isolation and enumeration are many, and may be found in the works cited; but there is considerable difference of opinion as to the number of B. colt which should condemn a sample of water. Prescott and Winslow state that if the colon bacillus is in “such abundance as to be isolated in a large proportion of cases from 1 c.c. of water, it is reasonable proof of the presence of serious pollution.” Savage suggests that B. coli should be absent from 100 c.c. in the case of water from deep wells and springs, and should be absent from ro c.c. in surface waters, such as rivers used for drinking purposes, shallow wells, and upland surface waters. The streptococcus examination is next in importance as an indi- cator of sewage. Streptococci should be absent from the amounts of water mentioned above for B. coli; and B. enteritidis sporogenes should not be present in 1,000 c.c. of water from deep wells, nor in roo c.c. from surface waters. SEDIMENTATION, FILTRATION, AND PURIFICATION OF WATER As areas become more and more thickly settled and towns and cities increase in population, the problem of obtaining sanitary con- trol over the water supply increases in importance. Very few towns and cities are fortunate to obtain their water supply from an unpol- ' MICROORGANISMS IN WATER 267 luted area. Consequently expensive installation must be made, in order to purify a suspiciously contaminated water by freeing it from organ- isms injurious to health. There are several methods of accomplish- ing such purification; and these will be briefly mentioned. SEDIMENTATION AND FitTration.—This method of purifying water “. has been used for nearly a hundred years; but the great impetus given to this hygienic measure was due to Koch, who showed in 18093 that the Fic. 111.—Section of a sand filter. proper filtration of Elbe water saved the town of Altona from an epi- demic of cholera which devastated Hamburg as a result of drinking un- filtered water. In this system of purification, the water is first stored in large reservoirs, where the effect of sedimentation and storage reduces considerably the number of bacteria. From the reservoir, the water is‘ filtered through sand, gravel, and pebbles, etc., arranged as shown in Fig. 111. This filtration removes from 97 to 99.5 per cent of the microérganisms. ; The action of the fiter bed is due to the mechanical obstruction of impurities, to oxidation of the organic matter, and to nitrification due 268 MICROBIOLOGY OF WATER AND SEWAGE MEAN oF MontTHLy EXAMINATIONS FOR THE YEAR Microérganisms per c.c. At source After storage After filtration London, Lambeth Works...... 16,138 7,820 75 London, Chelsea Works....... 16,138 1,067 34 Berlin, Lake Miiggel.......... r400 |... 60 Paris, Marne................. 79000 | ...... 630 Paris, Seine..............0000- 186,986 | ...... 400 to the living bacteria in the scum which forms on the top of the layer of | ' sand. Of these, the last is the most important; for until this gelatinous layer forms, the filter does not act properly—in fact, it has little filter- ing action, as the following figures show: BacTERIAL CONTENT OF WATER BEFORE AND AFTER CLEANING THE SAND FILTER Before cleaning, z.e., before removing the scum layer... 42 per c.c. One day after cleaning..................000005 estaaiats 1880 Two days after cleaning........... 0.2... cece eee eee 752 Three days after cleaning............. 0... cece ee eee 208 Four days after cleaning.....................000000- 156 Five days after cleaning.................. 0.020000 102 Six days after cleaning............... 0. cece eee eee 84 Thus provision must be made to permit the scum or film to form be- fore the filtered water is used for domestic purposes. The rate of filtration must be regulated; for if the water is allowed to exceed a certain rate (101.6 mm. or 4 inches per hour), inefficiency follows. ; CoaGULATING BASINS AND FILTRATION.—This method of purifica- tion consists in adding a coagulant, such as basic sulphate of aluminum, by means of a mechanical device which regulates the quantity, as the water is pumped into the coagulating basins or reservoirs, where it re- mains for six to twenty-four hours. The aluminum sulphate is decom- posed by the lime in the water and forms insoluble aluminum hydrate; and the sulphuric acid combines with the lime. The hydrate of alumi- num is precipitated in large flocculent masses, entangling all particles ‘ of soil or organic matter; and these, being deposited on the surface of the _ MICROORGANISMS IN WATER 269 sand, form the filtering layer. Such filters are very efficient; they re- move from 97 to 99.8 per cent of the bacteria from the water. Porous Firters.—(Fig. 112.) These filters are either made from unglazed porcelain or baked diatomaceous earth; the former are known .as Chamberland, and the latter as Berkefeld filters. These filters are usually candle-shaped, require considerable pressure to force water through them, and can be used only when a small supply of water is needed. Water which is forced through these filters is at first sterile; but with repeated use they allow bacteria to pass through the pores and Fic. 112.—Unglazed porcelain filters. Chamberland system; A, without pressure; B, fitted to main water supply; C, section of a porous porcelain filter. thus the filtering efficiency is impaired and will remain so, until the fil- ters are cleaned and baked to red heat in a muffle-furnace. Unless this is done regularly, no dependence should be placed on these filters, as they only put those who use them off their guard against the danger to which they are exposed. PURIFICATION BY OZONE.—The antiseptic properties of ozone are well known. It is used in the purification of the water supply of some towns—Nice, Chartres, etc. Ozone used for this purpose is usually _ obtained by means of the electric current; and a flowing film of water is 270 MICROBIOLOGY OF WATER AND SEWAGE brought into contact with an upward current of air charged with ozone, which current makes the water almost completely sterile. This method of purification is efficient, but rather expensive. PURIFICATION BY Hrat.—By bringing water to the boiling point, all harmful bacteria are destroyed; a few spores may resist this treatment, but they are harmless. Boiled water is of a flat, insipid taste, due to the driving out of the contained gases. The taste may be improved by cooling and shaking. The boiling of water is often resorted to as a hy- gienic measure in times of epidemic, and for the supply of armies in the field. PURIFICATION BY CHEMICALS.—The addition of a small amount of calcium hypochlorite, or potassium iodide, etc., purifies water; but these methods are seldom used, except for the use of soldiers on campaign. Hypochlorite, however, is now used more commonly in municipal water supplies where they can not be otherwise controlled. LocATION AND CONSTRUCTION OF WELLS Farms in many sections of this country are practically all supplied with surface water collected in shallow wells. Hence farmers should understand the principles involved in the location and construction of wells. Many farm wells are badly located—too near such sources of con- tamination as outhouses, cesspools, stables; or barnyards; and those who locate them give too little attention to the slope of the ground, and the nature and slope of the subsoil. There should be at least 22 to 30 M. (75 to 100 feet) between the well and all probable sources of contamination; and this distance is too small, if the soil is very porous, or if the surface and subsoil drainage is toward the well, or if the well is sunk in fissured rock—as it is obvious that there are serious chances of contamination in each of the above circumstances. In all cases, the surface drainage should be away from the well; and, as far as possible, the subsoil drainage also should be from the well. Sketches 113, 114, and 115 illustrate these points, the upper part of each drawing showing the plan and the lower portion a section through the dotted line marked on the plan. Fig. 113, shows that the surface drainage is from the house, privy, stables, and barnyard toward the well. The section through the line ‘“‘A”’ shows the relation of the impervious MICROORGANISMS IN WATER 271 v A- sa aa r Fic. 115. ; Fics. 113, 114, and 115.—In each figure—plan above—section through A B below. S =-Soil; B = Impervious subsoil or strata. 1, House; z, well; 3, outhouse; 4, piggery; 5, stables; 6, stable yard; 7, hen house; 8, sheep stable. Arrow heads indicate 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, improperly 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, = "Soil Fic. 116.—Construction of a model well. On the right is brick construction, on the left stone construction, as illustrated. (Original.) except the house, is in the direction of the well; and thus contamination of the water supply is liable to occur. Fig. 114 shows a well properly located as regards both surface and subsoil drainage. Such a well will supply pure water, if it is properly constructed. Fig. 115 shows the proper construction of ‘a well with brick or stone. Large vitrified drain pipes with cemented joints will answer equally well when there is an abundant supply of water; but in case the supply of MICROORGANISMS IN WATER 273 water is limited, a large area is needed, and a stone or brick well is necessary. Reference to the illustrations will show that every endeavor is made to prevent surface water from entering directly into the well. The walls are impervious; and the earth or clay is well rammed against the outer side of the wall. The curb is carried well above the surface of the ground. The waste water is conducted by means of a sloping platform, trap, and drain, away from the well; and the well opening is properly covered. All water entering such a well must percolate through a con- siderable depth of soil, and undergo purification by means of the aggre- gations of living bacteria in the soil spaces. Thus the soil around a well fulfils the same function in purifying the surface water as the scum layer that forms on the surface of gravel filters. 18 CHAPTER II* MICROBIOLOGY OF SEWAGE THE BACTERIAL FLORA OF SEWAGE CoMPLEXITY OF FLora.—Sewage is made up of the miscellaneous and varied wastes of human life and activity, and the bacteria which are found therein are the result of a haphazard and chance admixture of - substances of diverse origin and character. The resulting flora is not only of great diversity and variability, but it is with few exceptions non- characteristic. In brief, the medium with which we have to deal has had an origin too indefinite and a history too short to have permitted the establishment of anything approaching a constant or characteristic bacterial flora. TyprcaL Forms.—Our interest in this sewage flora is a very practical one, being confined to those organisms which carry on the work of bio- logical purification and to certain pathogens which for obvious reasons require special treatment. We are interested chiefly in what these bac- teria do rather than in what they are, and our classification is influenced accordingly. It is based, not upon the species or the genus nor even upon the group or type, that proves so convenient in general bacterial classification, but upon a sort of physiological or functional type, having to do solely with the activities of the organisms in sewage and in its puri- fication. Bacteria performing a common function or producing a com- mon result are members of one type. Individuals may belong to several of our types and there are doubtless a great many that belong to none These latter simply have no place assigned them as yet in the rdle of sewage purification, because they possess none of the recognized typical functions. Apparent exception may be taken to these general principles in the case of such organisms as the B. coli, sewage streptococci and B. enteritidis, These are, to a certain extent, characteristic sewage bacteria. But interest in them as individuals is confined to water “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(NHzp)s + 2H,0 = (NH,)2CO3. (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 réle in anaerobic reactions that is played by direct oxidation under aerobic conditions. The Liquefaction of Protein—One of the most clearly defined and useful types of bacterial activity to be seen in the various sewage disposal processes is that which we term liquefaction. This term is used to denote broadly all those changes by which solid and insoluble organic matter is converted into a soluble condition. The particular process known as protein liquefaction is in the main analogous to 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 favored by total immersion but the effect in both instances seems to be due 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 afew months. Wood itself is more resistant and withstands the action of the tank for years. This is largely due to the fact that the wood molecule is much more complicated than a simple cellulose molecule, and, among the conifers at least, to the further fact that antiseptic intercellular substances are present. - Chemically considered the action is hydrolytic and can be imitated 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 is most noticeable in the case of long sewers in which considerable veloci- ties are maintained. In quiescent tanks there is a tendency for the fats to rise to the surface and thus become removed from the influence of this action. Thus in small installations enormously heavy scums form upon the tanks and analysis shows a considerable percentage of fat in this material. In larger systems on the other hand there is less and less evidence of fatty material as such. It is true that there is a deposit upon the walls and tops.of such sewers and that small floating objects, like matches, rolling along such a wall will accumulate layers of grease and become eventually the familiar “grease-balls” found in the 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 proteolytic changes are accompanied by the freeing of alkaline products, ammonia and amines, which leads to some saponification, and which, in turn, leads to a further emulsification. It has also been demonstrated that bacterial activity is commonly associated with fat \ MICROBIOLOGY OF SEWAGE 279 oaniqeninis and decomp ction Whether specific enzymes are pres- ent which assist in this final process or not has never been determined. It is significant to note, however, that where sewages are slightly acid, unaltered fats are much more abundant, even though the acidity is insufficient to prevent vigorous putrefactive changes in the sewage itself. The Fermentation of Urea—The fermentation of urea has already been referred to as a typical and simple case of anaerobic decomposition. This reaction has great significancein sewage chemistry since a consider- able proportion of the nitrogen of sewage is present initially as urea. Owing to the ease and rapidity with which the reaction takes place, however, no special effort is necessary to bring it about in sewage treatment and it therefore receives brief attention in discussions of the chemistry of sewage. The change to ammonia takes place in the small sewers of the system and it is difficult and generally impossible to detect the presence of urea in sewage. It has even been suggested that certain enzymes present in fecal matter are instrumental in bringing about this change and that the bacteria are only indirectly concerned. It is known, however, that a large number of bacteria of general occurrence have the power to produce this fermentation. Of these the Bact. uree (Miquel) may be cited as an example. The Reduction of Sulphates and Nitrates—The production of sul- phuretted hydrogen during the anaerobic decomposition of sewage is commonly noted. This substance may arise in at least two ways. Sulphur, being a constituent of most protein substances, is split off from the molecule in this form during certain types of fermentation. Its formation in these cases is analogous to that of ammonia from protein. The amount so produced is small and is usually neutral- ized and precipitated by the small amounts of iron and other metals always present in sewage. There is therefore no liberation of the gas itself and it is often said that sulphuretted hydrogen is not formed normally in a septic tank. This conclusion is readily disproved by a simple test of the black residue found at the bottom of such tanks. A second and more important source of this substance is the sul- phate normally present in many sewages. Throughout many parts of the country the water supply contains material quantities of mag- nesium or calcium sulphate, and upon the sea coast the sewage gener- .ally: receives more or less salt water. 280 ~ MICROBIOLOGY OF WATER AND SEWAGE ~ In these cases the reduction of sulphates to sulphuretted hydro- gen is not only of interest bacteriologically but probably exerts an influence upon all the reactions that are going on simultaneously. In fact this example serves excellently to illustrate the great complex- ity of these anaerobic reactions and the mutual interdependence of each upon all the others. Sulphates, under anaerobic conditions, are a source of oxygen and it is upon oxygen that the course of all these reactions depends. Therefore the presence of sulphates and the possibility of their yielding oxygen may alter the course of the other reactions involved. The products of the protein hydrolysis for ex- ample may be profoundly modified by the presence of this additional source of oxygen. The effect upon the bacteria themselves is also to be considered as a factor quite distinct from the purely chemical effect just de- scribed. It has frequently been observed, and in fact would be ex- pected, that the products of anaerobic putrefaction are themselves detrimental to the activity of the organism producing the changes in question, The nature of sulphuretted hydrogen makes it appear quite probable that we are dealing here with a toxic substance that would at least inhibit the activities of certain bacteria and in this way further modify the final result. The same might be said of almost all the reactions with which we have to deal but this example is cited as a typical one. It is known in practice that the presence of sulphates in a sewage does lead to a distinct type of anaerobic change which is characterized by the marked blackening of the sewage, the formation of secondary reaction products which precipitate after the removal of the suspended matter of the sewage, the evolution of hydrogen sulphide, an excessive amount of mineral or non-volatile residue in the sludge and the forma- tion of free sulphur upon subsequent aeration of the sewage. “Here again, as in the other types of reaction, it is useless for the pres- ent to attempt to ascribe this reaction to any particular species. Sp. desulphuricans and B. sulphureus have been isolated. A non-liquefy- ing anaerobic bacillus, which reduced sulphates strongly, was isolated. from Boston sewage in the writer’s laboratory by G. R. Spaulding. Others have been described and there is undoubtedly a large group of organisms capable of bringing about the reaction. Just as the reduction of nitrates is a function performed by many, MICROBIOLOGY OF SEWAGE 281 ii perhaps most, anaerobes, so the reduction of sulphates, although a less common function, is still common to many forms. In fact ni- trates, sulphates, and phosphates form a series in regard to their reducibility and the effect of their presence upon the reaction as a whole. The phosphates so far as has been recorded are not ordinarily reduced. Oxipizinc BactErtA. The Production of Nitrate and Nitrite—A long series of investigations upon the organisms which oxidize nitrogen began with the Franklands and Winogradski, and has continued to the present day. These have given us much in- formation concerning the habits and functions of the nitrifying organisms. Winogradski’s original types were Nitrosomonas and Nitrobacter, the former oxidizing ammonia to nitrite, the latter completing the oxidation to nitrate. Work upon these organisms constitutes such an important factor in soil bacteriology to-day that more detailed discussion of this nitrifying function is left for another place. In the earlier days of sewage purification great stress was laid upon the work of these organisms, which was believed to be fundamental. ‘The degree of nitrification was accepted as a measure of the work of the filters and little thought was given to the possibility of oxidizing reactions by other forms. With the development of modern sewage disposal methods, the work of this latter type of bacteria has assumed a more important réle and the actual work of the nitrifying organism has been found to be of only minor and incidental importance. Other Oxidizing Reactions—The great groups of aerobic and 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. PatHocenic 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 1 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 generality can be extended to sewage is debatable. Our best informa- tion leads to the belief that any reduction in numbers of typhoid bacteria which may take place within the sewer before discharge is of minor importance and of slight sanitary significance. oe Discussion of other pathogens must be in even more general terms. Information is almost wholly lacking and it can only be assumed for purposes of safety that, in.so far as organisms of these various types are discharged into the sewer, they will persist to a certain extent in the sewage until it is finally disposed of. If such disposal be by discharge into a stream without purification; then the waters of that stream become polluted with infectious material. Studies recently made by Sedgwick and McNutt have indicated the possibility that many dis- eases, other than the oft-quoted typhoid fever, may be transmitted ' in this way. Life in Septic Tanks and Filters—With the introduction of the septic tank at Exeter, England, in 1893, the question of the fate of pathogenic bacteria in such a tank was raised. It was even suggested that bacteria, such as the typhoid organism, might multiply in the. tank. The question was investigated by Professor Sims Woodhead, who concluded that no organisms capable of setting up morbid changes in animals were discharged from the tank. This negative evidence, however, has little weight in the light of more recent experiments. Pickard introduced an emulsion of typhoid bacteria into this same tank and noted only a gradual decrease. After fourteen days he was able to detect 1 per cent of the initial number. He also reported a removal of go per cent of the typhoid organisms introduced into a contact filter. These data must be interpreted in the light of two established facts. The typhoid organism tends to die at a rapid but diminishing tate under any but the most favorable conditions. This results’ in a rapid decrease at first, with a prolonged survival of a few individuals. This process takes place in sewers, in streams, and, in fact, under most artificial conditions. The second fact of importance is the difficulty of recovering the typhoid organism under experimental conditions like those described. A thorough study of the bacteriology of sewage and of filter effluents led Houston to conclude that the biological processes at work in a filter or tank were not strongly inimical, if hostile at all, to the vitality of pathogenic germs. 284 MICROBIOLOGY OF WATER AND SEWAGE A conservative study of all the evidence bearing upon this impor- tant question including the vitality and fate of certain non-pathogenic species, such as B. coli, leads to the conclusion that the removal of pathogenic bacteria in purification methods is due to two allied causes, the efficiency of which can be approximately determined. There is first the time element and the known rapid decrease in the numbers of certain bacteria such as B. typhosus when placed under conditions that preclude multiplication. The rate of decrease varies but is roughly about 50 per cent in twenty-four hours. : The second factor, acting in reality in conjunction with the first, is the mechanical hindrance that is offered to the free passage of sus- pended materials through the body of a filter. Even fine sand offers_ little straining action as such, since the open channels are thousands of times as big as the bacterial cell, but surface tension phenomena tend to make all solid material adhere to the medium and thus its passage is delayed. This action is prominent although of less impor- tance in coarse-grained filters. Actual experiments by the writer have indicated that while the liquid may pass through a trickling filter in half an hour, small suspended particles such as ultramarine and B. - prodigiosus cells require an average of over twenty-four hours. In this way the actual time of passage is greatly delayed even when coarse broken stone is the filter medium, and the times that are now known to be necessary for the passage are ample in themselves to account for the reductions that have been noted. It may therefore be stated as a conservative view of the efficiency of purification processes in the removal of pathogenic bacteria, that there are no strongly inimical processes at work in the tanks or filters, and that the rate of decrease is not materially greater than would be observed in the same period of time under the conditions of a running stream. THE CULTIVATION OF SEWAGE BACTERIA There are two general methods employed for the cultivation of those bacteria which are of assistance in sewage purification. They may be cultivated in so-called filters of sand or coarser material, or in specially constructed tanks such as the septic or the hydrolytic tank. Jn the former case the bacterial growth occurs upon the special medium provided, the sand or stone; in the latter, it takes place in the liquid MICROBIOLOGY OF SEWAGE 285 itself and a continuous life history within such a tank is possible only when the rate of flow is sufficiently slow to permit of the inoculation of the incoming stream by the contents of the tank. Fitters.—The filtering media most commonly employed are sand or crushed stone or other coarse material. In natural sand beds a Fic. 117.—Sewage Experiment Station, Mass. Inst. Technology. Trickling filter in' front, sand filter just behind filter, dosing tank just behind sand filter, and septic tank just behind dosing tank. brief period of treatment with sewage suffices to produce an active state of “nitrification.” By this term is indicated all the complex processes of oxidation one index of which is the formation of nitrates. After such a filter has once become active in this way it will continue, with proper care, to oxidize sewage almost indefinitely. Improper care, such as an overdose of sewage or continued flooding of the surface due to poor drainage, will soon destroy the activity of the filter. The addi- tion 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 filter is a biological culture medium upon which the various types of bacteria are growing and carrying out their functions and that such a medium requires careful control and is sensitive to unfavorable changes in environment (Fig. 117). ~ The other filters are similar to this and illustrate the true function of filtration. In the case of the sand filter it might be maintained that filtration or straining was an essential element in the process, but in the case of these coarse-grained media straining action is eliminated. Here there is nothing but a pile of stones, varying from 1 tog in ches or more in diameter, upon the surface of which the bacteria grow. The Fic. 118.—Sketch of septic tank, (Original.) sewage trickles slowly over the surfaces, or is held in contact with them temporarily, according as we are dealing with trickling or contact filters. Solids adhere to the stones or settle upon them, and soluble material is “‘absorbed”’ by the surface growth and removed from solution. Within these gelatinous growths to which the air also has free access, the proc- esses of oxidation take place and the products, the semi-oxidized " organic material, are later “‘shed’’ from the stones appearing apatite in the effluent as humus or stable organic matter. ANAEROBIC TANKS.—The cultivation of bacteria in anaerobic tanks is not quite as simple a matter as that which has just been described. \ MICROBIOLOGY OF SEWAGE 287 The sewage is allowed to flow slowly through the tank and after some time, from a few days to a month or more, a normal and constant flora will have become resident there. This flora will soon have be- come so well established that the incoming sewage laden with a flora of its own mingles with a liquid in which the established flora is so greatly in excess that the former in large measure gives way to the latter. In this way, while the sewage itself moves onward and is gone within a few hours, the flora is constant and persistent. A further aid in preserving this constant flora is the sludge at the bottom, in which the bacteria lodge and multiply and from which they are carried upward by the ever moving eddies and constantly re-inoculate the liquid above (Fig. 118). THE DESTRUCTION OF SEWAGE BACTERIA By BrotocicaL Processes.—Reference has already been made. to the effect of biological processes of purification upon pathogenic bacteria. What was stated in regard to the pathogens is equally true of the sewage bacteria asa whole. Their destruction is due to time and an environment unfavorable to growth, rather than to any specific cause. Further evidence of these facts may now be given. Bacteria as a whole do pass even the fine-grained filters in large numbers, Careful analyses of their types show them to be a haphazard mixture -from the original sewage flora with little or no observable selection. Houston pointed out the relative abundance of the streptococci, sup- ‘posédly delicate organisms, and found on the whole that the relative abundance of the different kinds of bacteria seemed to be much the same in the effluent as in the crude sewage. On the whole we may conclude that the biological processes remove bacteria not by any specific antagonistic action but by delaying their passage and permitting the natural decrease that occurs when multi- . plication is prevented. The more efficient the mechanism of the filter in producing this delay the more complete will be the removal. By CHEMICAL PRocEssEs.—A much more reliable and economical method for bacterial destruction is now available in chemical disin- fection of sewage effluents. The writer’s studies at Boston, Baltimore and elsewhere have shown that the application of hypochlorite of calcium in amounts depending upon the character of the effluent, and 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 way, by Justus von Liebig in 1840. In his “Organic Chemistry in its Applications to Agriculture and Physiology” he developed important theories on the circulation of carbon and nitrogen in nature, and on the function of the so-called mineral constituents of plants. When Liebig’s book appeared, many of the leaders and students of agriculture still believed that humus, the partly decomposed residues of ' plants and animals in the soil, was the direct food of crops. They believed that soils could yield poor or rich harvests in proportion to the amount of humus present in them; they believed, in other words, that plants, like animals, used organic substances as food. _ Liebig rendered a great service to agriculture in emphasizing the significance of decay processes. He made it evident that humus as such is of no use to plants, and that it becomes valuable only in so far as it is resolved into the simple compounds carbon dioxide, ammonia, nitric acid and various mineral salts. To be sure, he regarded the decomposition of organic matter as a phenomenon purely chemical, nevertheless he succeeded in showing that decay, putrefaction and fermentation are fundamental facts, connecting links between the world of the living and the world of the dead. The research of the following decades brought to light the intimate relation existing between microérganisms and the decomposition of «a *Prepared by Jacob G. Lipman with exception of sub-chapter on “ Soil Inoculation” which has been prepared by S. F. Edwards. © 19 289 290 MICROBIOLOGY OF SOIL ' organic matter. In the realm of soil fertility the new discoveries re- vealed the vastness of the task assigned to soil microérganisms in providing available food for crops. It was shown that under the attack. of bacteria and of other microdrganisms the various organic débris in the soil is split into relatively small chemical fragments; that the carbon is restored to the air as carbon dioxide; that the nitrogen is changed into ammonia, nitrites and nitrates. It was shown, further, that in this breaking down of organic matter the various cleavage products, and, particularly, carbon dioxide, hasten, to an amazing extent, the weathering of the rock particles and make available thereby the mineral portion of plant food. It was shown, likewise, that apart trom accomplishing the transformation of unavailable into available plant food, microdrganisms are concerned also in the addition of nitrogen compounds to the soil. The evidence gathered slowly by many investigators made it plain, therefore, that microbes are an important factor in the growing of cultivated and uncultivated plants. Hence, the important place assigned to microdérganisms in the study of soil fertility problems. THE Sor aS A CULTURE MEDIUM Arable soils present so wide a range of conditions as to modify, materially, the development and predominance of different species. ‘Variations as to moisture, temperature, aeration, reaction, food supply and biological relations are important, in each case, in determining the survival or disappearance of any particular species. For this reason, the study of soil microérganisms must reckon with the mechan- ical composition of soils, their ability to retain water and their content of inert and soluble plant food. MoIstuRE RELATIONS IN THE SOIL AMOUNT AND DISTRIBUTION OF RAINFALL.—Precipitation in different regions of the earth’s surface varies from practically nothing to more than 1,524 cm. (600 inches) per annum. A portion of this water runs off the surface into the nearest stream, another portion is rapidly changed into vapor and is returned to the atmosphere, and the remainder passes downward, into the soil and becomes the medium in which plant food is dissolved. It is estimated that only about half LU MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 291 the total rainfall percolates through the soil. Where the sae are open and nearly level the proportion of percolating water is relatively greater; where the soils are fine-grained and more or less impervious, or the topography broken, the proportion is relatively smaller. Bacteria and other microérganisms, as well as the higher plants, are directly influenced by the amount of moisture available for their various needs. Hence soil microbial activities are affected not alone by the amount of rainfall, but also by its distribution. It is obvious, for instance, that an annual rainfall of 762 mm. (30 inches) distributed rather uniformly throughout the year would produce different soil- moisture relations than the same amount of precipitation confined to only two or three months. As is pointed out by Abbe, a daily pre- cipitation of 2 mm. (.079 inch) distributed throughout the three summer months would be quickly changed into vapor, and would hardly wet the soil; whereas the total quantity of 180 mm. (7 inches) evenly divided into ten or twelve rains would penetrate the soil to a considerable depth, and would furnish very favorable conditions for microbial development. In a similar manner it is pointed out by Hil- gard that Central Montana, and the region in the vicinity of the bay of San Francisco, have each a total precipitation of 610 mm. (24 inches). But while in Montana the rainfall is distributed over the entire year and irrigation becomes necessary, the precipitation near San Francisco is limited to the portion of the year that nearly coincides with the growing season, and crops are enabled to mature without irrigation. Rance oF Sort Motsture.—Any given volume of dry soil consists of solid particles separated by empty spaces. The sum of these spaces is known as the “‘pore-space.”’ It varies from about one-third of the entire volume in coarse sands to more than two-thirds in pipe clay. In peat and muck it may amount to as much as 80 or go per cent of the entire volume. Under air-dry conditions each soil grain is surrounded ‘by a very thin film of moisture designated as hygroscopic water. When air-dry soil is moistened the films around the soil particles become thicker and finally cease to be isolated. A continuous liquid membrane, as it were, is stretched from particle to particle, and the surface tension that thus comes into play is capable of lifting large amounts of water to the surface. The continuous film of soil water that can hold its own against the pull of gravity is known as capillary water. Finally, when the liquid films around the soil grains increase in thickness be- 292 MICROBIOLOGY OF SOIL yondfa certain point, the attraction between the molecules in the soil grains and the more distant molecules of water is no longer great enough to overcome the force of gravitation, and the excess of water percolates downward. The water more or less readily moved by gravitation is called hydrostatic water. For any given conditions of:the soils the amount of hydrostatic, capillary and hygroscopic water is directly dependent on the mechanical structure. It is evident that the aggregate surface of the particles in a fine-grained soil is much greater than that in a coarse-grained soil. Actual determinations have shown that the aggregate inner surface of .02832 c.m. (1 cu. ft.) of coarse sand may be but a fraction of an acre; whereas the same quantity of the finest clay may have an inner surface equivalent to 1.2i41-1.6188 hectares (3 or 4 acres). These differences are to be expected, since, as is shown by Lyon and, Fippin, 1 g. of fine gravel may contain 252 particles; 1 g. of medium sand, 13,500 particles; 1 g. of very fine sand, 1,687,000 particles; 1 g. of silt, 65,100,000 particles, and 1 g. of clay, 45,500,000,000 particles. Since the soil water is spread as a film over the solid particles and varies in amount with the fineness or coarseness of the soil, and since the quantity of plant food going into solution is determined largely by the amount of water in contact with the soil particles, it follows that * clay soils will, under the same conditions, contain more plant food in solution than loam soils and still more than sandy soils. From the standpoint of soil microbiology this is important, for the microérganisms live and multiply in the film water surrounding the soil particles. The concentration of salts in this film water as well as their composition must of necessity affect bacterial activities. In the same way, methods of tillage and cropping affecting the concentration and composition of the film water will modify the chemical changes caused by bacteria and other microérganisms. Errect oF DroucHT AND oF Excessive Moisture.—Optimum conditions for plant growth and the development of many important soil bacteria are furnished when about half of the entire pore space is filled with water. In light sandy soils the optimum moisture content may be reached when the wet material contains sarcely more than 8 to ro per cent of water by weight; while in silt and clay soils the optimum may reach 16 to 20 per cent. or even more. ; Continued depletion of soil moisture by plant roots and evaporation MICROORGANISMS AS A FACTOR IN SOIL FERTILITY, 2903 at the surface causes the film of capillary water to stretch move and more. Finally it becomes very thin, breaks, and ceases to be con- tinuous. The soil then becomes air-dry and contains only hyrgo- scopic water. It is estimated by Lyon and Fippin that, under average conditions of humidity, light sand will contain 0.5 to 1 per cent of hygroscopic moisture; silt loam, 2 to 4 per cent; and clay, 8 to 12 per cent. The amount of water present in air-dry muck or peat may range up to 40 per cent, or even more. According to Hall the film of hygro- scopic moisture is about 0.754 (0.00003 inch) thick. As the soil dries out bacterial activity is suspended and many vegetative cells undoubtedly perish. Nevertheless, it will be seen that the moisture film even in air-dry material is deep enough to allow the bacteria a reasonable degree of protection. This will account for the survival of non-spore-bearing bacteria in dry soil for a long time. Indeed, in- ‘stances are on record of the isolation of Azotobacter and Nitrosomonas from soils that had been kept in a dry state in the laboratory for several years. It may be noted, in this connection, that in the process of drying the soluble salts in the soil the moisture may be sufficiently concentrated in the films to cause plasmolysis and the destruction of individual cells. On the other hand, excessive moisture in the soil is not only directly unfavorable to aerobic species in that it limits their supply of oxygen, but is objectionable because it encourages the formation of reduction products that are toxic to these species. It is apparent, therefore, that favorable conditions for the formation of available plant food by bacteria are created when a certain relation is established between the volumes of moisture and air in the soil. The shifting of this relation in one direction or another is bound to react on species relationships and numbers. : AERATION MeEcuantcaL Composition oF Sorts.—Soil ventilation is an impor- tant factor in crop production. It provides for the proper supply of elementary oxygen so essential to decomposition processes in normal soils; for the supply of elementary nitrogen required by nitrogen-fixing species; for the removal of excessive amounts of carbon dioxide; and for the destruction of various toxic substances, The intimate relation “a 294 MICROBIOLOGY OF SOIL existing between soil ventilation and the mechanical composition of the soil niaterial is bound to react on the microbial factors involved. It is well known that the rate of flow of air through soils is inversely propor- tional to the fineness of the material; in other words, the fine-grained soils, notwithstanding their greater pore space, will not allow air to pass through them as rapidly as coarse-grained soils. King shows, for instance, that 5,000 c.c. of air passed through a column of fine gravel in thirty-seven seconds, whereas in similar columns of medium sand, fine sand, loam and fine clay soil the same amount of air required for its passage 1,178, 44,310, 282,200, and 2,057,000 seconds respectively. AEROBIC AND ANAEROBIC ACTIVITIES——The more rapid diffusion of gases from open soils naturally leads to a.more frequent renewal of their oxygen supply. In its turn, the latter affects the ratio of aerobes - to anerobes; it follows, therefore, that in clay soils and clay loam soils the activities of aerobic species are retarded to a greater extent than they are in sandy loams or sandy soils. It follows, also, that in fine- grained soils the activities of the aerobes are confined to a shallower soil layer than in coarser grained soils. The reverse is true of anaerobic species. Methods of soil treatment tending to improve soil ventilation react both on the amount of chemical change produced by definite species, as well as the numerical ratio of different species to one another. Among such methods may be included drainage, liming, manuring and tillage. ~ RATE OF OXIDATION OF CARBON, HYDROGEN AND NITROGEN.— Experiments carried out by Wollny proved conclusively that the pro- duction of carbon dioxide in soils is directly affected by the amount of ‘oxygen supplied; that is, by the more or less thorough aeration of the soil. In one of these experiments air containing varying proportions of oxygen and nitrogen was passed through columns of soil. When this air contained 21 per cent of oxygen there were produced for every 1,000 volumes of air 12.51 volumes of carbon dioxide; while with 2 per cent of oxygen in the entering air there were produced only 3.62 volumes of carbon dioxide. Similar observations were made by Schloesing in connection with the formation of carbon dioxide and of nitric acid. Dehérain and many others have recorded the favorable influence of aeration on the rate of nitrate formation, while Lipman and Koch have observed an increased fixation of nitrogen by Azotobacter, consequent upon a better supply of oxygen. - MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 2905 Tae MINERALIZATION OF ORGANIC MaTreR.—Conditions that favor the intense activities of decay bacteria lead to a relatively rapid restora- tion of the phosphorus, sulphur, calcium, magnesium and potassium that had been made fast in plant tissues, to the stock of available plant food in the soil; indeed, in extremely well-aerated soils the decomposition of organic matter and its ultimate mineralization proceed too fast. It often happens that the farmer is unable to maintain a proper supply of humus in these soils because of their openness and is forced to adopt measures that will retard soil aeration. He resorts therefore, to rolling, marling, manuring and green manuring. On the other hand, heavy, fine-grained soils are not sufficiently well aerated to allow a rapid mineralization of the organic matter. Under extreme conditions the decomposition processes do not keep pace with the process making toward the accumulation of organic matter, and a more or less considerable increase in the amount of the latter takes place. This occurs in low lying meadows, and, more particularly, in bogs and swamps. Hence the farmer attempts to intensify aeration and the resulting mineralization of the humus by more thorough tillage, drainage, liming and manuring. TEMPERATURE INFLUENCE OF CLIMATE AND SEASON.—An illustration of the differ- . ences that may exist in the soil temperatures of different regions is given by a comparison of the mean temperatures of 1901 recorded at Moscow, Idaho, and New Brunswick, New Jersey. The soil temperatures were taken to a depth of 152 mm. (6 inches). Sort TEMPERATURE,* 1901 T pate Feb. | Mch.) Apr. | May | June | July | Aug. | Sept.] Oct. | Nov.| Dec. Moscow, Idaho...| 32.0] 30.0) 35.0} 40.0 aol 58.0) 68.0; 72.0] 57.0) 50.0] 40.0] 34.u New Brunswick,| 31.5| 28.6] 35.3) 47.9 57.9} 72.t| 76.4) 73.4| 68.5) 56.0] 41.1] 33.4 N.J. ! AIR TEMPERATURE,* 1901 Moscow, Idaho... New Brunswick, N. J. | 30.0] 30.5) 38.3) 44.0! 56.9) 55.0} 65.5] 69.6] 50.3] 50.5) 39.5/39.0 30.8] 24.8! 39.1| 48.3! 59.2) 70.9] 77.4} 74.6) 67.6| 54.6) 38.6/32.6 * Recorded in Fahrenheit scale. 296 MICROBIOLOGY OF SOIL It will be observed that in the months of November to March the soil temperatures in the two places were nearly the same. On the other hand, in April to October the average temperatures at New Brunswick were for soil 14.5° (58°F.) and for air 22.5° (72°F.), re- spectively; and in July they were 20.0° (68°F.) and 24.5° (76.4°F.) respectively. It will also be observed that there is an unmistakable relation between the corresponding air and soil temperatures. As a further illustration of the relation of climate to temperature a comparison may be made of the average daily mean temperatures at Bismarck, North Dakota, for the period 1873-1895, and at Key West, Florida, for the period 1872-1895. Dairy Mean TEMPERATURES* (ArR) Jan. | Feb. | Mch.; Apr. | May | June | July | Aug. | Sept.| Oct. | Nov.] Dec. ’ Bismarck, N. D...| 4.5 9.5| 22.6] 42.1] 54.2] 63.8] 69.5) 67.5] 57.0] 43.8] 25.9] 14. Key West, Fla....| 69.7} 71.4] 72.7] 76.1] 79.4] 82.5] 83.9} 83.9] 82.5] 78.5] 74.2] 7 of om It is obvious from the figures given here that, because of the im- portant temperature variations of different soil regions, the micro- biological activities must be profoundly modified. But apart from the climatic variations already indicated there are seasonal variations in any particular locality that are of great moment for soil microbiological activities. Such differences are demonstrated by the temperatures of 1898 and 1902, taken to a depth of 152 mm. (6 inches), at New Brunswick, N. J. Sort TEMPERATURES*™ Jan. | Feb. | Mch.| Apr. | May | June | July | Aug. | Sept.| Oct. | Nov.| Dec. New Brunswick, N. J. (1898) ...} 33.2] 33.1] 45.1] 48.9! 59.1] 76.0] 79.3] 77.8} 72.0] 60.1] 44.6) 33.6 New Brunswick, : N. J. (1902)....| 30.7) 28.9] 41.3] 49.5] 60.4) 68.0) 72.6] 70.5] 65.9] 56.4) 48.6] 34.1 In this instance, the season of 1898 was not only earlier, but the temperatures of June to September were sufficiently higher to favor more intense bacterial growth and activity. * Recorded in Fahrenheit scale. MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 207 Earty AND Late Sorts.—Under any given climatic conditions the warming up of soils in the spring will depend on their chemical and mechanical composition, color, tillage and topography. Because of the high specific heat of water, fine-grained soils containing a relatively large amount of moisture will warm up more slowly than coarse-grained soils containing a relatively small amount of moisture. The differences in the specific heat of humus, sand, clay and chalk are less important, yet .they introduce appreciable variations in the soil temperature according to the proportion of each present. The topography of the soil introduces a factor of some importance for it affects the inclina- tion toward the sun’s rays as well as the drainage conditions. Tillage operations are of considerable moment, since they influence the rate of evaporation, that is, the rate at which heat is lost from the soil by the transformation of liquid water into vapor. Finally the color of soils exerts an influence on their temperature in that it affects the absorption and reflection of heat. Taking all of the factors together, it is found that sandy soils and sandy loams are early soils, because they part readily with their excess of water. Clay soils and clay loams are, on the other hand, late soils; it means, therefore, that in the more open soils microbial activities be- come intense earlier in the spring. Market gardeners usually attempt to improve matters still further by the use of large quantities of readily fermentable manure that develops enough heat to raise slightly the soil temperature. PRODUCTION AND ASSIMILATION OF PLtant Foop.—It was already observed by Mller that slight amounts of carbon dioxide may be evolved from frozen soil. Kostychev could detect a considerable pro- duction of carbon dioxide at 0° to 5°. Ina series of experiments carried out by Wollny the amounts of carbon dioxide produced were as follows: CO, IN 1,000 VoLs. OF AIR Water in soil 10° 20° 30° 40° 50° 6.79 per cent....... 2.03 3.22 6.86 14.69 25.17 26.79 per cent....... 18.38 54.22 63.50 80.06 81.52 46.79 per cent....... 35-07 61.49 82.12 91.86 97-48 The increased production of carbon dioxide at the higher temperatures, as shown 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 0°, 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 that at 12° nitrification is already quite perceptible; that urea bacteria grow slowly at 5°; Ps. radicicola at 4°; members of the B. subtilis group at 6° 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 render available compounds of phosphorus, potassium, calcium and mag- nesium. It is likewise evident that in warm countries bacterial activities are not only more intense at any one time, but they continue through a longer period. For this reason, the soils of the South can furnish both relatively and absolutely a greater amount of available plant food than the soils of the North. The production of plant food is necessarily followed by more vigorous growth of bacteria and of higher plants. More food is, 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 manner the soil may be dried out sufficiently to retard seriously the growth of 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 oF Sort Acipity.—Acid soils are very common in humid regions, The older soils of Europe include extensive areas whose lime MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 299 content has been restored repeatedly by the application of wood ashes, marl, oyster and clam shells, and various grades of burned or crushed limestone. In the United States acidity is becoming prevalent in many of the cultivated soils, as is shown by the investigations of the Rhode Island, Ohio, Illinois, Oregon and Florida experiment stations. These investigations, confirmed by experiments in other states, show that there is a marked removal of lime and of other basic materials from the soil as cultivation and the use of commercial fertilizers become more thorough. Knisley shows, for instance, that 38.75 per cent of the Oregon soils examined were acid, and that 16.25 per cent were strongly acid. Similarly, Blair found that of 189 samples of different Florida soils and subsoils, examined, 68.22 per cent of the former and 51.35 per cent of the latter were acid. He also found that virgin soils were less acid than cultivated soils. Causes or Sort Aciprry.—Soil acidity may be due to acids or acid salts, both inorganic and organic. Under ordinary conditions the latter are of much greater importance than the former as a cause of soil acidity. This is demonstrated by the extremely acid conditions of peat and muck soils that are particularly rich in organic acids. In soils left to themselves the formation of basic substances in the break- ing down of silicates and other compounds keeps pace with their neutralization by acid and their removal in the drainage water. When soils are placed under cultivation, lime and other bases are removed more rapidly and the inert humic acids are left behind. The loss of bases is intensified by application of acid phosphate, potash salts and ammonium sulphate, commonly used as fertilizers. This accounts for the less extensive acidity in and among virgin soils as compared with cultivated soils. Arid soils lose scarcely any of their basic sub- stances by leaching and are seldom acid. Residual limestone soils may be alkaline, neutral or acid, according to the loss of bases they have suffered by leaching. Low-lying soils, including meadows and swamps may accumulate large amounts of organic acids because of their imperfect aeration. EFrFect OF REACTION ON NUMBERS AND SpEcirs.—Some of the important groups of soil bacteria including nitro, azoto and ammonify- ing species will develop slowly or not at all, when the amount of acid in the medium is increased beyond a certain point. Hence it is realized by progressive farmers that a proper supply of lime is essential for the 300 ; MICROBIOLOGY OF SOIL satisfactory decomposition of organic matter in the soil, and the abund- 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 several years the numbers averaged about 7,000,000 per g. and reached a maximum of 22,132,000 per g. Foop SuppPLy Orcanic Matrer.—It may be said truly that a soil devoid of organic matter is practically devoid of bacteria. To the fresh and the partially decomposed organic matter (humus) the soil organisms must look for most of their food and energy. Being largely of plant origin this organic matter contains starches, fats, organic acids, higher al- cohols, proteins and amino-compounds. Because of the different relations that these vegetable substances bear to the several species of soil bacteria, a high or low proportion of starch, of cellulose, or protein must necessarily modify both numbers and species relationships. For instance, observations have been made by Coleman and others that small amounts of dextrose favor nitrification, whereas larger quantities retard it; similarly, it has been noted that in the spontaneous de- composition of protein bodies bacteria are prominent and molds absent or relatively few in numbers. But where dextrose is added to the decomposing proteins molds soon appear in large numbers. There may also be cited, in this connection, the observation of Hilgard that humus should contain at least 4 per cent of nitrogen if it is to furnish a sufficient quantity of available nitrogen compounds; otherwise, the soil bacteria seem to be unable to decompose it, so as to meet the needs of the growing plants. Many similar facts could be cited to show that as a culture medium the soil is influenced by green manures, barnyard manure, commercial fertilizers, lime, tillage and any other treatment that will modify the quantity as well as the quality of its organic matter. Tue MineraL PortTION OF THE So1Lt.—The moisture films sur- rounding the soil grains contain in solution substances derived from MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 301 these soil grains. A particle of calcium carbonate will be surrounded by a moisture film containing some calcium bicarbonate. In the same way particles of feldspar may give rise to a solution of potassium bicarbonate; particles of apatite to a solution of calcium phosphate; particles of selenite to a solution of calcium sulphate; particles of protein to a solution of ammonia, etc. In view of the fact that these reactions are more or less localized and diffusion slow, there are, un- doubtedly, in the soil minute zones where individual species are more prominent than they are in others. For example, Heinze has found it convenient to isolate Azotobacter by inoculating suitable culture solu- tions with particles of calcium carbonate picked out from the soil. Evidently these organisms were present in much greater abundance on these particles than on others of non-calcareous origin. Indeed, he occasionally obtained in this manner Azotobacter membranes that constituted almost pure cultures. The more general significance of this relation is apparent when it is remembered that nitro-bacteria are particularly favored by magnesium carbonate; tubercle bacteria by gypsum and calcium carbonate; Azotobacter by calcium phosphate and calcium carbonate; photo-bacteria by sodium chloride, etc. Considerable as must be the local differences in any one soil, they are undoubtedly even more pronounced when different soils are com- pared. Extreme conditions are met with in certain irrigated soils in which a marked concentration of salts occurs. In so far as crop production is concerned, it is stated by Hilgard that the upper limit is practically reached when the concentration of soluble salts in the irriga- tion water is about 4.55 g. (70 gr.) per gallon. Nevertheless, in Egypt and the Sahara region irrigation water is occasionally used that con- tains more than 13 g. (200 gr.) of soluble salts per gallon. Further differences are introduced by the quality of these salts, e.g., the pro- portion of sodium sulphate, magnesium sulphate, sodium chloride, sodium carbonate, etc. Again, instances are on record, as in the investi- gations of Headden in Colorado and California, where the concentration of nitrates in the soil water is so great as to kill even relatively resistant plants like alfalfa. It is to be shown by future investigations what the effect of the concentration and composition of such salts may be on the soil bacteria. , In humid soils conditions are less extreme, yet even here the variable concentration and composition of the soil solution are of direct moment 302 ; MICROBIOLOGY OF SOIL for the different microérganisms. 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. _IIl- 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 Mo tps.—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 in 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 of soil was shaken with water, and only a certain dilution. was used for inoculation; and by the direct method, wherea 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 and in Europe seem to point to the fact that many of these fungi are universal in their habitat, since the same species are recorded to have been isolated from different soil types and in different localities. Most -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, Penicillium, Cladosporium, Fusarium, Trichoderma, ' Cephalosporium, Alternaria, Zygorrhynchus, Monilia, Rhizopus, and Acrostagmus. Many other genera have been isolated, but to a more limited extent. As to the individual species occurring in the soil, Hagem, having isolated about 30 mucors from the soil, states that certain Aspergilli occur in larger numbers than all the mucors taken together. As to quantitative relations, no exact data are available. Some investigators report only several hundred fungi per g. of soil, while others record as many as 1,000,000 per g. of soil; that is the total number of spores and pieces of mycelium that develop on suitable ‘ ' MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 303 media. As to the numbers and types in relation to depth, Goddard concluded that there does not seem to be any appreciable variation in numbers at the different soil depths. Unpublished data of the New Jersey Experiment Station bring out the fact that there are very few fungi in the soil below 8 inches, and that one of the most common forms at these depths is Zygorrhynchus vuilleminii. It was formerly thought , that soil fungi are abundant only in acid soils, but recent investigations make it appear that also limed and well-cultivated soils have an abundant fungus flora. Ammontfication——Miintz and Coudon, and after them Marchal, working with pure cultures, proved conclusively that fungi decompose organic matter and cause an accumulation of ammonia in the soil. Wilson and McLean found that the forms of Moniliaare the most active ammonifiers among the several groups of organisms studied, while the Aspergilli showed the least ammonifying power. More recent work has confirmed the earlier findings and has proved that fungi may play an active part in the decomposition of organic matter, and the accumulation of ammonia. Nitrogen-fixation Experiments on nitrogen-fixation by fungi were carried on by Jodin as early as 1862. He observed a rich fungus growth on nitrogen-free media, supplied with sugar, tartaric acid, or glycerin. Berthelot, Saida, Ternetz, and others also reported fixation of atmos- pheric nitrogen through the activities of fungi, such as Aspergillus niger, Alternaria tenuis and several species of Monilia, Penicillium, Mucorini and others. But other investigators among them, Wino- gradsky, Czapek and Heinze were unable to confirm these observa- ‘tions. The careful work of Goddard has also given negative results. The entire question is therefore still an open one with the weight of evidence on the negative side. Cellulose Decomposition.—The destruction of cellulose in the soil is due to a large extent, to the activities of soil fungi, as has been demon- strated by several investigators. Cellulose decomposition by fungi was first observed in the study of plant diseases. Van Iterson used filter paper for the isolation of fungi, by exposing this medium to the air for twelve hours. Thirty-five species of fungi were isolated thus proving that a large number of cellulose-destroying fungi may be present in the air. Appel found that certain species of Fusarium destroyed in fourteen days 80 per cent of the filter paper used. Marshall Ward my 304 ‘ “MICROBIOLOGY OF SOIL and othefs recorded that a number of fungi are economically impor- tant as wood-destroyers. Spores of a pure culture of Penicillium sown on sterile blocks of spruce wood, germinated and grew normally. Sections of the wood showed that the hyphe had entered the starch- bearing cells of the medullary rays of the sapwood and consumed the whole of the starch. MacBeth and Scales found that when the medium ‘is slightly alkaline, certain aerobic bacteria will play the principal réle in the destruction of cellulose. When the medium is acid, molds and higher fungi become the active agents of destruction. They also found that the cellulose-destroying forms multiply with great rapidity in alkaline soils when cellulose in the form of filter paper is added. The power to destroy cellulose is reported for a number of species of Penicill- tum, Aspergillt, Trichoderme and other organisms which belong to the common soil forms. Though the fungi may play an important part as cellulose destroyers also in alkaline soils, in acid soils where the - activity of bacteria is greatly inhibited, fungi probably play a pre- dominant réle. This fact led Marshall to conclude in 1893 that fungi take an active part in the mineralization of the organic matter in acid humus soils. M ycorrhiza.—Apart from the so-called soil fungi, there exists another group known as mycorrhizal fungi. These live symbiotically on the roots of the higher plants. Many roots of forest trees, when examined carefully, show that there is a union between the mycelium of certain fungi, usually belonging to the fleshy fungi, and the root of the plant. This union is called a “mycorrhiza.” The fine filaments of the fungus - enter the cells of the root. These organisms were thought at first 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-called endotrophic Mvycorrhize, there are also the ectotrophic Mycorrhize which probably live only parasitically upon the roots of plants. Actinomyces.—The study of soil Actinomyces is nearly all of very recent origin. Several years ago but two soil Actinomyces had been definitely described, viz., Act. albus and Act. chromogenus. The work of Krainsky, of Conn and of Waksman and Curtis has demonstrated that Actinomyces are widely scattered in cultivated soils. The last- named investigators have shown that while the absolute numbers of Actinomyces decrease with depth of soil, their relative numbers are MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 305 materially increased so that if at a depth of 25 mm. (x inch) there are only 6 to 10 per cent of Actinomyces and 82 to 93 per cent of bacteria, at a depth of 750 mm. (30 inches) the Actimonyces form 40 to 80 per cent of the total micro-drganic flora of the.soil. The numbers of Actinomyces in the surface soil vary greatly with the types of soil and abundance of plant food. In one instance 1,300,000 Acii- nomyces were found in a total of 15,000,000 bacteria per g. of rich meadow soil. As to the activities of Actinomyces in the soil, Beyer- . inck has shown that the Acé. chromogenus produces an oxidizing sub- stance, quinon (CsH.O2) which may play an important part in the oxidation of organic matter in the soil. Munter, Krainsky and Scales have demonstrated that many Actinomyces are able to decompose cellu- lose in the soil, and that in some instances this ability is very marked. Krainsky records that soil Actinomyces need very little nitrogen for their life activities, and that they can get it from any available source. If nitrates are present, these are reduced first to nitrites, and then utilized. Waksman and Curtis working with soil sterilized by steam, did not find any great accumulation of ammonia through the activities of Actinomyces, although different species seemed toshow marked varia- tion in their power to accumulate ammonia. ALG&.—At times the influence of alge in changing the character of the soil as a culture medium for bacteria is quite considerable. As chlorophy]l-bearing organisms they are enabled to manufacture sugar and starch with the aid of sunlight, and to favor thus the development of Azotobacter and of other microdrganisms dependent for their energy on the organic matter in the soil. Investigators both in France and ‘in Germany have found that the fixation of nitrogen in sand used for pot culture experiments occurs in the surface layer possessing a growth of alge. The advocates of bare fallows attribute the greater pro- ductivity of fallowed land to the growth of alge, the accumulation of nitrogen through their influence and to other changes affecting the soil bacteria. Protozaa.—It has been shown for a long time that certain species of protozoa are common in soils and that their food consists of bacteria. To what extent protozoa play a part in soil fertility has not yet been fully explained, even though Russell and Hutchinson and of the Rothamsted Experiment Station have maintained that these minute .animals 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 fact 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 is made possible by the destruction of the protozoa (which have a lower ‘ power of resistance to heat and antiseptics than bacteria) that normally check the increase beyond a certain point. Under the conditions re- corded a causal relationship obtains between an increase in numbers of bacteria and the rate of ammonia production, which is considered to be an index of fertility. This theory has been the basis of considerable investigation, much of which has failed to corroborate the above conclusions. The fact that there is.an increase in bacterial numbers and in consequence, enhanced fertility of the soil may not be due to the elimination of protozoa but may rather be ascribed to such effects of the partial .Sterilization process as (1) increase in available food for bacteria; (2) rendering soil toxins insoluble; (3) destroying bacterio-toxins; (4) acceleration of the biological processes. It has even been noted in some instances that partial sterilization has been responsible for a decrease rather than increase in the produc- ‘tion of ammonia. Such considerations, among others, have been in- strumental in stimulating investigation in another branch of soil fertility, namely, soil protozodlogy. There has been difficulty in establishing suitable methods and technic, as for example the development of media favorable for isolation and the culture of soil protozoa; although blood meal solution, hay infusion and soil extract have been used to advantage. The organisms have been counted in the same manner as bacteria, namely, by the dilution method, or by means of a standard platinum loop. An adaptation of the apparatus used in the counting of blood corpuscles has been successfully employed by Kopeloff, Lint and Coleman. ; A study of the morphology and life history of soil protozoa reveals the fact that encystment occurs under most conditions which are not immediately favorable, as for example slight variations in moisture content, orfood. In point of fact this period of the protozoan life cycle which is analogous to the spore-forming stage of bacteria forms the basis for the question which arises as to the existence of protozoa, in their trophic form, in’ field soils. Of the well-defined groups of pro- MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 307 tozoa (page 1 30), namely, flagellates, ciliates and amoebe, many types have been described. Among those occurring frequently are: Colpoda cucullus, Boda ovatus, Colpidium colpoda, Amebe terricola, Monas, etc. The requirements for maximum development in the soil for these organ- isms are: (1) A high degree of moisture, closely approximating saturation; (2) an abundant supply of organic matter;.(3) moderate temperature. The thermal death point of active forms has been found by Goodey to be 40° to 50°, and for the cyst forms of the same organisms about 72°. The optimum temperature for most forms is about 22°. Encystment of protozoa occurs within wide limits in an alkaline medium containing up to .18 per cent NaOH, and in the presence of an acidity represented by .og per cent HCl. 5 Protozoa are found in many greenhouse soils, due no doubt to the fact that they contain a high degree of moisture and organic matter. However, in dealing with field soils some investigators have failed to isolate active forms of protozoa, whereas others record the presence of large numbers of these organisms. Their distribution appears to parallel that of bacteria, namely, the greatest number of protozoa occurs within the upper 100 mm. (4 inches) of soil, with a decrease down to 300 mm. (12 inches), which represents the lower limit of their activity. As regards the occurrence of the various groups of soil protozoa, flagellates are found to be dominant over ciliates and ameebe. G. P. Koch has found that the development of soil protozoa in artificial culture solutions varies (1) with the kind of media employed; (2) the quantity of soil used for inoculation; (3) drying of the soil; (4) different kinds of soil and different soils of the same kind; (5) the temperature of incubation. While it is generally accepted that protozoa feed upon bacteria, until the relation that obtains between the various types of protozoa and the different species of soil bacteria has been more fully investigated the direct effect of protozoa upon bacteria must remain, to a degree, indeterminate. Soil sterilization has had a practical application in eliminating various diseases in greenhouses and infested fields. Partial steriliza- tion as employed by Russell and Hutchinson while not so drastic, involves serious changes in the soil, which might be considered in much the same light as the phenomena attending complete sterilization by means of heat and antiseptics. It is an established fact that 308 MICROBIOLOGY OF SOIL _ sterilization is responsible for increased plant growth, and to explain this phenomenon the following theories have been advanced: 1. R. Koch’s theory of direct stimulation to plant. growth—a physiological effect of the sterilizing agency. 2. Hiltner and Stérmer’s theory of indirect stimulation—an altera- tion of the bacteriological equilibrium resulting in a marked develop- ment of numbers after decimation. 3. Liebscher’s view that soil sterilization may be regarded in the 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 in chemical composition is largely responsible for increased plant growth. 6. Greig-Smith and others adhering to the bacterio-toxin hypothesis. HIcHER Priants.—Higher plants modify the soil as a culture medium for bacteria in at least three ways. The root-hairs come into contact with the moisture films surrounding the soil grains and not only modify the composition of the film water, by withdrawing a portion of the dissolved matter, but also change its character by secre- tions from the roots. The changes thus effected must, necessarily, modify the character of the soil and the soil solution as a culture medium. Again, the rapid removal of water from the soil by growing crops causes the film water to become more concentrated in so far, at least, as some salts are concerned. Modifications, are, also, introduced thereby in the proportions of oxygen, nitrogen and carbon dioxide in the soil air. Finally, higher plants modify the soil environment for bacteria by their root and stubble residues. For example, residues of leguminous plants, being richer in nitrogen and possessing a narrower carbon-nitrogen ratio than the corresponding residues of non-legumes, will affect the soil somewhat differently than the latter. BactTERIA.—Occupying, as they do, the leading réle, bacteria demand a more detailed consideration, in fact,-most of the biological discussions of soil are based upon a knowledge of these organisms. Numbers and Distribution (Bacteria in Productive and Unproductive Soils).—The numbers of bacteria in soils well supplied with organic matter usually range from 3,000,000 to 15,000,c0c0 per g., as shown by the agar plate method. These numbers vary from soil to soil, and ‘from season to season for any particular soil. The numbers of fungi are also variable and may reach a total of 1,000,000 per g., although MICROORGANISMS AS A FACTOR: IN SOIL FERTILITY 309 _it still remains to be demonstrated whether the large numbers thus found represent organisms which lead an active life in the soil or only spores of fungi brought in by external agencies. The numbers of Actinomyces may reach 1,000,000 or more per g. of soil. The fungi almost disappear below 20 to 30 cm., while the actinomyces do not decrease rapidly at depths lower than 30 cm. Distribution at Different Depths —Most of the soil bacteria are found in the stratum in which the organic residues are concentrated, that is, in the surface soil. Immediately at the surface the rapid evaporation and the germicidal effect of direct sunshine act as disturbing factors, hence the numbers in the uppermost 25 to 50 mm. (1 to 2 inches) are smaller than.in the layer of soil immediately below. Beyond the depth of 20 cm. or 22 cm. (8 or g inches) the numbers diminish rapidly. Material from a depth of .6 m. to .g m. (2 to 3 feet) is nearly sterile in humid regions. Differences occur, however, in keeping with the mechanical composition of the soil. In light, open soils the bacteria are not only carried down to greater depths by the percolating water, but can also multiply there, thanks to better aeration. On the con- trary, fine-grained compact soils are more effective in holding back suspended material and do not allow the bacteria to pass downward as readily. Moreover, the less thorough aeration of these soils and the accumulation of toxic reduction products in the subsoil serve as an effective check in the increase of bacteria in the deeper layers. In irrigated soils of the arid and semi-arid regions bacteria are dis- tributed at much greater depths. Their occurrence 2 m. to 3 m. (8 or to feet). below the surface is made possible not only by the better aeration of these soils, but by the penetration of roots to great depths and the accumulation there of considerable amounts of organic matter. The practical significance of distribution appears, among other things, in the use of soil for inoculation purposes; for instance, it is reported by Salstrém that in making peat soils arable the addition of small amounts of fertile loam increases to a very marked extent their crop-producing power. The efficiency of the inoculating material decreases as it is taken from the deeper soil layers. Similarly, in the use of alfalfa soil for the inoculation of new fields the most efficient material is secured at a depth between 7.62 cm. and 17.78 cm. (3 and 7 inches). Seasonal Variations of Bacterial Numbers and Activities—Conn has reported an apparent increase of bacteria in frozen soil. This increase 310 MICROBIOLOGY OF SOIL =f t seems to be due to an actual multiplication of the organisms rather than to a mere lifting of the bacteria from lower depths by capillary action. The greatest increase was found to occur during the winter in the slow- . growing bacteria and not in those that liquefy gelatin rapidly or in the Actinomyces. Conn tries to account for the phenomenon by assuming the existence of two groups of bacteria, winter and summer bacteria. The latter, he thinks, prevents the former from multiplying rapidly in warm weather. Hence, the increase in frozen soils is not to be ‘p&cribed directly to the low temperature, but to the depressing effect of the cold upon the summer bacteria. Brown found that the soil bacteria diminish during the fall season with the lowering of the temperature, but, when the soil is frozen, an increase in numbers occurs. He also found frozen soils to possess a much greater ammoni-. fying, denitrifying and nitrogen-fixing power than non-frozen soils. According to him, the lowering of the freezing-point of the capillary water, due in part to the concentration of salts at the time of freezing, may account for the abnormal bacterial activities. Morphological and Physiological Groups (Morphological Groups).— Rod-shaped organisms are numerically the most prominent among soil bacteria. They occur at times to the extent of 80 or go per cent of the total number. Spherical organisms usually constitute less than 25 per cent of the bacterial flora. Spirilla and sarcine are present in slight numbers. Conditions may occur, however, when the proportion of spherical organisms is markedly increased. This happens, par- ticularly, when large quantities of composted manure (rich in spherical organisms) is added to the soil.- Among the rod-shaped species B. mycoides, B. subtilis, B. mesen- tericus, B. tumescens and other members of the subtilis group are quite prominent. Members of the amylobacter group are seldom absent. Members of the proteus group and various fluorescens are always present, while Bact. erogenes and allied species are common inhabitants of the soil. (Physiological Groups).—In the decomposition of organic matter in the soil certain important changes in both nitrogenous and non-nitro- ~ genous material are accomplished by definite groups of bacteria. The breaking down of protein substances is accomplished by the forma- tion of ammonia, nitrites and nitrates. These in turn may be trans- formed back into more complex amino-compounds, peptones, and pro- MICROORGANISMS AS A FACTOR IN SOIL FERTILITY air teins, or they may be destroyed with the evolution of free nitrogen’. Moreover, there are groups of bacteria capable of joining non-nitro- genous organic matter to elementary nitrogen and of producing thus nitrogen compounds. Again, there are groups of bacteria bearing distinct and important relations to the decomposition of cellulose, or the transformation of its cleavage products, methane and hydrogen. There are, likewise, definite groups of bacteria concerned in the transformation of sulphur and its compounds, and of ferrous compounds.. METHODS OF STUDY QUANTITATIVE RELATIONS.—Since the early work of Koch in 1881 many investigators have determined the number of bacteria in soil samples, by means of the plate method. It is well known, however, that on ordinary gelatin or agar plates kept under aerobic conditions but a fraction of the soil organisms produce visible colonies. The anaerobic species do not appear, nor do aerobic Azotobacter, and nitro-. bacteria, while other common soil organisms form colonies sparingly or not at all. By employing synthetic agar media instead of beef broth gelatin or agar, Lipman and Brown have succeeded in securing the growth of a much larger number of colonies from any given quantity of soil, yet even these larger numbers were incomplete for reasons mentioned above. H. Fischer recommends a simple medium of agar to which nothing has been added but soil extract (prepared by extracting with a .1 per cent solution of Na2CO;) and potassium phosphate. Following the path of Lipman and Brown in reducing the content of organic matter, Temple employed 1 g. of peptone per 1. as a culture medium and obtained satisfactory results. Brown has further modified the formula of Lipman and Brown by replacing the .o5 g. of peptone with 1g. of albumin, and obtained results which were somewhat superior. In a comparison of culture media, Conn considers the former media undesirable for quantitative purposes because they contain substances of indefinite chemical composition, and offers an agar medium con- taining no organic matter except agar, dextrose and sodium asparag- inate, and also a soil-extract gelatin which is valuable for qualitative purposes. Another medium that has been suggested, after a com- parison 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 results secured in the counting of soil bacteria have only a relative value. With the same media and methods some information may be secured concerning the influence of fertilization, tillage, liming, etc., on certain of the soil bacteria. But even this information must be properly discounted, since equal numbers do not necessarily mean equal amounts of chemical work accomplished; for example, there is no certainty that 1,000,000 of decay bacteria derived from one soil will accomplish exactly as much decomposition as the'same number of siniilar organ- isms from another soil. Otherwise stated, individual cells differ in their physiological efficiency from other cells of the same species. QUALITATIVE REAcTIOoNS.—By modifying the composition of the culture media different physiological groups may be favored in their development. In this manner the silica jelly medium proposed by Winogradski, or the gypsum plates proposed by Omelianski may be em- ployed for making numerical comparisons of nitro-bacteria in different soils. In like manner Beijerinck’s mannit agar may be used for the numerical comparison of Azotobacter, and other media could be adapted for the quantitative-qualitative determination of urea, denitrifying, methane, and still other physiological groups of microérganisms. There is no doubt that the quantitative-qualitative method just out- lined may be made to yield valuable information. Yet it, too, possesses defects already noted in connection with the more strictly quantitative method. Apart from the vast amount of work involved in the prepa- ration of a large number of media and in the counting of colonies on many plates, this method fails to indicate differences in physiological efficiency. Furthermore, the colonies of the specific organisms sought are almost invariably accompanied by those of foreign species not always easily distinguished. With these limitations properly recognized and with further improvement in the constitution of special media the method may be made useful in supplementing data secured by other methods. TRANSFORMATION REACTIONS.—Instead of counting soil bacteria in accordance with colonies produced in general or special media, soil investigators have attempted to measure the bacteriological functions of soils by comparing more or less definite quantities of the latter under known conditions. This method was employed by Wollny and others in studying the factors that affect the formation of carbon dioxide in MICROORGANISMS AS A FACTOR IN SOIL FERTILITY 313 soils. It was also used by Schloesing and Miintz and their followers in similar studies on nitrate formation. A method somewhat similar in principle but different in its application was proposed by Remy in 1902. He suggested the use of special media for the quantitative estimation of different physiological reactions; thus, making a 1 per cent solution of peptone and inoculating with equivalent quantities of soil; he caused the decomposition of the peptone and the formation of ammonia, and secured comparisons of the ammonifying power of different soils. In a similar manner he used special solutions for com- ' paring quantitatively the transformation accomplished by nitrifying, denitrifying or nitrogen-fixing bacteria. Remy’s method has been extensively tested by Léhnis, Ehrenberg, Lipman and others. It has been shown to possess a serious defect in that it deals with conditions unlike those occurring in the soil itself. For this reason more recent investigations have been carried on in weighed portions of soil rather than in culture solutions inoculated with to per cent of soil as is done in Remy’s method. RATE OF OXIDATION OF CarBon.—The rate of decomposition of humus or of other organic matter in the soil may be measured, as was done by Wollny, by determining the amount of carbon dioxide evolved in weighed quantities of material kept under definite conditions. The influence of temperature, moisture, aeration, organic matter, anti- septics, etc., has been determined in this manner. The same method may be used in studying decay, and factors influencing decay, in soils in the field. a: | More recently Russell and his associates have modified the method in that they have determined the rate of oxidation of carbon not by measuring the carbon dioxide evolved, but by estimating the amount of oxygen absorbed. In either case decay is measured from the carbon standpoint. The method based on this principle should find wide application in future soil fertility investigations. RaTE OF OXIDATION oF NirroGEN.—Another method or series of methods for studying decomposition processes in the soil may be based on the determination of nitrogen compounds formed in the breaking down of proteins. ‘Two of the derivatives of protein, namely, ammonia and nitrate, have been used successfully to gauge the decomposition of organic matter in the soil. The recent results secured by Lipman and ‘his associates demonstrate that ammonia formation from dried blood in 1 314 MICROBIOLOGY OF SOIL weighed quantities of soil may serve as a very accurate measure of decay from the nitrogen standpoint. Corresponding determination of nitrates may similarly be employed in tracing protein cleavage and transformation as influenced by the various factors of season, soil and cultivation. ADDITION oF NirrocEN.—At least one other bacteriological factor in soils should be mentioned here as deserving attention in a systematic study of soil fertility from the nitrogen standpoint. It is known that Azo-bacteria are widely distributed in arable soils, and that they are more prominent in some regions than they are in others. The student of soil fertility finds it desirable, therefore, to study azotofication in ‘different soils, and employs (for this purpose) mannit solutions like those proposed by Beyerinck, sand cultures supplied with sugar solu- tions like those proposed by Fischer, or weighed quantities of soil mixed . with sugar as suggested by Koch. The methods referred to above make possible thus the study of ammonification, nitrification and azotofication under controlled con-. ditions and permit, thereby, the measure of bacteriological factors in soil fertility from the nitrogen standpoint. Reactions ConcerNninc Catcrum, MaGNEstuM, SULPHUR AND ° PuHosPHoRvs.—In addition to the purely chemical methods available for the study of these constituents, microbiological methods have also been suggested. In some of his still unpublished experiments with A zoto- bacter Lipman employed solutions of mannit in distilled water, provided -with small quantities of sterile soils which were to supply the organisms with the essential mineral constituents. In’ this manner interesting data were secured on the availability of phosphorus compounds in different soils; similarly, Christensen has suggested the use of Azoto- bacter for determining the lime requirements of soils, and Butkevich has experimented with cultures of Aspergillus niger in determining the availability of the mineral constituents. j CHAPTER II THE DECOMPOSITION OF ORGANIC MATTER IN THE SOIL CARBOHYDRATES Origin ——The sugars, starches, vegetable gums and allied pectine substances, as well as the cellulose, contained in roots and other crop - residues add large quantities of carbohydrates to the soil. The crop ' residues are augmented still further by green manures’ and animal manures whenever these are used. A good growth of timothy, for ‘example, may increase the content of organic matter in the surface © soil by 250-500 kg. (500 or 1 ,000 pounds) per acre, and three-quarters of this consists of carbohyrdates. In the same manner, a green ma- nure crop, or an application of barnyard manure may add to the land as much as 1,500 pounds, or even more, of carbohydrates per acre. These carbohydrates contain a large proportion of cellulose. The Decomposition of Cellulose—-Pure cellulose (page 167), (CeHi00s), is a rather inert substance, as exemplified by the resistance of cotton and flax fiber to decomposition processes. It is well known, nevertheless, that even cellulose is in the end decomposed and resolved into simple compounds. Plant roots, leaves and stems, as well as the trunks of fallen trees, gradually disintegrate and vanish. Under favor- able conditions this may proceed rapidly, as is indicated by the process in septic tanks, or in manure heaps on the one hand, and in open sandy soils on the other. The disappearance of cellulose may be ac- complished by (a) anaerobic organisms, (b) by aerobic organisms, (c) by denitrifying bacteria, and (d) by molds. The Production of Methane and Hydrogen—The decomposition of pure cellulose and the. formation of methane and hydrogen mixed with other gases was first noted by Popov in 1875. Some years later Tappeiner and also Hoppe-Seyler confirmed Popov’s observa- | tions that nearly pure cellulose in the form of Swedish filter-paper, or cotton fiber may be fermented by bacteria with the evolution of methane, carbon dioxide and occasionally also of hydrogen. These 315 316 MICROBIOLOGY OF SOIL investigators ascribed the decomposition of cellulose to ‘an organism found by Trécul in decomposing vegetable materials, and named by. him Amylobacter in 1865, because of the blue color assumed by it when stained with iodine. Subsequent investigations by Omelianski begun in 1894 and con- tinued through a period of years demonstrated the presence of specific anaerobic organisms in decomposing cellulose. He described two dis- tinct species of long, slender bacilli, assuming the clostridium form when in the spore stage. Morphologically the organisms can hardly be dis- tinguished, but physiologically they show important differences in that one causes the fermentation of cellulose with the production of gases consisting of carbon dioxide and methane, while the gasés produced by 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 not belong, therefore, to the butyric bacilli designated as Amylobacter by ___ earlier investigators. Omelianski’s investigations make it appear that the butyric organisms are not capable of fermenting cellulose proper. In culture solutions containing mineral salts and nitrogen in the form of ammonium compounds the decomposition of filter-paper and other forms of cellulose proceeds with considerable rapidity. Calcium car- bonate must be added to neutralize the acids formed, otherwise the fermentation soon comes toa standstill. In one of Omelianski’s experi- ments begun in October, 1895, and ended in November, 1896, 3.3471 g. of cellulose was decomposed by a nearly pure culture of hydrogen bacilli. The products consisted of 2.2402 g. fatty acids, .9722 g. carbon dioxide and .0138 g. of hydrogen, a total of 3.2262 g. which nearly accounts for all of the cellulose destroyed. The fatty acids were made up of butyric and acetic acids with a slight proportion of some higher homologue, probably valerianic acid. In a similiar experiment with an apparently pure culture of the methane bacillus, begun in December, 1900, and ended in April, 1901, fermentation began after an incubation period of about one month, and the entire volume of gas gradually evolved was 552.2 c.c. This mix- ture consisted of 190.8 c.c. methane and 361.4 c.c. carbon dioxide. The products formed from the 2.0065 g. cellulose consumed included 1.0223 g. fatty acids, .8678 g. carbon dioxide and .1372 g. of methane, or a total of 2.0273 g. The slight difference in weight in favor of the DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 317 fermentation products falls within the limit of error. These’ experi- ments shew that about one-half of the fermentation products is gaseous and that the other half consists of acetic and butyric acids. The Oxidation of Methane, Hydrogen and Carbon Monoxide.—Aside from cellulose, methane may also be produced from various other carbo- “hydrates, organic acids and proteins. Large amounts of methane are thus contributed to the atmosphere by swamps, manure heaps and low- lying meadows. In a purely chemical way methane may also be set free from volcanoes and mineral springs. The constant additions of methane, ethane, hydrogen and carbon monoxide represent a consid- _ erable amount of potential energy. It is important to know, therefore, whether these materials are at all utilized. That methane may be utilized by bacteria as a source of energy was first shown by Sdéhngen in 1905. He isolated an organism named by him B. methanicus that showed itself capable of growing in inorganic solutions confined over an atmosphere of methane, oxygen and nitrogen. The methane gradually disappeared and there were formed considerable quantities of organic matter. The ability to oxidize methane has been claimed for a number of other organisms by Sdéhngen and others. Early observations on the ability of moist soil to cause the oxidation of hydrogen are credited to de Saussure (1838). Many years later (1892) Immendorff called attention to the same fact. It was not, however, until 1905 that the oxidation of hydrogen was shown to be a specific biological process. In that year papers by Séhngen and Kaserer reported experiments wherein inorganic solutions confined under an atmosphere of hydrogen, oxygen and carbon dioxide and inoculated with very small quantities of soil developed a bacterial membrane at the surface. The hydrogen was oxidized and organic matter produced at the expense of the energy set free. The observations just noted have been confirmed by other investigators, by means of mixtures and single species of soil bacteria. Finally it should be added here that B. oligocarbophilus previously isolated by Beijerinck and Van Delden is able, according to Kaserer, to oxidize also carbon monoxide. THE CLEAVAGE AND FERMENTATION OF SUGARS, STARCHES AND Gums.—Sugars (page 163) are a very acceptable source of food and energy for soil bacteria. A culture solution containing’ suitable mineral salts and sugar ferments readily when inoculated with a small amount of fresh soil. When no combined nitrogen is added, Azotobacter, or B. 318 MICROBIOLOGY OF SOIL t (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-butyl’ alcohol are common cleavage products. , In the case of starch, pectins and pentosans, similar conditions hold good. Diastatic enzymes seem to be produced by various bacteria, as well, as molds and streptothrices. Members of the subtilis group and B. fluorescens seem to be able to transform starch into sugar with- out difficulty. It needs hardly be added here that the vast quantities of organic acids and of carbon dioxide thus formed must play, an im- portant réle in the breaking down of the mineral constituents in the soil. Fats AND WAXES , Origin and Decomposition—Plant substances contain varying proportions of fats and waxy materials. In the dry matter of grasses and cereal straw crude fat is usually present to the extent of 1.5 to 2.0 per cent. In hay made from clover and other legumes the propor- tion of crude fat is rather more than 2 per cent. In cereal grains it may range up to 4 or 5 per cent while in soy beans the content of crude fat is 19 per cent, in germ oil meal 22 per cent and flax seed meal 34 per cent. Under the influence of enzymes produced by molds, yeasts and bacteria the fatty acids occurring as glycerides are decomposed into glycerin and fatty acids. The extent of fat decomposition, brought about largely by molds in the opinion of some, is shown by numerous experiments with peanut cake, olive press cake, cottonseed meal, almond oil, corn meal, etc. In a number of these experiments Asper- gillus niger seemed to be particularly efficient in decomposing fats. Analogous decomposition processes may occur in the soil as proved by the experiments of Rubner. té DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 319 ORGANIC ACIDS Source.—The cleavage products of proteins include large quantities of amino-acids. The latter are still further transformed and yield a variety of fatty acids. The carbohydrates being present in larger quantities than the proteins are still more important as a source of organic acids. Finally, the fats, gums, and higher alcohols contribute additional quantities of the latter. Among the more simple acids, acetic, propionic, butyric, succinic and lacticare common. The extent of acid production was already indicated in connection with cellulose decomposition by the methane and hydrogen bacilli. Apart from these organisms, organic acids are formed by nearly every important species of soil bacteria; moreover, the tissues of dead plants and animals are not the sole source of organic acids in the soil. According to Stoklasa conditions may occasionally occur in the latter, especially when atmospheric oxygen is excluded, that favor the excretion by plant roots of appreciable quantities of acetic acid. Transformation and Accumulation.—Salts of organic acids are suitable as food for a wide range of soil bacteria. Azotobacter will readily make use of acetates, propionates and butyrates. A number of denitrifying bacteria will grow vigorously with citrates as the only source of organic nutrients. The fermentation of lactates by butyric bacteria has been known for a long time. The decomposition of malates, succinates, tartrates and valerates may be accomplished by various species, and even simple compounds like formates may yield food and energy to certain soil bacteria, among them B. methylicus studied by Loew and his associates. It is evident, therefore, that organic acids are not liable to accumulate in well-ventilated soils. Molds, as well as bacteria, destroy them rapidly, and carbonates, carbon dioxide and water are the final products of the decomposition of non-nitrogenous organic matter. Notwithstanding the ready decomposition of the more simple organic acids in the soil, it is well known that arable soils are frequently acid. This acidity is largely due to the so-called “humic acids,” organic compounds whose composition is not well understood. Théy are composed, to some extent, of rather complex organic acids or of their acid salts. Continued cultivation seems to favor the accumulation of these acid compounds, partly on account of the diminished supply of lime and of other basic materials in older soils. When these soils are 320 MICROBIOLOGY OF SOIL ¥ limed the humic acids and acid humates are changed into neutral com- pounds and are then subject to more rapid decomposition by micro- érganisms. According to the investigations of Blair the average acid -soil in Florida requires 1,500 pounds of lime (CaO) per acre to neutralize the acidity to a depth of 84 mm. (9 inches). This means an acidity equivalent to more than one ton of hydrochloric acid per acre. In peat and muck soils the acidity is equivalent to many times this amount of hydrochloric acid. PROTEIN BoDIES Amount and Quality—The protein content of farm crops that leave residues in the soil is variable, but in all cases quite considerable. Dried corn stalks contain 5 per cent of protein, timothy hay 6 per cent, red clover hay 12 per cent or more, alfalfa hay 15 or 16 percent. Even wheat and rye straw may contain as much as 3 per cent of protein. Cotton-seed meal and other oil cakes, tankage, ground fish, hair and wool waste and dried blood (all used more or less extensively as sources of nitrogen to crops) are made up in a large measure of protein compounds. Being derived from plant residues, from microérganic, insect and animal remains, and from fertilizers and manures applied, the nitrogen in the soil humus exists, for the most part, in the form of protein com- pounds. Hilgard reports the following humus and nitrogen content, as based on the analyses of a large number of samples of humid, semi-arid and arid soils. (Nitrogen in | (Nitrogen in - ae ee Arid uplands:: s sce cecus yews cece eeoes execs 0.91 15.23 0.135 Sub-irrigated arid soils................0004. 1.06 8.38 0.099 Humid soils from humid and arid regions CCAlUOTHIA) joe ctaiaicind ie haat Rasavreses ancuene 2.45 5.29 0.135 Humid soils from other states............... 7.01 3.78 0.295 Taking the weight of an acre-foot of dry soil at 2,000,000 kg. (4,000,000 pounds) and multiplying the nitrogen by 6.25 (the factor usually employed for converting nitrogen into protein) we find the protein content of these soils to range from about 11,339 kg. (25,000 DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 321 pounds) per acre to nearly three times as much. Similarly, the Illinois Experiment Station reports quantities of nitrogen equivalent to 3,175 to 4,989 kg. (7,000 to 11,000 pounds) per acre to a depth of tor.6 cm. (40 inches) in gray silt loams, of the lower Illinoisan glacia- tion. In the brown silt loams the amount of nitrogen to the same depth is usually more than 4,535 kg. (10,000 pounds) per acre; occasionally it is more than 9,071 kg. (20,000 pounds) per acre. In one instance a black clay loam of the late Wisconsin glaciation is reported to have about 13,154 kg. (29,000 pounds) of nitrogen per acre, to a depth of to1.6 cm. (40 inches). This would be equivalent to more than 81,646 kg. (180,000 pounds) of protein; of course, not all of the nitrogen in the soil exists in the form of protein, some of it occurring as amino-com- pounds, and a small portion as ammonia and nitrates. Nevertheless, by far the greatest part of it occurs as protein compounds. The protein compounds of the soil humus must be considered from the standpoint of quality as well as from the standpoint of quantity. It is well known that fresh plant residues are attacked more readily by microérganisms than older plant substances. For this reason soils frequently supplied with fresh organic material supply greater amounts of available food to crops than similar soils whose organic matter con- sists, largely of older residues. ‘ Carbon-nitrogen Ratio—The decomposition of organic matter is readily influenced by the relative content of nitrogenous and non-ni- trogenous compounds. Substances of animal origin yield relatively and absolutely more available nitrogen in a given length of time than sub- stances of plant origin. The difference noted is due largely to the greater proportion of protein in the animal materials; in other words, to the narrower carbon-nitrogen ratio. On this basis Hilgard attempts to explain the adequacy of the small proportion of humus in arid and semi-arid soils. Because of the narrower carbon-nitrogen ratio the humus compounds in these soils are decomposed with greater rapidity and yield a sufficient amount of ammonia and nitrate to supply the needs of the crop. But when plant substances alone are considered the statement just made requires qualification. It is true that cotton-seed meal or linseed meal, having a narrower carbon-nitrogen ratio, will decay more readily than corn-meal or wheat flour. It is also true that any given plant sub- stance, as it undergoes decay, will lose in proportion more carbon than BT 322 MICROBIOLOGY OF SOIL nitrogen. Older humus has, therefore, a narrower carbon-nitrogen ratio than humus of recent origin. The former is more resistant to. decay, however, than new humus. In a concrete way, on the other hand, it may be stated that fresh vegetable material of a narrow car- bon-nitrogen ratio will decay more rapidly than fresh vegetable material of a wide carbon-nitrogen ratio. The reverse, nevertheless is true of vegetable materials in advanced stages of decay. Under any given climatic conditions and in any given soil type, the carbon-nitrogen ratio may give important indications only as to the availability of the humus nitrogen. Lawes and Gilbert, as quoted by Hall, found the following carbon-nitrogen ratio in the organic matter of different soils: Cereal roots and stubblev.es ss asena coun a niew ey stemed esas 43.0 Leguminous stubble........ 0... cece eee e ee eee eee eens 23.0 DUNG ite 55 a sacerone cal ecaees Giaversal d ete Readies s BW Ridiisas hoon uae Ree 18.0 Veeryold grass Lads cc sini. 2acepucceus. tt oka birth nix ued ean aeea 13-7 Manitoba prairie soils........ 0.0... cece eee eee 13.0 Pasture recently laid down....... 00... cece eee eee eee eee! 11.7 Arable Soles ijcass's's's ae avgdiiew nave aah eatda Pugni a aeauee mes 10.1 Clay subsolli socio uga-s wsvmea see ete ve 2a eA ete te eds bes 6.0 Hall concludes, therefore, that humus with a wide carbon-nitrogen ratio is more valuable than humus with a narrow carbon-nitrogen ratio, since the latter will beattacked more easily by the soil bacteria. Brown and Allison indicate that there might be a possibility of applying ma- terials of a wide carbon-nitrogen ratio to supply the deficiencies of organic matter on the basis that the former may have the same or better effect on bacterial activities such as azofication, or non-symbiotic nitrogen fixation. THE TRANSFORMATION OF NITROGEN COMPOUNDS AMMONIFICATION. Experimental Study—By ammonification is meant the production of ammonia by bacteria out of protein substances or their cleavage products. That ammonia production in the soil is a biological process was first demonstrated by Miintz and Coudon in 1893. These investigators showed that no ammonia is formed in sterile soils. They also showed that ammonia may be produced out of nitro- genous organic matter by molds as well as by bacteria. Marchal not only confirmed these observations, but proved that various micro- drganisms differ markedly in their ability to produce ammonia. Of the several species of bacteria tested by him, B. mycoides (one of the DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 323 common soil bacteria) proved itself particularly efficient in the breaking down of nitrogenous materials and the production of ammonia. Since the publication of these experiments a large number of investi- gators, both in Europe and America, have studied ammonia production in culture solutions as well as in the soil itself. It has been shown that under favorable conditions the breaking down of protein compounds and the formation of ammonia may be very rapid; for instance, im some ex- periments carried out by Lipman and his associates the following pro- portions of nitrogen were transformed into ammonia in the course of six days: | De1Ed: BLOGs <5.5::4 wiainse see his ave aed Siewsbans osha odd alauapncben 16.74 per cent Concentrated tankage................ sce ee ee ene 56.66 per cent GO uiT ASIN 25. erst dase cavitcS Sodas ipee eca-hee ovate 47.16 per cent Cotton-seed meal... 2... cece eee eee eee eee 4.95 per cent Bone meal ss ciaanienne grace nndeg nedoaas aodew en 16.65 per cent Cow manure, solid and liquid excreta.............. 32.60 per cent Cow manure, solid excreta..............0. ee eeee 5.39 per cent The experiments were carried out in equal quantities of soil and with equivalent quantities of nitrogen in the different substances. It will be observed that more than 56 per cent of the nitrogen in the con- centrated tankage was transformed into ammonia, whereas under the same conditions cotton-seed meal yielded less than 5 per cent. Mechanism of Ammonia Production —The relatively large protein molecules are readily broken into larger or smaller fragments. This may be accomplished by purely chemical means, as, for instance, by boiling with acids or alkalies, or by biological activities. Among the first cleavage products albumoses and peptones are quite prominent. These in turn undergo further cleavage and the various amino-acids and their derivatives, as well as ammonia, make their appearance. In so far as the different species of bacteria are concerned, ammonia pro- duction seems to depend, to a marked extent, on the ability to secrete proteolytic enzymes. With the aid of such enzymes the proteins are more readily hydrolyzed and further changed into amino- and hydroxy acids, ammonia and carbon dioxide. Influence of Soil and Climatic Conditions —Ammonia production in the soil is affected by (a) its mechanical and chemical composition; by (6) the amount and distribution of rainfall; by (c) the prevailing tem- peratures; by (d) fertilizer treatment; and by (¢) methods of tillage and ’ 324 MICROBIOLOGY OF SOIL cropping. The mechanical composition of the soil influences the pro- portion of aerobic and anaerobic species, while the chemical composi- tion, particularly that of the humus, influences the rate of multiplica- tion and the character of the chemical transformation accomplished. It is well known, for example, that additions of fresh organic matter intensify the rate of decomposition of the soil humus, and, likewise, ammonia production as has been already demonstrated by Breal. Ina more general. way it was proved by Lipman and his associates that, with a constant bacterial factor, ammonia production in soils varies with the chemical and mechanical composition of the latter. In some of these experiments 100 g. portions of different soils were each mixed with 5 g. of dried blood, sterilized in the autoclave, cooled and inoculated with equal quantities of infusion from fresh soil. The following amounts of ammonia nitrogen were produced in six days: Soil Ammonia nitrogen found Are a AN aN due oD uneieh des SUNDER Mae ANAT aay an 31.62 mg. TB yachserecatess taste emcee chai states the a eneaiate Su de thee 68.29 mg. Cotenig texte a Ae Gane Rie Slap a Reon eS 117.06 mg. . Dingoes Gite socee aes oe ais Md econ aes eta a cere 107.16 mg. Hide eased esos SARE Re Rea MBE eaeess DARE EaR EAE 156.47 mg. With all other factors constant, chemical and mechanical differences ‘in the soil used were responsible for striking variations in ammonia production, as indicated by the figures given above. The influence of temperature and moisture conditions is fully as important as that of the chemical and mechanical composition of the soil. The following data secured by Lipman may be cited in this connection as showing the effect of moisture: One-hundred-gram quantities of air-dried soil were each mixed with ' 3 g. of dried blood and varying amounts of water added. The ammonia formed was distilled off and determined at the end of eight days. The amounts of ammonia nitrogen found were as follows: Water added Ammonia nitrogen found O CG@iive veae es 2 WR) tiee ee be pees ele aa ee oe 4.13 mg. T OSies ves vues: One Ree R ARR eRe LOD WORT Hs 4.13 mg. Bie ConA RAST GA DEE st Gamer adda, ermtandicts. aa uee Merigtus 5-40 mg. OG, Coens rn cee th RAD dete hata M aistse AUC MURA san ae wala 10.64 mg. PCC piss CRG RRL TR Se aE A Rea Dente Rob 26.37 mg. TOW eg peek ck dea g we ceeoheecnree ds hae gree 49.57 mg. 12 CGiex i eaghe ee dened eee exer e eee e eed Ee eR 70.71 mg. 15 GCracecoseeacers eee awed esse eee yea gees 93.90 mg. DECOMPOSITION OF ORGANIC MATTER IN THE SOIL ' 325 It appears, therefore, that ammonia production in soils rises or falls as the rainfall or irrigation is increased or decreased, or as the soil water is more or less thoroughly conserved by proper methods of tillage. In the same way, seasons of high temperature favor ammonification while seasons of low temperatures discourage it. This point is well illustrated by the observations of Marchal that at 0° to 5° only traces of ammonia were formed in his culture solutions; that at 20° ammonia production was quite marked, and that at 30° the maximum was reached. More- over, apart from the seasonal variations in any one locality, there is a wide range in ammonia production, as we pass from the torrid to the temperate and from the latter to the frigid zones. Species and Numbers ——Ammonia production is a function common to most soil bacteria. Already in the earlier experiments of Marchal, seventeen out of the thirty-one species tested were found capable of producing ammonia. Prominent among these ammonifiers were B. mycoides, B. (Proteus) vulgaris, B. mesentericus vulgatus, B. janthinus, and B. subtilis: Of a considerable number of soil bacteria tested by Chester all but one were observed to produce ammonia. In Gage’s experiments with sewage bacteria, seventeen out of twenty species tested proved to be ammonifiers. Similarly, a number of species tested by the writer, among them B. coli, B. cholere suis, B. (Proteus) vulgaris, B. subtilis, B. megaterium, etc., all produced ammonia in meat infusions. A-mass of additional data, accumulated by different investigators, furnish further proof that ammonia production is a common function of soil bacteria. é The more prominent ammonifiers, including members of the B. subtilis group and certain streptothrices, are numerically important in all arable soils. Their numbers are affected, however, by the amount _and composition of the soil humus. It has been found, for instance, that additions of straw and of strawy manure increase markedly the numbers of B. subtilis and of other members of the group. An increase in the numbers of certain ammonifiers is caused also by additions of lime or of green manure. For example, in experiments carried out by Lipman and his associates portions of fertile soil inoculated with B. mycoides were found to contain, a month later, 2,000,000 of bacteria per g. of soil. In similar soil portions that had also received additions of grass the number was twice as great. 326 MICROBIOLOGY OF SOIL Relative Efficiency of Different Species—In Marchal’s experiments already referred to, the species employed showed marked differences in their ability to produce ammonia out of egg albumin. The following proportions of the protein nitrogen were converted into ammonia in twenty days: . B. mycoides.........4.. 46 per cent B. subtilis....... Tegteeg es 23 per cent B. (Proteus) vulgaris..... 36 per cent B. janthinus..........006 23 per cent B. mesentericus vulgatus.. 29 per cent B. fluorescens putidus..... 22 per cent Sarcina lutea........... 27 per cent B. fluorescens liquefaciens . 16 per cent Furthermore, apart from the variations from species to species, differ- ences have been observed by Marchal and many other investigators between one strain and another of any single species isolated from the same or different soils. It must be remembered, therefore, that in the study of ammonification in soils and culture solutions, due considera- tion should be given to differences in physiological efficiency as they are manifested by strains and species of microdrganisms. Apart from the ammonifying bacteria already mentioned there is a group of organisms studied by Miiller, Pasteur, van Tieghem, Leube, _ Miquel, Beyerinck and others. These are the so-called urea bacteria, capable of intensive transformation of urea and allied compounds into ammonium carbonate, by means of the enzyme urease. CO + 2H.0 = (NH,)2CO; | NH, Morphologically these organisms include spherical and rod forms, spore-bearing and non-spore bearing species. Most of the urea bacteria are particularly prominent in the transformation of animal manures. Ammonifying Efficiency—Lipman and Burgess have found marked differences in the ammonifying efficiency of fifteen organisms in pure cultures using peptone, bat guano, sheep and goat manure, dried blood, tankage, cottonseed meal and fish guano. The nature of the soil as well as the nature of the nitrogenous material markedly modify anorganism’sammonifying power. B. twmescens on the whole appears to have been the most efficient organism tested, Comparing these findings DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 327 with those of Marchal the former have obtained results in soils, while the latter’s work was with solution cultures, the application of which to soil conditions is not always permissible. In point of fact the am- monifying efficiency of organisms is greater in sandy soil and possi- bly in others than in solutions, as Lipman and Burgess have obtained a transformation of 41.98 per cent of peptone in nitrogen and 36.06 per cent of bat guano nitrogen into ammonia by Sarcina lutea and B. mycoides, respectively, in twelve days at temperatures between 27° and 30°., while Marchal. obtained similar transformations in thirty. days at 30°. in albumen solutions. f It is also of interest tonote that investigations with soil fungi have revealed the fact that certain species are even more efficient am- monifiers than B. mycoides. McLean and Wilson, Waksman, Cole- man and Kopeloff have worked with organisms like Trichoderma koeningi which is capable of transforming more than 50 per cent of the nitrogenous material added in such experimentation. NITRIFICATION. Experimental Study.—The term nitrification refers to the oxidation either of ammonia or of nitrites to nitrates. In a broader sense nitrification may be defined as the production of nitrates from decomposing organic matter. Saltpeter or niter, the terms formerly applied to potassium nitrate, possessed, for a long time, a peculiar interest because of its relation to gunpowder. Whether it be true or not that gunpowder was known to the Chinese before the be- ginning of the present era, there is no doubt that for several centuries it played an important part in the political and economic history of Europe. The large quantities of gunpowder consumed in the almost incessant wars created a steady demand for saltpeter that was not readily met by the saltpeter refiners of India, Hungary and Poland. European nations, particularly France, were therefore thrown on their own resources and were forced to develop the domestic production of saltpeter. The industry came under government control and experts were appointed to study the so-called saltpeter plantations and the conditions affecting the appearance and increase of nitrates in com- post heaps and in the soil. Much knowledge was thus gained about nitrification even though it was not suspected that living organisms were concerned in the process. With the rapid development of chemistry in the latter half of the eighteenth century a nearer approach was made to the understanding 328 | MICROBIOLOGY OF SOIL of the true character of nitrification. The observations of Cavendish in 1784 that potassium nitrate is formed when electric sparks are passed through air confined over a solution of potassium hydrate formed the starting point for various theories that attempted to account for nitrate: formation on the basis of purely chemical reactions. The formation of nitric acid and of its salts was thus assumed to be due to electric dis- charges in the atmosphere, to combustion processes in nature, or to the: oxidation of organic matter and of calcium, magnesium, iron and man- ganese compounds in the soil. Much credence was given to the latter explanation because of the almost universal occurrence of nitrates in: arable soils. The first indication that nitrate production in the soil and in de- caying organic matter is due to biological activities was given by Pasteur in 1862. A few years later Miiller expressed his belief in the biological origin of nitrates and nitrites in sewage and drinking water. It was not, however, until 1877 that the true character of nitrification was made clear. In that year Schloesing and Miintz demonstrated that dilute solutions of ammonia could be changed into nitrate by being passed slowly through long tubes filled with soil. The amounts of nitrate nitrogen found in the leachings corresponded almost exactly to the amount of ammonia nitrogen used up. When the soil in the tubes was first sterilized by heating or by means of chloroform and other germicides, the ammonia passed through unchanged. But when soils sterilized by heat or chloroform were reinfected with small quantities of fresh soils nitrification again proceeded in a normal manner. The biological nature of nitrification having been thus established numerous investigators tried to isolate the specific organisms in pure culture. A large amount of work in this direction was done by Schloesing and Miintz, Celli and Marino-Zuco, Munro, Warington, the Franklands and many others. A large number of bacteria, yeasts and molds were tested with negative results. Warington, who gathered a great mass of valuable information about nitrification, almost succeeded in securing pure cultures of nitrifying bacteria. Finally, Winogradski showed in 18go not only that nitrification is caused by specific bacteria, but explained’also why the others failed in securing pure cultures. He proved that these organisms do not develop colonies on the ordinary gelatin and other organic media, a fact whose recog- nition was largely responsible for his successful solution of the problem. \ rf DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 329 The medium subsequently employed by him consisted of silica jelly properly supplied with inorganic nutrient salts. After him other in- vestigators proved that agar, deprived of its soluble organic matter, gypsum and ‘sandstone disks, filter-paper pads, etc., could be used effectively as solid media. Nitrous and Nitric Bacteria—Winogradski’s investigations led to the conclusion, foreshadowed by the earlier work of the Franklands and Warington, that the oxidation of ammonia proceeds in two stages, viz., (1) 2NH; + 302 = 2HNO, + 2H20 (2) 2HNO,z + Oz = 2HNO; The organisms oxidizing ammonia to nitrites, and designated as nitrous or nitrite bacteria, were called by Winogradski Nitrosomonas and Nitrosococcus. The former include species or varieties isolated from soils in Europe, Asia and Africa, and the latter those isolated from soils in America and Australia. The organisms oxidizing nitrites to nitrates and known as nitric or nitrate bacteria, were included by Winogradski in the genus Nitrobacter. Apart from these bacteria there is an organism, according to Kaserer, that can oxidize ammonia directly to nitrate. He named it B. nitrator. The reaction is illustrated by the following equation: NH; + HCO; + O2 = HNO; + H.0 + CH:0 — 41 Cal. CH20 + O2 = H2CO; + 132 Cal. Enough energy for the completion of the reaction is obtained by the oxidation of .the formaldehyde (CHO). Beyond the preliminary announcement of Kaserer’s there are no experimental data to prove the existence of this organism, even though other evidence of an indirect nature may be construed to lend support to his theory. But whether it be proved or not that ammonia may be oxidized to nitrate by a single species, it is evident that the number of species concerned in nitrate production is relatively small. Relation to Environment.—The conditions that affect nitrate forma- tion in soils may be classified under the following heads: -(@) supply of oxygen; (b) range of prevailing temperatures; (¢) amount and dis- tribution of moisture; (d) quantity of lime and of other basic materials; (e) quantity of soluble mineral salts; (f) character and amount of 330 MICROBIOLOGY -OF SOIL organic matter; (g) presence of toxic substances; (%) physiological efficiency of the nitrifying bacteria. The rapid disappearance of organic matter from sandy soils is due in large measure to their better aeration. On the other hand, the decom- position of vegetable and animal substances in heavy, ill-ventilated soils is materially retarded by the limited supply and very gradual renewal of oxygen. An intimate relation exists here between air and water in that the latter replaces the former to a more marked extent in heavy than in light soils. The influence of both aeration and the range of moisture is illustrated by an experiment of Lipman’s in which equal quantities of soil were kept in large boxes under different moisture conditions. At the end of a year the following quantities of nitrate nitrogen were found: oe { 6.52 per cent 14.75 per cent 18.62 per cent 22.05 per cent 22.12 per cent Nitrate nitrogen j 697 mg. 823 mg. 720 mg. Trace Trace found In examining the figures recorded above, we find that moisture was the controlling factor in the development of the nitrifying bacteria, when the proportion of water in the soil was 6.52 per cent. As the amount of water increased to 14.75 per cent there was a marked increase in the amount of nitrate produced. Beyond that, however, the further in- crease in the amount of water began to limit the supply of oxygen, and the production of nitrate nitrogen with 18.62 per cent of water in the soil was somewhat decreased. A still further addition of water up to 22.05 per cent led, practically, to saturation, and the encouragement of reduction rather than oxidation processes. Hence, no nitrate was al- lowed to accumulate in the soil. The data in question thus help to explain why care was taken, on salt-peter plantations, to keep the compost heaps moist, yet not too wet. The influence of temperature on nitrate formation has been observed by many investigators. Already Schloesing and Miintz recorded that at 5° nitrification is quite feeble, at 12° marked and at 37° at its best. Other investigators have obtained substantially the same results, except that the optimum has been found to be considerably lower, often be- tween 25° and 30°. Under field conditions nitrification seems to take place at relatively low temperatures, as is indicated by the rapid DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 331 oxidation of ammonium salts in the Rothamsted experiments in Eng- land; and the rapid decay and nitrification of clover and of other legume residues in the experiments at the New Jersey Experiment Station. These facts have, therefore, an important bearing on the nitrogen feeding of crops in tropical, subtropical and temperate zones. The influence of lime and of other basic substances including the carbonates of magnesium, potassium and sodium, and of the oxides of iron is of far-reaching importance in all nitrification processes. It is well known that applications of magnesian and non-magnesian lime, marl or wood ashes promote nitrification in the soil and in compost heaps, a fact that was well recognized by theancient niter refiners. The favorable action of lime is readily explained by its ability to neutralize organic and mineral acids and to render, thereby, the soil reaction favorable for the rapid growth of ammonifying, as well as of nitrifying bacteria. Furthermore, the reserve of basic material serves to neutral- ize the acid formed by the bacteria and prevents thus the accumulation of an undue amount of acidity. ‘The réle of certain mineral salts in promoting nitrification is quite significant. Small amounts of sodium chloride have been found to favor nitrification in the experiments of Pichard and also those of Lipman. The former showed also that sulphates not only promote nitrification, but that different sulphates display marked variations in this respect. In the same manner nitrate formation was shown to be favorably affected by phosphates in bone meal, Thomas slag, and acid phos- phates. Generally speaking, therefore, nitrifying bacteria are stimu- lated in their development by a proper supply of available mineral nutrients. The exact relation of organic matter in the soil to the activities of nitrifying bacteria is but beginning to be properly understood. Earlier observations made it manifest that heavy applications of animal manures, or of green manure may not only retard nitrification, but may actually cause the disappearance of a part or of all of the nitrate in the soil. Subsequent experiments by Winogradski and by Winogradski and Omelianski showed that in pure cultures the presence of even slight amounts of soluble organic matter may depress or even suppress the development of the nitrifying bacteria. It was, therefore, concluded by these authors that relatively small amounts of soluble organic matter may inhibit nitrification. These conclusions, hased on the 332 MICROBIOLOGY OF SOIL study of liquid cultures only, were given a very broad application by many writers on agricultural topics. More recent experiments make it certain, however, that in the soil itself small amounts of soluble organic matter, ¢.g., dextrose, are not only harmless, but may really stimulate nitrification. It was shown, likewise, that humus and extracts of humus may, under suitable conditions, stimulate nitrifica- tion to a very striking extent. Certain substances in the soil may exert a toxic effect on nitrifying bacteria. Ferrous sulphate, sulphites and sulphides may thus act in- juriously, as may also calcium chloride and excessive concentrations of sodium carbonate, sodium bicarbonate, sodium chloride, magnesium sulphate, etc. Injury by ferrous compounds, as well as by organic acids, is not uncommon in low-lying fields and bogs; while injury from excessive concentration of soluble salts may occur in the so-called alkali lands. Finally nitrification in the soil should be considered from the stand- point of the organisms themselves. There is no doubt that continued growth under extremely favorable conditions leads to the develop- ment in the soil of nitrifying bacteria, possessing a very marked phy- siological efficiency. On the other hand, in ill-aerated, sour soils the environment would depress the physiological efficiency of the nitrify- ing bacteria. Differences are thus undoubtedly established under actual field conditions, as is made probable by the variable behavior of soils from different sources when used as inoculating material in recently reclaimed or peat swamp lands. Accumulation and Disappearance of Nitrates——As shown above, the rate of formation of nitrates in the soil is dependent upon moisture, temperature and aeration, as well as on the presence of organic matter and basic substances. On the other hand, the accumulation of nitrates depends, under any given conditions, largely on the character of the growing crop. Observations on the rain gauges at Rothamsted showed an average annual loss 14 kg. (31.4 pounds) of nitric nitrogen per acre in the drainage water from uncropped soil. In one of King’s experi- ments, land that had been fallowed contained 137 kg. (303.24 pounds) of nitric nitrogen per acre, to a depth of 4 feet. Adjoining cropped land contained only 26 kg. (57.56 pounds) of nitric nitrogen per acre to the same depth. Stewart and Greaves found in limestone soil in’ Utah 64 kg. (142 pounds) of nitric nitrogen per acre, under corn; DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 333 98 pounds under potatoes, and only 12 kg. (27 pounds) under alfalfa. Under the same conditions fallow land contained 74 kg. (165 pounds) of nitric nitrogen per acre. Thesmaller amount of nitric nitrogen found under alfalfa bears out the observations already made by a number of other investigators that the accumulation of nitrates under legumes is smaller than it is under non-legumes. While several explanations have been offered to account for this fact, it is generally agreed that legumes assimilate nitrate nitrogen more rapidly than non-legumes. Unusual circumstances may favor, at times, the accumulation of quantities of nitrate large enough to destroy all vegetation. It is reported, for instance, by Headden that he has found in limited areas in Colorado as much as 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- ing crop not alone because of the nitrogen absorbed by the latter, but because of the moisture relations as affected by growing plants. It is quite apparent that a large crop dries out the soil more rapidly than a small crop. When the soil moisture is sufficiently depleted, nitrifica- tion stops and the further accumulation of nitrates becomes impossible, while their disappearance is hastened by the constant demands of the crop. The disappearance of soil nitrates is, likewise, hastened by the leaching action of rain and by certain species of bacteria that transform them into other nitrogen compounds. ‘DENITRIFICATION. Experimental Study.—Denitrification may be defined as the reduction of nitrates by bacteria, involving the evolu- tion of nitrogen gas or of nitrogen oxides. In a more general way, denitrification has been defined as the partial or complete reduction of nitrates by bacteria. The term direct denitrification has been sug- gested for complete reduction, and indirect for the partial reduction to nitrites or ammonia. The term denitrification should not be em- ployed to designate losses of nitrogen gas due to the oxidation of ammonia, or to the disappearance of nitrates following their conversion into proteins by microdrganisms. The reduction of nitrates in the presence of fermenting organic matter was noted by Kuhlmann as early as 1846. The same fact was recorded many years later by Froehde and by Angus Smith. In 1868 Schoenbein expressed the belief that nitrates may be reduced to nitrites by fungi. For more than a decade after that, data were rapidly accu- mulating in support of Schoenbein’s contention, until in 1882 Gayon 334 : MICROBIOLOGY OF SOIL and Dupetit made it certain that nitrate reduction with the evolution of nitrogen gas may be caused by a “ferment.” Finally, in 1886, the same investigators described two organisms, B. denitrificans a, and B. denitrificans 8, capable of completely reducing nitrates. Subsequently the studies of Giltay and Aberson, Burri and Stutzer, Severin, van Iterson, Jensen, Beyerinck and of many others not only greatly in- creased the number of known denitrifying bacteria, but added much to our knowledge concerning the development and activities of these organisms. It has been shown that a very large number of species can reduce nitrates to nitrites and ammonia; moreover, a considerable number of organisms are already known that can cause the complete destruction of nitrates with the evolution of nitrogen gas or nitrogen oxides. The following reactions illustrate diagrammatically the com- plete or partial reduction of nitrates. 2HNO3 = 2HNO2z + Oz HNO; + H.O = NH; + 202 4HNO, = 2H20 + 2Ne + 302 In the soil, manure or other culture media the denitrifying bacteria which are, for the most part, aerobic develop also under anaerobic conditions and transfer the oxygen of nitrates and nitrites to carbon compounds. This is illustrated by the equations suggested by van _ iterson: 5C + 4KNO; + 2H.0 = 4KH CO; + 2N2 + COz 3C + 4KNO, + H,0 = 2KH CO; + K.CO3 + 2Ne When nitrates are reduced to nitrites in the presence of amino- compounds, or even of ammonium compounds, elementary nitrogen may escape as shown by the following reactions: C.HsNH2 + HNO: = C2H;OH + Nz + H20 NH.Cl] + KNO, = KCl + 2H.0 + N; An organism has been described by van Iterson that can decompose nitrates in the presence of cellulose: 5CeH100s + 24KNO3; = 24K HCO; + 6CO, + 12Ne + 13H20 Still more interesting is Thiobacillus denitrificans described by Beyerinck as capable of reducing nitrates in inorganic media. The nitrate oxygen is used to oxidize elementary sulphur: 6KNO; + 5S + 2CaCOs = 3K2SO4 + 2CaSO4 + 2CO2 + 3Ne DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 335 The Actinomyces reduce nitrates to nitrites, but they do not cause any loss of free nitrogen, for the nitrites are utilized by the organisms, and complete denitrification does not take place. Thus these organ- isms may prevent the leaching out of nitrates and nitrites in the soil, or the active denitrification by other organisms, Relation to Environment.—Nitrate reduction is.favored by insufh- cient aeration, as well as by an abundance of readily decomposable organic matter. In fine-grained, compact soils nitrate formation and nitrate reduction may alternate, depending upon the more or less complete replacement of soil air by water. Similarly, in soils receiving excessive amounts of animal manure denitrifying bacteria may cause the reduction of nitrates. In greenhouse soils excessive moisture, as well as excessive amounts of organic matter, may be present and may prevent the accumulation of nitrates. It has also been shown by Niklevski that, contrary to opinions previously held, denitrification may occur in manure heaps. In the better aerated surface portion of manure heaps conditions are favorable for the oxidation of ammonia to nitrites and nitrates. The nitrous acid may combine with ammonia to form ammonium nitrite, the latter decomposing, spontaneously, into water and nitrogen gas. It is very likely, also, that the nitrites and nitrates are reduced by the denitrifying bacteria in manure. On the other hand, in manure kept moist under the feet of cattle nitrite and nitrate formation is prevented and losses by denitrification are not likely to occur. The economic significance of denitrification was overestimated at one time, on account, largely, of the assertion of Wagner in 1895 that in all soils receiving applications of horse manure, the nitrates in the soil itself as well as those added in commerical fertilizers are almost certain to be destroyed by denitrification. Subsequent experiments by many investigators demonstrated that under field conditions, deni- trification is a factor of slight moment; however, in the greenhouse, in the manure heap (under certain conditions) and in market gardening where manure is used at the rate of 45,359 kg. to 54,431 kg. (50 to 60 tons) per acre, the danger of denitrification is real. ANALYTICAL AND SYNTHETICAL REACTIONS AMOUNT OF BACTERIAL SUBSTANCE IN THE SOIL.—Various decom- position processes in the organic matter of the soil may be designated 336 MICROBIOLOGY OF SOIL as analytical in that protein, carbohydrates and fats are split into more simple compounds. At the same time, the microérganisms concerned in the decomposition processes multiply very rapidly and fashion the complex compounds of their cell-substance out of the simple cleavage products in their medium. In other words, analytical and synthetical reactions proceed hand in hand in the soil. While it is not definitely known how large a proportion of the soil humus consists of the dead and living cells of microérganisms there is a mass of indirect evidence to show that these cells form a very con- siderable proportion of the total quantity of organic substances in the soil. For instance, it has been demonstrated that a large proportion of the dry matter of solid animal faeces may consist of bacterial cells. At various times and by different investigators the proportion of bacterial substance has been estimated at from 5 to 20 per cent or more of the total dry weight of feces. A heavy application of barnyard manure may introduce, therefore, several hundred pounds of bacterial cells per acre of soil. Moreover, because of the extensive changes in the soil humus itself, as is evidenced by the rapid formation of nitrates, large masses of bacterial substances are constantly being formed and dis- integrated. AVAILABILITY OF BACTERIAL MaTTER.—Substances of microdrganic origin are decomposed more or less rapidly, according to their com- position. The extent of transformation under favorable conditions_is indicated by an experiment performed by Beyerinck and van Delden, in which 50 per cent of the nitrogen in Azobacter cells was transformed into nitrate in seven weeks. On the other hand, the humus of peat and muck soils, or that of worn-out soils, may contain microdrganic residues of so inert a character as to yield but little available nitrogen to crops. TRANSFORMATION OF PEPTONE, AMMONIA AND NITRATE NITROGEN. —The cleavage of protein compounds into peptones, amino-acids and ammonia, and the oxidation of the latter into nitrites and nitrates, may be properly included among analytical reactions. It should not be forgotten, however, that in the accompanying synthetical reactions the compounds just mentioned may be transformed back into complex proteins. It happens, thus, that large quantities of the available nitrogen compounds may be withdrawn from circulation by micro- érganisms that use these as building material. Under extreme con- DECOMPOSITION OF ORGANIC MATTER IN THE SOIL 337 ditions microdrganisms may become serious competitors of higher plants for available nitrogen food. Manure stored in heaps not infrequently deteriorates in quality, even when losses by leaching are excluded. This deterioration is largely due to the change of the water-soluble ammonia and amino-compounds into insoluble protein substances. While the extent of the change into protein compounds is variable it may range from less than a tenth of the water soluble material to more than three-quarters or four-fifths of it. Also in the soil the same processes take place, but not so intensively. A large number of species of molds and bacteria have been isolated and tested as to their ability to transform ammonia, amino- and nitrate nitrogen into protein compounds. Among the more recent investi- gations in this field those of Lemmermann and his associates testify that _ in three weeks 5 to 6 per cent of the nitrate added to the soil was changed into protein. In the presence of barnyard manure the proportion transformed was increased to 15 per cent. In the case of ammonium compounds the transformation may be even more far-reaching, amount- ing, at times, to more than 25 to 30 per cent of the material originally present. Generally speaking, molds will assimilate ammonia nitrogen more readily while bacteria and alge will assimilate nitrate nitrogen by preference. However, the preference of molds for ammonia nitrogen is often more apparent than real, because of the rapid formation of acid residues in culture media rich in certain ammonium compounds. ‘Similarly, some species of bacteria will assimilate ammonia nitrogen in preference to nitrate nitrogen. 22 CHAPTER III FIXATION OF ATMOSPHERIC NITROGEN THE SouRCE OF NITROGEN IN SOILS Earty THEORIES.—When chemistry had made sufficient progress to allow the analysis of soils and plants it was recognized that nitrogen is always present in both. It was also recognized that the soil nitrogen is almost wholly confined to the surface portion and is evidently of atmospheric origin, since the unweathered, underlying rock is devoid of this constituent. The vast accumulations of nitrogen, known to exist in all arable soils, were ascribed, therefore, to the residues of many generations of plants; and the assumption seemed to be justified that the atmosphere, 79 per cent of whose bulk consists of nitrogen gas, is the direct source of this element to plants. It was not long, however, before plant physiologists demonstrated experimentally that nitrogen gas as such could not directly serve as food for plants. There thus arose one of the most interesting and, for a long time, one of the most puzzling problems in agricultural research. Among the earlier in- vestigators de Saussure believed, at the beginning of the nineteenth century, that nitrogen is taken up from the soil in combined form. Liebig in 1840 advanced his well-known “mineral theory” according to which plants secured their nitrogen from the air, in the form of “ammonia. He assumed, thus, that plants cannot use elementary nitrogen, and that the supply of atmospheric nitrogen in the form of ammonia was great enough to meet the needs of growing vegetation. The latter view was not accepted by Lawes and Gilbert of the Roth- amsted Station in England. By a series of elaborate and carefully controlled experiments they demonstrated in 1858 that nitrogen in the elementary form cannot be used by plants. They further demonstrated that the amount of combined nitrogen brought down in the form of ammonia, nitrites and nitrates, by atmospheric precipitation was but slight when compared with the quantities annually removed by crops. Hence the problem as to the source and maintenance of combined nitrogen in the soil seemed to be more puzzling than ever. 338 FIXATION OF ATMOSPHERIC NITROGEN 339 CHEMICAL AND BrioLocicaAL REtations.—The second and third quarters of the nineteenth century saw the birth of a number of theories dealing with this problem. It was suggested that nitrogen compounds may be formed in the soil by the oxidation of nitrogen to nitric acid. Compounds of iron, manganese and lime were supposed in some way to make such oxidation changes possible. It was likewise suggested that nascent hydrogen may be generated in the decomposition of organic matter in the soil, and reacting with elementary nitrogen, may give rise to ammonia. The various hypotheses were not supported by experimental proof; moreover, the situation was complicated by the knowledge, based on empirical observations, that crops of the legume family seemed to be more or less independent of the supply of combined nitrogen in the soil. Indeed, clovers and other legumes had, appar- ently, the ability to increase the content of combined nitrogen in the soil as was indicated by the experiments of Boussingault and of Lawes . and Gilbert. Finally, the mystery was solved by the investigations of Berthelot and Hellriegel and Wilfarth who furnished the proof that elementary nitrogen may be utilized by plants when certain biological relations are met. These relations involve the presence and activities of microérganisms that by themselves, or in conjunction with higher plants, make available to growing vegetation the great store of atmospheric nitrogen. NON-SYMBIOTIC FIXATION OF NITROGEN HistoricAL.—Non-symbiotic nitrogen fixation, or Azofication, has already been defined as the production of nitrogen compounds out of atmospheric nitrogen by bacteria independently of higher plants. The part played by bacteria in this process was not recognized until 1885, when Berthelot published some of his data on the accumulation of com- bined nitrogen in uncropped soils. His results seemed to explain a number of scattered observations, made since the middle of the century, on the apparent increase of the nitrogen content of cultivated soils. While Berthelot’s experiments proved that the nitrogen gains occurred only in unsterilized soils and were, therefore, due to micro- érganisms, it remained for Winogradski to demonstrate, in 1893, that the formation of nitrogen compounds by certain types of bacteria may be accomplished in culture media nearly or quite devoid of com- 340 MICROBIOLOGY OF SOIL bined nitrogen. Soon after that he succeeded in isolating his organisms in pure culture, and described them as anerobic bacilli allied to those of the butyric group. In 1901 our knowledge of Azobacteria was | enriched by Beyerinck’s discovery of a group of large, obligate aerobic bacteria that he designated as Azotobacter. Since that date it has been found that the ability to fix atmospheric nitrogen is possessed also by certain molds and by various species of bacteria. However, this ability is-not only extremely variable, but is also very feeble as compared with that of the members of the two groups described by Winogradski and Beyerinck. These two groups may, therefore, be designated as including the nitrogen-fixing bacteria par excellence. ANAEROBIC SPECIES.—The species isolated by Winogradski was named by him B. (Clostridium) pasteurianus (Fig. 119). It was found to Fic. 119.—B. (Clostridium) pasteurianus, a non-symbiotic nitrogen-fixing organism. , (After Winogradski from Lipman.) grow readily under anaerobic conditions in culture solutions contain- ing dextrose and the necessary mineral salts, but no combined nitrogen. The products of growth included protein, butyric and acetic acids, carbon dioxide and hydrogen. In the presence of other bacteria B. (Clostridium) pasteurianus was found to develop also under aerobic conditions. Subsequently studies by Winogradski and other investi- gators showed that B. (Clostridium) pasteurianus, and varieties of this species are very widely distributed in cultivated soils. More recently Bredeman made a thorough and extended study of anaerobic Azo- bacteria and demonstrated their almost invariable presence in a large number of soil samples from Europe, Asia and America. In his opinion they correspond more or less closely to B. amylobacter described many years before by van Tieghem. AEROBIC SPECIES.—A more or less pronounced power to fix at mos- FIXATION OF ATMOSPHERIC NITROGEN 341 pheric nitrogen is apparently possessed by a considerable number of aerobic species. Lipman has demonstrated the fixation of small amounts of nitrogen by Ps. pyocyanea and Lohnis secured similar results with Bact. pneumonie, B. lactis viscosus, B. radiobacter and B. prodigiosus. Gottheil has detected fixation by B. ruminatus and B. simplex; Pillai has described a nitrogen-fixing aerobic bacillus, B. malabarensis; Westermann studied a similar organism that he named B. danicus; while Beyerinck and van Delden observed, some years earlier, that. certain strains of B. mesentericus could fix relatively large amounts of nitrogen. Similarly Ps. radicicola has been found to possess a slight, but nevertheless an appreciable power to fix elementary nitrogen in culture solutions or in the soil. Fic. 120.—A zotobacter vinelandi, a non-symbiotic nitrogen-fixing organism. (After Lipman.) But while nitrogen fixation among aerobic soil bacteria is not as uncommon as was at one time supposed, this function is so feeble and so variable in most instances, as to be of negative, or, at best, of doubt- ful economic significance. On the other hand, the aerobic, Azotobacter, first described by Beyerinck in rgor, may be regarded not only as pos- sessing a very pronounced ability to fix atmospheric nitrogen, but as playing a réle of some moment in maintaining the supply of combined nitrogen in the soil. To the two species of Azotobacter, A. chroococcum and A. agilis described by Beyerinck and van Delden, Lipman, added A. vinelandii 342 MICROBIOLOGY OF SOIL (Fig. 120), A. beyerincki and A. woodstownii, and Léhnis and Westermann, A. vitreum. Of these species A. chroococcum and A. beyerincki are most common and are widely distributed in cultivated soils of Europe and America, and probably also of the other continents. They are absent in acid soils deficient in humus, and most common in limestone regions and in irrigated soils rich in mineral salts. Their food requirements are covered by solutions containing potassium phosphate, magnesium sulphate, calcium chloride and ferric sulphate, and some organic nutrient, such as dextrose, saccharose, xylose, mannit, acetate, pro- pionate, butyrate, malate, ethyl alcohol, etc. An alkaline or neutral reaction and the presence of salts of iron are essential for the vigorous development of Azotobacter, while humates have been shown by Krzemieniewski to exert a stimulating influence on the growth of these organisms, even though not acting directly as a source of food and energy. As shown by Lipman and others, Azotobacter may gain an increased power of fixing atmospheric nitrogen in the presence of other organisms. It is resistant to drying, notwithstanding the fact that it produces no spores, and has been successfully isolated from soil samples that had been kept in a dry state for several years. For some reason it may be detected in the soil most readily in the fall and winter months. As to the nitrogen-fixation by fungi, it has been shown elsewhere that the evidence is, if’ anything, of a negative character. Some alge are able to fix atmospheric nitrogen, especially those that live symbiotically with azotobacter. ENERGY RELaTIons.—In the fixation of nitrogen by bacteria the necessary energy for the process is furnished by the carbohydrates, organic acids, alcohols or other organic nutrients employed in the culture media. Since any given quantity of organic nutrient possesses a definite amount of potential energy the fixation of nitrogen is neces- sarily limited by the supply of such potential energy. This limitation was already recognized by Winogradski in his experiments with B. (Clostridium) pasteurianus. For every gram of dextrose used up there was produced, on the average, 2 to 3 mg. of combined nitrogen. In the experiments of Bredeman with B. amylobacter, and of Pringsheim with “Clostridium americanum” the amounts fixed were, at times, con- siderably larger. On the whole, however, it has been proved by a number of investigators that Azotobacter can fix much larger quantities FIXATION OF ATMOSPHERIC NITROGEN 343 of nitrogen than the anaerobic bacilli. The extended investigations of Lipman showed that A. vinelandii has the ability to fix more nitrogen per unit of organic nutrient consumed than either A. chroococcum or A. beyerincki. Under favorable conditions A. vinelandii may at times fix 15 or even 20 mg. of nitrogen per g. of mannit used up. Krze- mieniewski found in experiments with A. chroococcum that additions of humates to the culture solutions increased the nitrogen fixed from a maximum of 2.4 mg. to a maximum of 14.9 mg. The practical bearing of the foregoing data lies in the fact that the fixation of nitrogen in cultivated soils is limited, among other things, by the energy available, that is, by the quantity of readily decomposable organic residues. An indication as to the extent of these is given by the amount of humus present; nevertheless, this must remain an indication merely, for most of the humus is too inert to serve as a source of energy to Azotobacter. From the data at present available different investi- gators have estimated the quantity of nitrogen fixed by Azotobacter at 6.8 kg. to 18 kg. (15 to 40 pounds) per acre, per annum. Assuming favorable conditions for fixation, so that 500 g. (1 pound) of nitrogen could be fixed for every 125 g.(100 pounds) of carbohydrate consumed, it would still take an equivalent of 680 kg. to 1,814 kg. (1,500 to 4,000 pounds) of sugar to produce this quantity of combined nitrogen. Itmay be noted in this connection that Azotobacter have been demonstrated to live in symbiosis with alge, obtaining thereby the necessary energy for their activities. This may explain, perhaps, the remarkable facts observed by Headden in Colorado, relating to the accumulation of such enormous quantities of nitrate in the soil, as to destroy all vegetation. In some instances the nitrates were found to be present to the extent of 90,718 kg. (100 tons), or more (per acre), to a depth of a few inches. If the accumulation of combined nitrogen was due to Azotobacter, as is claimed by Headden, and the bacterial residues oxidized by nitrifying bacteria to nitrates, it is difficult to account for the source of the 1,000 or 2,000 tons of carbohydrates necessarily used up in the process of fixation, unless it could be proved that the energy was furnished by alge. SYMBIOTIC FIXATION HisToricaAL.—Empirical observations extending well back into ancient 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 other hand, he points out that luzerne (alfalfa), lupins and vetches improve | the land and act as manure. He points out, also, that it was the practice of Roman farmers to plow under lupines in order to enrich the soil. In the centuries following the fall of Rome the use of legumes for soil improvement persisted to some extent in Italy, France and other countries; yet the practice was not followed consistently and the 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 century helped to restore the fertility of many farms, and led, ultimately, to the recognition of the peculiar place held by legumes in the maintenance of soil fertility. But while practical farmers knew of the soil-enriching power of legumes, and while they retained their belief in it even when it seemed contrary to scientific authority, they did not know the secret of this power. It remained for Hellriegel and Wilfarth to demonstrate in 1886, and more fully in 1888, that this power, already hinted at by the investigations of others, is the resultant of the combined activities of the plants and of bacteria that enter their roots, and produce there the well-known nodules or tubercles. They showed in no uncertain manner that legumes can improve the soil only in so far as they add nitrogen to it with the aid of the bacteria in the tubercles; in other words, legumes were shown to enter into a symbiotic relationship with certain bacteria and to acquire, thereby, the ability to fix atmospheric nitrogen. The presence of tubercles on the roots of leguminous plants was first recorded by Malpighi in 1687. He regarded them as root galls. The ‘botanists who studied them in the first half of the nineteenth century classified them as modifications of normal roots or as pathological processes. In 1866 the Russian botanist Woronin found that. the tubercles were filled with minute bodies resembling bacteria and con- - cluded that they were pathological outgrowths. Some years Jafer Frank, in 1879, not only showed that tubercles are almost invariably present on the roots of legumes, but that their formation may be pre- vented by sterilizing the soil. Frank was thus in possession of -facts that might have revealed to him the true nature of. the root-tubercles. FIXATION OF ATMOSPHERIC , NITROGEN 345 However, he later modified his belief in the origin of tubercles as due to outside infection, and accepted the interpretation of his pupil Brunchhorst who claimed that’ the, bacteria-like bodies in the tubercles were merely reserve food materials. Because of their resemblance to bacteria Brunchhorst named them bacteroids. The studies of Marshall Ward, published in 1887, proved not merely that tubercle formation is due to outside infection, but that such infec- tion may be brought about at will by placing the roots of young plants in contact with pieces of old tubercles. Hellriegel in his preliminary communication of 1886 also showed that outside infection is necessary , for the production of tubercles, and called attention: to the true func- ‘Fic. 121.—Ps. radicicola. .1, From Melilotus alba;:2 and 3, from Medicago sativa. 4, from Vicia villosa. (After Harrison and Barlow from Lipman.) tion of the. latter as. laboratories wherein. nitrogen compounds are manufactured out of elementary nitrogen. The true worth of: Hell- tiegel’s investigations was brought out more clearly in another paper that he published jointly with Wilfarth in 1888. The authors showed that in sterilized soils legumes behaved precisely like non-legumes, and died. ultimately. of nitrogen hunger when not provided with nitrates or other suitable nitrogen compounds. On. the other hand, when the sterilized soil was later infected with a few drops of leachings from fresh Soil that had. supported a normal growth of legumes, the starving plants -tecovered and grew vigorously. Under the same conditions non- legumes + did not recover. The recovery of the starving legumes was found to be coincident with the formation of tubercles. 346 MICROBIOLOGY OF SOIL Hellriegel and Wilfarth’s studies were soon confirmed by the inves- tigations of others. Wigand showed in 1887 that the tubercles con- tained within them were truebacteria. Inthe following year Beyerinck reported the successful isolation of these bacteria on artificial media, and named the organism B. radicicola (Fig. 121). Prazmowski also isolated pure cultures of Ps. radicicola, and followed the entrance of the organisms into the root haits of young plants, their passage through the cell-walls, and their transformation into bacteroids. These facts were all confirmed by other investigators, and it was further shown by Schloesing and Laurent that properly inoculated legumes not only can grow in soils devoid of combined nitrogen, but that when growing in such soils in a confined atmosphere they decrease the quantity of nitrogen gas surrounding them by transforming it into nitrogen com- pounds. It was, therefore, made clear by these investigations, and by Fic. 122.—Sections through root tubercles. 1, Cell from tubercle of Pisum sativum, showing bacterial filament; 2 and 3, cells with bacterial filaments from tubercle of Trifolium pannonicum. (After Stefan from Lipman.) others not mentioned here for lack of space, that the belief of practical farmers in the soil enriching qualities of legumes was amply justified. It was shown, further, that the later experiments of Boussingault, as well as those of Lawes, Gilbert and Pugh failed to solve the problem because these investigators treated their soil so as to prevent the survival and subsequent entrance of Ps. radicicola, and deprived the leguminous plants of the ability to utilize atmospheric nitrogen. Mopes oF ENTRANCE AND DEVELOPMENT.—Tubercle bacteria con- sisting of small motile rods usually enter the legumes by way of the root- hairs. For this reason young tubercles, with but few exceptions, are found on young roots. The organisms multiply at the point of infection and penetrate into adjacent plant-tissue by means of a hypha-like FIXATION OF ATMOSPHERIC NITROGEN 347 hollow thread or tube that seems to, consist of a gelatinous material (Fig.122). Thetubes branch out as they passfrom cell to cell and carry the invading organisms with them. The bacteria which may be readily detected within the tubes and cells are the involution forms of Ps. radicicola and assume various irregular shapes. They are designated as bacteroids. Stefan has suggested that bacteroids may be produced within the tubes and, possibly, from the buds or swellings that appear on the tubes. While still young, the bacteroids are capable of dividing, but as they grow they swell up and finally degenerate. RESISTANCE, IMMUNITY AND PuysIoLocicaL EFFICIENCY.—The invasion of legumes by Ps. radicicola and the acquisition by the plant, thanks to this invasion, of the power to fix elementary nitrogen are cited as a case of symbiosis. However, some writers would regard the pres- ence of Ps. radicicola in legume roots as a case of parasitism. According to them symbiosis presupposes the living together of two organisms with resulting benefit to both. In the present instance, however, conditions may arise when the host plant is injured, rather than bene- fited; and similarly, conditions may arise when the invading bacteria are suppressed by the plants. Making due allowance for the ob- jections raised we still find, nevertheless, that in the broad relation of the two groups of organisms there is an apparent benefit to both plants and bacteria. The former gain an adequate supply of nitrogen and the latter a supply of carbohydrates and of mineral salts. A more detailed study of this relation shows that the plants resist the entrance of bacteria. When an abundance of available nitrogen compounds is supplied tubercle formation may be largely or wholly suppressed. In that case the plants secure their nitrogen from the soil and are not only independent of the bacteria,.but are strong enough to resist their entrance. It is further claimed by Hiltner that tubercle bacteria differ in their virulence, and that the more virulent the organ- isms, the more readily will they penetrate the root tissue. Moreover, he believes that when a plant is invaded by organisms of any degree of virulence, the host plant becomes immune to a large extent and can keep out all but the most virulent bacteria. The use of the term virulence, in this connection, has been objected to, since it is borrowed from animal pathology and is likely to be misleading. It is better to employ the term physiological efficiency as implying not only a more pro- nounced ability to enter the plant roots, but also to fix atmospheric 348 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 fixation of nitrogen is accomplished by the bacteria within the tubercles.. The claim, at one time, advanced by Stoklasa, that the fixation is accom- plished by the plants themselves with the aid of enzymes produced by the bacteria in their roots, has been disproved. It is known that the period of active nitrogen assimilation by the plants coincides with the appearance of the bacteroids in the tubercles, and it is supposed that the microérganisms fashion nitrogen compounds out of atmospheric nitrogen by using the carbohydrates and organic acids in the plant juicesas a sourceof energy. The plants then seem to utilize the soluble | nitrogen compounds that pass out of the bacterial cells. It is further supposed that bacteroid formation is an attempt on the part of the microorganisms to adjust themselves to the drain caused by the activities of the host plant. VARIATIONS AND SPECIALIZATION.—Apparent differences in bacteria from different legumes were noted by Hellriegel. Some of his experi- ments indicated that bacteria from clovers could not produce tubercles on lupines and serradella. Analogous differences were found by Nobbe and his associates, nevertheless they were finally led to conclude that the root invasion of legumes is caused by a single species. -How- ever, continued association with any particular legume accomplished in the end a certain modification, or specialization, as it were, of the microdrganisms, and they were then no longer able to invade the roots of other legumes. Later, Hiltner and Stérmer have been led to modify this view and have arranged the tubercle bacteria in two groups, possessing, according to them, well-defined morphological and physiological differences. One of these groups is included under the species ‘‘ Rhizobium radicicola” and the other under “ Rhizobium beyerinckii.”” The former comprises the organisms from lupines, serra- della and soy beans while the latter comprises all of the others. RELATION TO ENVIRONMENT.—Nitrogen fixation .by leguminous vegetation is readily influenced by soil conditions, particularly the supply of lime and of other basic: substances; the supply of organic matter and the aeration of the soil. . As to the first of these it is well FIXATION OF ATMOSPHERIC NITROGEN 349 known that all legumes, with the exception of lupines and serradella, are stimulated in their growth by generous applications of lime. Fic. 123.—These two pea plants were grown in clean quartz sand to which had been added small quantities of all the necessary elements of plant food except nitrogen. The conditions were exactly identical except that plant A was without root nodules (see Fig. 124) and plant B had numerous nodules well developed (see Fig. 125). (Mich. Exp. Station.) The top dressing of lawns with lime, marl or wood ashes encourages the 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 bacteria are favorably affected in their survival and multiplication by an abundant supply of organic matter. On the other hand, acid soils or those deficient in humus and inadequately aerated are but ill suited to the activities of Ps. radicicola. Fic. 124.—Roots of Plant A without nodules (Fig. 123). Sort INocULATION* By soil inoculation is now understood the adoption of some artificial method for supplying suitable quantities of nitrogen-fixing organisms to soils deficient in these types. The first attempts at soil inoculation were made in 1886 by Hellriegel and Wilfarth during the * Prepared by S. F. Edwards. FIXATION OF ATMOSPHERIC NITROGEN | 351 course of their studies on the cause of nitrogen accumulation by legumes. They found that when leguminous plants were grown in sterile sand, nodules were formed on the roots only after the addition of a small portion of aqueous extract of fertile soil, or an extract of crushed nodules, or in some cases (lupines and seradella) by soil itself from a field on which these crops had been grown. The first successful artificial production of nodules by the aid of pure cultures was made Fic. 125.—Roots of plant B with nodules (Fig. 123). in 1889 by Prazmowski in the course of studies on the method of entrance of the organism to the root hairs of the host plant. The first inoculation experiments in a large way were those made in 1887 at the Moor Soil Experiment Station, Bremen, Germany, where earth taken from fields that had borne luxuriant crops of various legumes was scattered over reclaimed heath or swamp soils upon which legumes had not previously grown, with the result that in every instance the yield on the inoculated portions of land was greater than on the 352 MICROBIOLOGY. OF SOIL. uninoculated plots. After such favorable results, it was but a natural 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 there was no increase in the vigor or amount of the crop as a result of the inoculation. ‘ MeEruHops oF Sort InocuLation.—From these early experimental results there evolved two general methods of inoculation, namely, the application of soil from an already inoculated field, and’ the application: of pure cultures of the module toonunig bacteria to the. Besa. before sowing. : Inoculation with Legume- intl —The'use of soil as’ creaulbdae material was tried by various experiment stations of the*United States, “with results : ‘not varying: widely from those secured in*the pioneer experimental . work at’ Bremen.. It’ was found in general that the. commonly ‘grown'crops, such as the common clovers, peas.and beans, made little or no increase as a result of inoculation with old legume-soil. With new crops, however, such as alfalfa and ‘soy beans when they-were first’ introduced, it was-found impossible in many places to.secure a successful stand until the fields on which these crops were to.be grown had received a top-dressing of soil from land tht: had CaCOs3 + (NH,4)2SO.4 Moreover, calcium sulphate may be reduced to sulphide and may react with carbon dioxide as follows: CaS + CO. + H:0 = CaCO; + HS Magnesium would be subject to similar reactions and Nadson has observed the formation of a mixture of calcium and magnesium car- bonates (corresponding to dolomite in composition) in media inoculated with a pure culture of B. (Protets) vulgaris. Lime as A Base.—The carbon dioxide generated in vast amounts in the life processes of most soil bacteria, the nitrous and nitric acids formed by the nitro-bacteria, the sulphuric acid produced in the oxidation of hydrogen sulphide and of sulphur by the so-called sulphur bacteria, and the great variety of organic acids formed in the decom- position of carbohydrates, fats and proteins all react with basic sub- stances in the soil. Of these basic substances calcium carbonate is by far the most prominent. Combining with the different acids it maintains a favorable reaction for microdrganic life in the soil. ' The calcium salts thus formed are more or less soluble. In this manner enormous amounts of lime are annually carried to the ocean - as bicarbonates, and to an appreciable extent also as nitrate and sulphate. Thus soil bacteria help to furnish shell fish and other forms of marine life, the material necessary for the building of their skeletons: In the course of ages the latter become a portion of the solid land and as coral reefs, chalk cliffs and marl beds offer to microdrganisms a new opportunity to start calcium carbonate on its migrations. EFFECT OF CALCIUM AND MAGNESIuM CoMPOUNDS ON BACTERIAL Activit1es.—Being basic in character calcium and magnesium car- bonates are of great service in maintaining a suitable reaction in the soil, But somewhat apart from this service calcium and magnesium compounds seem to be particularly important for the growth of certain organisms. It has already been observed by Winogradski and Ome- lianski that magnesium carbonate is especially useful in facilitating the CHANGES IN INORGANIC CONSTITUENTS 357 isolation and culture of nitrate bacteria. Heinze and others have noted the favorable action of calcium carbonate on the growth of Azotobacter, while the beneficial influence of calcium carbonate and sul- phate on the development of Ps. radicicola has been repeatedly observed by different investigators. PHOSPHORUS AVAILABILITY OF PHOSPHATES.—Phosphorus exists in the soil largely in the form of phosphates of calcium, magnesium, iron and aluminum. A small portion of it occurs in organic combination in lecithin, phytin ’ and other compounds. The soil phosphates possess a very slight degree of solubility and often fail to become available rapidly enough to meet the demands of the growing crop. Fortunately the presence of carbon dioxide generated from decaying organic matter hastens the solution of the inert phosphates, thus: . Ca3(PO,)2 + 2CO» + 2H2,O — CagH2(PO,4)2 + Ca(HCOs3)s For this reason a maximum supply of available phosphates may be secured by plants in the presence of readily decomposable organic matter. Apart from carbon dioxide as a means for making available inert phosphates, bacteria produce organic and inorganic acids that are of direct service. The influence of nitrous, nitric and sulphuric acids, all of them products of bacterial activity, is undoubtedly of some im- portance. The influence of lactic, acetic and butyric acids, as well as of the more complex humic acids, must be of considerable moment. For instance, in the decomposition of bone meal by B. mycoides, Stoklasa found that 23 per cent of the phosphoric acid had become soluble, whereas in similar uninoculated portions of bone meal only 3 per cent of soluble phosphoric acid was found. The significance of organic acids produced by microérganisms is brought out even more strongly in the loss of phosphates from acid soils. In so far as the organic phosphorus compounds are concerned bac- terial activities are important in that the processes of decay restore the phosphorus to circulation. Hence, it will be seen that microdrganisms are directly concerned in the migration of phosphorus from the soil to the plant and from the plant back to the soil. RELATION OF PHOSPHORUS TO DECAY AND NITROGEN FIxaTION.— Just as bacteria influence the transformation of phosphorus compounds 358 MICROBIOLOGY OF SOIL in the soil, so phosphorus itself affects the growth and activities of bacteria. As one of the essential constituents of living cells it reacts on the growth of microérganisms and influences species relationships. There are undoubtedly species whose phosphorus requirement is greater than that of other species. Indeed, conditions may arise that favor the rapid assimilation of soluble phosphates by bacteria. In that case the microérganisms would act as competitors to the higher plants. Among the species favorably affected by an abundant supply of phosphates Azotobacter is quite prominent. Hence nitrogen fixation is in a meas- ‘ure dependent upon a proper supply of phosphorus compounds. SULPHUR SULPHUR COMPOUNDS IN 1HE So1Lt.—Sulphur occurs in the soil in the form of sulphates and in that of organic compounds. In ill- aerated soils the reduction products of sulphates; viz., sulphites, sul- phides and even elementary sulphur may be present in small amounts as a transition stage. According to Berthelot.and André the protein compounds of the soil humus are quantitatively more important than the sulphates. However, this is not true of arid and semi-arid soils in which sulphates represent a larger store of combined sulphur than is contained in organic substances. SULPHUR BactTEriA.—In the decomposition of protein compounds with a limited supply of air, hydrogen sulphide and mercaptans are evolved. The quantities of hydrogen sulphide produced may be large enough to become perceptible to the sense of smell, as happens in the putrefaction of eggs. At the bottom of seas, rivers, lakes and ponds (in canals, ditches, swamps, etc.) as well as in finer-grained soils the production of hydrogen sulphide goes on almost uninterruptedly owing to the activities of a great variety of bacteria. The hydrogen sulphide thus generated serves as a source of energy to a group of organisms known as sulphur bacteria. The oxidation of the hy- drogen sulphide by these bacteria may be expressed by the following equations: 2H2S + O2 = 2H20 + Se Se + 202 = 280. The sulphur dioxide produced is further changed into sulphuric acid in the presence of oyxgen and water. In its turn the acid reacts with CHANGES IN INORGANIC CONSTITUENTS » 359 some base, usually calcium carbonate, resulting in the formation of calcium sulphate. Thus: SO2.+0-+ H20=H2S0,4 H.SO4+ CaCO; = CaSO.+H,O + COz We owe much of our knowledge concerning the sulphur bacteria to Winogradski. This investigator showed that in places where hydrogen’ sulphide is generated in considerable quantities sulphur bacteria grow vigorously and accumulate granules of sulphur within their cells. When the cells containing sulphur granules are removed to suitable media, in which no hydrogen sulphide is present, the sulphur seems to be gradually oxidized and disappears and the bacteria finally die of starvation. Thanks to the sulphur bacteria, the higher plants are enabled to utilize again the sulphur once locked up in plant and ani- mal tissues, and liberated thence by decay bacteria. The circulation of sulphur is thus made possible and the cycle is completed when the sulphates are again used by plants to build protein compounds. It may also be noted in this connection that “ Thiobacillus denitrificans,” described by Beyerinck, may also oxidize elementary sulphur. In this case, however, the oxygen is derived from nitrates instead of the atmosphere. Thus: . 6KNO; + 55 + 2CaCO;3 = 3K2SO,4 + 2CaSO, + 2COz + 3Ne SULPHOFICATION.—Lint has found that under optimum temperature and moisture conditions, sulphur applied at the rate of 600 pounds per acre was almost completely oxidized within ten weeks. Boullanger and Dugardin in explaining the fertilizing action of sulphur on the basis of its effect on the supply of available nitrogen found that am- monification was increased by small amounts of sulphur, nitrogen- fixation was not affected and nitrification was depressed. It has been pointed out by Kossovitch, Brioux and Puerbet that the mechanism of sulphur fertilization is very complex and that the oxidation of free sulphur occurs entirely by bacterial and not by chemical means. Brown and Kellogg have recently advanced evidence to prove that soils have a definite sulphofying power which is determinable in the laboratory by a newly devised method. They claim that the process of 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 REpucTION.—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 microdrganisms. Two species isolated by these investigators have been named Sp. desulphuricans and Msp. e@stuanii. When grown under anaerobic conditions in culture media 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) 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. + H.O = CaCO; + H2S 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 PoTasstUM COMPOUNDS IN THE SOIL.— Potassium occurs in the soil largely in the form of silicate minerals. CHANGES IN INORGANIC CONSTITUENTS (361 Smaller amounts occur as nitrate, carbonate and in organic compounds. The portion present as silicates is often very large in clay-loam soils, amounting not infrequently to 22,679 kg. to 34,019 kg. (50,000 to 75,000 pounds) per acre-foot. Unfortunately for the farmer, the grow- ing crops fail, in many cases, to secure sufficient quantities of available potash for their rapid development, notwithstanding these enormous stores of potassium compounds. However, when sufficient quantities of readily fermentable organic matter are present and the generation of carbon dioxide is rapid the silicates weather sufficiently fast to meet the demands of maximum harvests. The part played by carbon dioxide in the transformation of inert potash compounds may be illustrated by the following reaction: Ale03K,0 6Si0., + CO, + 2H.O = Al,O3 28102 2H.O + K.CO3 + 48102 Under actual conditions it is the aim of the farmer to stimulate bacteria] activities (and, therefore, the production of carbon dioxide) in his land by the use of animal manures or green manures and of com- mercial fertilizers. Apart from the influence of carbon dioxide avail- able potash compounds may likewise be formed on account of nitric, sulphuric, acetic, lactic, butyric and other acids produced by different soil bacteria. OTHER MINERAL CONSTITUENTS Tron.—The investigations of Ehrenberg, Winogradski, Molisch, Adler, Ellis and others have accumulated a mass of data relating to the so-called iron bacteria. These organisms belong to the class of higher bacteria and recently forms, such as rod-shaped bacteria, have been isolated which have a marked ability to precipitate iron oxide out of solutions of iron salts. Winogradski believed that the reaction is a physiological one in that the microdrganisms oxidize ferrous to ferric compounds, and utilize for their growth the energy thus made available. The investigations of Molisch, Adler and Ellis show, however, that the iron bacteria can exist very well without iron compounds; and that the precipitation of iron oxide is due to mechanical rather than chemical influences. But whether physiological or mechanical the influence of these microdrganisms is felt in the formation of bog iron, and in the filling up of iron pipes; in the latter instance much annoyance is occa- ‘sionally experienced by those in charge of municipal water supplies. 362 MICROBIOLOGY OF SOIL Compounds of iron are of considerable significance in the life processes of many bacterial species. For instance, it was shown by Lipman and after him by Koch, that Azotobacter will not develop in cul- ture media devoid of iron compounds. In field practice small applica- tions of ferrous sulphate often seem to exert a favorable effect on crop growth. and there is reason to suspect that soil-microbial activities are of some moment in bringing about the results noted. ALumINuM, MANGANESE, CoppER.—Weathering processes and the relation of carbon dioxide to these processes have already been dis- cussed in connection with calcium and potassium compounds. To a great extent aluminum is affected by these reactions, for in the decompo- sition of feldspar, kaolinite is one of the important products formed. Hence, bacteria become a factor of considerable importance in the forma- tion of hydrated silicates of aluminum, at least, in the presence of organic matter. Moreover, it is recognized in the ceramic industries that after it is dug clay must undergo ripening in order to be suitable . for certain purposes. The ripening process involves the activities of bacteria. Unfortunately very little is known about the reactions that occur in the ripening of clay. _ As to manganese and copper there is scarcely any experimental evi- dence available as to the part played by their compounds in the soil, particularly in so far as they affect microérganic life. To some extent, it is known that where Bordeaux mixture has been employed for spray- ing potatoes, cranberries, fruit trees, etc., plant growth is subsequently stimulated to a striking extent. In view of the very slight quantities of copper that are actually added to the soil by these sprays, it is possible that the effects noted are caused by stimulated or changed microbial activities. This view finds.some support in the influence exerted by copper sulphate on the growth of alge in lakes, ponds, and shallow streams. It has also been reported that the decomposition of complex silicates has been effected from powdered minerals by nitrite bacteria. ANTAGONISM A subject which bids fair to become a fertile source of investigation is the application of certain biochemical laws, as established by Loeb and Osterhout in the animal and plant worlds respectively, to the CHANGES IN INORGANIC CONSTITUENTS "363 effect of salts on the physiological efficiency of soil bacteria in pure and . mixed cultures, as well as in the soil. C. B. Lipman has advanced in- formation concerning the antagonism between anions as related to nitrogen transformations in soils, with special reference to the reclama- tion of alkali lands. Antagonism exists to a more or less marked extent between anions of alkali salts (as for example between NaCl and NazSOu, NaeCO3 and NaeSO, and between NaCl and NasCO;) when the ammonifying or nitrifying powers of the soil are employed as criteria. The nitrogen-fixing flora, however, is not similarly affected, apparently offering greater resistance. The practical sug- gestion carried out of such data then, involves the addition of salts to the toxic salts already contained in a given soil, and thereby im- proving 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 Foop Fresh normal milk is one of the most important of human foods. It has a pleasant taste and aroma and is generally liked as a food or drink; but unless properly cared for will not long remain in its normal condition. No article of human diet is more susceptible to undesir- able changes, due to the delicate nature of the milk itself and to the conditions naturally surrounding its production and handling. The injurious changes which commonly occur in milk are of two kinds. ABSORBED TAINTS AND ODORS Milk is very quickly affected by odors of any sort. The foreign odor may be absorbed before the milk leaves the udder if the cow has eaten strong feeds, such as cabbage, onions, etc., or it may be absorbed after the milk is drawn from the cow. If milk is exposed to any -strong odor, such as silage or foul air, resulting from lack of ventila- tion in the stable at milking time, these odors will be taken up by the milk with surprising rapidity. If placed in an ice chest with fresh strawberries or pineapple, or foods like cabbage or turnips, the milk will very quickly absorb the odor of these foods. The absorption of any foreign odor gives to milk a decidedly disagreeable taste. This is true even when the odor which is absorbed is pleasant in itself as _ in the case of strawberries or pineapples. When the “‘off” flavors are due to absorption they are strongest at the outset and become less * Prepared by W. A. Stocking with the exception of the paragraphs treating the acid-forming bacteria, prepared by E. G. Hastings. 364 THE RELATION OF MICROORGANISMS TO MILK 365 pronounced as the milk becomes older, especially if it is subjected to -~ some method of aeration. CHANGES DuE TO MICROORGANISMS While absorption of foreign odors is not uncommon, probably most of the undesirable flavors, found in milk when it reaches the consumer, are caused not by absorption but by the growth of microérganisms in the milk. In this class the changes are slight at first and increase with the age of the milk. Changes of this sort include the common phenomena of souring and curdling, the -so- called sweet curdling, ropy or slimy milk, bitter flavors, gassy milk and a large variety of changes usually known as barny or cowy odors and flavors. If milk {could be kept free from microdrganisms, it might be kept for some time without showing perceptible changes in appearance or taste. No other food product will undergo fermenta- tion changes as rapidly as milk because it is an ideal culture medium for the growth of most kinds of microérganisms, especially bacteria and yeasts. Not only does milk contain the needed food elements but, being in liquid form, they are easily available for the use of micro- ~ organisms. The proteins and milk sugar are most easily attacked and it is the breaking down of these which causes most of the changes in the milk. MicropiaAL CONTENT OF MILK When we recognize the extreme ease with which milk undergoes bacterial changes, we are not surprised to find that ordinary milk, when delivered to the consumer, contains relatively large numbers of bacteria. The amount of cate exercised in the production and handling is a most important factor in determining the bacterial contamination of milk. On this basis milk may be roughly divided into three classes. Common Mirx.—Age is one of the chief factors in determining the germ content of milk. We are, therefore, not surprised to find the milk in large cities having a much higher germ content than ‘in smaller cities and towns. The normal germ content of ordinary milk as it is found in the cities may be shown by the following tables. 366 MICROBIOLOGY OF MILK AND MILK PRODUCTS ‘ BACTERIA IN Boston MiLk* Average taken from 2,394 Samples From June to September Per cent Below 100,000 bacteria per C.C........ 0... eee eee 42.0 Between 100,000 and 500,000 per C.C............... 000 20.75 Between 500,000 and 1,000,000 per C.C........... 000 ee eee 9-75 Between 1,000,000 and 5,000,000 per C.C.........-..000 12.75 ADOVE 5,000,000 PEF C.Ciesi dass ce od swe ve eee BEE ome OO 5.0 Uncountable plates. .......... Mi sodicss db soiblisdacocsoss stated coy hoe 0.75 BacteriaL Counts or Cuicaco (Raw) Mitxt Date Number of Average Lowest Highest samples count count count January, 1910....... 64 1,067,000 27,000 5,500,000 April, 1910......... 43 5,948,000 14,000 150,000,000 July, 1910.......... 183 12,548,000 8,000 I90,000,000 Bacteria IN Mitx oF Connecticut Crrtest Bacterial count Number of samples Did er SOj00 0c fhe ete ls eaten ae etaartarn dle Wipes aaa ae 1,707 SO;OOS=1GOJO0O Nish venue alaiomngedunstaiiels gee stuaulalerereup rate 130 100,000-500;000ss skis gueen net eee yest ae none ees ys aee 459 §00,000-1,000/000h sss anes de ams cue ete ea 2 See e aan 98 OVER- 1,000,090 5.386 ssc nachna BA Blea T ASIEN dieon ease Ma 73 These figures give the results of 2,467 samples collected in seventy-five different towns in the State from October 1, 1908 to October 1, 1909. Goler gives the average bacterial count for 1,057 samples of market milk collected in Rochester during the year 1909 as 446,099 perc.c. Of these samples 1.79 per cent were above 5,000,000 and 38.4 per cent below 100,000, In Montclair, N. J., the average bacterial count for the year 1909 from samples representing fifty-seven dairies was 53,000 per c.c. In Ithaca, N. Y., 148 samples were taken for the year beginning April 1, 1909, and ending March 31, 1910. The average bacterial count of these samples was 221,000. The immense numbers of bacteria found in milk in the large cities are usually the result of the rapid growth of the Bact. lactis acidi group resulting from the age of the milk and the temperature at which it has * Data given by Hill and Slack. + Data given by Tonney. + Data given by Conn. THE RELATION OF MICROORGANISMS TO MILK 367 been kept. Such milk may also contain large numbers of those sapro- phytic organisms which occur in the atmosphere and about the stables and milk-house. The number of this group depends largely upon the sanitary conditions of production and the initial contamination. In ordinary milk organisms of the Bact. lactis acidi type will constitute a very large percentage of those present when the milk reaches the city even before it shows any perceptible signs of souring. During the past: few years great progress has been made in the production of clean milk, and at present quite an important part of the general milk supply of our cities has a very much lower germ content than it had a few years ago. SprctaL Mitxs.—In this class may be considered those milks known as Selected, Inspected, or Guaranteed. As commonly used these terms mean milk which has been produced and handled with considerably more care than ordinary market milk but not with the extreme care required for certified milk. Guaranteed milk is produced by herds’ which have been shown by the tuberculin test to be free from tubercu- losis. Considerable care is exercised in all the operations of handling the milk. The result is that these milks usually have a much lower germ content than the ordinary milk supply of the same city. Some- times the germ content of such milk compares favorably with that of certified milk. These milks may contain various types of normal milk organisms but they should not contain any tubercle bacteria. CERTIFIED Mitx.—Certified milk means milk which has been pro- duced according to the regulations of and under the supervision of a medical milk commission. The stables and cows are kept extremely clean. No dust is allowed in the stable at milking time. The cow’s flanks and udder are washed just before milking, the milkers wear white suits and wash their hands before milking each cow. Small-top pails are used and the milk is cooled as soon as drawn from the cow. The extreme care exercised i in the production and handling of this milk has © a very marked effect on the number of bacteria found init. The follow- ing counts are typical of certified milk. 368 MICROBIOLOGY OF MILK AND MILK PRODUCTS BacTEeriaL Counts oF CERTIFIED MILK IN DIFFERENT CITIES Boston, Oct. 1, 1909 to Sept. 30, 1910* Dairy number Number samples | Average bacteria tT 17 53794 2 13 4,176 3 30 6,825 4 12 1,475 5 7 2,204 New York City, Oct., i909 to Sept., 1910 Farm number | Average count | I | 11,132 2 | 10,516 3 | 8,504 4 16,193 5 2,863 6 11,246 7 23,795 8 51379 9 15,062 ro ~ 459 Chicago t Dairy number | Number counts | Average number bacteria I 51 5,012 Z 60 4,078 3 43 6,502 4 | 17 2,553 Brooklyn Moak gives the average of 321 counts for certified milk delivered in Brooklyn during the first six months of 1910 as 4,095 bacteria perc.c. The best average from any one farm was 561 bacteria per c.c. * Data given by Arms. + Data given oy Park. t Data by Heinemann. THE RELATION OF MICROORGANISMS TO MILK 369 SOURCES OF MICROORGANISMS IN MILK The sources from which bacteria get into the milk have been the sub- ject of much investigation during the past few years, until now the chief sources of contamination are pretty well understood. ‘These sources may be grouped in a general way under the following heads: Fic. 126.—Vertical section of one quarter of udder showing teat, milk cistern, and larger milk ducts. (After Ward and Hopkins.) INIERIOR OF THE Cow’s UppDER. Healthy Udders.—-Milk as it is secreted by the normal udder of a healthy cow is probably free from bacteria. It is very difficult, however, to obtain milk from the udder 24 370 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 microérganisms. 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 the teat where they find suitable conditions for growth and, once inside, work up through the milk cistern to the larger milk ducts and finally though all parts of the udder (Fig. 126). Thenumber of bacteria found in the udder varies widely in different cows as may be seen by the following figures: . BACTERIAL CONTENT OF ENTIRE MiLk oF DIFFERENT Cows Cow No. 2... eee eee eens so acne Bane 850 bacteria per c.c. Cow: NO. 2). aang, take alonaiedenk caiaeu.es Race 750 bacteria per c.c. COW NG: 3545 a0sd Genus aie teiee a Beh ess . 25 bacteria per c.c. Cow NO: Assssueran eis cena peg pganneyas 112 bacteria per c.c. Cow NOs Sinasath 35 35 Experiment No} 4s.aais oyna adhe oe ce eal: 89 54 Experiment: No: Seicses voce sewn teeing Gan 50 50 It will be seen that in no case was the keeping quality of these samples increased by the straining process while in some cases it was materially injured. THE RELATION OF MICROORGANISMS TO MILK 389 Cotton filters are more efficient than cheese cloth and in some cases the keeping quality of the milk may be improved by this process. AERATION.—This is the process of exposing the milk to the atmos- phere by allowing it to run over the surface of the aerator in a very thin film. If milk-has been produced under such conditions that it has absorbed foreign odors, this process may be of value in getting rid of the absorbed odors, but from the bacterial standpoint the process of aerating is not desirable, since it gives one more opportunity for the milk to become contaminated with organisms from the atmos- phere and from the aerator itself. It is possible to aerate milk under such conditions that. the germ content will not be increased, but if aeration takes place in the cow stable or other place where the atmos- phere contains dust the number of organisms will be greater after aeration than before, the amount of increase being proportional to the sanitary conditions under which the aeration is done. It is even possible that the milk may absorb foreign odors during the proc- ess of aeration and be of poorer quality than it was before. It is thought by many that the process of aeration is necessary in order to get rid of the so-called animal odors commonly found in milk. These odors are, however, not normal to the milk but are absorbed from the foul air in the stables or other sources. This is shown by the fact that some of the very finest quality of certified milk is bottled while still con- taining the animal heat with the least possible exposure to the air, tightly sealed at once and plunged into ice water. Such milk contains no suggestion of animal odor. Aeration may be of value in removing undesirable odors from milk which is not produced under good sanitary conditions, if done in an atmosphere free from all dust and odors, but it is not necessary for milk of good quality. The common belief that aeration is valuable is probably due to the fact that most aerators are coolers as well, and the beneficial results are due to the cooling and not the aeration. CENTRIFUGAL SEPARATION.—It is a common practice in some dairies to pass the milk through a centrifugal separator or clarifier to remove any dirt which it may contain. This operation is effective for the removal of much of the insoluble dirt which may be in the milk, but it is of undetermined value as yet from the standpoint of the bacterial content and the keeping quality of the milk. In spite of the fact that the separator slime is very rich in bacteria, the milk and 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 Passtnc MILK THROUGH A CENTRIFUGAL SEPARATOR UPON THE GERM CONTENT OF THE Skim Mitk AND CREAM Bacteria in Bacteria in Par whole mill skim mill Bacteriaiia-cream Sample No. r............ 39,000 69,000 75,000 Sample No, 2............ 44,000 76,000 790,000 Sample No. 3............ 56,000 75,000 820,000 Sample No. 4............ 200,000 336,000 330,000 EFFECT OF A CENTRIFUGAL CLARIFIER UPON THE GERM CONTENT oF MILK Sample Bacteria before Bacteria after Numerical Percentage number clarifying clarifying increase increase I 6,000 9,000 3,000 50 2 15,000 22,000 7,000 46 3 60,000 156,000 96,000 160 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 in- 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 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 at temperatures of 10° or lower. There are, however, certain species THE RELATION OF MICROORGANISMS TO MILK 391 which will grow with considerable rapidity at temperatures below 10°, especially some of the spore-bearing non-acid forms. If the tempera- ture of the milk is allowed to rise above 10°, the growth of the common species increases rapidly. The influence of temperature upon the development of bacteria may be seen in the following experiment where a given lot of milk was thoroughly mixed and divided into seven portions, which were then held at the temperatures indicated for twelve hours, at the end. of which time they were plated for the total germ content. EFFecT OF DIFFERENT TEMPERATURES UPON THE DEVELOPMENT OF BACTERIA In MILE Temperature maintained Bacteria per c.c. at end Hours to curdling for 12 hours of 12 hours at 21° Cc. FL 4.5° 40° 4,000 75 7 45° 9,000 75 10° 50° 18,000 72 12.5° 55° 38,000 49 15.5° 60° 453,000 43 21° 70° 8,800,000 32 26.5° 80° 55,300,000 28 Thefresh milk showed a count of 5,000 per c.c. and curdled in fifty-two hours at a temperature of 21°. The curdling time of these samples was determined by placing them at a constant temperature of 21° at the close of the twelve-hour period and holding them at this temperature until coagulation took place. The difference in time of curdling therefore is due to the maintenance of the special tempera- ture for twelve hours only and not for the entire period up to the time of curdling. PASTEURIZATION.—The term pasteurization is used to designate the process of heating milk to a temperature sufficient to destroy a portion of the bacteria and then cooling it to a temperature which will prevent the rapid development of the organisms that are left. The temperatures commonly used for this purpose vary from 60° to 85°. The length of time the milk is exposed to the high temperature may also vary from a few seconds to thirty minutes, depending upon the method employed. The two chief purposes for the pasteuriza- 392 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 materially increase its keeping quality, but the temperature and time to which any particle of milk is exposed cannot be accurately controlled, and this method cannot be depended upon to kill all of the disease-pro- ducing organisms which may be in the milk. This method has been 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 is employed. This consists in raising the temperature of the milk to about 60° to 63° and holding it at this temperature for a period of twenty to'- thirty minutes. If this method is properly done, most of the organisms except certain spore forms should be killed and the milk at the end of the pasteurizing process contain only a small percentage of its original germ content. Formerly it was believed that heating milk to a high temperature killed all the lacticacid organisms, and favored the subsequent growth of other more undesirable species, but more recent studies on the bacterial flora of milk, pasteurized by the ‘‘holding”’ method, have shown that some strains of the lactic acid bacteria can survive the relatively lower temperatures used in this method, and that the later development of the different groups of bacteria is similar to that in raw milk of equal bacterial grade. Pasteurization at the temperatures used in the holding process does not seem to cause any injurious chemical changes in the milk constitu- ents, or affect its digestibility. Proper pasteurization gives a valuable means of rendering the milk 1 THE RELATION OF MICROORGANISMS TO MILK 393 supply for our cities reasonably free from pathogenic microérganisms, but, in order to insure this safety, the work must be carefully done, and all later contamination avoided. Preferably, the work should be done under expert, municipal supervision. Undoubtedly the ideal method is pasteurization in the sealed bottle which is to be delivered to the consumer, since this method reduces to the minimum the danger of subsequent contamination. Pasteurization must not be regarded as a substitute for care and cleanliness or a means of renovating old or dirty milk otherwise unfit for use, but rather as an additional means of protecting the consumer against disease-producing microdrganisms in the milk supply. Tue UsE or CHEmICALS.—The addition of certain chemicals to milk will retard the growth of bacteria. The chemicals most commonly used for this purpose are calcium hypochlorite, borax and formalin. While the keeping quality of milk may be materially increased by the use of such chemicals, their use has been opposed by health authorities and is contrary to the Pure Food Laws. If milk is handled with any degree of care, there should be no need for the use of chemical preservatives. They are simply a means of counteracting the unsanitary conditions of the production and handling. The same results can be obtained by cleanliness in the production of the milk and the use of low temperatures for preventing the contamination and subsequent growth of the bacteria in the milk. The developments in the production of clean milk of the past few years have illustrated very clearly that the use of chemical preservatives is not necessary. NorMAL DEVELOPMENT OF MICROORGANISMS IN MILK The flora of any particular sample of fresh milk is determined by the conditions under which it is produced. In stables where extreme cleanliness is practised the flora may be practically limited to those - species which occur in the udder of the cows, but under ordinary condi- tions there will be in addition to the normal udder types such others as may occur on the cow’s body and in the dust and atmosphere of the stables. Market milk, therefore, when first obtained from the cow ordinarily contains a mixed flora, the different types present depending upon the sanitary conditions under which the milk is produced. The future development of this initial flora is largely dependent ' 394 MICROBIOLOGY OF MILK AND MILK PRODUCTS upon the temperature at which the milk iskept. If the milk is held at temperatures between 10° and 21° there will result what may be con- sidered as the normal development of milk fermentations. These changes may be divided for convenience into four periods or stages. First Stace. GERMICIDAL PERIop.—It has been shown by a num- ber of investigators that instead of an increase in the numbers of bacteria in fresh milk there is normally a decrease in the number during the first few hours after its production. The rapidity of this decrease and the length of time over which it extends seem to be determined largely by the temperature at which the milk is kept. The higher the tempera- ture the more rapidly the number of organisms decreases and the more quickly the end of the germicidal period is reached. If the tempera- tures are kept fairly low the rate of decrease is much slower but the de- cline will extend over a considerably longer period. This is shown by the following examples given by Hunziker. TABLE SHOWING THE GERMICIDAL ACTION IN Cow’s MILK Name bose Temp.* After | After | After | After | After After After | After of, ant of 3 6 9 xe Is 24 32 48 cow fresh milk hours | hours | hours | hours | hours} hours hours | hours 40° 1,080] 1,220] 1,040] 1,020} 1,120 1,360) 1,040 400 May....... 1,212 55° 1,260} 1,400] 1,500] 1,462} 1,360 1,080! 3,500] 17,740 70° 1,000} 1,340| 1,860] 3,460] 3,460 64,000] 800,000 40° 4,400] 4,260] 3,620] 3,700] 3,900 4,000] 3,900) 3,840 Tae cies eeu 5,120 55° 3,900) 3,460] 2,980] 2,800] 2,920 3,260] 3,220| 3,240 70° 3,560| 2,120) 1,880} 1,880] 1,240 4,960] 58,400 40° 1,170} 1,070] 1,120 ? 870! 1,120 * 990 1,060| 1,080 Julia....... 1,345 55° 1,080 990 980} 1,400; 1,080 1,080] 3,110] 68,800 70° 1,000] 1,000} 1,200! 5,600] 17,720! 1,600,000 The exact reason for this decline is at present not well understood. Some investigators believe that milk possesses 4 certain germicidal ac- tion or property which results in the destruction of a portion of the organisms found in the milk at the outset. The work of other investigators seems to show that the so- -called germicidal action is felt by certain species and not by others as is indi- cated by the following sample. * Fahrenheit. THE RELATION OF MICROORGANISMS TO MILK : 395 Age of mill bastante jacana |i Wee | ween \ Presb visa siciarede 83 12,550 1,250 Io 200 3 hours............ 12,250 2,000 16 200 6 hours............ 19,650 2,250 23 800 g hours............ 56,900 20,250 36 550 z2 hours............ 114,250 68,400 60 1,900 This would seem to indicate that the decrease in number is due not so much to a definite germicidal property possessed by the milk as to the gradual dying out of certain species which for some reason do not find the milk a suitable environment for development, while other types, . finding the milk suitable to their needs, develop uniformly from the start. Rosenau and McCoy found that the germicidal properties of milk were destroyed by boiling or by heating it above 80° and that lower temperatures destroyed it for certain organisms. These workers also found that there was marked agglutination of the organisms in raw milk and conclude that this accounts for the decreased number of colonies developing in plate cultures and that the germicidal action is therefore * more apparent than real. SECOND STAGE. PERIOD FROM END oF GERMICIDAL ACTION TO Time or CurDLING.—The period following immediately after the ger- micidal action is characterized by the rapid development of the lactic organisms. Under normal conditions this group develops much more rapidly than any other type. Not only do they increase rapidly in actual numbers but their percentage also rises rapidly. There may be a continual increase in numbers in the other species, but their growth is much less rapid than that of the Bact. lactis acidi type. As this period advances certain of the miscellaneous types may cease to grow entirely. During this time the gas-producing acid organisms of the B. coli and Bact. lactis aerogenes type may develop more or less rapidly, but if the milk is held at temperatures not much above 20°, the Bact. lactis acidi type will develop much more rapidly, so that by the time the milk be- comes sour and curdles, this type will constitute 99 per cent approxi- mately of the total number in the milk. From the standpoint of the milk consumer milk ceases to be of value when the end of this period is 396 MICROBIOLOGY OF MILK AND MILK PRODUCTS reached, but there are further developments which are of importance in certain lines of dairy manufactures, notably cheese making. Tuirp StacGE. PERIOD FROM TIME OF CURDLING UNTIL ACIDITY 1s NEUTRALIZED.—At the time milk curdles it contains enormous num- bers of the lactic bacteria. The number usually runs into the millions and may be even higher than one thousand million per c.c. By the time the coagulation takes place the acidity of the milk is so high that the growth of the lactic organisms is checked and from this time on their number decreases with more or less rapidity. During the period following the curdling certain other types of organisms which have existed in the milk during the earlier stages now begin to grow. The organisms especially important in this stage are Oidium lactis, certain species of molds, and yeasts. These organisms are able to grow in a highly acid medium, and as a result of their development the acid is decreased until the milk finally presents a neutral or alkaline condition resulting from the decomposition of the proteins in the milk. FourtH Stace. Frnat Decomposition CHaNncEs.—The reduction of the acidity affords favorable conditions for the growth of certain types of organisms which have remained in the milk during the earlier stages but have been practically dormant. In this fourth stage the conditions are suitable for the growth of the liquefying and peptonizing bacteria and-they now grow rapidly, causing the decomposition of the casein. The changes resulting from this type of organisms are of special signifi- cance in cheese making and are discussed more fully in another chapter. ABNORMAL FERMENTATIONS IN MILK me ‘ Gassy FERMENTATION.—It frequently happens that instead of the normally rapid development of the Bact. lactis acidi type of organisms in the milk, other acid producers develop rapidly, with the production of more or less gas. The organisms most prominent in this type of fer- mentation are the B. coli communis and the Bact. lactis aerogenes types. This group of organisms contains a number of varieties, some of which produce little or no gas while others develop large amounts. Their action in milk is usually accompanied by disagreeable odors and flavors. They grow readily in the presence of air and therefore develop abundant colonies on the surface of plate cultures. This distinguishes the mem- THE RELATION OF MICROORGANISMS TO, MILK 307 bers of this group quite clearly from those of the true lactic group which grow chiefly below the surface of the medium. The members of this group do not form spores, but certain varieties are quite resistant to heat and will oft times survive pasteurizing temperatures which com- pletely destroy the Bact. lactis acidi group. They grow most rapidly at high temperatures, between 20° and 37°. SWEET CURDLING FERMENTATION.—This phenomenon is caused by a variety of organisms which cause the milk to coagulate without the production of acid. The coagulation is brought about by a rennet-like enzyme produced by this type of bacteria. The resulting milk is either neutral or alkaline in reaction. Usually the coagulation of the milk is followed by the digestion of the casein as a result of another enzyme which is also produced by these bacteria. The coagulation caused by these organisms is slower than in the case of the acid formers and the curd is usually soft and mushy as compared with the curd formed in the normal acid fermentation. -The members of this group get into the milk from and along with dust and dirt associated with unsanitary conditions. Some of the species produce spores and are not killed by the ordinary methods of pasteurization. This fact accounts for the occurrence of sweet curdling of pasteurized milk. This group of or- ganisms is unable to develop rapidly in the pres- ence of the lactic bacteria and for this reason we do not commonly get the sweet curdling of raw milk. The presence of these organisms is evi- dence of insanitary conditions. Frequently they develop very disagreeable flavors in the milk. ; Ropy or Stivy FERMENTATION.—One of the Fic. 131.—Ropy most common milk infections causing trouble to le ae — a fork. the milk dealer is that which causes a ropy or ; slimy fermentation of milk. This is sometimes spoken of as stringy milk (Fig. 131). Several species of organisms are capable of pro- ducing this condition. These organisms grow most freely in the presence of an abundant supply of oxygen and for this reason the cream usually becomes slimy before any changes are apparent in the under- lying layers of milk. B. lactis viscosus is perhaps the most common species 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 organisms (Fig. 132). Representatives of this group are quite resistant to heat and frequently pass uninjured through the methods of cleansing and scalding used under ordinary dairy conditions. Because of this, dairy utensils once infected become a constant source of infection. Fic. 132.—Bacillus lactis viscosus from a milk culture. (After Ward.) This trouble can be effectively stopped by a thorough scalding of all utensils coming in contact with the milk. BirTeR FERMENTATION.—Bitter flavors in milk may be the result of bacterial changes after the milk has been drawn, or due to certain feeds which the cows have consumed. If the cows are allowed to eat certain kinds of vegetation, such as “rag weed’ and certain other plants, they may impart a bitter taste to the milk, in which case the abnormal flavor will be apparent when the milk is fresh and usually becomes less pronounced as the milk becomes older, because of the volatile nature of the substances causing the bitterness. Most of the cases of bitter milk and cream, however, are due to the growth of certain types of bacteria in which case the bitterness increases in in- tensity with the age of the milk. Some of the species capable of producing bitter milk grow at quite low temperatures, which accounts for the fact that the most trouble with bitter flavors is found in milk and cream which has been held at low temperatures for some time. ae THE RELATION OF MICROORGANISMS TO MILK 399 ALCOHOLIC FERMENTATION.—The. bacteria as a group are not able to act on the milk sugar and produce alcohol, but it sometimes happens that yeasts get into the milk in sufficient numbers to ferment the milk sugar, producing appreciable amounts of alcohol. To the milk handler this trouble is not usually serious but the action of the yeasts is frequently of considerable importance in the cheese industry. OTHER FERMENTATIONS.—It frequently happens that a consider- able variety of disagreeable flavors and odors develop in milk. These may be due to the direct absorption of odors from the foul stable atmosphere or strong-smelling feeds, such as silage; or they may be, and no doubt frequently are, the result of the growth of certain types of bacteria which have entered the milk from dirty surroundings. The growth of some of these organisms is frequently the cause of the so-called cowy and stable odors and flavors. : CoMMERCIAL SIGNIFICANCE OF MICROORGANISMS IN MILK RELATION OF Dirt CONTAMINATION TO GERM CONTENT.—To the commercial milkman bacteria are of importance only as they influence the length of time the milk will keep in a salable condition. The consumers do not want milk that is sour or has unpleasant flavors and odors. In order to sell his milk, therefore, the milkman must avoid the presence of these undesirable conditions, and in proportion as he recognizes the relation between germ life and the quality of his product, will he pay attention to the presence and development of microérganisms in his milk. In like manner, the presence or ab- sence of dirt contamination is important from the commercial stand- point since it bears a relation to the bacterial count, and, therefore, affects the keeping properties of the milk. Under normal conditions there is a fairly direct relation between the amount of visible or soluble dirt and the number of bacteria found in any given lot of fresh milk. This relation may be shown by the following samples taken from four different milk producers: Average number ‘ Number of Average mg. r Average hours Producer samples dry dirt per liter ene per to time ef curdling Assy exten 5 51.5 115,000 | 175 Bass: ar 16 58.8 273,600 78 Cys on ces 21 70.0 428,600 75 Dh esos fais 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 of one kind of dirt may contain infinitely more organisms than an equal amount of some other kind. The difference in the solubility of various forms of dirt always causes apparent discrepancies in this normal relation. In the majority of cases, however, the relation shown in the above examples will hold reasonably true in the case of fresh milk. There is also a general relation between the number of bacteria in ‘fresh milk and the length of time it will keep before souring and curd- ling. In this case the relation is in inverse ratio, the smaller the initial contamination, the longer the keeping time, and vice versa. This re- lation is also shown in the table given above. There are many ir- regularities, however, in this relation because of differences in the flora of fresh milk. It may frequently happen that a sample of milk containing a relatively high number of organisms will not sour as quickly as another sample with a smaller original germ content. The associative action of the different species of organisms is an important factor here. In making comparisons of this sort, it is, of course, necessary that the different samples be held at the same temperatures. MILK AS A CARRIER OF DISEASE-PRODUCING ORGANISMS It is not the purpose of this chapter to discuss in detail the diseases which may be carried by milk, but a chapter on bacteriology of milk would be incomplete without a brief discussion of this important subject. From the standpoint of their relation to the health of the con- sumer the microdrganisms in milk may be divided into three groups on the basis of whether they are beneficial, inert or injurious to health. Acid Forms—The preservative properties of sour milk have been known since very ancient times. Its use as a preservative for meat, eggs and other perishable food products demonstrates the value of sour milk as a means of preventing decomposition. It has also been known for a long time that sour milk has a certain therapeutic value because of the action of the lactic bacteria in preventing harmful fermentations in the digestive tract. More recently the work of Metchnikoff has shown the usefulness of sour milk both for the treat- ment and prevention of intestinal disorders by inhibiting the develop- ment of the putrefactive bacteria in the digestive tract. In view of the value of sour milk for preventing certain forms of disease and its THE RELATION OF MICROORGANISMS TO MILK 401 inhibiting action on certain undesirable organisms the Bact. lactis acidi type of bacteria must be regarded as beneficial organisms, and . from the standpoint of the health of the consumer their presence in the milk is to be welcomed rather than discouraged. As the value of sour milk drinks becomes better known the importance of this group of milk bacteria will be more fully recognized. Neutral or Inert Forms——In ordinary milk there is a large class of bacteria which, so far as known, have no appreciable effect either upon the composition of the milk or the health of the persons consuming it. This group includes a number of species, many of them being coccus forms, some of them appearing in plate cultures as chromogenic colonies. They grow more or less freely in milk, depending upon the conditions, but they are usually held in check by the acid-forming bac- teria and do not constitute a very important part of the flora of normal milk. They are, therefore, of little significance from the practical standpoint except as they indicate the conditions under which the milk has been produced and handled. Injurtous Organisms.—The diseases which may be carried by milk are of two classes. Epidemic Diseases—The human diseases most commonly carried by milk are typhoid fever, diphtheria and scarlet fever and occasionally other diseases such as septic sore-throat, cholera and foot-and-mouth disease. The first three are by far the most important of this group. The outbreaks of typhoid fever which are traceable to milk occur most frequently. There is a large accumulation of data showing the occurrence of epidemics caused by infected milk. An epidemiccaused by the milk supply has certain characteristics which distinguish it from epidemics resulting from other causes. A considerable number of cases of the particular disease will appear almost simultaneously and will be distributed along some particular milk route. Usually the epidemic stops as suddenly as it began except for a few secondary cases contracted from those first taken. The source of the disease organisms is a human patient suffering from the disease. The infection of the milk may be direct, as when a sick person handles a milk, or it may be indirect as when a person caring for a patient also works about the milk. In other cases it may be caused by contamination of the water used in washing the utensils or by cows wading in water of infected streams and getting the organisms on their body whence 26 402 MICROBIOLOGY OF MILK AND MILK PRODUCTS they fall into the milk pail at'milking time. The return of milk bottles from the sick room sometimes is the means of infecting the milk supply. CITY OF ROCHESTER NW ‘Average Dedihs wader 5 Years of ee ond after the esta stent ef Monicip dl Mk Stations Fic. 133. Unfortunately the specific organisms of these diseases grow readily in milk and a small infection is all that is necessary to render the milk dangerous by the time it reaches the consumer. THE RELATION OF MICROORGANISMS TO MILK 403 Non-epidemic Diseases—There is another class of diseases which may be carried by milk .which are not characterized by a sudden out- break, and for this reason are not so readily recognized as being asso- ciated with the milk supply. One of these diseases, namely tuber- culosis, is caused by the specific, well-known organism, Bact. tuberculo- sis, which may get into the milk from the udder of a tuberculous cow or by the organisms which have been given off from the digestive . tract of the animal becoming scattered about the stable and finally , getting into the milk with particles of dust and filth. In some -cases the milk may become infected by persons having the disease being permitted to handle the milk. Fortunately for mankind Bact. tuberculosis does not multiply in milk. Regarding the danger of contracting tuberculosis from the use of milk there is at present some difference of opinion, but the con- sensus of opinion at the present time seems to be that there may not be very great danger for healthy adults, but that a considerable percentage of the cases of tuberculosis of children may be traced from the milk supply. Fortunately the temperatures used in the process of pasteurization by the “holding” method are sufficient to destroy any of the disease bacteria known to be carried by milk. There is another class of disorders not so well defined as the above but which are nevertheless of great importance from' the standpoint of public health, especially of young children and also to some extent of adults. This group includes such disorders as infantile diarrhoea, sum- mer complaint, cholera infantum and other disorders of the digestive tract. The organisms producing these troubles doubtless belong to the group of putrefactive bacteria which come from filth. Some of the gas ‘producers and some of the peptonizers are probably responsible for these troubles. Shiga isolated from a large number of cases of infant diarrhoea a bacterium which he named Bact. dysenterig, but in general the specific organisms responsible for these intestinal troubles are not well known. Their importance, however, is shown by the relation of the germ content of milk to infant mortality (see Fig. 133). \ BACTERIOLOGICAL ANALYSIS OF MILK The development of our knowledge of the relation of bacteria to the wholesomeness of foods has led to a study of the bacterial content of 404 MICROBIOLOGY OF MILK AND MILK PRODUCTS milk as a means of determining its purity. The methods used for this purpose have followed quite closely those of the water bacteriologists. For many years, dairy bacteriologists have endeavored to determine the number of organisms in milk by plating it into nutrient agar or gelatin. By this method the number of colonies developing in the plates is assumed to represent the germ content of the milk. But even when the best methods are employed, the plate count represents only the approximate and not the exact number of bacteria in any lot of milk. It should also be borne in mind that such counts are always underestimates, because of the fact that not all species will develop in any given medium or incubation temperature. The careful worker can recognize certain types of bacteria in plain media, but the addition of blue litmus solution to either agar or gelatin, greatly ‘ assists in the differentiation of types and species. Tue Direct Microscopic MetHop.—The plating method is expensive because of the large amount of time and materials needed. It is not possible for one person to handle a large number of samples at one time. In routine work in the city labora- tories this labor has been a serious drawback to this method. In order to decrease the labor and give greater possibilities to the work Stewart devised a method by which the bacterial condition of milk can be studied by direct microscopic examina- tion. His purpose was to determine only the species present, but later Slack and still more recently Breed developed the method for determining the approximate numbers as well as the general species present in a given sample of milk. LrvucocyTEes.—The microscopic examination of milk sediment revealed the fact that frequently a sample would be found which showed the presence of leucocytes in greater or less numbers. The presence of these cells was regarded as important because it was assumed that they showed the presence of inflammation and pus for-’ mation in the udders of the cows producing the milk. Several methods have been used for determining the leucocyte content of milk. “The Smear Sediment” and ‘Blood Counter’’ are methods which more strictly belong to laboratory practices and will not be considered in this place. BACTERIOLOGICAL MiLK STANDARDS The relation of the bacterial content of milk to its wholesomeness has led to the adoption of certain standards by the boards of health in our cities. These standards recognize the fact that the germ content of milk in the large cities is greater than in the smaller ones because of the greater distance from which it is shipped and its age on arrival to the city. New York City in 1900 adopted a maximum limit of 1,000,000 per c.c. Later Boston established a limit of 500,000 Chicago 1,000,000 from May to September, inclusive, and 500,000 from October to April, inclusive and Rochester 100,c0o. Other cities have made similar standards. / THE RELATION OF MICROORGANISMS TO MILK 405 Stokes’ standard for the number of leucocytes permissible in normal milk was 5 per field of the 142 objective in his smeared sediment prepa- ration. Bergey found so many samples running above this number that he made the limit ro cells per field and felt that no milk containing more than this number should be used for food. Later Slack raised the limit to 50 cells per field. The reason for changing the stand- ard was due partly to the larger numbers found as a result of improved methods but more especially to the discovery that milk from appar- ently healthy cows normally contains leucocytes in excess of the first standards set. With the development of the dairy score card, there was a decided tendency to place emphasis on the sanitary conditions at the farm rather than on the germ content of the milk. But it was soon discovered that the farm score did not necessarily show the true condition of the milk, and at present, the tendency seems to be toward placing more confidence in the germ content as the best measure of the true conditions of pro- duction and handling. However, the fact must be recognized that our methods of bacteriological analysis are not sufficiently accurate to justify the bacteriologist in passing judgment concerning the quality of any milk supply on a single analysis. In order to secure results which are at all trustworthy, a series of analyses must be considered. It is held by some that a numerical standard is of little value since the actual number of organisms present in a given lot of milk may not be ‘a correct measure of its wholesomeness. For this reason some cities — pay little attention to the numbers of bacteria. present but base their standards wholly on the species and the quality of the milk is judged on the presence and numbers of streptococci, B. coli, leucocytes, sediment. Milk is passed or condemned on the basis of any one or combination of these conditions. In recent years there has been a tendency to combine these two standards using the total germ content as a measure of the care the milk has had and the presence or absence of certain groups or species as an indication of the occurrence of pathological conditions in the cows producing the milk. The practice in most city laboratories now is to make use of both the numbers and the species present in determin- ing 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 is still some difference of opinion among milk bacteriologists. The germ content of any lot of milk is largely dependent upon three factors: the number of organisms getting into the fresh milk; the temperature at which it is kept; the age of the milk when analysis is made. The high bacterial count in any lot of milk may be the result of any one of these conditions or a combination of them. A high count means that there has been carelessness either in the production, resulting in high initial contamination, or in the subsequent handling permitting a rapid multiplication of the organisms, or that the milk is old. On the other hand, milk with a low germ content can be obtained only where the original contamination is small and the milk has been held at low temperatures. A low count, therefore, means care both in the production and later handling of the milk. While the germ content may be regarded as a general index to the care the milk has received, it may not at all indicate its wholesomeness. Ahigh count may be the result of the rapid growth of the lactic bacteria, in which case the milk may be perfectly safe and wholesome. On the other hand, the count may be quite small but contain pathogenic species. The bacteria count is valuable as showing the sanitary conditions of production and handling, but much care should be used in the inter- pretation of such results. In some ways a direct microscopic examina- tion of the milk sediment is much more satisfactory. The skilled analyst can recognize certain types which may indicate the sanitary quality of the milk. With sufficient experience one can recognize strep- tococci, certain other groups and leucocytes. The presence and abun- dance of one or more of these groups may indicate the nature of the original contamination and the existence of diseases in the udders of cows. Tf rightly interpreted the information thus obtained is of much value. The weakness of this method lies in the fact that it is not always possible to recognize the above types of organisms. In a smear prepa- ration it is not possible to differentiate between pathogenic and non- pathogenic streptococci or between B. colt and certain other types. The presence of unusual numbers of streptococci and pus cells may indicate the existence of disease in the cows and when this condition is found in the milk it is often possible to trace it back to the farm and lo- THE RELATION OF MICROORGANISMS TO MILK 407 cate the diseased cow and prevent her milk from being used for human consumption. te The tendency at present is to combine the quantitative and quali- tative analyses and the results thus obtained in the hands of the careful worker are of much practical value in controlling the quality of a city’s milk supply. CHAPTER II* THE RELATION OF MICROORGANISMS TO BUTTER Butter is the fat of milk that has been largely freed from the other constituents of milk by the processes of creaming and churning. If milk is allowed to stand, the fat, which is in the form of minute globules, accumulates in the upper layers of the milk because its spe- cific 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 milk through a cream separator. In therapidly revolving bowl of thesepara- tor the centrifugal force exerted is many times greater than that of grav- ity and the fat is rapidly and efficiently removed. The cream, which is obtained by these methods, contains varying amounts of fat which is further concentrated, by subjecting it to agitation in the churning process. The globules of fat cohere to form larger and larger masses until the entire amount of fat is brought into a single mass, the butter. TYPES OF BUTTER SwEET-cREAM Butrer.—lIf little or no increase in the acidity of the milk or cream develops, previous to churning, the butter will have certain marked characteristics and is called sweet cream butter. It is especially characterized by its low flavor, since it has only the flavor of the fat of milk which is not marked. This is usually known as the primary flavor of butter. Sweet-cream butter is also marked by the rapidity with. which it undergoes decomposition changes, especially when it is made from raw cream. SouR-CREAM ButtEer.—If the cream is allowed to undergo the acid fermentation, the butter will differ markedly both in degree and kind of flavor from that prepared from sweet cream, and as arule its keeping qualities are much better than those of sweet-cream butter. This type of butter is made throughout northern Europe, England and her * Prepared by E. G. Hastings. 408 THE RELATION OF MICROORGANISMS TO BUTTER 409 colonies, and in America. It.may be said to be the standard butter of the world since it is the type made in all the great dairy countries. Sweet-cream butter is made especially in southern Europe, and in limited amounts in other countries. The intensity and kind of flavor of butter is thus dependent on the acid fermentation of the milk or cream. It is not believed that the fat undergoes any changes during the acid fermentation of the milk which could produce the flavor of sour-cream butter, but rather that the increase in flavor is due to the absorption by the butter fat of certain of the compounds formed in the acid fermentation. It is not essential that the fat be present during the acid fermentation in order to impart flavor to the butter. If sweet cream is mixed with sour milk and churned at once, the flavoring compounds are absorbed by the fat from the fermented milk, and the butter will have much the same flavor, both ‘as to intensity and kind, as though the fat had been present during the fermentation. The churning of a mixture of sweet cream and sour milk is used commercially and is identical with the methods employed by the manufacturers of oleomargarine and renovated butter to impart flavor to the flavorless fats they employ. It is impossible to recognize these substitutes for butter by their flavor since it is identical with and derived from the same source as the flavor of butter. In the past many ideas have been expressed as to the source of the flavor of butter; some have asserted that it is due, in part, to the decomposition of the proteins of milk by proteolytic bacteria. Both practical experience and experimental work have demonstrated the connection between the acid fermentation of milk and the flavor of butter, and it is certain that what is now considered the finest type of butter can be made from cream in which only acid-forming bacteria (see Chap. I) have grown. FLAVOR OF BUTTER ContRoL oF BuTrer FrLavor.—The commercial value of any sample of butter is largely determined by its flavor. If it is lacking in flavor and aroma, or if it has a poor flavor; it brings a lowprice. The importance of being able to control the flavor, both as to degree and kind, 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 has largely supplanted the making of butter on the farm. The financial success of any creamery is largely dependent upon the ability of the butter maker to control the flavor of the product, so that it shall be uniform from day to day. It is asserted that one of the factors in the remarkable invasion of Denmark into the butter markets of the world is the uniformity of the Danish butter, not only from a single creamery, but from all the creameries of the country. To the Danes we owe the most improved methods for the control of the flavor of butter. The other points, texture, color and salt, which the judge of butter takes into consideration, can be easily controlled, since they are due to mechanical operations. The flavor, on the other hand, is due to the by- products which are formed by microdrganisms in the fermentation of the milk and cream, and which are absorbed and held by the fat. If any of the products formed possesses a disagreeable taste or an offensive odor, the flavor and aroma of the butter will be impaired. It is thus evident that the control of the flavor of butter is dependent on the control of the acid-forming bacteria thatferment the milk andcream. Thisis the prob- lem of the modern butter-maker and the modern methods seek to give him this control, to enable him to eliminate the undesirable bacteria, B. coli and Bact. aerogenes, the second group,* and to insure the predomi- nance of the desirable bacteria, Bact. lactis acidi. This general statement is not to be interpreted as meaning that all bacteria that injure the flavor of butter are to be included in the group mentioned, for many other types of bacteria, when present in milk in large numbers, may injure the flavor of the butter prepared from it. The acid fermentation of the cream is most frequently called the ripening of cream and sour-cream butter is frequently called ripenéd- cream butter. The ripening of the cream not only increases the flavor of the product, but it enhances its keeping quality. The ripening of the cream also aids in the mechanical process of churning, the sour cream churning more easily and with less loss of fat in the butter milk. Kinps AND NumBERS OF BACTERIA IN CREAM.—The number and kinds of bacteria found in cream are dependent upon the number and kind in the milk from which the cream is obtained. The cream will, however, contain a greater number of bacteria per unit volume than the * See Chap. I, Div. IV, in which the groups of bacteria are considered. THE RELATION OF MICROORGANISMS TO BUTTER 4II milk, since the immense number of fat globules passing through the milk serum carry mechanically a considerable proportion of the bacteria of the milk into the cream. This phenomenon is to be noted in gravity creaming, but to a much greater extent in the removal of the cream by use of the separator. SPONTANEOUS RIPENING OF CREAM.—By this expression is meant the fermentation of the cream by those acid-forming bacteria that have, from one source and another, gained entrance to it, but which have not been intentionally added. Under these conditions the butter-maker can exert but little control over the fermentation. A very considerable part of the butter made from such cream has an excellent flavor, because at the temperature at which cream is usually kept, Bact. lactis acidi and related organisms are the primary factors concerned in its fermentation and their by-products produce desirable flavors in butter. It has often -been asserted that the highest type of butter can be made only from spontaneously ripened cream. . As the cream from many farms was assembled at a creamery for the manufacture of butter, it became evident that some means of controlling the type of fermentation in the cream was needed. If the milk had been produced under clean conditions, and had been received at the creamery before the acid fermentation had gone on to any extent, and if the cream was then kept at temperatures most favorable for the lactic bac- teria, the product was likely to be of good quality, but such ideal condi- tions did not always obtain. Cream containing a large proportion of harmful bacteria, or in an advanced state of fermentation, or possessing an undesirable flavor was often received, and the butter-maker could not control the quality of the product under such conditions. UsE oF CULTURES IN BUTTER MAxinc.—As the science of micro- biology progressed and the réle of microdrganisms in all kinds of fermen- tation became known, it was evident that the control of the causal or- ganism is an important factor in determining the quality of any product of the fermentation industries. In the manufacture of butter, the first step in this direction was the addition of some fermented milk, cream, or of buttermilk to the cream to be ripened. In this manner the number of acid-forming organisms in the cream was greatly increased, and the fermentation went on more rapidly and in a more definite direction than without such additions, as the bacteria added were largely of the desirable group, Bact. 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 the science of bacteriology. The next logical step in the Sey elopment of the process was the use of the same types of bacteria from day today. Cultures of these were ob- tained by allowing a quantity of milk to sour, and if it had the desired flavor, a small amount of it was added to another quantity of milk that had been heated, in order to destroy the acid-forming bacteria it con- tained. By the daily preparation of some heated milk, and the inocula- tion of it with the soured milk previously prepared, the butter-maker could use the same types of bacteria for an indefinite time for addition to the cream. It had been found by Hansen that, in order to control the flavor of peer, pure cultures of yeasts must be used for the fermentation of the wort. The success of this method in the brewing industry led to the introduction of pure lactic cultures for the fermentation of cream. The - use of such cultures was suggested independently by Storch, a Danish bacteriologist and by Weigmann, the director of the dairy experiment station at Kiel in Germany, in 1890. Many cultures were isolated and tested as to their effect on the flavor of butter. Those found to be desirable could be maintained in the laboratory, and could be furnished to butter-makers to be used and propagated in a manner similar to the method employed with the impure and less constant home-made starters. The pure cultures of lactic bacteria are widely used at present in the butter-producing countries of the world and their use is being constantly extended, as butter makers come to recognize the importance of con- trolling the ripening of cream. It was found that the butter made from cream ripened by pure lactic cultures did not possess as high a flavor as did the finest butter made from naturally ripened cream. This led to the search for organisms that could be used alone, or together, with the lactic bacteria, and which should give the high flavor desired. Such cultures were found, but their use did not prove practical, either because they did not maintain their properties on continued cultivation, or because of their effect on the keeping quality of the product. The difference in flavor in the case of butter made from naturally ripened cream and that from cream rip- ened by pure lactic cultures is undoubtedly due to the products of the B. coli-aerogenes group. The acid’ in spontaneously soured milk is very evident to the taste THE RELATION OF MICROORGANISMS TO BUTTER 413 when the acidity is 0.6 per cent and above; the volatile acids formed by the members of the colon-aerogenes and coccus groups impart a sharp, pungent taste. In milk of like acidity fermented by pure cultures of Bact. lactis acidi, the acid is scarcely evident to the taste and there is no sharpness, due to the absence of volatile acids. This same difference appears in the butter made from the two kinds of milk. The low flavor of the butter made from cream ripened by pure cultures was one of the factors that prevented the rapid introduction of the cultures in this country. The demands of the butter market have changed and the mild flavored butter, which is now considered to be the finest, can be made by the use of pure cultures in the fermenta- tion of pure sweet cream. CoMMERCIAL CULTURES.—In this country the preparation and distribution of cultures for the ripening of cream is largely in the hands of commercial firms; hence, the term ‘‘commercial culture” is applied to them. The different pure cultures are propagated in the laboratory of the maker; they are sent out either as liquid cultures, a small mass of milk or bouillon inoculated with the organism, or in a dry form, the latter being prepared by mixing a culture of the organism with an inert substance, such as milk sugar, milk powder, or starch, and drying at a low temperature. In a liquid the organisms are exposed to the effects of their own by-products, and the vitality of the culture is rapidly lost. Such cultures must be used when fresh in order to give good results, and they cannot be kept in stock by the manufacturer or dealer. The resistance of Bact. lactis acidi to desic- cation is great; it thus lends itself to the preparation of the dry cultures, in which the organisms remain in a dormant condition and retain their vitality for long periods. Most of the cultures now sold are pure, as this term is used in bac- teriology, still others contain non-acid-forming organisms, intentionally added or introduced accidentally during the process of preparation. If the lactic bacteria are present in such cultures in large numbers, the impurities are usually of small practical significance. In the past so-called “duplex” cultures have been sold which were supposed to contain an acid-forming organism and a second organism that was to enhance the flavor of the product. Such cultures are no longer sold. For the propagation in the creamery the contents of the container purchased are added to a small mass of milk that has been heated 414 MICROBIOLOGY OF MILK AND MILK PRODUCTS to destroy all non-spore-forming bacteria and other microtrganisms; the milk, after being inoculated, is incubated at favorable tempera- tures and when curdled can be used for the inoculation of the second and larger quantity. The process of inoculating a quantity of milk is carried out daily. It is impossible for the butter-maker to propagate the culture so as to maintain the original purity, but with care in the heating of the milk the sterilization of all utensils and the maintaining of proper temperatures, the contamination that occurs will not injure the culture for practical work. The cultures propagated under such conditions gradually deteriorate and recourse must be had sooner or later to a fresh culture. The contamination that is of the greatest practical significance is undoubtedly that with other acid-forming bacteria rather than with the forms that remain in the milk after heating. ‘Many of the cultures gradually lose their fermentative properties, and do not form acid rapidly and in sufficient amounts to insure exhaustive churning and to produce the desired degree of flavor in the product. Cultures frequently become slimy or ropy on propaga- tion. This is not necessarily due to contamination with specific slime-forming organisms but rather to a change in the lactic organism itself. Such an abnormality usually persists for only a short period and the conditions that govern its appearance and disappearance — are not known. It is asserted by practical butter makers that the development of too high an acidity in the cultures as they are propa- gated in the creameries permanently impairs the value of the culture. The cultures are propagated in skim-milk. Where this is not avail- able, unsweetened, condensed milk or milk powder have been employed. Efforts have been made to grow the bacteria in some other kind of medium than milk, but without success. The starter is said to be ripe or in the best condition for use soon after curdling, or when the © acidity is 0.5 to 0.7 per cent, as at this time it contains the maximum number of living cells. The practical man thus uses the curdling as an indication of the ripeness of the starter. The curdled milk should show no free whey, and the curd should be easily broken up to form a creamy mass that can be uniformly incorporated with the cream. The temperature of incubation and the amount of initial inoculation determine the rapidity with which the acid fermentation will progress, THE RELATION OF MICROORGANISMS TO BUTTER 415 the maker seeking to regulate these so that the culture shall be ripe at the desired time each day. UsE oF Pure CULTURES IN Raw CrEAM.—The cream as it reaches the creamery contains a greater or less number of acid-forming bac- teria that ultimately will cause it to ripen and the flavor of the butter will be due to the by-products of the mixture of bacteria. If, through the addition of a pure culture, the relative number of organisms that are known to be favorable is greatly increased, the flavor of the product should be improved. This has been found to be true in practice and it is now believed that pure cultures are of value not only in the ripen- ing of sweet cream, but that the addition of a relatively large amount of starter to cream that is already fermented will enhance the value of the butter. UsE oF Pure CULTURES IN PASTEURIZED CREAM.—It is evident that the maker has but imperfect control over the fermentative proc- esses when raw cream is treated with a pure culture. To insure more perfect control the destruction of the contained bacteria and the subsequent inoculation of the cream with a pure culture is indicated. The introduction of the process of pasteurization of cream for butter making was due to Storch. In Denmark this method is used almost exclusively. It has been introduced into the other dairy countries of the world and is constantly spreading. Pasteurization combined with the use of the pure culture represents the highest type of modern butter making, and where the raw product can be obtained in a fresh condition the butter-maker has perfect control over the bacteria that cause the ripening; hence he can control the flavor of the butter, both qualitatively and quantitatively. The intensity of flavor of butter is dependent upon the amount of acid that is developed in the cream or more correctly on the ratio between the amount of fat and the by-products of the acid fermenta- tion. If these by-products are small in amount, as in cream having a low acidity, the flavor of the butter will be low. If the acidity is allowed to reach the maximum, the flavor will be much higher. Thus the maker can control the intensity of flavor. of butter as accurately as he can the kind of flavor. With rich cream, the acidity that can be developed is small and the ratio between the fat and the products of fermentation is low; thus, the flavor of butter made from very heavy cream is certain to be low. § 416 MICROBIOLOGY OF MILK AND MILK PRODUCTS PuRE CULTURES IN OLEOMARGARINE AND RENOVATED BUTTER.— It was previously mentioned that the manufacturer of butter substi- tutes employs the same methods to impart butter flavor to his prod- ucts as does the butter-maker. The oleomargarine manufacturers employ pure cultures of lactic bacteria for the fermenting of milk that is mixed with the fats they employ. The same practice is followed by the manufacturer of renovated butter. Many of the creameries of the western states receive cream that is shipped long distances, and is collected from the farms but once or twice a week. It is thus in an advanced state of fermentation when it reaches the creamery. In order to prepare from this grade of cream, which often has a most undesirable flavor, a merchantable product, various means are em- ployed to remove the flavoring substances and to replace them with desirable flavors from the pure cultures. The acidity may be reduced by the addition of lime so that the cream can be pasteurized; the cream may be aerated by passing air through it, or it may be mixed with water and reseparated. After such treatment it is mixed with a large proportion of milk fermented by a pure culture and churned. The resulting product is constantly sold as the highest grade of creamery- butter. ‘ ~ ABNORMAL FLavors oF BUTTER.—Most of the abnormal flavors of butter are traceable to the partial replacement of the desirable acid- forming bacteria with other types of microdrganisms Many samples of butter having abnormal flavors have been examined, and the organ- isms believed to be the cause isolated and studied but it cannot be said that any particular group of microérganisms can be associated with any of the abnormal flavors met. It is asserted that “‘oily”’ butter, z.¢., that having the taste of machine oil, is caused by bacteria and by microdrgan- isms that decompose the fat, as Oidium lactis, yeasts, and liquefying bac- teria. Organisms of the B. coli group that produce a turnip-like flavor in butter have been described by Weigmann. The flavors of putrid ~butter, fishy butter and also many other abnormal flavors have been ascribed to bacteria. Other abnormal flavors may be due to the presence in the milk of certain aromatic principles contained in the feed and excreted in the milk. Cabbage, turnips, and other plants impart their characteristic 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- ern churn and all subsequent changes are likely to cause more or less deterioration. The specific causes of these changes are not well known but it is very evident from a study of the conditions that favor or retard the appearance of the flavors, characterizing these changes, that biolog- ical factors areconcerned. Whenraw cream is used, sweet-cream butter has very poor keeping qualities. As the proportion of acid-forming ' bacteria in butter is increased, either by the fermentation of the cream, by the addition of pure cultures, and through the use of the latter in “connection with pasteurization, the keeping qualities are enhanced. Of the butter made from ripened cream, that prepared from cream, handled in a clean manner, and thoroughly pasteurized and ripened with a pure culture of Bact. lactis acidi, has the best keeping quality. If fresh, sweet, clean cream is pasteurized, the butter will have better keeping quality than when made from the same cream pasteurized and ripened with a pure culture. This is evidence that not only the bac- teria other than Bact. lactis acidi are harmful, but that this organism, that has usually been considered without influence on the keeping quality, must be classed as one of the factors in the decomposition of butter. It has been shown that the bacterial content of the water used for the washing of the butter has an influence on the keeping quality. If the water is of surface origin and contains the bacteria peculiar to these types of waters, its influence may be marked and some method of treat- ment must be followed. Filtering or heating the water has been re- sorted to, the latter with marked success. A pure water will contain so few bacteria that they will not exert any noticeable influence on the keeping quality of the butter. Storage temperature also has a marked influence on the deterioration changes in butter. Modern butter-storage rooms are kept below o°F.; the butter deteriorates slowly during storage at these tempera- tures, but on removal undergoes change much more rapidly than would have been true beforestorage. Another factor that is of influence in the keeping of butter is the amount of salt used. In salted butter, the contained water is a concentrated brine; in such a medium most forms of bacteria are unable to grow. Small packages deteriorate more 27 418 MICROBIOLOGY OF MILK AND MILK PRODUCTS rapidly than large ones, because the proportion of the mass of butter, exposed to the air is relatively greater. Exposure to light is also claimed to exert a harmful influence. ‘Antiseptic substances such as borax and boric acid have a marked effect on the deterioration changes. The New Zealand and Australian butter exported to'the English mar- kets is treated with preservatives. A large amount of experimental work has been done in order to determine the effect of specific organisms on the keeping quality of butter. The results obtained have not been definite and it is not certain that the organisms employed are constantly concerned in the deteriora- tion changes. It is very probable that both bacteria and molds exert an influence. The chemical changes that take place in the spoiling of butter are no better known than are the causal factors. It has been asserted that there is a decomposition of the glycerides with a resulting increase in free acids. It has been shown that this does not always occur; that a butter may be in an advanced state of decomposition and its content in volatile acids not be higher than when fresh. Two types of changes are usually distinguished, rancidity and the appearance of a tallow-like odor. The latter may be due to purely chemical factors, while the former is quite certainly biological. Moldy butter is a frequent trouble encountered by the butter-maker. If the butter is not salted, molds may develop just below the surface. The most usual form of mold to appear is one with black hyphe; the slightest development of which will be evident on the butter. In the case of salted butter, mold on the butter itself is very rare, due appar- ently to the concentration of the brine in the butter. The parchment paper in which print butter is wrapped and with which the butter con- tainers are lined is an excellent substratum for mold growth. If the papers and containers are badly contaminated with mold spores, or they have been kept under such conditions as to permit of a limited amount of growth before they are used, the development of the mold on the paper after it is brought into contact with the butter is likely to be rapid, even at low temperatures, and the butter is likely to reach the market in an objectionable condition. The paper may be rendered free from molds by placing it in water which has been heated to at least 80°. Butter tubs are scalded, steamed, or soaked in brine or treated with a dilute solution of formalin in order to destroy the mold spores that may be present. The most efficient manner of preventing trouble \ THE RELATION OF MICROORGANISMS ‘TO BUTTER 4i6 is to coat the inside of the butter tub with paraffin. This prevents trouble from the container but not from the paper. PatHOGENIC BACTERIA IN BUTTER 1 If the milk contains pathogenic bacteria, they are certain to pass into the cream and be incorporated in the butter. It is not believed that butter is an important agent in the distribution of the organisms of tuberculosis and typhoid fever, although both are able to exist in salted butter for over two months. Foot-and-mouth disease is said be caused in humans by the use of butter made from the milk of infected animals, but this may still be regarded as a mooted question. CHAPTER III* THE RELATION OF MICROORGANISMS TO CHEESE GENERAL Cheese consists of the fat and casein of milk, together with the insoluble salts; however, along with these constituents are carried some of the moisture of milk, in which are dissolved small quantities of sugar, albumin, and salts. The amount of moisture and soluble constituents found in cheese is determined by the amount of whey incorporated in the curd. In the process of making cheese, it is necessary to curdle the milk, thus enabling the separation of the casein and fat from the milk serum. Two methods are employed to accomplish this purpose, and, as a result, two types of cheeses are produced. TypEs oF CHEESE These types may be designated as “‘Acid-curd Cheeses” and “‘Rennet- curd Cheeses.” Acip-curD CHEESES.—The curdling may be accomplished by allowing the milk to undergo acid fermentation, either spontaneously through the action of the normal flora of the milk, or through the addition of pure lactic cultures. Most acid-curd cheeses are ready for use as soon as the whey has been removed by draining and the curds salted. Acid-curd cheeses are not commercially important. They are made for local consumption and are to be classed as a form of sour milk. They owe their flavor to the products of the acid 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 cheesés are made by the use of rennet for the curdling of the milk. Over * Prepared by E. G. Hastings. 420 THE RELATION OF MICROORGANISMS TO CHEESE 421 four hundred kinds of rennet-curd cheeses are made, but only twelve to fifteen are of great commercial importance. With few exceptions, they are made from cow’s milk. From the same raw material—milk, rennet, and salt—therefore, a wide variety of products, differing in texture, taste and odor, is obtained. This fact indicates the im- portance of biological factors in the changes the curd undergoes during the ripening process. The rennet-curd cheeses may be divided into: (1) hard cheeses; (2) soft cheeses; the initial difference is largely in the amount of whey left in the curd during the making of the cheese. The two great groups of rennet-curd cheeses gradually merge into each other in varieties that 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 in texture. The curd, which is not easily digested, is quite in soluble in water and is devoid of flavor and aroma. The.curd must pass through a complete series of chemical and physical changes, which alter its texture, solubility, and digestibility, and give to it a flavor and aroma by which the different kinds of rennet-curd cheeses are especially to be differentiated. In the hard cheeses the factors concerned in these changes act in a uniform manner throughout the entire mass of the cheese, making it possible to manufacture such cheeses in any desired size. In the case of the soft cheeses, the ripening changes are largely due to agents which grow only on the surface; the products of such agents by means of diffusion gradually affect the entire mass. In order that this may take place within a reasonable time, it is essential that these cheeses be made in small sizes. Then, too, the soft texture of such cheeses makes it impossible to handle them commercially in large sizes. ConDITIONS AFFECTING THE MAKING OF CHEESE Quatity oF Mitx.—In the curdling of milk by rennet the solid bodies present in the milk are retained in the curd, thus the fat globules are held, as are also the bacteria. The latter continue to ‘grow as they would have done in the milk except that growth must take place in the form of colonies as in the solid culture media of the bacteriologist. The bacteria, however, produce the same fer- mentation 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 use of pure lactic cultures, the fermentation of the cream. The pasteuri- zation may be so efficient as to destroy all non-spore-forming bacteria since the quality of the product will not be impaired by the use of temperatures approximating the boiling point. The cheese-maker Fic. 134.—The type of curd obtained from milk in which the acid-forming flora consists largely of organisms of the B. coli-aerogenes group. Many gas holes and few irregular shaped, angular, mechanical holes due to imperfect “matting.” (Original.) is much more dependent on the original quality of the milk, since it has not been found possible to make most of the important varieties of cheeses from pasteurized milk. If undesirable forms of microérganisms are present in the milk, they will pass into the cheese and there produce their harmful effects. Through the addition of pure cultures of THE RELATION OF MICROORGANISMS. TO: CHEESE 423 Bact. lactis acidi to the milk, the proportion of desirable bacteria can be increased and a partial control of the fermentation thus secured. TESTS FOR THE QuaLITy oF MitxK.—Methods by which the cheese maker can determine, in a rough manner, the kinds of bacteria present Fic. 135.—The type of curd obtained from milk in which the acid-forming flora consists almost wholly of Bact. lactis acidi. No gas holes and no marked mechanical holes as the curd has “‘matted” almost perfectly. (Original.) have been devised. The bacteria most dreaded and most frequently present are those of the B. coli-aerogenes group. The method most frequently used for their detection consists in incubating a sample of the milk to be tested at temperatures ranging from 35° to 40° for a few hours and noting the type of curd that is formed. Milk suitable for cheese making should show the solid curd characteristic of the Bact. lactis acid group, while gassy curds or soft 424 MICROBIOLOGY OF MILK AND MILK PRODUCTS and partially digested curds are indicative of bacteria that are likely to be harmful in the cheese. An improvement over the fermentation test of foreign origin has been devised by Babcock and Russell and is known as the Wisconsin Curd Test. It has for its basis the same principle as the simple fermen- tation test; however, a modification is introduced; the milk is curdled by the addition of rennet and the curd is cut and drained to free it from the whey as completely as possible. The undesirable organisms most likely to be present in milk are those of the B. coli-aerogenes group; therefore the jars containing the curds should be kept at temperatures, 35° to 40°, that will favor their development. The great advantage of the Wisconsin Curd Test is its greater delicacy, since the bacteria are concentrated in a small volume, and thus their presence is more evident than would be the case in the larger mass of curd obtained when no rennet is added. The curd can also be removed from the jar, cut, tasted, and its texture determined, all of which aid in judging the quality of the milk. The curd should have a clean acid odor and taste; it should be free from sliminess on the sur- face, and possess a uniform texture. Such a curd can be obtained only in the presence of a considerable number of lactic bacteria. Very clean, fresh milk is likely to give an undesirable result, since milk always contains microérganisms which will grow rapidly at the high temperature in the absence of the acid-forming bacteria and which will usually produce undesirable flavors in the curd. This fact should be. kept in mind in the testing of market milk. RIPENING OF Mitx.*—The methods for the determination of acidity in milk have very considerable limits of error. It is not possible to detect any increase in acidity until the number of acid-forming bacteria has increased to hundreds of thousands per c.c. Originally it was thought that no acid was produced by the growth of the acid-forming bacteria during the initial stages of their development. This period during which bacterial proliferation was taking place, but without an apparent increase in acidity, was known as the “‘period of incubation.” It is now certain that this rests upon our inability to detect small *In order to illustrate the rdle of microérganisms in the making and ripening of cheeses, a somewhat detailed summary of the present knowledge concerning their action in Cheddar cheese will be given. Many of the factors concerned in the ripening of this kind of cheese also function in the ripening of other rennet cheeses. In their description only such additional factors need be considered as are not active in Cheddar cheese. THE RELATION OF MICROORGANISMS TO CHEESE 425 " increases in acidity. The Cheddar cheese-maker desires milk that shall contain such a number of acid-forming bacteria that during the opera- tions that are carried on in the first part of the cheese-making process large amounts of acid shall be formed in the curd. He thus wishes to know, as accurately as can be determined under the conditions found in the factory, the number of bacteria in the milk which he is to use. This information is gained either by the titration of the milk with a standard alkali solution or by determining the time required for the curdling of a definite quantity of milk at a definite temperature by a known quantity of rennet. Very much smaller increases in acidity can be detected by the so-called rennet test than by titrating the milk. If the milk shows the desired acidity when it reaches the factory, the making process is immediately begun. If the milk is too sweet, or in other. words, too low in its bacterial content,’ bacterial growth is favored by warming the milk to temperatures most favorable _ for the lactic bacteria, 30° to 32°, and by the addition of pure cultures of Bact. lactis acidi which are identical in nature and the method of propagation with those used in butter making. The development of a slight acidity is known as the “ripening” of milk. In order to insure proper rennet action the maker of Cheddar cheese desires the milk to have an acidity of about 0.2 per cent. He thus wishes milk that has passed through the period of incubation and in which the acidity has begun to increase. CurRDLING oF Mitx.—Under the influence of a favorable tempera- ture and the slight acidity, the milk is quickly changed by the rennin* to a firm, jelly-like mass that is cut, with appropriate knives, into small cubes. The curd encloses over 80 per cent of the bacteria in the milk. The same factors that favor the curdling of the milk favor the shrinking of the curd and the expulsion of the whey from the cubes. The develop- ment of acid within the curd is rapid, due to the concentration of large numbers of bacteria in a small volume and to the favorable environ- ment. During the six to eight hours that elapse between the curdling of the milk and the pressing of the curd, the increase of acidity is over o.1 per cent per hour. The following table gives the acidity of milk and the whey expressed from the curd at various stages in the making of a typical Cheddar cheese. * The rennet used in cheese-making is obtained by extracting the abomasum, the true diges- tive stomach of the calf, with a solution of sodium chloride. The extract céntains two enzymes a clotting or curdling enzyme, rennin, and a proteolytic enzyme, pepsin. 426 MICROBIOLOGY OF MILK AND MILK PRODUCTS Acidity of milk before adding rennet.......... ©.20-0.21 per cent. Acidity of whey immediately after cutting curd. 0.14-0.145 per cent. Acidity of whey when removed from the curd.. o.16-o.18 per cent. Acidity of whey when curd is packed.......... ©.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........... ©.90-1.10 per cent. -MAnIpuLaTIoN oF THE CuRD.*—The curd particles at first show little tendency to cohere; but, as the acidity increases, the nature of the curd changes, and, when the whey is removed, the pieces of curd soon cohere and ultimately form a single mass in which the original cubes of curd cannot be detected. The fusion of the curd particles is known as “matting” and is an important, step in the Cheddar process. The lack of acid formation within the curd prevents matting while the curd is in the vat, and may even render difficult the fusion of the particles under pressure. The nature of the change which the curd undergoes at this stage in the manufacture is not well understood, but probably is due to a combination between the paracasein and the lactic acid, the resulting compounds differing from the paracasein in physical properties and in solubilities. RIPENING OF CHEESE.—Cheese in ripening undergoes profound physical and chemical changes under the influence of a number of factors, which for purposes of discussion may be divided into two groups: those by which the content of soluble nitrogen in the cheese is increased and the digestibility enhanced; and those which cause the formation of flavoring substances. During the ripening of the cheese the maker can do little toward the control of the factors which ultimately deter- mine its commercial value. As in butter, the flavor is the most impor- tant characteristic of the ripened cheese and the most difficult to control. Theories of Cheese Ripening.—Many theories have been advanced to explain the changes that occur during theripening process. Duclaux, a French microbiologist, studied the bacterial flora of Cantal cheese by aid of the crude methods available before the introduction of the gelatin-plate method. By the use of the dilution method, using bouillon as the nutrient medium, he isolated a number of kinds of spore-forming bacteria. The organisms formed two enzymes, one a curdling enzyme related to rennin, the other a proteolytic enzyme * Cheddar cheese. THE RELATION OF MICROORGANISMS TO CHEESE 427 to which was given the name casease. A chemical study of the by- products of the organisms, when growing in milk, revealed a number of compounds that had previously been found in ripe cheese, such as. leucin, tyrosin, and the ammonia salts of acetic, valeric and carbonic acids. The cultures often possessed a cheese-like odor. These facts led Duclaux to believe this class of organisms were responsible for the ripening of the hard cheese in question. The generic name Tyrothrix was applied on account of the supposed relation to cheese. This term is still found in current bacteriological literature. The methods employed by Duclaux were such as favored the growth of the liquefying, rather than the acid-forming bacteria. To the latter more recent investi-. gators have devoted attention. The theory that the proteolytic bacteria function in the ripening of hard cheese has been more recently emphasized by Adametz. It is sufficient to say that the number of spore-forming proteolytic bac- teria in cheese is not sufficiently large, nor is their presence so constant that any importance can be attached to them. Any agent to be con- sidered as a factor in the ripening process must be present in every cheese in sufficient numbers to account for the change for which it is considered responsible. Such agents should be capable of demonstra- tion. It should be remembered that, by following the rules laid down by the practical maker, a normal cheese can invariably be made, hence the factors of importance in the ripening must be constantly present in the milk or rennet. It is doubtful whether the liquefying bacteria will satisfy this requirement. It has been shown by de Freudenreich that such organisms, even when added to milk in large numbers, exert no influence on the ripening of hard cheese, since the conditions within the cheese are not such that growth can occur. De Freudenreich, a Swiss microbiologist, by the aid of modern methods, demonstrated the constant presence of certain classes . of acid-forming bacteria in Swiss cheese, and to them ascribed an impor- tant réle in the ripening of this hard cheese. He was led to this con- clusion by their great numbers in the fresh cheese, and by the fact that cheese made from milk drawn under aseptic conditions, which thus contains no lactic bacteria, do not ripen; through the discovery, also, that certain of the lactic bacteria, predominating in Swiss cheese, those of the Bact. bulgaricum group, exert a solvent effect on the casein of milk, although they are devoid of action on gelatin. 428 MICROBIOLOGY OF MILK AND MILK PRODUCTS Babcock and Russell demonstrated the presence of an inherent proteolytic enzyme in milk, to which the term galactase was applied. This enzyme can be demonstrated by preserving a sample of fresh milk with chloroform or other mild antiseptic. At 37° curdling occurs in three to four weeks; the content of soluble nitrogen in the milk is slowly augmented. The presence of this proteolytic enzyme, together with the fact that a normal cheese cannot be made from milk in which this enzyme has been destroyed by heat, led these investi- gators to consider this inherent enzyme of milk an important factor in cheese ripening. Present Knowledge of Causal Factors.*—The réle of certain factors in the ripening of Cheddar cheese has been established beyond doubt by the chemical and bacteriological investigations of recent years. It is certain that acid-forming bacteria are essential factors in the ripening of this kind of hard cheese, and probably all kinds of rennet cheeses. ‘ As has been shown the growth of acid-forming bacteria is rapid during the making of Cheddar cheese. The growth continues during the pressing and subsequent thereto; the maximum number of lactic bacteria is found when the cheese is one to five days old. As many as 1,500,000,000 per g. of the moist cheese have been demonstrated. Causes of Proteolysis.—The proteolytic action of rennet extract on the paracasein of cheese was demonstrated by Babcock and Russell, and by Jensen. This property is due to the fact that rennet extract also contains the enzyme pepsin, which for its action outside the body requires conditions similar to those which obtain in the stomach; in other words, the presence of sufficient acid toactivate it. Thehydro- chloric acid secreted by the walls of the stomach acts as the activating agent in the body. The acidity resulting from the fermentation of the sugar in the curd is sufficient to activate the pepsin. Under its influence the paracasein is partially converted into soluble decomposi- tion products such as albumoses and peptones. In the absence of acid-forming bacteria no acid is formed; consequently the pepsin does not become active and no proteolytic effect is produced. Under these conditions the curd remains tough and elastic and the solubility is not increased. It is thus evident that acid-forming bacteria are essen- tial factors in cheese ripening. The pepsin of the rennet extract and *Cheddar cheese. THE RELATION OF MICROORGANISMS TO CHEESE 429 the galactase suffice to account for the initial proteolysis of the para- casein. Since neither of these enzymes forms ammonia, which is always found in ripe cheeses, some other factor must be responsible for the production of this compound. It may owe its origin to micro- organisms not yet discovered. Prevention of Putrefaction—The various stages in the decomposition of milk have been outlined in a previous chapter. Briefly they are as follows: The first evident change is the curdling due to the acid-forming bacteria. Succeeding this, the acid, semi-solid mass or curd is a favor- able substratum for the characteristic mold of milk, Oidiwm lactis, which A B Fic. 136.—Proteolytic action of rennet extract in the absence and in the presence of acid-forming bacteria. A, sterile milk agar; a strip of filter-paper treated with rennet was allowed to remain on the medium for one hour at 37°. No digestion of the casein. B, milk agar inoculated with Bact. lactis acidi; incubated for twenty-four hours at 37°, then treated as A. True digestion of the casein is indicated by the clearing. (Original.) soon forms a white, velvet-like layer over the surface of the milk. Like other molds, this form can use acids as a source of energy. The acid is then oxidized to carbon dioxide and water, and thus the reaction of the milk is slowly changed until a point is reached which allows the putre- factive bacteria, that have remained dormant during the period of un- favorable environment, to develop. The curd is accordingly pepton- ized and putrefaction occurs. If the acid reaction is maintained through 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 an unlimited time. The second réle of the acid-forming bacteria in cheese is to protect it against the putrefactive organisms that are constantly present in milk and hence in cheese. The acid reaction of the cheese, due to the persistence of lactic acid, or to the formation of volatile acids after the initial fermentation, is sufficient to prevent the growth of the putrefac- tive bacteria within the cheese. If the cheese is made from milk which contains no acid-forming bacteria and few putrefactive ones, or if the - sugar is removed from the curd by washing it with water, the cheese will not ripen since there is no acid to activate the pepsin; the curd will re- main in much the same condition as when it was removed from the press. Cheese made from milk containing no acid-forming bacteria but many putrefactive bacteria is likely. to undergo putrefaction, since the latter class of organisms finds conditions for growth in the absence of an acid reaction. Such a condition is rarely noted in a hard cheese under normal conditions, but may be produced experimentally. ‘The biological acid may be replaced by other acids added to the curd in appropriate amounts, since these will activate the pepsin and protect the cheese against the attacks of putrefactive bacteria; but it is not cer- tain that the cheese will develop a normal flavor when lactic acid is teplaced by mineral acids. Other Groups of Bacteria in Cheese.—It has been shown at- the Wis- consin Experiment Station that other groups of bacteria are constantly present in Cheddar cheese. The development of certain members of the Bact. bulgaricum group occurs somewhat later than that of the Bact. lactis acidt group. It occurs largely after the sugar has disappeared. | Their numbers approximate those of the Bact. lactis acidi group. Coccus forms are also found in great numbers in the cheese. It is probable that these two groups may be responsible for the ammonia production, since typical cultures of both groups are able to produce small amounts of ammonia in sterile milk. Flavor Production in Cheese—The factors that have been discussed are undoubtedly the most important ones concerned in the proteolysis of the curd, and are thus the factors concerned in the changes of texture, solubility and digestibility. The flavor, which develops during the ripening process, has been regarded as due to the proteolysis of the para- casein. A thoroughly ripened cheese contains a large amount of am- THE RELATION OF MICROORGANISMS TO CHEESE 431 monia and relatedcompounds. It was thusnatural to consider the flavor due to these simple products of protein degradation. More recently it has been discovered that the intensity of flavor does not necessarily cor- respond to the content of the cheese in these products; indeed a cheese may have a high content of nitrogen as ammonia and yet be low in flavor. The Wisconsin Experiment Station has found that the volatile fatty acids of Cheddar cheese increase as the ripening progresses. In the following table are given the data obtained from the detailed study of a normal Cheddar cheese. ~ Acips In 100 G. or Dry MATTER c.c. of N/10 alkali neutralized zi | 3 days 42 days 3 months 54 months | 10 months Lactic acid.......... 84.09 90.28 124.00 "| 103.70 74.10 Acetic acid.......... II.50 29.44 24.25 25.86 12.64 Propionic acid....... 0.41 2.15 3.42 1.07 2.63 Butyric acid......... 1 (0.93 2.17 3-50 4.82 | 5.45 Caproic acid......... | 9.00 0.36 0.96 1.25 | 2.23 It will be noted that the content of the higher volatile acids, those especially marked in odor, continually increases. It is possible to sepa- rate other volatile compounds found in cheese from the volatile fatty acids by distilling with steam, neutralizing the distillate with an alkali and redistilling; the second distillate will contain the alcohols and esters present in the cheese. Such a distillate prepared from Cheddar cheese is found to possess the characteristic aroma of the cheese in question. The esters it contains are largely those of ethyl alcohol. The acid-form- ing bacteria, as stated previously, produce varying amounts of volatile acids and slight amounts of alcohols and esters. It is likely that the larger part of the volatile compounds found in the ripening cheese is formed in fermentations which take place subsequent to the initial fermentation of the lactose. The flavor of Cheddar cheese, therefore, owes its origin very probably to the fermentation of the lactose, and to the further change which the products of the initial fermentation un- dergo under the influence of biologicalfactors yetunknown. Thatsome 432 MICROBIOLOGY OF MILK AND MILK PRODUCTS biological factor is concerned in the production of flavor in Cheddar cheese is indicated by the fact that if changes are made in the methods of manufacture, changes in flavor are likely to result. If the salt is omitted, the typical flavor does not appear. This can be explained only by the action of the salt on certain types of bacteria, which, in its absence, are able to grow and produce compounds that are not found in a normal cheese. Apparently the methods of manufacture establish a certain equilibrium in the bacterial life which results in the production of definite substances in amounts varying within certain limits. If any condition is varied too widely, a deviation in the micro- bial balance is produced and the products formed in the cheeses are changed in kind or in amounts, either of which may result in a change of flavor. ABNORMAL CHEESES The development of a normal texture and flavor in Cheddar cheese is largely dependent -on the presence of definite types of bacteria. If these are replaced, wholly or in part, by other kinds, the product is likely to suffer in texture, flavor or both. As has been emphasized previously, the bacterial content of the milk is of the greatest importance in cheese, since the organisms in the milk pass into the cheese and there produce the same products as they would have done in the uncurdled milk. All abnormalities of the cheese so far as they are occasioned by bacteria are due to the abnormal flora of the milk. To the raw material the maker must direct his attention if a fine product is to be prepared. Gassy CHEESE.—The most frequent trouble encountered and the one of greatest economic importance is the fermentation caused by organisms belonging largely to the B. coli aerogenes group. It has been seen that these produce in milk gases, such as carbon dioxide and hydrogen, and offensive smelling and tasting compounds. In cheese similar compounds are formed by these organisms; the gas causes the more or less abundant formation of holes which give to the cheese judge an indication of what may be expected with reference to flavor. All milk contains some of the gas-forming organisms, but it is only when they are numerous that marked injury is done. Gassy cheese may also be due to the presence of lactose-fermenting yeasts which are usually found in milk in such small numbers that they THE RELATION OF MICROORGANISMS TO CHEESE 433 cannot compete with the lactic bacteria in the fermentation of the sugar in the cheese. At times the number may be increased to such an extent that the major part of the sugar is fermented by them, alcohol and car- bon dioxide being produced. An outbreak of gassy Swiss cheese was found by Russell and Hastings to be due to such yeasts that had gained entrance to the milk from the whey-barrels because of careless washing of the milk cans. The cheese makers of the country are realizing the importance of the contamination of the milk from the transportation of whey and milk in the same can. The most practical means of prevent- ing trouble from this practice is to heat the whey to 68° as it passes from the cheese vat to the storage tank. This temperature destroys the harmful microédrganisms, and if the storage tank is kept in a sanitary condition the whey is sweet when returned to the farm in the milk can. It has been demonstrated that such a treatment of the whey results in a marked improvement in the quality of the product. MISCELLANEOUS ABNORMALITIES OF CHEESE.—Bitter cheese is produced by bacteria that form a bitter principle. An outbreak of bitter cheese investigated by Hastings was found to be due to the re- placement of the normal acid-forming flora by a lactic organism which produced such an intense bitterness as to mask the acid taste in the milk and cheese. Colored cheese is produced by chromogenic bacteria. In case the colonies are not numerous and the pigment formed is not soluble in any of the constituents of the cheese, the color will appear as colored specks, such as the rusty spot investigated by Connel and Harding, which is due to red forms of B. rudensis. If the colonies are very numerous, or if the pigment is soluble, the curd may be uniformly colored. Puirid cheese is caused by the absence of sufficient acidity to hold the putrefactive bacteria in check. This trouble is rare in cheddar cheese, since such cheese is made from ripened milk. Fruity flavors are asserted to be due to yeasts which form fruit esters. Moldy Cheese.—In the moist air of the curing-room the cheese forms an excellent substratum for the growth of common molds whose pig- mented spores discolor the surface of the cheese and thus impair its value because of the appearance rather than by any effect in the flavor. Cheddar cheese is protected effectively from molds by dipping the cheese, when two or three days old, in melted paraffin which excludes the air from the spores on the surface of the cheese. 28 434 MICROBIOLOGY OF MILK AND MILK PRODUCTS SpEcrIFIC KINDS OF CHEESE There are cheeses made in this, and especially in foreign countries, which are of great commercial importance. Only a few can be men- tioned. It has been found possible to manufacture a few so-called “foreign cheeses” in this country; however, with some “foreign cheeses” the manufacture has been successful only in such localities where such types originally developed, and where the climate and other conditions are favorable to a normal ripening. CHEDDAR CHEESE.—Cheddar cheese, treated in much detail in the foregoing considerations because it is the most important American cheese, is made in England and her colonies and in the United States. Fic. 137.—Typical development of ‘‘eyes” in Swiss cheese. (Original.) It appears in many varieties and by the American consumer is often called American cheese in distinction from the foreign cheeses, This distinction is not wholly applicable at the present time. EMMENTHALER CHEESE.—Swiss or Emmenthaler cheese originated in Switzerland, but is now made in various other countries. A large amount is made in Wisconsin, Ohio and New York (Fig. 137). It is characterized by its sweetish flavor and by the so-called “eyes,” which are holes formed by gas, produced in a fermentation that occurs’ subse- quent to the fermentation of the lactose. The number of eyes is not large and they are evenly distributed throughout the mass of the cheese except near the surface. The cheese is made from as fresh milk as it is possible to secure. The rennet used is prepared by placing a piece of the dried rennet in whey and incubating the same for twenty-four to thirty-six hours at 30°. THE RELATION OF MICROORGANISMS TO CHEESE 435 This is employed in place of the commercial extract used by the Cheddar maker. It serves not only to curdle the milk, but adds to it a large number of acid-forming bacteria that have grown in the rennet solution during the period of incubation. The number is not, however, suffi- cient to cause any development of acid during the making process which differs from the preparation of Cheddar cheese in the method of firming the curd. This is accomplished by heating the curd to 52° to 60°, and by cutting it into pieces scarcely larger than grains of wheat. The salt is applied to the exterior of the cheese by immersion in brine for one to four days and by sprinkling salt on the surface. The fermentation of the lactose proceeds rapidly during the pressing and subsequent thereto, so that within a few days the sugar has disap- peared. Thelack of the development of acid during the making probably results in a somewhat different relation between the acid and protein from that existing in a Cheddar cheese, which, together with the ab- sence of salt gives a somewhat different environment, thus making possi- ble the development of a different flora. Thereisno ground for believing that the agents concerned in the proteolytic changes are other than those that function in Cheddar cheese. The flavor must, however, be due to other factors; this is indicated by the fact that if the milk is ripened as in the Cheddar process, or if salt is added to the curd the flavor will approximate the Cheddar flavor. The formation of the eyes is inhib- ited by salt, as is indicated by their relative scarcity in the outer layers of the cheese. Jensen has shown that the eyes are due to the fermenta- tion of lactates with the formation of propionic and acetic acids, and carbon dioxide. The causal organism is found in the milk and the whey rennet. It is believed that lactic bacteria of the Bact. bulgaricum group are important factors in the ripening of Swiss cheese. They are pre- sent in large numbers in the rennet and cheese. Mixed cultures of an organism of this group and a mycoderma are used with success for the inoculation of the whey in which the rennet is to be soaked. The exact réle of this form of lactic organism is not known; de Freudenreich con- sidered them to be concerned in the proteolysis of the paracasein, since he had found that the content of sterile milk in soluble nitrogen increased when inoculated with the organism. It is probable that the formation of eyes and the flavoring compounds are due, in part at least, to the same factors. In the other kinds of cheeses to be described, the réle of the acid- 436 MICROBIOLOGY OF MILK AND MILK PRODUCTS forming bacteria is similar, if not identical, to their réle in Cheddar cheese, i.¢., in activating the pepsin of the rennet and in preventing the growth of putrefactive bacteria. The factors concerned in flavor development are different. RoQquEFort CHEESE.—This cheese, which is prepared almost exclusively in the Department of Aveyron in southern France, is made from sheep’s milk. Its most striking characteristic is the marbled or mottled appearance of the interior, due to the growth of a mold, Penicillium roqueforti, Thom. The curd is inoculated with the mold, when it is placed in the press, by sprinkling the curd with bread crumbs on which the mold has grown. The growth and sporulation of the mold in the interior of the cheese are favored by piercing it with small needles, thus admitting air. The characteristic flavor is due, partially at least, to the mold. This cheese is cured in caves having a teinperature below 15°. The fermentative processes are apparently closely dependent on the moisture and temperature conditions of the curing room. This emphasizes the importance of biological factors in the ripening process. GORGONZOLA CHEESE, prepared in Italy from cow’s milk, and STILTON CHEESE, made in England are similar to Roquefort in appearance and contain the same mold—Penicillinm roqueforti. CAMEMBERT CHEESE.—The soft cheeses are best represented by this important French cheese made from cow’s milk by the addition of rennet. The milk is ripened to an acidity of 0.20 to 0.25 per cent before the addition of the rennet. The curd, which thus contains many acid-forming bacteria, is neither cut nor heated in order to re- tain the maximum amount of whey. The curd is placed in small hoops and allowed to drain without pressure. Salt is applied to the surface of the cheese. The milk sugar is rapidly fermented and the resulting acidity is high, for the cheese contains 60 to 70 per cent of moisture when fresh and 50 per cent when ready for consumption. The high moisture content of the cheese and the humidity and temperature conditions of the curing room favor the rapid development of microérganisms on the surface of the cheese. Both molds and bacteria thrive under the influence of these favorable conditions, changing the cheese to a soft, smooth and butter-like mass, while a characteristic flavor is developed. THE RELATION OF MICROORGANISMS TO CHEESE 437 In three or four days the cheese becomes covered with the growth of Oidium lactis; the characteristic mold of Camembert cheese, Peni- cillium camemberti, appears later, within five to six days. These molds reduce the acidity of the curd, and through the enzymes, which they produce and which gradually diffuse into the cheese, proteolyze the curd very completely. The appearance of the cheese when cut in- dicates the depth to which the enzymes have penetrated; when the entire mass is acted upon, the cheese is ready for use. The reduction of the acidity by the molds exposes the cheese to the attacks of putre- factive bacteria and it soon becomes unfit for use after it is completely ripened. A number of different kinds of bacteria are found in the slimy surface layer, but their réle is not known. The development of the characteristic flavor and aroma is dependent on a certain relation between the various biological agents concerned in theripening. This balance is dependent on very narrow conditions of temperature and humidity; slight changes in these environmental conditions favor or retard. the individual types in varying degrees. If the equilibrium essential for the development of typical flavor is destroyed this cheese fails to ripen properly and is of low value. The manufacture of Camembert cheese is a delicate problem in the ecology of microdrganisms, and because of this fact the manufacture is attended with greater difficulties than is the case with most types of hard cheese. CHAPTER IV* RELATION OF MICROORGANISMS TO SOME SPECIAL DAIRY PRODUCTS GENERAL There is a number of special dairy products which do not normally come into a discussion of market milk, butter or cheese, but which are of considerable importance. A book of this sort would not be complete without a discussion of some of these products from the bacteriological standpoint. Some of these special products have been developed as commercial enterprises and the processes of manufacture have been zealously guarded as trade secrets. The result is that there is very little available data on the manufacture of these products and very little authoritative knowledge about their bacteriological condition. It is, therefore, difficult to give a full discussion of the microbiology of these products. A few of the more important ones will be discussed, however. CoNDENSED MILK There are at least three quite distinct kinds of condensed milk made under conditions which result in an entirely different bacteriolog- ical condition in the finished product. These different products must, therefore, be considered separately. Condensed milk means simply milk from which a large part of the water has been removed, thus decreasing its bulk, the purpose being to lessen the cost of transportation and to increase the keeping quality of the product. Water is removed from milk by some process of heating, either with or without vacuum, the heating process being more or less equivalent to pasteurization. SWEETENED CONDENSED MiLx.—This product is made by reducing cow’s milk at the ratio two and one-half to two and three-fourths parts of fresh milk to one part condensed milk, by means of heat and the addition of cane sugar. It is then put up in sealed cans. * Prepared by W. A. Stocking, 438 RELATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 439 It is not intended to be sterile. The degree of heat to which it is subjected is not sufficient to kill all of the microdrganisms present and it is also subject to infection after the condensing is completed. Cane sugar is added to the milk, making the final product contain about 25 per cent of water, 35 per cent milk solids and 40 per cent cane sugar. The low percentage of moisture together with the added sugar tends to preserve this product against the action of microérganisms. There may be some bacterial growth, the rapidity depending upon the temperature at which the product is kept, but it is usually slow and milk prepared in this way will keep for a considerable time without undergoing marked bacterial changes. Some gas producing bacteria exist in the milk and if cans containing the organisms are allowed to remain at warm temperatures, they will develop in spite of the large percentage of sugar, producing suffi- cient amounts of gas to cause the ends of the cans to bulge out. Such cans are known commercially as ‘‘swell-heads.” : UNSWEETENED CONDENSED OR EvaporateD Mitx.—In this form of condensed milk approximately the same amount of moisture is removed as in the sweetened product but no sugar is added. The decreased amount of moisture tends to prevent the rapid growth of bacteria, but this is not enough to guarantee the keeping quality of the product. After the milk is condensed it is put into the can, hermetically sealed, and then placed in steam sterilizers and subjected to temperatures some- what above the boiling-point. In this way the milk is heated a suffi- cient length of time to insure perfect sterilization of the contents of the cans. If this process is properly done, the finished product contains no living microdrganisms and from the bacteriological standpoint the milk should keep indefinitely. . .. \ Sometimes the unsweetened product is sold in bulk in cans. In. this case it is subject to more or less contamination after heating and is not sterile, but because of the small amount of moisture and the concen- tration of the milk solids, the bacteria do not develop rapidly and if kept at a cool temperature, the milk will keep several days without undergoing appreciable biological fermentations. CONCENTRATED Mirx.—There is now on the market ‘a form of con- densed milk prepared by a different process, which is commonly known as concentrated milk. By this method the water in the milk is removed by means of dry air 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 forced through it. By this process the milk is heated to a temperature of 60° (140°F.), and this temperature maintained for two hours, during which time air is forced through the milk causing violent agitation and 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 a pasteurized milk, with a marked reduction of the original germ content. Investigations by Conn failed to show the presence of B. coli in milk prepared by this process. The reduction in the bacterial content of the milk is similar to that secured by other methods of pasteurization. No additional sugar is added to this milk so the product is, therefore, a pas- teurized milk containing a small amount of moisture. Because of the small amount of moisture and the concentration of the milk sugar, the bacteria which survive the heating process do not grow rapidly at low temperatures. The following figures will serve to illustrate the effect of this process upon the bacterial content of milk: Number of bacteria per c.c. in finished Number of bacteria per c.c. in original milk product 1,250,000 15,000 3,000,000 21,000 518,000 26,000 894,000 9,950 796,000 10,000 150,000 5,000 The rate at which the bacteria develop in this milk is shown by the following counts: Number of bacteria per c.c. Number of sample - 2 days old 4 days old 6_days old I 18,000 39,000 46,000 2 55,000 28,000 39,000 3 3,500 II,000 10,000 4 4,400 55270 4,630 The lack of moisture and concentration of milk sugar prevents the rapid growth of these organisms so that bacterial changes do not take * Data furnished by H. W. Conn. RELATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 441 place as rapidly as in ordinarily pasteurized milk retaining its normal moisture. . / PowbERED Mitx.—This product is produced by carrying the extrac- tion of the water farther than in the case of the condensed milks. The water is removed to a point where the milk solids can be reduced to a powdered form. ‘This product contains the original milk solids with a very small percentage of moisture usually not more than 214 per cent. There are several forms of powdered milk now on the market produced by somewhat different methods. In some cases the moisture is removed from the milk by its being exposed to a heated surface in a thin layer. Sometimes the drying is done in vacuum. The resulting product is dry and can be ground to the form of flour. Another process is to remove the moisture by spraying the milk by means of an atomizer into the top of a hot chamber, the moisture being removed while the fine particles of milk are falling to the floor. By this process the product accumulates on the floor as a very dry flour and does not require any grinding. In the first process the heat is sufficient to pasteurize the milk while in the latter process it is pasteurized before being subjected to the drying process. The powdered milks do not claim to be sterile but are preserved against subsequent action of micro- érganisms because of the very low percentage of moisture which they ‘contain. It is probable that there is no appreciable increase in the number of bacteria in milk flour and the product will keep for along time without undergoing bacterial fermentations. CANNED BUTTER AND CHEESE Some effort has been, made to put up butter and cheese in hermet- ically sealed cans, the purpose being to increase the keeping qualities of the products and influence the flavor by controlling the development of the aerobic bacteria. Only a limited amount of bacteriological work has been done on these canned products and the biological changes which take place in them are not very well known. SpeciAL Mitk Drinks MaAbDE BY THE ACTION OF MICROORGANISMS From time immemorial fermented or sour milk has been used as an article of food. We are told that Abraham* placed “‘curdled milk”’ * Genesis 18:8. The Hebrew word ‘themah”’ translated in the English authorized version of the 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 milk was one of the blessings which Jehovah had given to his chosen people.* History also tells us that the wandering tribes of Arabia used fermented milk as a beverage. For centuries many of the tribes of eastern Europe and western and middle Asia and parts of Africa have used sour milk for food. Each of these regions appears to have had its own particular milk beverage resulting from the particular bacterial flora of the region. The sour milk products which are now on the market under a variety of names have been derived from these original sour-milk drinks of antiquity. Fermented milk beverages have become very popular during the last few years among all the civilized peoples, partly because they make a pleasant drink but more especially because of their supposed therapeutic value. f Kumyss (Kovumiss, Kumiss, Erc.).—Kumyss derives its name from the Kumanes, a Russian tribe which lived along the river Kuma. This drink was prepared from mare’s milk by placing it in a leather bag and adding a small amount of old kumyss as a starter.t In this country kumyss is made from cow’s milk. This product is now placed upon the market by a number of companies who keep their methods, so far as possible, from their rivals by maintaining strict secrecy in regard to the methods of preparation. Dr. Piffard§ who has done special work on this product states that kumyss is fermented by the action of yeasts and lactic bacteria. This fermentation produces approximately 1 per cent of alcohol and about 0.75 per cent of acid. Kumyss is strongly effervescent. The lactic organisms used in the preparation of this material appear to be a strain of the common Bact. lactis acidt. Whether or not the yeasts are the common forms used by bakers cannot be stated with certainty. Kumyss can be easily prepared in the household by the addition of cane sugar and baker’s yeast to fresh, warm milk which should be kept at a temperature of about 38° (z00°F.) until gas begins to form. It should then be bottled and be kept at a cool temperature. In one or two days a slight amount of alcohol will be formed and a sufficient amount of carbon dioxide to cause marked effervescence. * Deut. 32:14. ft Metchnikoff's Prolongation of Life. tf Milch Zeitung, September, 1889. § New York Medical Journal, January 4, 1908. RELATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 443 Kerir (Keryr, Kepuir, Kerr, Erc.).—Kefir was originally made and used by, the inhabitants of the Caucasus Mountains. It was made from the milk of goats, sheep or cows and was fermented by the addition of “kefir grains” to the milk. The origin of these kefir grains is unknown but the natives believe that they were the gift of Mahomet and are carefully preserved by them. | Kefir was prepared by the natives by placing milk in a goat-skin bag and shaking it at intervals until it began to ferment. The kefir grains were then removed, dried and preserved for future use. The fermented kefir was also used as a starter for inoculating new lots. This beverage is now commonly made by more scientific methods.* The principal points to be observed in the preparation of kefir are cleanliness and proper temperature for fermentation and the regulation Fic. 138.—A large-sized kefir grain and the three species of bacteria of which it is composed. (From Conn, after de Freudenreich.) of the fermentation so that not the acid but the alcoholic fermentation will prevail.t Good kefir should be highly effervescent, should be free from lumps and contain about 1 per cent. of acid but show no marked tendency to whey off. According to Kern, kefir is fermented by a mixed culture of yeasts and bacteria in symbiosis. He found but one form of bacteria present in the cultures he studied. De Freuden- reich{ made an extended study of the flora of kefir. He prepared the kefir from the kefir grains and isolated the organisms present, putting these organisms together in different combinations in order * Milch Zeitung, 1885, p. 209. { F. Stohman, Milch and Molkerei Products, p. 1906 to 1013, } Centr. far Bakt. Abt. 2, Vol, 3, 1897. 444 MICROBIOLOGY OF MILK AND MILK PRODUCTS to determine which were necessary for the proper fermentation of the kefir. He found the kefir contained four different organisms: yeasts, streptococci, micrococci, and bacilli. The yeasts and strepto- cocci were plated in gelatin without difficulty but it was very difficult to grow the other two organisms present on any artificial media. He concluded that the yeasts present in kefir are not identical with the species commonly used in making beer and named it Sacchar- omyces kefir. The streptococcus curdled milk in less than forty-eight hours at a temperature of 37° but the micrococcus did not curdle milk at all, although it produced a considerable amount of acid. De Freudenreich changed the name of the bacillus from Dispora caucasica, given it by Kern to B. caucasicus, because it did not produce spores as Kerp sup- posed. He also found that this organism would not grow at all on media without sugar, very slightly on milk, serum, agar, and best of all in milk, in which it produces both gas and acid without curdling the milk. This organism is sy or 6y in length by ru in width, is slightly motile and retains Gram’s stain. It has a thermal death- point of 55° for five minutes. The preparation of good kefir seems to depend upon the combined action of the four types of organisms described. Kefir is sometimes prepared without the use of the kefir grains* by placing milk in bottles to which is added a small amount of compressed yeast and sucrose. The bottles are then held at a temperature of 10° to 15° about fifteen hours and shaken occasionally. Kefir prepared in this way gives an effervescent mild flavored drink. Lepen.—For centuries the Egyptians have used a fermented milk drink known as leben or leben raib. This was prepared from the milk _ of cows, buffaloes, and goats. In general it resembles the other fer- mented milk drinks in the fact that the fermentation is. produced by yeasts and a variety of other microérganisms working together. At least one yeast and three species of bacteria seem to be normal to this product. A fermented milk drink very similar to leben is also used in Algeria. Just the action of each microérganism concerned in the fermentation of this product is not certain, but it is probable that all of the species are essential for the production of the particular flavor and consistency of the fermented product. It is claimed that the fermentation that takes place in the milk renders it more digest- ible than raw milk. For this reason it is recommended for the use of invalids and persons having weak digestion. * Milch Zeitung, 1888, p. 393. RELATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 445 YAHOURTH OR Matzoon (Yocurt, YanourD, Mapzoon, Etc.).—A fermented milk drink known by one of the above names has been used by the Bulgarian tribes for a long time. It has recently been studied and brought to public notice by the investigations and writings of Metchnikoff,* who was struck by the longevity of the tribes using this product as a part of their regular diet. As a result of his investigations, Metchnikoff has advanced his theory regarding the antiseptic power of certain strains of lactic bacteria in the digestive tract. His theory is that certain species or types of bacteria which are able to resist the action of the stomach and can, therefore, pass through into the intestines have the power of checking the growth of the putrefactive bacteria existing there and thereby prevent the production and ab- sorption of bacterial toxins which cause autointoxication. As a result of his experiments, Metchnikoff came to the conclusion that the acid organism (Bact. bulgaricum){ found in yahourth was able to establish itself in the intestinal tract and produce enough lactic acid to hold in check the putrefactive processes which otherwise exist there. Yahourth is made by the Bulgarians in skin bags in the same way that the Russian tribes prepare kumyss. It is similar to the other fer- mented drinks already described in the fact that it is produced by a mixed flora of microédrganisms. At least one yeast is present and two or more species of bacilli capable of producing lactic acid in relatively large amounts. These two organisms are known as Bact. bulgaricum and Bacillus paralacticus. Herter states that Bact. bulgaricum is 4p to 6 in length by ry in width and grows singly or in pairs and occasionally in chains. It stains with ordinary aniline dyes and by Gram’s method. . It grows with difficulty on ordinary laboratory media and is therefore hard to obtain in pure cultures. These organisms producea much higher percentage of acid than the common Bact. lactis acidi and also grow at a much higher temperature. This makes it possible to secure it in practically pure cultures by growing it in milk at a high temperature. Grown in pure cultures, the Bact. bulgaricum will produce from 1 to 2 or more per cent of acidity. It grows well at temperatures between 37° and 4o° and even higher. Recently a number of fermented milk drinks have been put upon the market which have evidently been derived from the yahourth. These * El. Metchnikoff, Prolongation of Life. + Hastings has found this organism also common in cow’s milk in this country. 446 MICROBIOLOGY OF MILK AND MILK PRODUCTS are sold under such trade names as Zoolak, vitalac, yogurt, fermentactyl, etc. The flora of these preparations appears to be practically the same as that of the original yahourth. All of the fermented milk drinks thus far discussed are similar in that each contains a variety of microdrganisms, made up of at least one species of yeast with one or more species of bacteria, capable of produc- ing greater or less amounts of acid. In some, as in the case of kear, the yeast fermentation is allowed to predominate, while in others, like ya- hourth, the action of the yeasts is held in check by the rapid develop- ment of the acid by the Bact. bulgaricum. All of these drinks are com- monly recommended by physicians because of their beneficial effect upon the digestive tract. ARTIFICIAL BUTTERMILK.—In recent years there has developed an important industry in the manufacture of artificial buttermilk. This is usually made by inoculating skim-milk with a culture of lactic bacteria, either Bact. lactis acidi, or Bact. bulgaricum or a combination of these two types. In making the artificial buttermilk, yeasts are not commonly added. After the milk becomes coagulated, it is then churned in order to give it a smooth, creamy consistency, after which _ it may be bottled and kept for some time by holding at low tempera- tures. Sometimes a small percentage of whole milk is added at the time of churning to make the finished product more closely resemble natural buttermilk. In making artificial buttermilk, the skim-milk is fre- quently pasteurized in order to get rid of the miscellaneous flora which it contains. The finished product, therefore, differs from ordinary but- termilk in the fact that it contains nearly pure cultures of the lactic organisms while the natural buttermilk will contain a more or less miscellaneous flora in which the acid organisms predominate. It is possible to obtain a more uniform product in the artificial buttermilk than in the natural product, and this is perhaps responsible for the rapid development of this industry. All of these fermented milk drinks contain enormous numbers of microérganisms, usually millions per c.c. Frozen MILK Some effort has been made to put upon the market milk which has been frozen into cakes or bricks. This has been tried both in Europe and in this country. Some difficulty has been met in satisfactorily freez- RELATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 447 ing the milk and holding it in a frozen condition. The process has proved to be rather expensive and not very satisfactory. One difficulty with this process seems to be that the quality of the frozen milk after it has been melted is not as good as it was before it was frozen. Froma bacteriological standpoint, this process is of some interest, but it is doubtful whether it becomes of much importance commercially. Ick CREAM Ice cream is one of the important manufactured dairy products and ‘its use seems to be increasing steadily. Its bacterial flora varies with the materials used in its manufacture and the conditions under which it is made. It may be made from fresh cream which is only a few hours: old and under good sanitary conditions. On the other hand, it may be made from cream which has been produced and handled under unsani- tary conditions, kept in storage for a number of days and finally manu- factured in surroundings not, conducive to a low bacterial content. We are not surprised, therefore, to find a very wide variation in the germ content of ice cream, as it is placed upon the market. An examination of 263 samples of ice cream collected in the city of Washington* showed an average germ content of over 26,600,000 per c.c. The lowest count ‘obtained was 37,500 and the maximum was 365,000,000. A similar study of commercial ice cream in Philadelphia showed the average bacterial content to be very high. The lowest count found was 50,000 per c.c., while the highest count was 150,200,000. In this work it was found that the bacterial content of the ice cream was in quite direct relation to the sanitary conditions of the establishment where the ice cream was manufactured. When ice cream is manufac- tured in a city from materials which have been shipped in from consider- able distances and frequently held for several days in cold storage, it is not surprising that the germ content of the manufactured product should be high. In some establishments the cream is pasteurized be- fore manufacturing, while in others it is used in its raw condition. In normal cream held for sometime, the lactic bacteria should exist in considerable numbers, but when cream is held at low temperatures these organisms do not develop rapidly. Pennington found that cer- * Results of work done under the direction of G. W. Stiles. t+ Work 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 at refrigerator temperatures. Streptococci were found in fifty-five (80 per cent) of the sixty-eight samples examined. It was found that at refrig- erator temperatures the relative growth of these organisms was greater than at higher temperatures, a fact which may account, in part at least, for the frequency with which these organisms occur in ice cream. Frequently ice cream is held for a considerable time in a frozen con- dition before it is sold. It has generally been supposed that there is no bacterial growth in material which is held below the freezing tempera- ture. This, however, did not seem to be the case in samples examined by the investigators already mentioned. They found in samples held about a month that there was normally a decrease in the bacterial count and also in the amount of gas production for a number of days, after which there was frequently a marked increase in the bacterial counts. These results would seem to indicate that even in the frozen condition there may be some increase in the number of bacteria present. The number of these experiments, however, is not sufficient to justify very general conclusions. The work of Conn and Esten* in holding milk at 1° may throw some light upon this question. If the cream from which the ice cream is made has been produced and handled under sanitary conditions, the bacterial content should consist chiefly of organisms of the Bact. lactis acidi type, in which case the high count in the ice cream might not be objectionable. If, on the other hand, the cream has been held in cold storage for some time under conditions which inhibit the growth of the lactic organisms and permit the development of putrefactive types, bacterial poisons may be developed in the cream, which will be highly objectionable. There seems to be little doubt that this is the cause of the cases of ptomain poisoning, resulting from the use of ice cream. - It is known that certain types of bacteria, especially those belonging to the so-called putre- factive group, are capable of developing at very low temperatures and can, therefore, produce considerable quantities of toxic products in the cream. Whether or not these products are developed before the cream is manufactured or whether they may develop in the frozen product cannot at present be stated. In general it can be said that the total bacterial count does not indicate the wholesomeness of the ice cream any more than does a similar count in buttermilk or in the com- * Annual Report, Storr’s Experiment Station, 1901. RELATION OF MICROORGANISMS TO SPECIAL DAIRY PRODUCTS 440 mercial fermented-milk drinks. The kinds of organisms present is a far more important question from the standpoint of the wholesomeness of the ice cream. However, the results obtained by many ice-cream manufacturers has demonstrated the fact that the germ content of this product can be quite definitely controlled by the same methods of care and sanitation as are required in the handling of other forms of dairy products. 29 DIVISION V MICROBIOLOGY OF SPECIAL INDUSTRIES CHAPTER I* DESICCATION, EVAPORATION, AND DRYING OF FOODS Factors THat Brinc ABout CHANGES IN DRIED Foops The factors that bring about changes in dried foods may be considered under two general heads, chemical and microbial. Enzymes, although the product of living cells, may represent the chemical changes, and the activity of bacteria, yeasts and molds, the microbial changes. Enzymes are normally present in food stuffs derived from animals or plants which have not been subjected to heating. All living cells contain enzymes, and these may remain active for a considerable time after the death of the cell. Some of these enzymes attack carbohydrates, some fats, some proteins, and some other compounds. Enzymes are responsible for the stiffening of the muscles after death (rigor mortis), others later break down the tissues and bring about a ripening of meat whereby it becomes more tender. Autolytic enzymes may in some instances- produce rancidity in food products by a splitting of the fats. Bacterial enzymes are known that duplicate the action of practically all those produced by higher animals and plants. Some of the changes pro- duced are desirable, others undesirable, particularly if action is allowed to continue for too long a time. In foods dried for preservation, it is therefore important that sufficient heat be used to destroy the enzymes or that enough water be removed to inhibit their activity. Ordinarily the activity of enzymes will be inhibited by the removal of water sufficient to prevent the growth of microdrganisms. The action of enzymes is characterized as reversible, that is, after a certain concen- tration of enzymic products has been reached, the transformation ceases * Prepared by R. E. Buchanan. 450 - DESICCATION, EVAPORATION, AND DRYING FOODS 451 until a part of the accumulation has been removed by diffusion or other- wise. Since many of these actions are hydrolytic in nature, water for both diffusion and hydrolysis must be present before the enzyme can act. Bacteria are introduced in large numbers when food is handled and probably constitute the most important factor in its destruction. If moisture and temperature conditions are favorable, they bring about undesirable changes. The amount of water present in foods may be used as a basis for their classification into four groups: first, those in which moisture is present in appreciable quantities in the interstices, that is, those which seem wef. Under these conditions bacteria not only multiply but spread rapidly through the medium by actual space growth, by diffusion currents, and by their own motion. Second, some foods may contain sufficient moisture for the abundant growth of bacteria, but not free water for diffusion and distribution. In these the spread of infection must be largely by direct growth of the organism and will necessarily be slower than in the preceding. Third, the sub- stratum may be so dry that little or no growth of organisms may take place, yet there is sufficient moisture so that they remain viable. Fourth, the food may be so dry that only those organisms that can withstand relatively complete desiccation will survive. These groups cannot be differentiated entirely upon the basis of the percentage of water present, for the character of the food itself and of the material in solution are also important. Yeasts usually require sugars for their best development and are : therefore commonly present in foods containing this substance. They are of importance therefore in fewer foods than bacteria. In nature, they are frequently found upon fruits, particularly those which contain considerable quantities of sugar in the sap. They will be found also upon the cut ends of twigs or grass culms where sugary sap has oozed out. Colonies of considerable size may sometimes be seen upon corn stubble during damp weather. They are commonly distributed by flies and other insects which feed upon the sugary plant juices. They are not motile, hence the spread of infection in any food must be by direct growth. Molds, like bacteria, are ubiquitous and under proper conditions will destroy almost any food. They grow readily in solutions and on saturated substrata, but ordinarily are prevented by the bacteria which 452 MICROBIOLOGY OF SPECIAL INDUSTRIES find the optimum condition for their growth in such conditions. For example, it is commonly observed that wet silage rots when exposed to air supports a luxuriant growth of bacteria, while drier silage becomes moldy. Unlike bacteria, the molds extend through and over food when there is no visible water film. The spores are much better adapted to air dispersal than are bacterial cells, and the hyphe penetrate more rapidly than will the bacterial colony. In certain foods, therefore, as meals and flours, molds are more destructive than are the bacteria. Usually they will multiply with less moisture. INHIBITION OF GROWTH OF MicROORGANISMS IN DRIED Foop Tn a few cases, the development of microdrganisms is prevented by the absence of sufficient moisture in the medium to support growth. This isnot nearly so common as might appear at first thought. It occurs in some foods as olive oil, starches, meals, cane sugar, etc., that have little or no free water. Frequently the drying results in a con- centration of the solutes, beyond the point to which microdrganisms can adapt themselves to the osmotic pressure. When it is remembered that a 50 per cent solution of cane sugar is capable of exerting a pressure of about 226 kg. (500 pounds) per square inch, it will be realized that considerable readjustment is necessary in the cell of a yeast plant that can grow in such a medium. Drying also sometimes changes the former relationship of cells and tissue constituents so that protective layers may be formed. For example, in curing pork, the fat which is structurally isolated in distinct cells for the most part becomes diffused throughout the outer layers of the tissues and forms a water-free and water-proof exterior. Foods are sometimes subjected during the process of drying to sufficient heat to destroy the microdrganisms con- tained. At other times they are exposed to the germicidal action of the direct rays of the sun or to the fumes of some disinfectant or bleaching agent as sulphur dioxid or smoke. MetHops OF DRYING The reduction of the water in foods below the minimum required for the growth of microérganisms is accomplished i ina variety of ways. Most'commonly heat is employed, either the sun’s ray or some artificial DESICCATION, EVAPORATION, AND DRYING FOODS 453 source. In localities where the humidity of the air is low, as in many of the irrigated fruit districts of the western United States, exposure to the rays of the sun results in rapid drying. With other types of foods and in more humid regions, artificial heat is used to reduce this relative humidity. Some foods cannot be dried at high temperatures because of their instability. In most cases such foods must be dried quickly for they are readily attacked by microdrganisms. These are usually dried at a low temperature and in a partial vacuum. Other foods are dried without recourse to evaporation by the use of the hydraulic press or by centrifugal action, the latter in the manufacture of cane sugar. The water available for the growth of microdrganismis may be reduced by the addition of some crystalline substance such as sugar or salt. The usefulness of the latter depends largely upon their ability to create a concentration of solutes too great for the growth of bacteria. At the same time a considerable proportion of the water from that part of the food into which the solutes will not penetrate, is abstracted by osmosis. Many food products do not require any additional drying, as they naturally contain little moisture. Such are the grains and the products manufactured from them, as flour. The drying in this instance has occurred during the ripening process of the grain. When for any reason this does not occur, the grain will mold. It has been found necessary in many instances to kiln-dry corn. Grain, nuts, etc., are by their nature adapted to keep under normal conditions for considerable _ periods. Other foods require artificial drying. In these we have the intergrading classes, which have been discussed above, those which contain a very small percentage of water and those which, have con- siderable water but a high concentration of solutes. The absolute amount of water in the food is by no means an index to the amount that is available for the growth of microérganisms. Many foods are hygroscopic. Foods having the same water content and percentage of solutes will behave very differently with reference to delivering up the water to any organism present. The effect of the concentration of solutes by drying is perhaps the most important factor in the preservation of food. These substances dissolved in the water may be actually antiseptic when concentrated, as the acids ofthe juices of certain fruits. More often the sugars reach 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 a maximum concentration reached sooner or later, beyond which growth is impossible. Dried foods may be divided into three groups, using the relative abundance of carbohydrates, fats, and proteins as a basis for classification. Carbohydrate foods are usually preserved by drying. Many, such as grains and nuts and the flours and meals prepared from them, do not require artificial heating. They are, however, somewhat hygro- scopic and in damp climates enough moisture is taken up to allow the growth of injurious molds and bacteria. Moldy corn has long been 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 dried by simple exposure to the air and sun until most of the water has been evaporated. Fodder that has become moldy through the presence of too much moisture is a prolific cause of trouble in horses and less fre- quently in cattle. The many deaths due to the so-called cerebrospinal meningitis are suspected many times to be due to the consumption of moldy hay. In localities where the air is too moist or it rains so fre- quently as to make it difficult to dry hay, curing is effected by a process of self fermentation. ‘The hay is piled in a mass while still green and undergoes a process of heating. The temperature rises usually to about 70°. The causes of this rise are somewhat uncertain, but it is prob- ably due to the combined action of enzymes and microérganisms. Just how much of this keeping quality is due to the heating, how much to the loss of water, and how much to the accumulation of products of fermentation is uncertain. In other cases, the heated hay is spread out and quickly dries sufficiently so that it may be stored. A certain small percentage of the nutriment of the hay is necessarily lost in the development of the heat energy. Fruits are quite generally preserved by drying. In many instances, as in peaches, apples, and berries, it is probable that enough moisture is usually removed to prevent organisms from growing, but in many other cases, as in the preparation of currants and raisins, the concen- tration of the sugar and other soltites is the controlling factor. Fre- 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 cases artificial heat or even hydraulic presses are used. 4 DESICCATION, EVAPORATION, AND DRYING FOODS 455 Many manufactured products, particularly baker’s goods, as crackers, biscuits, dried -yeast cakes, etc., are preserved by the. elimination of water. Macaroni and vermicelli are prepared by forcing a thick paste of especially pre- pared flour and water through openings of different sizes. The product is then dried in the air until it is brittle and may then be kept indefinitely. Copra, one of the principal exports of certain of the islands of the Pacific and Indian Oceans, is prepared by cutting the meat of the cocoanut into pieces and drying them in the sun. From this copra, much of our desiccated and powdered cocoanut is prepared. , ‘Syrups, molasses, jellies, jams, and many other carbohydrate foods are preserved through the concentration of the solutes. Many of these are partially sterilized by the heat used in the process of manufacture, but there is usually plenty of opportunity for subsequent infection. They are more frequently attacked by molds and yeasts than by bacteria. An exception may be noted in Streptococcus mesenterioides which sometimes causes considerable trouble by a gelatinous fermenta- _ tion in syrups from which sugars are manufactured commercially. Foods with considerable quantities of fat usually contain little water. Cottonseed, olive and other vegetable oils, the plant and animal fats, as lard, tallow, and butter, are quite resistant to change by bacteria unless water is present and considerable traces of nitrogenous im- purities remain in them. With these foods the water is necessary for the growth of the organism and also for the activity of the lipolytic enzymes, which might hydrolyze fats and aid in the development of rancidity. Butter forms an exception to the rule that fat foods contain little water, as it usually has from 12 to 16 per cent. Where it is necessary to keep butter-fat for long periods or under unfavorable conditions, it is melted, the water and the nitrogenous impurities removed, and the clear fat preserved. Bacteria, enzymes, and a few molds have been described that attack fats. In the process of prepara- tion or manufacture of any fat foods, sufficient heat is used to sterilize the material and infection thereafter spreads to the interior very slowly. The heat destroys the enzymes as well as the bacteria. The third class of foods preserved by drying includes those that contain a high percentage of protein, in large part flesh foods and flesh derivatives. 456 MICROBIOLOGY OF SPECIAL INDUSTRIES Desiccation, however, is only one of the agencies acting to preserve the flesh. Jerked meat is sometimes prepared in localities with a hot dry climate. Lean meat is cut into thin slices and exposed to the direct rays of the sun until dry. The bactericidal action of the sunlight and the rapid extraction of moisture prevents microérganisms from producing undesirable changes during the curing process. Dried beef is lean meat which usually has been treated with certain condiments or smoked and salted and then dried. Dried fish such as cod, mackerel, and herring, is prepared by the use of condi- ments, salt, and smoke in addition to the drying. Pemmican is prepared by drying lean meat, grinding it, and mixing it with sugar and fat, dried fruits, spices, etc. It is highly nutritious, not unpalatable, and com- pact, and will keep for a long period. It is frequently used as a concentrated form of food by Arctic explorers, etc. Beef extract is prepared by cooking minced beef and water in a receptacle under a slight steam pressure. The digestion is continued for several hours. The liquid is filtered off and concentrated in a partial vacuum to the desired consistency. Gelatin is prepared by boiling bones and tendons, sometimes also horn and hide scraps and concentrating the gelatin which dissolves from these. Somatose, sarco-peptone and related so-called predigested protein foods are mix- ‘tures of albumoses and peptones prepared by the artificial digestion and drying of 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 warm compartment from which the air is partly exhausted. It dries immediately, in the form of a very fine powder. -This powder, if thoroughly dry, will keep well and is finding an extensive use. The high sugar content of this powder is instrumental in ‘ preventing the development of microérganisms. Eggs are dried in much the same manner as milk and the product is being used extensively at the present time by bakers. Meats are frequently preserved by a combination of drying and the action of certain antiseptics or preservatives. The salting of meat owes its effectiveness in part to the abstraction of water. In most cases, the surface of the meat and probably even the other portions are protected in large measure by the diffusion of the fat and the satura- tion of tissues and by the formation of water-proof fat films. The autolytic, enzymes are active in the fresh meat and soon become inert upon the removal of water. The organisms responsible for decay of preserved meats and flesh foods are usually bacteria. Some of these, break down the protein into simpler chemical compounds, of which a few are poisonous. CHAPTER II* HEAT IN THE PRESERVATION OF FOOD PRODUCTS HisToricaL R&SUME The principle involved in the preservation of food by heat may be said to have had its origin in the experiments of Spallanzani, who in 1765 boiled meat extract for an hour and hermetically sealed the flasks, after which treatment no change occurred in the material. An applica- tion of this principle was suggested as early as 1782 by the Swedish chemist, Scheele, who advised the exposure of vinegar in bottles to the temperature of boiling water in order to effect its preservation. Some years later the principle was applied to the conservation of food by a French confectioner, Nicholas Appert, who in 1811 published an exhaustive treatise on ‘The Art of Preserving Animal and Vegetable Substances.”’ His method was to enclose the food in a glass jar which was then corked tightly, and placed in boiling water, the length of time of heating varying with the article treated. In 1810 Peter Durand secured a patent from the English govern- ment for the preservation of fruits, vegetables and fish in hermetically sealed tin and glass cans. He did not claim to be the discoverer of the process, but said it had been communicated to him by a “foreigner residing abroad.”’ Although the secret of the process was jealously, guarded, the employees of different establishments became familiar with its essentials, and in this manner the industry found its way to America. One of the first to introduce the process was Ezra Daggett, who, with his son-in-law Thomas Kensett, in 1819 engaged in the manu- facture of hermetically sealed goods, the principal foods packed being salmon, lobsters, and oysters. In 1820, William Underwood and Charles Mitchell, emigrant employees from a canning factory in England, opened a factory in Boston where they canned plums, quinces, cran- berries and currants. , In the earliest days of canning, glass jars were used exclusively, but * Prepared by S. F. Edwards. 457 458 MICROBIOLOGY OF SPECIAL INDUSTRIES were gradually abandoned as it was found that they could not readily withstand the extremes of temperature, and were expensive, bulky, ~ and costly in transportation. In 1825, Thomas Kensett secured a patent on the use of tin cans in preserving food, and in the same year began using the process in his factory. The early manufacture of tin cans was by hand and crude, the bodies being cut with shears and the side seams made with a plumb joint (that is meeting but not over- lapping), and then soldered together. Heads were made to set into the body and were soldered in place in a very crude manner. The making of 100 cans was considered a good day’s work for one man. Improvements were gradually made, however, in their manufacture, until finally can making became a distinct industry and now all the parts are made and put together by mechanical devices. In the original Appert process, the goods were cooked in open kettles, the highest temperature obtainable by this method being the boiling point. A little later common salt was used to aid in securing a higher temperature, and this was followed later by the use of calcium chloride which made possible a temperature of 115°. In 1874, a closed kettle was invented for superheating water with steam, and this was imme- diately followed by another improved kettle in which dry steam was used, the principle employed being that of the modern autoclav, by which method any desired temperature may be obtained and modified to suit the requirements of different classes of food. Economic IMPORTANCE From STANDPOINT OF HEALTH AND DiEtetics.—The value of a variety of foods, especially fruits and vegetables, is recognized by dieticians. Unfortunately, however, the season for fresh fruits and vegetables is comparatively short. Moreover, many foods grown exclusively in one section of country will not withstand shipping in a fresh condition to other sections. In spite of improved methods of refrigeration, it is not practicable to ship fresh sea foods to far inland towns, or to send some perishable products of warm climates to cold countries. The canning and perserving industry overcomes these difficulties by supplying pure, clean, wholesome fruits, vegetables, meats, and fish to any region the year round, and at prices com- paratively low. HEAT IN THE PRESERVATION OF FOOD PRODUCTS 459 From STANDPOINT OF CommeERcr.—In its commercial aspect, the importance of the industry can scarcely be estimated. Canned products make possible the carrying of larger stores of provisions by armies and navies and expeditions for exploration than would otherwise be possible. In fact, the stimulus which prompted the investigation of Appert was a prize offered by the French Navy Department for a method of preserving foods for provisioning ships more satisfactory than by pickling, drying, or other methods in use up to that time. “ Although the preserving industry was established in three great commercial centers in the United States as early as 1825, it did not become of much importance until the last decades of the nineteenth century. There were many hindrances to the progress of the industry, such as the secrecy observed in the process, skepticism of the public regarding the healthfulness of canned foods, the general prejudice ~ against them, and the high cost of production. These obstacles have gradually been surmounted, and at the present time the several branches of the industry have collectively assumed large proportions. An idea of the magnitude and importance of the industry in the United States may be gained from statistics for 1914 compiled by the. National Canners’ Association, and here reproduced by permission. The pack of tomatoes was 15,222,000 cases; of corn, 9,789,000 cases; and of peas 8,847,000 cases. This does not include fruits, the pack of which in California alone in 1912 was 4,833,900 cases. Nor do these figures include the great variety of other vegetables, fruits from other states than California, preserves, oysters, meats, or fish. The average case holds two dozen cans, and sells at an approximate average price of $2.40. It is apparent from these data that the canning and preserv- ing industry is one of immense value, and that it constitutes a large factor in the feeding of the race.” ALTERATION OF Foop PHysICAL CHANGES.—A ppearance-—Some physical changes attend the conservation of foods by heat, approaching more or less closely the changes incident to the ordinary preparation of fresh foods for the table. In the preserving of some vegetables, notably peas and asparagus, the canner subjects them to a blanching process which con- sists 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 the outside and a part of the green coloring matter so as to have a clear liquor in the can, and second, to drive water into the vegetables so that all will be tender. The blanching process also improves the color of these vegetables. Mechanical Disintegratton.—In the case of very soft fruits or vege- tables, the high temperature essential for sterilization causes a slight amount of mechanical disintegration, which is not objectionable, how- ever, unless excessive, as there is little deterioration in appearance, and none in food value. In the case of meats, practically the only physical change is the shrinkage during the parboiling previous to placing in ‘the cans. CHEMICAL CHANGES.—A ppearance.—The chemical changes in foods preserved by heat may be considered under two heads: first, those in which the appearance is modified; and second, those in which the food itself is altered. Some change of color sometimes occurs and results from various causes. In colored vegetables, such as peas, string beans, — and asparagus, a part at least of the loss of color is due to the oxidation of chlorophyll. With a few foods, iron sulphides are occasionally formed by a combination of sulphur of the foods with the iron of the container. This seldom occurs however, and is not of great impor- tance. Some fruits packed in glass gradually lose their color by oxida- tion on exposure to the light. Chemical Change.—So far as chemical alteration of the food itself is concerned, there is little change and none other than would occur in the ordinary preparation of the food for the table. The albumins are coagulated. The fats probably remain unchanged. Of the carbo- hydrates, the chief action is on the sugars. The cane sugar is wholly or partly inverted by the combined action of the heat and the fruit or vegetable acids. The starch undergoes little if any cleavage, inasmuch as this change occurs only in the presence of acids, and in foods with a high acid content, the proportion of starch is relatively low. The other amyloses probably undergo little if any change. Palatability and Digestibility—lIt is often contended ‘that canned foods are less palatable than fresh foods of the same kind. This lack of agreeableness to the taste is, however, more seeming than real, and arises largely from the prejudice of the consumer against food conserved in tin cans, rather than from any actual change. When the preserving HEAT IN THE PRESERVATION OF FOOD PRODUCTS 461 is properly done the product should be no less attractive to the eye, no less pleasing to the palate, and of no less value from the standpoint of digestibility than the same food when served in the fresh condition. BroLocicaL CHaNncEs. Vital Disorganization—The entire industry of conservation of foods by means of heat is based on a microbiological process. It isa universally recognized fact that the ordinary spoilage of food is a microbiological change; hence the individual desirous of protecting food from spoilage must give consideration to the microbial agents responsible for the change. : Normal Flora and Fauna.—Aside from a few types of organisms causing disease or specific poisonous conditions, we are unable to desig- nate definite species as those causing canned goods to spoil. Consider- ing the great variety of foods preserved by heat, and the different con- ditions under which they are grown and secured, it naturally follows that the normal flora and fauna of food to be preserved in this manner would embrace a wide variety of species, including some higher fungi, molds, yeasts, bacteria, and low animal forms, Generally speaking, the microbial flora of fruits consists mostly of molds and yeasts, although bacterial forms may also be present. In the case of vege- tables, and of fruits coming in contact with the earth, more species of bacteria are apt to be present, many of them spore formers able to withstand a high temperature. Finally, in meats and fish the living forms may include not only molds, yeasts, and bacteria, but animal forms as well, such as the organisms of teniasis (tapeworm) and trichinosis. In the preserving industry, therefore, consideration must be given to all these forms, not only from the standpoint of the suc- cessful preservation of the various foods, but also from that of pro- tecting the health of the consumer from possible poisonous substances present or produced in the conserved food, and from possible infection with pathogenic organisms present in the food before it is packed. ‘ PASTEURIZATION Economic ConsmDERATIONS.—In the preservation of food by heat, two processes are applicable, pasteurization, and sterilization. In pasteurization, the aim is not to effect the permanent preservation of foods or drinks by destroying all life present, but rather to destroy - certain species of organisms, thus checking the natural fermentation, and effecting a temporary preservation. 462 MICROBIOLOGY OF SPECIAL INDUSTRIES The principle of pasteurization may be said to have originated in the early work of Spallanzani and Scheele, already mentioned, and. was employed by Appert in his later investigations. The operation as carried out by Appert does not, however, appear to have found general application until Pasteur revived the method, and as a result of his activities in attempting to secure a general adoption of the practice to prevent the spoiling of wine, the process was named from him. Speciric APPLICATION. Beer.—Pasteurization is of economic im- portance particularly in the dairy and fermentation industries. In brewing, ‘‘ The process of pasteurization is in use with even the smallest brewers in the United States, beer being pasteurized even for local consumption.” The beer is pasteurized in bottles by being subjected to a temperature of 58° to 63° for one-half hour. The entire process as practised in the large breweries, requires less than an hour, and in- cludes the warming of the cold bottles to pasteurizing temperature, the pasteurizing proper, and the cooling to a little above room tempera- ture. The process is a continuous one, the bottles being put into the machine at one end and taken out at the other. Experiments have been made in pasteurizing beer in large containers, steel, copper, aluminum, and tin having been tried, but without complete success as yet. Fruit Juices —The essentials in the pasteurization of wine and fruit juices are similar to those for beer. There is, however, no universal rule of application. Details of the process must be arranged to suit the character of the different liquids under treatment. Cream and Milk.—Pasteurization as employed in the dairy industry varies in its method of application according to the purpose for which it is used. In factory butter making, it must be employed to secure the best results. Milk as ordinarily received at creameries contains a widely variant microbial flora, many of the species exerting a greater ‘or lesser influence in determining the flavor of the finished product. By pasteurization of the cream, the butter-maker destroys most of the organisms present; and by the use of a culture starter of lactic acid bacteria, he is able to control the fermentation, and is assured of a uniform quality of product from day to day throughout a season. An added value of pasteurization is that all pathogenic organisms are destroyed, thus aiding in the prevention of such diseases as might be HEAT IN THE PRESERVATION OF FOOD PRODUCTS 463 conveyed through this product. In creameries, the usual process of pasteurization is what is known as the continuous or flash process, in which the milk is subjected to a momentary heating to about 85°, the flow through the pasteurizing machine being so regulated as to bring all the milk up to the desired temperature, the heating being immediately followed by rapid cooling, and subsequent addition of the lactic starter. In the pasteurization of milk for infant feeding, a lower temperature is employed. A temperature sufficiently high to kill the organism of tuberculosis (the standard for pasteurization) by momentary heating, imparts to the milk a cooked flavor, making it less palatable, and coagu- lates some of the protein constituents making it less digestible. The desired end may be reached however, by using a lower temperature for a longer period of time, and the method generally recommended is to heat the milk to 60° to 65° for thirty minutes. This heating is suffi- cient to render harmless any pathogenic organisms likely to be present in the milk, without the objectionable features attendant on heating to a higher degree. Condensed Milk.—It is commonly stated that Gail Borden invented the process for preparing condensed milk, in 1856. Previous to this however, milk had been condensed in France, Germany, and England as early as 1825 to 1835. While he cannot, therefore, be called the inventor of condensed milk, to Borden belongs the credit of having first prepared it by a rational process, and in a practicable form. In the manufacture of condensed milk, good fresh milk is evaporated in a vacuum pan similar to those used in sugar factories, at a tempera- ture of 40° to 50° until the volume is reduced a little more than half, cane sugar being added so that the finished condensed milk usually contains 4o per cent. cane sugar. The evaporation must be conducted with great care, otherwise the lactose crystallizes out, and this causes the product to feel ‘‘sandy”’ to the tongue. When the evaporation of the milk is complete, the yellowish white syrup is sealed up in tins which hold about 450 g., and this quantity is equivalent to about 114 1. of normal milk. The addition of cane sugar acts as a preservative, and although the finished product may contain some living organisms, ‘itissaid to keep indefinitely if unopened, and will even keep for a number of days after opening. Occasional losses do occur by spoilage of the 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 from yeasts. , STERILIZATION Economic CoNsIDERATIONS.—For certain classes of food products, pasteurization is widely applicable, and is of immense value from an economic standpoint. Preservation by pasteurization is at best, how- ever, temporary. Spores of spore-forming bacteria are certain to be present on many kinds of foods, and these, unharmed by pasteurizing temperatures, develop vegetative cells, and spoilage occurs. For permanent preservation, therefore, sterilization must be adopted and it is upon the principle of sterilization, coupled with prevention of future contamination by hermetically sealing the container, that the whole canning and preserving industry is based. The method is applicable to nearly every class of food, and with less alteration in the food than any other method of conservation. The principle employed to-day is essentially the same as that used by Appert over 100 years ago. Although he knew nothing of microdrganisms or their relation to the spoilage of food, Appert’s experiments taught him that not only must the food to be conserved be heated thoroughly, but that it must be so sealed as not to allow air to enter the container. In the light of microbiological science, it is clear that the success of the process depends, not upon keeping out the air, but upon keeping out organisms which are carried in the air. SPEcIFIC APPLICATION.—The process of preservation by steril- ization is not so extensively practised for fruit juices and fermented liquids as that of pasteurization. If too high temperature is employed for.fruit juices, certain compounds of agreeable taste and aroma are destroyed, with a consequent deterioration in the flavor of the product. Fruit juices may, however, be sterilized by heating at a low temperature for several successive days. The method of Appert has its widest application in the conservation of fruits and vegetables, meats and fish. Whatever modifications are made in the handling of the different classes of foods the essentials are the same. The raw material after thorough cleaning and removal of waste if any, is filled into the cans and submitted to the sterilizing process, the degree of heat and time of processing varying with different products. From the character of the flora, fruits as a rule require a com- paratively low temperature for sterilization, while some vegetables and meats re- HEAT IN THE PRESERVATION OF FOOD PRODUCTS 46 5 quire a very high temperature to destroy the bacterial spores sure to be present. Briefly, the methods employed in canning some foods follow: Meat.—In the meat-canning industry, lean meat is largely selected for two reasons. Fat, well-finished carcasses bring a better price when offered for sale in the fresh con- dition; and in the second place, lean meat has a better appearance in the canned state than fat meat. The selected meat is cut into pieces of approximately from x to 4 pounds in weight, according to the size of the tins in which it is to be preserved. The pieces are cut as nearly as practicable the same size, not only for purposes of appearance in the cans when opened, but also that the process of sterilization may be more uniformly carried out. If the pieces were of different sizes, the smaller ones would become thoroughly cooked and disintegrated before the larger ones were sterilized. After the pieces have been selected and dressed they are parboiled before being placed in the containers, the time ranging from eight to twenty minutes, according to the size of the pieces of meat. The object of parboiling is to secure the shrinkage which always takes place on heating. Meats put into tins in the fresh state and sterilized shrink to about two-thirds of their original volume. When the meat is put directly into boiling water, there is less loss of protein than when the meat is placed in cold water and heated gradually. During parboiling, the meat loses about 1 per cent of the protein content, about one-third of the total meat bases, and 50 per cent of the mineral matter. This shrinkage by parboiling tends to make a more concentrated article, thus favoring transportation, and, pound for pound, the nutritive value is not lowered. Practically, the nutritive value of a pound of properly canned beef is about one-third greater than that of 1 pound of fresh beef of the same kind. After parboiling, the meat is placed in tins, by hand or by machinery, and to each can is added a small quantity of ‘‘soup liquor,”’ the manner of preparation of which is not disclosed by the packers. It may be regarded as a thin soup, the object of which is to fill up the spaces between the meat, and to add condimental substance to render the meat more palatable. After the cans are filled, they are closed and processed in suitable retorts by steam under pressure, as previously described, the temperature ranging from 110° to 120°. A modification of the usual method consists in exhausting the cans in vacuo, and automatically sealing them in the exhausted state, thus removing all the air and other gases, after which they are placed on an endless conveyor and dipped into an oil bath at a temperature of 115°, the speed of the conveyor being so regulated that the cans remain in the bath a sufficient length of time to complete sterilization before they are carried out at the oppositeend. They are next carried automatically into a solution of carbonate of soda, and finally into pure water, after which they are dried, painted with a shellac or lacquer and labelled. Fresh meats other than beef or pork are canned in a fresh state. When game and wild fowl, as well as domesticated chickens, ducks, geese, turkeys, and pigeons are used, the general process is as already described. Horse meat is used more or less commonly in some European countries, but probably rarely in the United States. Fish.—The process of fish canning does not differ materially from that of other 30 466 MICROBIOLOGY OF SPECIAL INDUSTRIES meats. .On account of its proneness to rapid decomposition, especial care must be observed that the fish are in a perfectly fresh state before canning, and that the sterilization be most thorough. Salmon is the principal fish for the preservation of which dependence is placed on sterilization alone, most fish being preserved by other methods. Vegetables and Fruits. Corn—The young tender ears of sweet corn are picked from the stalk, preferably in the early morning, keeping the husks on, and are taken in this condition to the factory. They are husked and the silks removed and passed through machines with sets of knives which cut the grains evenly from the cob, care being observed not to cut the corn so closely as to cut off particles of the cob with the corn. After the corn is cut off the cob, some canners add a ‘“‘syrup” of water, salt and sugar, and cook the corn for a few minutes at 80°, after which it is filled into the cans, sealed, and sterilized. Another method is to fill the uncooked corn directly into the cans, fill them with ‘‘syrup,”’ hermetically seal and sterilize. The temperature employed varies somewhat, but usually lies between 115° and 121° for thirty minutes for No. 2 cans holding 20 ounces. Proportionately longer time is required for larger cans. Most of the operations are carried on by machinery. The sterilization is sometimes done in the ordinary canners’ retort, or the cans may be placed on an endless conveyor, dipping into water or brine of a proper tem- perature, the speed of the conveyor being so regulated that the cans are sufficiently ~ heated to sterilize them during the passage. Peas.—In the pea-canning industry the vines are cut with a mower, loaded onto racks like hay, and hauled to the vining machines. The viner is a machine consisting of an outer and an inner cylinder revolving in opposite directions, the inner one bearing paddles or beaters so arranged that as the vines pass through the machine the paddles break open the pods. As the peas are thrown out, they pass through perforations in the outer cylinder, while the vines are discharged at the opposite end. The shelled peas are next washed to remove all dirt and also the mucous ‘substance from the surface, thus insuring a clearer liquor in the can. Grading for size is done by passing the peas over sieves, or into a revolving cylinder having four sections with perforations of different sizes. The peas are next blanched in hot water to remove the mucous covering and to drive water into the peas so that all will be tender. The time of blanching varies from one-half to five or more minutes, large mature peas requiring more time for the blanching than smaller ones.- After blanching, the peas are filled into the cans by special machines, the cans are filled with “liquor” consisting of water, salt, and sugar, sealed, and sterilized. The time of processing varies, the average being 115° for thirty to thirty-five minutes, for the ordinary sized can. i Fruits —The essentials in the canning of fruits do not differ from those for vegetables. Stone fruits may be canned either with or without the pits. In the case of such fruits as cherries, or other acid fruits, the tin can is coated on the inside with a laquer or enamel which protects the tin from erosion by the action of the acid juices. The time and temperature of processing of fruits is usually less than that required for vegetables, for the reason that in the presence of the fruit acids, the organisms are more easily destroyed than in foods in which acids are not present. HEAT IN THE PRESERVATION OF FOOD PRODUCTS 467 CoNTROLLING Factors IN SUCCESSFUL CANNING CLEANLINESS.—Too much emphasis could hardly be placed upon the importance of cleanliness throughout the whole preserving process, and especially in the preparation of the product for preserving. Vege- tables that have come into contact with the soil are pretty certain to harbor many spores of bacteria, and if as many of these are removed as possible by a thorough preliminary cleansing, sterilization may be effected with greater ease and certainty. The necessity of cleanliness on the part of factory employees is needful only of mention, not only from the esthetic standpoint, but also from that of good health. THE SOUNDNESS OF RAW MarTeERIAL.—The necessity of sound and wholesome raw material is fully as great as that of cleanliness in handling. Foods are never better than when they are fresh. It makes no difference how long nor by what method they may be cooked, the quality cannot be bettered, and if food is unsound when put into the containers for canning, it will never be wholesome for food; and this fact is equally true whether the unsoundness is the result of diseased conditions of meats, fruits, or other products, or whether it is due to ordinary decay. WatTER Suppiy.—Another essential for the success of the canner is an ample supply of pure water. It is a well-known bacteriological fact that outbreaks of spoilage have occurred in canneries, which could be traced to organisms getting into the goods from the water supply.’ RECEPTACLES.—The commercial canner recognizes two essentials for suitable containers for his goods. First, they must be tight, both to prevent the escape of the contained material and the entrance of con- taminating organisms. Second, they must be of a material which will withstand erosion or corrosion for a reasonable length of time, without giving up any notable quantity of foreign material to the food with which they may be in contact. Glass is most satisfactory from this consideration, but for reasons previously stated it is impracticable for - use on a commercial scale. The difficulty from erosion in tin cans has been largely overcome by the use of enamelled cans as mentioned above. DEGREE OF Heat REQUIRED.—The actual sterilization of food products after placing in the containers is termed processing by the commercial canner, and he appreciates fully that upon the care with which the processing is done depends the success of the entire pack. 468 : MICROBIOLOGY OF SPECIAL INDUSTRIES The degree of heat necessary to accomplish sterilization varies con- siderably with different products. - One factor lies in the chemical composition of the fruits or vegetables to be sterilized. It is, for example, well known that peas and asparagus are rendered germ-free with much greater difficulty than beans, not- withstanding the fact that the heating of the can contents of the former is accomplished much more easily than that of the latter. The higher acid content of the beans facilitates the sterilization, and this same prin- ciple holds true in a broad way for all products of an acid character. The canner and the housewife have long known that tomatoes were easy to preserve as compared with other vegetables. The canner also finds a variation from season to season. In some seasons the acid content of fruit and vegetable products will be higher than in others, and consequently a lower processing temperature will suffice for sterilization. In sterilization under pressure, as in the canners’ retort, it is impor- tant that the steam forced into the autoclaves should completely dis- place all the air, for otherwise at a certain pressure the corresponding temperature will not be obtained. Large cans require a longer time for thorough heating than small cans; closely packed cans are heated with greater difficulty than loosely packed ones; the inner temperature is frequently lower than that of the outer parts of the can. In addition to these factors, the canner must consider the possible presence or absence of bacterial spores, which may gain entrance to his factory, and necessitate a higher temperature than that usually em- ployed to accomplish the desired result. , Homer CANNING oF Foops The successful canning of foods in the home depends upon the same principles as those employed in commercial canning, namely, cleanliness, soundness of raw material, and complete sterilization. - Aside from the universally used open kettle method of handling the foods, two other methods are now widely used: the cold-pack method, and the vacuum-seal method. In the cold-pack method, the products are packed cold in their fresh natural state into the glass jars or other containers. To the fruits, hot syrup is applied; to the vegetables and greens hot water and a little salt is added. Then the sterilization is done in the jars after they are partially or entirely sealed. Steriliza- HEAT IN THE PRESERVATION OF FOOD PRODUCTS 469 tion may be effected by the intermittent or fractional method of heating on each of three successive days. The time of heating would depend on the nature of the product, and the size of the container. The extra time, labor, and fuel required for this method makes it impractical where a large amount of canning is to be done. The more common method is to sterilize in one period of heating by immersion of the con- tainers in water which is then brought up to the boiling point; or by the use of steam pressure outfits of which the market affords several types. These operate on the same principle as the laboratory autoclav. SPOILATION OF CANNED Foops MicropiaAL changes occur when the goods have not been proc- essed at a temperature sufficiently high to destroy all the organisms which may have been present in the uncooked food. In some instances, the organisms decompose the contents of the can with formation of gas, causing bulging of the ends of the cans sometimes to the point of burst- ing at the seams. Such cans are designated at the factory as “swells.’’ In other instances, the bacteria in the imperfectly sterilized cans cause an acid fermentation with consequent souring of the contents. The canner terms such cans “flat sours.” DETECTION OF SPOILED Goops.—lIn cases of spoilage accompanied by gas production, detection of the spoiled cans is easy from the bulged appearance of the ends of the cans. On account of the exhaus- tion of air from the cans during processing, the ends of Sound cans should be slightly concave. If the ends of the cans are convex, it indicates some abnormal condition of the contents, and such cans should be rejected. In the case of sours, detection isnot soeasy. The can may appear normal, and there may be no change in the contents apparent to the eye on opening the can. Taste, however, reveals a more or less pronounced disagreeable acid flavor. Canned meats, fish, or crus- taceans are likewise liable to spoilage if sterilization has been im- perfectly carried out. In these goods the change is generally but not always accompanied by gas production, hence detection is easy because of the swelled appearance of the cans. DISPOSAL OF FAcTORY REFUSE The disposal of factory refuse has at times become a serious problem for the commercial canner. Of late years, however, methods have been devised for utiliz- 470 MICROBIOLOGY OF SPECIAL INDUSTRIES ing much of the material that formerly was allowed to accumulate about the factory in fermenting heaps to the extent of sometimes becoming a nuisance to the neigh- borhood. . At pea canneries several methods of utilizing the vines are in use. They may be converted into silage, either by putting into silos or stacking in large stacks. In some sections the vines are cured for hay. They are also valuable as a fertilizer. Corn husks and cobs are also used for silage. Experiments were made by the United States Department of Agriculture in regard to the feasibility of using the refuse from the canning of corn for the production of alcohol. It was found, that on account of the expensive machinery and apparatus required in the manufacture, a small factory could not profitably utilize the corn waste for alcohol. It was shown that where several factories were located within a short radius of each other, by shipping their waste to a central plant, it might be used up to advantage. Apple cores, “chops” and peelings are usually either used’ for vinegar making, or are made up into apple jelly. From one factory visited by the writer, the apple cores and peelings were dried, baled, and shipped to Europe, “to be made up into champagne.” . ‘ Peach pits are sometimes sold to nurserymen for seed. Sometimes the pits are cracked and the meats used for almond meats. In many factories, no use is made of the peach stones. In the classes of foods in which the waste is not large, the refuse is either hauled away to a dumping ground near the factory, or is taken away by farmers for its manurial value. CHAPTER IITI* THE PRESERVATION OF FOOD BY COLD \ INTRODUCTION In recent times cold storage has become of very great importance in the preservation of perishable food stuffs, and foods preserved by cold usually command a higher market price than those preserved by other methods. This is probably due primarily to the fact that the general appearance of refrigerated food resembles that of the perfectly fresh article, in many instances very closely. Moreover, in many instances cold storage, for a reasonable length of time, preserves not only the appearance and the nutritive value, but also the chemical composition, and even the delicate flavors of the original articles, so important in determining market value. The great economic impor- tance of this industry is at once apparent, for it aims to preserve un- changed the over-abundance of one locality for transportation to another, and the over-production of one season of the year for subse- quent use. ‘ THE EFFECTS OF REFRIGERATION UPON Foops IN GENERAL The decomposition of foods depends upon the activity of their own intrinsic enzymes to some extent, but more especially upon the activity of foreign microdrganisms—bacteria, yeasts and molds. Cold acts as a preservative, not by destroying these microbes, but by retarding or inhibiting their activity. In general, cold not only retards the growth of the microérganisms but delays their death also, tending to preserve them as well as the food unchanged. In discussing the refrigeration of foods we may consider three periods of treatment, (1) the removal of the heat or chilling of the food, (2) the prolonged storage at low temperature, (3) the subsequent warming of the food before sale or consumption. * Prepared by W. J. MacNeal. 471 472 , MICROBIOLOGY OF SPECIAL INDUSTRIES CHANGES DURING CHILLING.—The period of cooling is a relatively short one, varying from a few hours to a few days in length. The chief physical change is the intentional removal of heat by conduction and . convection, but there is usually also some loss of water by evaporation. If cooled to a sufficient degree the water content of the food may crystallize, altering to a considerable extent the physical structure of the food substance (frozen food). Most foods are either actually living when chilling begins, or they are only recently dead and various chemical changes due to intrinsic enzymes continue at a diminishing rate as the heat is removed. Decomposition changes, due to microbes, may also be in progress and continue during the process of chilling. At this time the microbes living in the cold-storage chamber gain access to the newly arrived food and others areadded in the process of handling. The extent to which these will grow and multiply depends upon their ability to flourish under the storage conditions. In general the bacteria which flourish at ordinary temperatures, producing the familiar decom- position of the particular food, are greatly retarded in their activities and other kinds outstrip them under the new conditions. Thechanges taking place during chilling are very important in some special in- stances, and often a very definite procedure must be followed to obtain a satisfactory result. CHANGES DURING STORAGE.—This is often a relatively long period and causes acting very slowly may ultimately produce marked altera- tion. There is ordinarily some loss of water by evaporation, as well as the evaporation or diffusion of other volatile constituents, some of them at times important factors in the flavor of the food. Other vola- tile substances may be absorbed from the air of the storage room. The chemical changes of the chilling period continue at a greatly dimin- ished rate, or may be entirely inhibited if the food is frozen. The behavior of the microbic content of the food is the most important factor to be considered during this period. Besides those already present, various other microdérganisms, bacteria, yeasts or molds, may gain access to the food from time to time, either from the circulating air or by contact with other things. The fate of the implanted microbes will depend upon their nature and adaptation to the conditions exist- ing in the stored food. Many of them perish, but many also survive the entire period of storage, and some may actively multiply. There can no longer be any doubt that some bacteria can grow at the tem- [ THE PRESERVATION OF FOOD BY COLD 473 perature of zero, and many kinds multiply at a fraction of a degree above that point. In order definitely to inhibit microbic activity, the food must be frozen. When it is not frozen, bacteria continue to multi- ply slowly at the lowest temperature of storage, and small variations in the temperature and in the humidity of the atmosphere serve to ~ accelerate their activity. Such variations also accelerate diffusion currents in the food substance and so tend to distribute the microédrgan- isms and their products. The extent of the resulting chemical changes in the food will depend upon these factors and upon the nature of the food, the temperature and the length of the period of storage. CHANGES AFTER STORAGE.—This is a relatively short period, but in many instances a very important one as regards change in the food. If warmed too rapidly, vigorous currents may be set up in the food mass by the great difference in temperature between the outer portion and the interior, serving to distribute microdrganisms and their products. In the case of frozen foods rapid warming fails to restore the original physical structure. Dry cold foods are likely to condense moisture from the warmer atmosphere unless it is particularly dry, and this condensed water becomes another cause of diffusion currents. In frozen foods the water, in melting, may fail to reénter the food struc- ture, and exude and drip away, carrying a portion of the soluble con- stituents withit. At this time still more microbes are likely to be added to the food, and, together with those already present, they multiply with increasing rapidity as the temperature rises. As they may be already pretty well distributed throughout the mass of the food, the resulting chemical decomposition is the more rapid. It is well recog- nized that, in keeping qualities, foods removed from cold storage are much inferior to the corresponding fresh foods. REFRIGERATION OF CERTAIN Foops Meat, FisH AND PovuLttry.—Meat, in this sense the flesh of mammals, is preserved by cold in two ways, by storage above the freezing-point (chilled meat) and by storage at —10° to —4° (frozen meat). Fish and poultry are usually frozen for storage, often in the undrawn condition. Mammals killed for chilled or for frozen meat are slaughtered and carefully dressed, For chilled meat the temperature is reduced by 474 ; MICROBIOLOGY OF SPECIAL INDUSTRIES storage in a cold air chamber to about ++2° in forty-eight hours, and the meat is stored at a temperature between +1° and +2°. Under these conditions the enzymes of the dead flesh continue to act and bacterial decomposition proceeds slowly, bringing about a process of ripening which, up to a certain point, improves the market value of the flesh by making it more tender and giving to it a more desirable flavor. The extent to which the slow bacterial decomposition may proceed before the flavor becomes disagreeable varies with different tastes, but in general the beginning of proteolytic change which follows after the almost complete fermentation of the muscle sugar may be said to mark the desirable limit. ‘This point is reached in from a week to three months, depending upon the condition of the animal, skill and care in slaughter and dressing, especially the extent of bacterial contamina- tion at this time, and the accurate control of the storage conditions. In the production of frozen meat the carcasses are rapidly chilled in an air chamber at —20°, where the meat remains until frozen solid. It is then kept at a temperature below —4°. Freezing produces a marked change in the finer physical structure of the meat, as the water crystallizes, leaving the protein material, with which it was formerly intimately mixed, in a shrunken and shriveled state between the crystals. Enzymic and bacterial activities are practically if not ab- solutely suspended under these conditions, and, save for slight surface evaporation, such meat remains unchanged for long periods. The subsequent thawing presents certain difficulties and requires particular care. If warmed very slowly the melting water crystals are imbibed by the protein material and the original structure of the flesh almost com- pletely restored. The warmer air must be dry and must be kept in motion to avoid condensation of moisture on the exterior of the thaw- ing meat. Bacterial activity is likely to gain considerable headway during this process and the penetration of the microbes into the flesh is favored by the diffusion currents. The more prolonged the warm- ing process the greater the opportunity for bacterial decomposition. Ordinarily, in order to avoid this, the thawing is carried out rapidly and the finer structure of the meat is not restored. It is softer, darker, and more moist than fresh or chilled meat, and usually sells at a : lower market price. Fish and poultry are usually frozen for storage. As these foods are especially subject to rapid objectionable decomposition changes they are THE PRESERVATION OF FOOD BY COLD 475 rapidly chilled by immersion in ice water or packing in ice immediately after death, and are frozen as quickly as possible. During storage in the frozen condition microbic activity is suspended, but in the sub- sequent thawing the same physical and biological changes occur as in frozen meat. When fish and poultry are stored in the undrawn condi- tion there is an abundant supply of bacteria at hand in the intestinal contents ready to multiply energetically during the chilling and thawing stages. It would appear desirable that the poultry should be killed and dressed with great care previous to freezing and that the period of chilling should be shortened as much as possible. Practically, how- ever, it has been found that the dressing of poultry, as ordinarily done, previous to storage leads to such an extensive soiling of the edible flesh of the birds that their condition at the end of the storage period is often less satisfactory than that of undrawn frozen poultry, not only in gross appearance but alsoin respect to microbic content and chemical com- position. Most frozen poultry is, therefore, stored in- the undrawn condition. The tendency of such food to undergo decomposition after thawing should be clearly recognized. Its sale as fresh or as chilled food is a fraud upon the purchaser. In-fact many individuals seem to be pecu- liarly liable to suffer digestive disturbances after eating frozen poultry and such persons should avoid its use. The nature and source of the bacteria which produce poisonous changes in poultry are not definitely known, but there is some evidence indicating that they belong to the para-colon group and that they are derived from the intestinal contents of the fowls. Eccs.—The cold storage of eggs is an industry which has attained large proportions in recent years. A very constant storage temperature between ++o.5° and +1° is essential for the best results. The hu- midity of the atmosphere is also of very great importance, as a dry air causes extensive evaporation from the egg and a too moist air favors the development of microdrganisms on the exterior of the shell and the absorption of their products and even their penetration into the egg. A constant humidity of 70 per cent saturation has been found to be the best. Storage at this temperature and humidity greatly retards the ‘growth of microérganisms and definitely inhibits the ordinary putre- faction of eggs. The activity of the intrinsic enzymes of the egg are not necessarily inhibited by this temperature, nor is the growth of all . 476 MICROBIOLOGY OF SPECIAL INDUSTRIES microdrganisms prevented. Unquestionably there is a marked differ- ence between the ordinary cold-storage egg and the strictly fresh egg, but to what extent this deterioration may be due to errors in storage, such as inaccurate control of temperature and humidity, use of odorif- erous crates for packing, decomposition changes previous to storage, too rapid chilling of the eggs, or too rapid warming of them after removal from storage, and to what extent it is inherent in the most perfect cold-storage procedure, is still somewhat uncertain. Doubt- less a certain amount of deterioration, especially the loss of the peculiar flavor of the fresh egg, is unavoidable in any method of prolonged storage. The discrimination in price in favor of new-laid eggs in the market is an indication of difference in actual value, and the sale of cold-storage eggs for new-laid or strictly fresh eggs is generally recog- nized as a fraud by the purchaser and doubtless will in time be so recognized by law. The cold-storage egg is nevertheless a very valuable food and the economic importance of saving the over-abundant supply produced during the spring for use during the colder season of the year makes this industry a great benefaction to the public. Suitable regula- tion may be expected to remove its objectionable features. MILK AND Butter.—Milk as ordinarily sold at retail is not subject to sufficient seasonal change in market price to make its prolonged storage advisable. But milk is so rapidly changed by bacterial activity at ordinary temperatures that efficient dairy methods necessarily in- clude prompt cooling of the milk after it is drawn from the animal and the maintenance of a low temperature until it is delivered to the consumer. At the low temperature bacteria slowly multiply, unless the milk is actually frozen, but at a temperature slightly above the freezing-point very clean milk may be kept in perfect condition for a week, and it may be kept sweet for several weeks. Refrigeration of milk cannot compensate for unhealthy animals producing it, nor for careless and uncleanly methods of handling. The cold does not destroy the microbes in the milk but only retards their multiplication and chemical activity. In practice, especially in the transportation of milk into large cities, it is frequently most economical to freeze the milk and trust to insulation and the latent cold in the ice to maintain a low temperature during transportation. Such milk should arrive at its destination in a partly frozen condition. The cold storage of butter is essential even when it is kept for only THE PRESERVATION OF FOOD BY COLD 477 short periods, and the seasonal variation in price is sufficient to warrant its storage from summer to winter. The keeping qualities of butter depend upon many factors,* and the most efficient cold storage cannot compensate for previous deficiencies. In refrigerated butter there is a gradual diminution in the total number of living bacteria, with possibly a multiplication of a few particular kinds. There is a slow increase in acidity. In frozen butter the bacterial content and the chemical com- position remain practically unchanged. FRUITS AND VEGETABLES.—These foods are for the most part adapted to preservation for short periods at ordinary temperatures, and cold storage at a temperature slightly above zero is very effective in diminishing the rate of changeinthem. The humidity of the storage chamber should be kept constant at about 60 per cent saturation in order to diminish evaporation as far as possible without favoring the ‘development of molds. These foods generally remain alive during storage and the changes due to intrinsic enzymes are often important. Some fruits need to undergo further ripening in storage before they are ready for consumption and this change may be accelerated or delayed by changing the temperature of the storage chamber. The develop- ment of bacteria and molds with consequent rotting is best delayed by maintaining dry clean fruits and vegetables in an atmosphere of very constant humidity and very constant temperature slightly above the freezing-point. LEGAL CONTROL OF THE COLD-STORAGE INDUSTRY At present there is a rather widespread prejudice against cold-stor- age food products, and in some respects this is not without justification. Cold storage preserves so well the external appearance of fresh foods that deception in the sale of them to the consumer is too frequently practised. This is extremely unfortunate for all parties concerned in such transactions. The proper branding of all cold-storage foods, clearly indicating their character and the length of time held in stor- age, would ultimately benefit the producer, the consumer and also the cold-storage industry. Where cold storage is efficient such a practice would proclaim its efficiency. Where it is inefficient the cold-storage industry can ill afford to allow the consumer to be deceived concerning * See chapter on the microbiology of butter, 478 MICROBIOLOGY OF SPECIAL INDUSTRIES the food he is purchasing. The strict enforcement of laws compelling the proper labeling of such foods and prohibiting their sale except when branded as such would quickly remove unjust prejudice against cold storage, and would place this industry upon a secure foundation, greatly increasing the possibilities of its service to the food producers ‘and consumers, and at the same time promoting the legitimate interests of the cold-storage industry. i CHAPTER IV* THE PRESERVATION OF FOOD BY CHEMICALS The addition of preservative substances to foods is a very ancient practice, and as no extensive equipment is required it is one of the cheapest ways of preserving food, especially on a small scale. The resulting alteration of the food in appearance and composition is greater than when it is preserved by cold storage, for the preservative substance added becomes a more or less permanent constituent of the food, but the changes are not necessarily undesirable. The addition of chemical preservatives is often practised in conjunction with desiccation or cold, or sometimes even in canned or bottled foods sterilized by heat. All the substances employed as preservatives owe whatever efficiency they may possess to their ability to restrict the activity of microérganisms, that is, their antiseptic properties. THE EFFECTS OF PRESERVATIVES UPON Foops IN GENERAL In only a few instances are chemical preservatives added to foods to be sold as fresh foods, and these practices are generally regarded with disfavor. Their most important use is in the prepared foods, the pre- servative being incorporated with the food during the process of preparation for storage. THE Process or Curtnc.—The procedures employed necessarily vary with different foods. Physical alterations in the food, such as changes in form, texture and water content are usually involved, as well as the solution of the preservative in the juices of the food. Chemical changes due to the intrinsic enzymes of the food, to the various accessory procedures such as drying, cooking or soaking in pickling solution may produce marked alteration. In some cases the preservative reacts chemically with some constituent of the food. During the curing process microbic activity may be more or less * Prepared by W. J. MacNeal. 479 480 MICROBIOLOGY OF SPECIAL INDUSTRIES \ prominent at various times, playing its part in the chemical changes. Bacteria, yeasts and molds are likely to be introduced into the food by the various manipulations and some of these may find conditions favor- able for their proliferation. In some instances the activity of certain kinds of microbes appears to be essential to the proper curing and subsequent adequate preservation of the food; the preservative, the constituents of the food and the microdrganisms mutually reacting to ‘bring about the desired result. It is worth noting that the added chemical preservative is never sufficiently potent to destroy with certainty pathogenic microbes which may be present in the food. THE PERIOD OF STORAGE.—Unless the food has been sterilized and stored in sealed containers, slow changes in water content, in physical appearance and in chemical composition usually take place during storage. The added preservative may continue to react with the food _ substance and its decomposition products. During this period there is relatively little intimate manipulation of the food and therefore little opportunity for the penetration of new microbes. Some of those already present may continue their activities at a diminished rate, producing slow chemical changes often of a desirable nature rather than otherwise. Accessory conditions, such as desiccation, cold storage, or sterilization and sealing, may greatly retard or check altogether microbic activity. Tur AFTER-STORAGE CHANGES.—The immediate preparation of preserved food for consumption is frequently important. The pre- servative may be largely removed mechanically, or extracted with water. During cooking peculiar chemical reactions may occur, and cooking is also important in the destruction of microérganisms remain- ing alive in the food up to that time. THE CHEMICAL PRESERVATION OF CERTAIN Foops MEAarts AND Fisu.—The preservation of meat and of fish by salting depends upon the increase of osmotic tension in the food, a physical change sufficient to prevent or greatly delay the growth of micro- organisms. Sodium chloride (NaCl) probably owes its preservative value solely to this physical effect. In practice its action is often sup- plemented by the addition of a small amount of saltpeter (KNOs), and sometimes also cane sugar (Ci2zH22011). The fluids of the flesh THE PRESERVATION OF FOOD BY CHEMICALS 481 are in part removed by this treatment, carrying away a part of the soluble constituents. -The fluids which remain contain the added preservative substance in solution, and the ‘whole mass of food sub- stance is permeated by them. Potassium nitrate (saltpeter) reacts with the flesh, being reduced in part to nitrite. This enters into a combination with the coloring matter of meat, which upon cooking produces the characteristic red color of meat cured with saltpeter. The various manipulations during the process of pickling or dry curing serve to introduce numerous microérganisms. Many of these may flourish in the pickling fluids, but in a sufficient concentration of salt and at a sufficiently low temperature, decomposition ordinarily does not progress so as to become objectionable, and proteolytic decom- position (putrefaction) is effectually prevented. This protection of the protein depends to some extent upon the acidity of the medium, which in turn is due largely to the bacterial decomposition of the muscle sugar. The powerful putrefactive bacteria (B. edematis group) flourish only in an alkaline medium. On the other hand too high a degree of acidity becomes in itself objectionable on account of the sour or rancid taste, and it is, therefore, important that the acid- producing bacteria should be held in check somewhat. In practice, saltpeter has proved of value for this particular purpose, and its action apparently depends upon the antiseptic effect of minute quan- tities of nitric acid (HNO3;) and nitrous acid (HNO.) set free from the salt by the excess of organic acids produced by the bacteria. The. curing of meats by pickling solutions is often supplemented by desicca- tion and impregnation with the antiseptic substances of wood smoke. The dry-salting of codfish is an example of preservation by increasing the osmotic tension. The fish is cleaned and beheaded, split longitudinally, and the vertebral column removed. It is then carefully washed, and all visible blood is removed. The pieces are next covered with dry salt and packed in open casks. The salt rapidly extracts water from the flesh and a strong brine results. After a few days the casks are emptied out, and the pieces of fish, now smaller because of the loss of water, are again thoroughly washed and again packed in dry salt so that the adjacent pieces of fish are completely separated by an intervening layer of solid salt. The contents of the cask are subjected to high pressure to remove air, and the cask is finally closed. The curing of hams is an example of preservation by increased osmotic tension combined with the addition of chemical preservatives. After slaughter and chilling the hams are injected witha solution containing 25 per cent common ‘salt, T5 percent granulated sugar, and 12 per cent saltpeter, and are then stored at a low tem- perature, preferably between o° and +4", in a brine containing about 20 per cent 31 482 ° MICROBIOLOGY OF SPECIAL INDUSTRIES common salt, 5 per cent sugar, and 1 per cent saltpeter. The brine is renewed once or twice at intervals of a week or ten days. After about a month the hams are washed in warm water, dried and hung in wood smoke for several days. They are then stored in a cool place. The proportions of the various constituents of the pickling solutions are subject to rather wide variation, and in general it may be said that the higher the temperature of the storage room, the more concentrated must be the pickling solutions to insure satisfactory preservation. Dairy Propucts.—Butter is usually salted with sodium chloride to impart the desired taste, and this salt also acts to some extent as a preservative by increasing the osmotic tension of the moisture remain- ing in the butter. Antiseptics such as boric acid, saltpeter, salicylic acid and formaldehyde have been employed in the preservation of butter, ‘the first-mentioned appearing to be the most satisfactory. One half of 1 per cent of boric acid incorporated with high-grade butter ' previous to storage greatly delays rancid change. Fresh milk and cream are also sometimes treated with antiseptics such as formaldehyde, but the use of any chemical preservative what- ever in these dairy products is unnecessary and generally disapproved. PREPARED VEGETABLE AND Fruit Foops.—These foods are some- times preserved by vinegar, sugar or alcohol, the presence of which is of course very evident to the consumer. Other substances less readily detected, such as sulphurous acid and sulphites, boric acid, salicylic acid, benzoic acid and sodium benzoate, and formaldehyde, are also em- ', ployed in foods which must be kept some time after exposure to the air. These substances are incorporated with the food before it is packed, and serve to prevent the activity of microérganisms which gain access to it. THE NUTRITIVE VALUE OF PRESERVED Foops The nutritive value of a food depends upon the amount of utilizable food principles it contains. The food-principle content can be readily measured by chemical analysis, and in general there is no important difference between a preserved food and the corresponding fresh food in this respect. The utilization of the food principles, however, depends upon a number of factors and may be greatly influenced by individual peculiarities of the consumer. One important factor in the utilization of a food, and probably the most important factor in deter- mining its market value, is palatability. In general, preserved foods THE PRESERVATION OF FOOD BY CHEMICALS 483 are pleasant to the taste when eaten at intervals, but upon prolonged daily ingestion, the appetite for them fails and they may even become distasteful. It would, therefore, appear to be erroneous to regard pre- served foods as in every respect as valuable from the standpoint of nutrition as the corresponding fresh foods. The difference is not de- pendent upon a change in the food-principle content, but must be sought rather in slightly altered composition of the food and the specific’ effects of newly formed substances, and especially in the possible effects of the continued ingestion of the contained chemical preservatives upon the consumer. Tue EFFECTS OF Foop PRESERVATIVES The essential characters of a food preservative include antiseptic action to prevent decomposition of the food, and absence of evident poisonous or deleterious influence upon the consumer. It follows there- fore that the effects of food preservatives upon the consumer, if they exist at all, are at any rate not easily recognized, and on account of the economic importance of the questions here involved this field of scientific research has been energetically cultivated by investigators with different viewpoints, and the results of investigation have been discussed with some heat. The passage of the U. S. Food and Drugs Act was followed by considerable discussion of these questions. Gradu- ally. the practical administration of the law has become more settled and the use of many food preservatives is still permitted. ; SUBSTANCES WHICH PRESERVE BY THEIR PuysicaLt AcTION.—The preservative effects of sodium chloride seem to depend entirely upon the high osmotic tension of strong salt solutions, and the same may be said of cane sugar. When diluted so as to be eaten with relish, these substances are themselves properly classed as foods, without deleterious effects upon ordinary individuals. SUBSTANCES WHICH PRESERVE BY THEIR CHEMICAL ACTION.— These preservatives inhibit the activity of microdrganisms in a dif- ferent way, not by withdrawing water from the microbic cell, but by entering into chemical combination with the living substance in sucha way as to hinder its activity, or by entering into chemical reactions with the food to produce new substances capable of attacking the microbic protoplasm in this way. The ideal chemical food preservative 484 MICROBIOLOGY OF SPECIAL INDUSTRIES would be one which, without altering the food substance, would ex- hibit this poisonous property toward living protoplasm until the food was ready for consumption, and then would suddenly and permanently lose this property. None of the ordinary food preservatives approaches this ideal very closely. Inorcanic Foop PRESERVATIVES.—Boric acid and borax are weak antiseptics, practically a saturated solution of boric acid being neces- sary to inhibit ordinary bacterial growth. When employed as a dry ‘ powder on the surface of meats, boric acid prevents the growth of mold, and most of it is removed from the food before consumption. When incorporated with butter it is eaten, and 0.5 to 1.0 g. may be taken daily in this way alone. The effect of such amounts of boric acid upon the consumer is still a disputed question. Wiley,* after an extensive investigation, concluded that small doses of either boric acid or borax continuously administered for a long period create disturbances of health. Nitric acid and nitrous acid and their salts are food preservatives of some theoretical interest because it is well known that some bacteria readily decompose fairly strong solutions of nitrates, and also oxidize or reduce nitrites. Apparently, however, this is true only in neutral or alkaline solutions, and in the presence of free acid the activity of these microbes is quickly inhibited. The preservative effect of nitrates and nitrites is best ascribed to the liberation of minute quantities of free nitric and nitrous acids from these salts, and these substances are without value as preservatives in foods which are alkaline in reaction. The effects of the ingestion of nitrate or foods preserved with nitrate upon the consumer has been investigated by Wiley, who concluded that the deleterious effects are slight and less clearly detected than in the case of the other preservatives. Minute but variable amounts of nitrites occur in foods preserved with nitrates, but whether these amounts are sufficient to produce the specific nitrite effect upon the blood circulation of the consumer has not yet been definitely ascertained. Sulphurous acid and the sulphites are rather extensively used in chopped meat (Hamburg steak) and in cider and wines. The addition of sulphite to chopped meat serves a three-fold purpose, retarding bac- terial decomposition, producing a red color on the exposed surface, and removing odors of decomposition. It thus not only delays decomposi- * U.S. Dept, Agr., Bureau of Chemistry, Bull. No. 84, Part I, THE PRESERVATION OF FOOD BY CHEMICALS 485 tion, but also to a certain extent conceals the decomposition which has already occurred. The ingestion of moderate quantities of sulphites in food has at times been followed by acute gastric derangement in man, and prolonged feeding of meat containing sulphites has been followed by inflammatory changes in the kidneys of experimental animals. Fluorides have been used to a slight extent in beverages, but acute gastric derangement and depression of the heart are caused by rather small quantities, and probably on this account the salts of hydrofluoric acid have not come into very general use as food preservatives. Orcanic Foop PRESERVATIVES.—Formic acid (H:COOH) and acetic acid (CH3-COOH) are produced by microbic activity and their preservative action appears to depend more upon the degree of acidity than upon the character of the acid radical. Both these acids appear to be utilized as food in the body of the consumer. Benzoic acid and benzoates are rather extensively employed in prepared vegetable food products, such as jams and catsups. The antiseptic effect seems to be due wholly to free benzoic acid, even where it is added in the form of the salt, but the action is not due merely to the acidity (.e., the hydrogen ion). Benzoic acid is not utilized as a food in the body, but is excreted by the kidneys in the form of hippuric acid. It has been said to produce irritant effects upon the stomach and the kidneys, and to arrest the action of digestive enzymes in dilute solutions, but the Referee Board* of the United States Department of Agriculture, after extensive investigations, concluded that small doses of sodium benzoate mixed with food are not injurious to health, and do not impair the quality or nutritive value of the food. Salicylic acid and the salicylates have been used for much the same purposes as benzoic acid, and there does not appear to be much dif- ference between the two acids, either in their efficiency as preservatives or in their possible deleterious effects upon the consumer. Salicylic acid is more expensive. After extensive investigation Wileyt has concluded that the addition of salicylic acid and salicylates to foods is reprehensible’ in every respect, this conclusion corresponding to the results of similar work by the same investigator upon benzoic acid. *U.S. Dept. Agr. Report: No. 88, May, 1909. + U.S. Dept. Agr., Bureau of Chemistry, Bull. No. 84, Part II, 1906. t U.S. Dept. Agr., Bureau of Chemistry, Bull. No. 84, Part IV, 1908. 486 MICROBIOLOGY OF SPECIAL INDUSTRIES Formaldehyde is very efficient as an antiseptic, delaying microbial decomposition when added to foods in very small quantity. Its use for this purpose is generally condemned, partly because of its hardening or ‘“‘fixing” effect upon the protein constituents of the food, tending to make them more indigestible. Its use in milk and milk products, . though still practised to some extent, has been prohibited by law in some states. Alcohol (CH;-CH,OH), in sufficient concentration, is an excellent preservative, but its presence in foods is readily detected, and it gives rise to characteristic effects upon the consumer. Furthermore, such foods are subject to special taxation as alcoholic products. Its use as a food preservative is therefore limited. Wood smoke has been employed for centuries in the curing of meats. Its antiseptic properties probably depend for the most part upon creosote and pyroligneous acid, constituents of wood smoke which are antiseptic and also undoubtedly poisonous in sufficient doses. Smok- ing is a time-honored custom, however, and the amount of these substances actually consumed with the smoked meat is doubtless exceedingly minute. SUBSTANCES ADDED TO Foops To Improve THE APPARENT QUALITY. —Several chemical substances are employed in various foods to im- prove the appearance, or to simulate the taste of a higher-grade product. In some cases the presence of these agents is known to the consumer, and desired by him; in other instances they are employed to deceive - the purchaser. Butter coloring is quite generally used to produce the color of June butter the year around; nitrates bring about a pleasing red color in cooked pickled meats; copper sulphate is used to give a more brilliant green color to prepared vegetable foods; sulphites restore the red color of freshly cut meat to meat far from fresh; saccharine devoid of food value gives a taste resembling sugar to a variety of preparations at a great saving in cost to the manufacturer; carbonates of the alkalies or alkaline earths, added to milk, neutralize the acids resulting from bacterial decomposition and so keep the milk sweet; inorganic acids added to weak vinegar increase its acidity. Some of these practices are so universal and so well known that they are no longer criticized; others, such as the use of chalk in milk, are generally disapproved. THE PRESERVATION OF FOOD BY CHEMICALS 487 LEGAL CONTROL OF THE PRESERVATION OF Foops By CHEMICALS The desirability of legal regulation of the use of chemical food pre- servatives is now generally recognized, but there is still considerable diversity of opinion concerning what this regulation should be. Few, indeed, maintain that a substance exerting antiseptic action upon microdrganisms outside the body is wholly without influence, after ingestion, upon the enzymes and bacteria of the normal digestive tract, even if we disregard the possible effects of the substance after absorp- tion. It seems necessary to grant the existence of some effect, even though it be so slight as to have escaped detection. Over against the possible injury to the consumer must be placed the economic saving through the use of the preservative, often involving a considerable amount of money. In the absence of accurate and trustworthy knowledge concerning the actual influence of preservatives in the human body it would seem wise to prohibit all deception in regard to their presence. The principle advocated by Pasteur (1891) would still seem to be best, that is, to allow the use of preservatives, which are not known to be dangerous, upon the condition that their presence and the exact amounts be definitely and clearly stated on an appropriate label for the benefit of the purchaser and the ultimate consumer. Such regulation would not only protect the consumer against deception and fraud but would go far toward removing unjust prejudice against preservatives, for even now there is little or no objection to those preservative substances of which the presence and the amount can be detected and roughly measured by the senses, such as salt, sugar, spices, vinegar and wood smoke. CHAPTER V* MICROBIAL FOOD POISONING GENERAL CONSIDERATIONS Illness following the ingestion of food, more or less definitely ascribable to the food, has been long recognized. The Mosaic regula- tions in regard to foods forbidden to the Jews are evidently designed in part to avoid the occurrence of food poisoning. In recent times recog- nized instances of food poisoning have been sufficiently frequent to make the subject one of considerable practical importance, but there are undoubtedly many instances of actual food poisoning in which the causal relation of the food remains unrecognized or even unsuspected. Food poisoning is usually suspected at once upon the occurrence of sudden acute illness in a number of people at the same time, after they have partaken in common of some particular food or foods. The causal relation is especially evident when, as sometimes happens, a large number of people are affected in the same way immediately after eating together at a banquet, not having been associated with each other either before or after the meal. When a smaller number of indi- viduals is involved, the connection with food may be more obscure, For this reason most of the well-authenticated instances of food poison- ing are instances in which many persons have been affected at the same time. Acute food poisonings involving only a few persons probably occur very frequently in the home, but they receive little public notice unless fatal, and are often dismissed as mere “errors in diet,” or as ‘“indigestion.”’ A careful study of these cases is likely to be made only where there is suspicion of criminal poisoning, or some other practical end to be served by the investigation. Chronic forms of food poisoning are for obvious reasons very difficult to recognize with certainty, and some of the forms of disease now regarded as due to chronic food poisoning may eventually prove to be due to other causes. On the other hand chronic food poisoning may really be more impor- * Prepared by W. J. MacNeal. 488 MICROBIAL FOOD POISONING 489 tant than is recognized at present. The subject is still in a very doubtful state. To establish by laboratory investigation the poisonous character of foods requires toxicological training and experienced judgment, a dis- cussion of which would lead beyond the scope of the present chapter. For a general review of this field of work and references to further information the articles cited at the end of this chapter should be consulted. Several different classes of food poisonings may be recognized according to the source of the poisonous substance. The material of plants or animals may be naturally poisonous to man as a result of the physiological activity of their own living sub- stance. Poison of this kind may be constantly present throughout the tissues, or it may be confined to certain parts, or it may occur only at particular times or seasons. Some instances of poisoning with fish and with mushrooms belong to this class, and possibly also some of the instances of poisoning with potatoes of high solanin content. Plants and animals may feed upon substances not poisonous to themselves, and these substances may remain a constituent part of _their bodies to poison man when consumed by him. Some poisonings with freshly killed game are considered to be of this nature. Any food may contain foreign poison added to it by design or by accident, such for example as the salts of the various poisonous metals. The amount of tin or lead passing into solution in canned or tinned foods may conceivably be sufficient to cause poisoning, but there is no reliable evidence that it has ever occurred. Animals may be infected with pathogenic bacteria or with other parasites capable of infecting man, and the use of food products from such animals may cause disease. Tuberculosis, trichinosis, and tapeworm may be acquired in this way. Any food may serve as the passive carrier of infectious agents, such as B. typhosus, and some foods may even favor the multiplication of pathogenic bacteria gaining access to them. A food may undergo chemical changes due to microérganisms in- capable of infecting man, resulting in the production of poisonous sub- stances in the food. Undoubtedly the great majority of instances of food poisonings belong in this class. The bacteria causing these changes have been designated as pathogenic saprophytes. 490 MICROBIOLOGY OF SPECIAL SIGNIFICANCE The last three classes comprise the microbial food poisonings, and these are the kinds of food poisoning with which we are at present more particularly concerned. INFECTIONS OF FOOD-PRODUCING ANIMALS TRANSMISSIBLE TO MAN Animals dead of infectious diseases or slaughtered in the last stages of disease are not ordinarily used for food, nor is the milk of such animals ordinarily considered wholesome. This custom is certainly an ancient one, and is doubtless founded upon observation of un- favorable results following the consumption of such food. Exact knowledge of the nature of the diseases transmitted in this way is a more modern development, and this more exact knowledge is now being applied to some extent through food-inspection regulations to prevent the transmission of such diseases. Tuberculosis of cattle has been shown by Smith to be due to a germ somewhat different from that causing the ordinary human tuberculosis, and this discovery has called into question the necessity of avoiding the use of food products from tuberculous animals. After a con- siderable amount of controversy it may now be regarded as definitely established that the bovine type of tubercle bacillus is capable of infecting man, and that a very considerable proportion of cases of tuberculosis in children are due to this type of organism, the infection probably arising through the use of milk from tuberculous animals. Anthrax, glanders, actinomycosis, and acute enteritis of animals are also transmissible to man. Food products from animals afflicted with these diseases should not be used until they have been passed upon by competent authority. Further information concerning them will be found in the sections dealing with these particular diseases. The human disease known as septic sore throat may be due to in- fection with streptococci present incow’s milk. Whether these virulent streptococci are derived from an inflamed udder of the cow or from the throats of persons who handle the milk is not fully ascertained, but the inflamed udder is to be looked upon with suspicion. In this connection it may be mentioned that some of the animal parasites, especially trichine and various sorts of tapeworms, gain access to the human body with the food. Thorough cooking usually serves to kill these parasites, as well as the pathogenic bacteria, but \ MICROBIAL FOOD POISONING 491 ordinary cooking should not be too implicitly relied upon to accomplish this result. HuMAN INFECTIONS TRANSMITTED IN Foop Food may serve as the passive carrier of the germs of any human in- fectious disease capable of indirect transmission upon dead material. In some foods, especially milk, these infectious agents may actually multiply. Typhoid fever, diphtheria, and scarlet fever appear to be rather frequently disseminated through the agency of food, and para- typhoid fever seems to be commonly transmitted in this way. Especial precautions are advisable to prevent persons afflicted with dangerously communicable diseases and those who are chronic germ-carriers from engaging or continuing in occupations concerned with the immediate preparation of food for consumption, particularly such occupations as. milk producers, milk handlers, market-dairying, cooking, and serving food. Numerous serious epidemics have been traced to such sources in recent years. Foop PoIsonNING DUE TO THE GROWTH OF SAPROPHYTIC BACTERIA IN THE Foop Most food poisonings are due to food derived from perfectly healthy and wholesome animals or plants, which has subsequently undergone some bacterial decomposition giving rise to poisonous products. Our knowledge of the specific causes of the poisonous changes is, however, very incomplete, and on account of the difficult nature of investigation in this field, some of the conclusions reached by careful men are still open to question. The bacteria which have been most frequently identified with various epidemics of food poisoning are the following: B. enteritidis in meat poisoning; B. botulinus in meat and in sausage poisoning; B. paratyphosus in poisoning with meat, chicken, shell- fish, and vegetables; B. coli in cheese poisoning and in milk poisoning; B. vulgaris in meat and in vegetable food poisonings. Doubtless other microdrganisms, as yet unrecognized, play an important part in many food poisonings, and there is reason to believe that some of these important unknown forms are anaerobic bacteria. Porsonous MEAT AND SausaGE.—The flesh of a healthy animal is ordinarily free from bacteria at the time of slaughter, and bacterial 492, MICROBIOLOGY OF SPECIAL SIGNIFICANCE changes must begin at the surfaces of the pieces of meat and gradually extend inward. In diseased animals, bacteria more frequently circulate in the blood, and the flesh may be contaminated throughout when the animal dies of the disease or when it is slaughtered, not only with the specific germs of the disease but also with bacteria derived from the intestinal tract of the animal. It is a matter of observation that the flesh of diseased animals is more liable to undergo early putrefac- tive and poisonous changes than that derived from healthy animals. Hashed meat is, of course, much more prone to bacterial decomposition, because in it the bacteria have become well distributed throughout the mass, and ideal conditions are provided for the development of anaerobic as well as aerobic bacteria. Minced chicken and chicken pie appear to be very frequent sources of acute poisoning in the United States, and epidemics of sausage poisoning have repeatedly occurred, especially in Germany. The bacteria found to be concerned in these instances have been B. enteritidis, B. paratyphosus, B. coli, and B, botulinus. Some of these poisons, as for example the toxin of B. botulinus, are rendered inert by boiling, but occasionally bacterial poisons which are not destroyed by such high temperatures may be present in food. Moreover, meat rendered poisonous by these bacteria may’ show no evidence of putrefaction. B. (Proteus) vulgaris has also been found in some samples of poisonous meat, and this finding is usually associated with definite evidence of putrefaction. The symptoms of meat poisoning are usually those of acute gastro- enteritis, vomiting, cramps, and diarrhoea. The patients often recover very quickly, but occasionally the illness is rapidly fatal, or it may merge into a subacute form resembling or identical with paratyphoid fever. In those instances of poisoning due to the presence of B. botulinus the symptoms are of a different kind, consisting almost solely of nervous disturbances, secretory and motor paralyses, without fever, resembling in many respects poisoning with atropin. In this form of meat poisoning the death rate is relatively high, about 40 per cent of the cases ending fatally. FISH POISONING is of two general kinds, that due to poisons natural to the fish, and that due to poisons formed by bacterial activity in the flesh of the fish. Blanchard has applied the Spanish name “Siguatera” to the first kind and the term “Botulism” to the second. In the Japanese fish of the genus Tetrodon the roe is poisonous, giving rise MICROBIAL ‘FOOD POISONING 493 to severe gastro-intestinal irritation and convulsions. The remainder of the fish is not poisonous. In some other fishes the sexual glands are poisonous during the spawning season; others are provided with special poison glands connected with protective spines or barbs. These are examples of poisons natural to fish. Bacterial poisons are likely to be formed in any kind of fish, given the suitable conditions, and thus give rise to the kind of fish poisoning designated as botulism. Cases of this kind have resulted from eating (spoiled) canned salmon and sardines. Poisoning may also result from eating diseased fish, the effects being due to poisons elaborated by the infecting bacteria in the body of the fish before consumption. This appears to be a rather common form of fish poisoning in Russia. B. paratyphosus has been isolated from some poisonous fish, and certain toxicogenic an- aerobes have been found in others. POISONING WITH SHELL-FISH is so well recognized that this form of food is not customarily used at all during the warmer part of the year, May to August inclusive, the months without an r in their names. Shell-fish may serve as carriers of human infectious diseases, such as typhoid fever; they may be poisonous on account of actual disease or through serious contamination due to living in dirty water; or they may be poisonous because of decomposition which has taken place after removal from the water. According to the symptoms produced, there appear to be at least three distinct varieties of shell-fish poi- soning, one a purely gastro-intestinal disorder, the second an involve- ment of the nervous system with itching skin eruption and convulsions, and a third type resembling very closely alcoholic intoxication. The exact nature of the microbic agents concerned in these different types of poisoning is unknown. It is pretty well established, however, that the poisonous character of shell-fish is due either to their living for some time in dirty water, or to their too long preservation, especially at high temperature, after removal from the water. MILK, ICE-CREAM AND CHEESE sometimes give rise to poisoning, and although these instances are small in number in comparison with the enormous amount of milk and milk products consumed, yet in the aggre- gate they are numerous. That many human infections may be trans- mitted by milk has already been pointed out. In the summer, milk is undoubtedly a great factor in the infant morbidity and mortality, and this poisonous action is largely due to bacterial changes in the milk. 494 MICROBIOLOGY OF SPECIAL SIGNIFICANCE Extraordinary precautions are therefore essential in the production and care of milk to be used as food for children, particularly during the warmer season of the year. Severe poisoning of adults with milk, ice- cream, or cheese, is relatively less frequent. Cases which have been studied have been traced to the development of B. coli or B. paraty- phosus in these foods. There is some evidence that other bacteria, probably strict anaerobes, are also sometimes concerned. Strict cleanliness, proper refrigeration, and pasteurization of milk of uncertain character, may usually be relied upon to prevent milk poisoning. Ice-cream should be made only from wholesome materials and with due regard to cleanliness in making it. The causes of serious cheese poisoning are not definitely known, but such poisoning may be avoided, to a large extent at least, by using only standard varieties of cheese of the proper odor and flavor. VEGETABLE FOOD POISONING, in an acute form, has followed the use of sprouting and partly decomposed potatoes, and also various canned vegetables, particularly those of high protein content, such as beans. The large majority and possibly all of these cases are due to decomposi- tion changes in the foods, B. botulinus and B. proteus appearing to be the microbes most frequently concerned. There are also certain definite, more or less chronic diseases which have been attributed to the use of certain grains as foods. Ergotism, characterized by cachexia, gangrene, and convulsions, is caused by eating the fungus, Claviceps purpurea, which grows as a parasite upon rye. The grain of this parasite has a considerable commercial (medic- inal) value sufficient to pay for its separation from rye where it occurs, so there is little economic excuse for food poisoning from this cause. Beriberi or kakke is an acute or chronic nervous disorder which has been observed especially in the Orient, Japan and the Philippine Islands, although it has also been found in Brazil, in Labrador and rather frequently among sailors after long sea voyages. At one time the disease was ascribed to the use of fish as food, later to the use of rice. Modern studies, especially those of Chamberlain, Vedder and their associates in the Philippine Islands, have shown that beriberi may be prevented by including beans, unpolished rice or rice hulls in sufficient quantity in the diet and furthermore that those already afflicted with the disease usually recover completely when given these foods or when treated with an alcoholic extract of rice polishings. MICROBIAL FOOD POISONING 495 The curative principle of rice polishings has been studied by Funk who has named it vitamine. He ascribes the causation of beriberi to a lack of this supposedly necessary vitamine in the food and this theory has been very favorably received. It must be acknowledged, however, that the etiology of beriberi is still not convincingly proven. The discovery of a remedy which eradicates a given disease is not sufficient to prove that the lack of this particular therapeutic agent is the es- sential cause of the disease. Pellagra is a cachexia, characterized by a definite sort of skin erup- tion, which has been ascribed to the use of maize (Indian corn) as food. This disease is discussed in a separate section (page 813). THe CHEMICAL NATURE OF Foop Poisons The poisonous substances in foods are for the most part of the same nature as the poisons of the pathogenic bacteria. The simplest in structure of these poisons belong to the alkaloidal substances, substi- tuted ammonia and ammonium compounds, called ptomains (page 171). Several of these have been prepared in a pure state, for example, mytilo- toxin (CsHisNO2) from poisonous shell-fish, and neurin (C2:H3'N- (CH;);0H) from putrefied horse, beef, and human flesh. Although ptomains undoubtedly occur at times in poisonous foods, they are not now considered of so much importance in food poisoning as formerly, for in the majority of samples of poisonous food the search for ptomains has been in vain. The poisonous effects are believed rather to be due for the most part to much more complex bodies resulting from the earliest analytic changes in the food protein, or else to bodies built up by actual synthesis by the bacteria. Such substances are classed with the toxic proteins and the true toxins. Their chemical composition and structure are not definitely known. REFERENCES Dieudeomné, A., translation by Bolduan, C. F., Bacterial food poisoning. E. B. Treat and Co., New York, 1909. Novy, F. G., Food poisons, Osler-McCrae, Modern Medicine, 1914, Vol. II, PP. 450-471. ; Thresh and Porter, Preservatives in food and food examination. J. and A. Churchill, London, 1906. Vaughan and Novy, Cellular toxins. Lea Bros. and Co., Philadelphia, 1902. CHAPTER VI* MICROORGANISMS OF THE DIGESTIVE TRACT INTRODUCTION The digestive tube of the vertebrate animal is in communication with the external world and is the passageway for a great variety of materials constituting the food of the animal. This food brings with it various sorts of microbes, at times in considerable numbers. Within the digestive tube the food is more or less completely resolved by the processes of digestion into soluble nutritive split products, which furnish an excellent medium for microbic development. It is not surprising, therefore, that there is’an enormous multiplication of microérganisms within the intestine, both in health and disease, and that this multi- plication is most active during the digestion of the food. MICROORGANISMS OF CERTAIN PORTIONS OF THE ALIMENTARY CANAL The entire digestive tract is free from microbes during normal intrauterine life. After birth the canal is quickly invaded by bacteria, chiefly through the mouth and nose, but to a lesser extent also through the anal orifice. In the mouth, pharynx and intestine, some of these invaders establish themselves to remain throughout the life of the individual host. The species of microbes present and the numerical proportions of the different species of normal buccal and intestinal microérganisms vary somewhat with the age of the host and the charac- ter of his food. They are also considerably disturbed sometimes by the entrance and multiplication of pathogenic germs, giving rise to disease in their host, such as Oidium albicans in the mouth or the chol- era vibrio in the intestine. MicroORGANISMS OF THE Movuru.—The buccal cavity presents conditions of temperature, moisture, chemical reaction and a variety of food substances in its various parts, which are very favorable to the growth of many microbic species. Aerobic, facultative and anaerobic forms are found and the species are very numerous. Miller, in a few *Prepared by W. J. MacNeal. 496 MICROORGANISMS OF THE DIGESTIVE TRACT 497 weeks, was able to isolate more than a hundred different kinds of bac- teria from the mouth. Many of these are doubtless only transient residents, having gained entrance with food, water or air. Among the almost constant inhabitants of the mouth may be mentioned the streptococci, both the variety which produces a green color on bloodagar, the Strept. salivarius or Strept. viridans, and the hemolytic variety, Strept. hemolyticus; the M. pyogenes var. aureus and albus; the Iodococcus magnus and Iodococcus parvus of Miller, which may be cultivated upon a sugar-starch gelatin-agar medium and are stained blue by iodine; two or three species of spirilla, described by Miller, which may be cultivated with some difficulty upon ordinary nutrient agar; B. fusiformis of Vincent, which may be cultivated as a strict anaerobe in media containing blood serum or ascitic fluid; B. maximus (buccalis) of Miller, a bacillus forming threads 0.5 to 1.5u wide and 2oy or more in length, cultivable upon maltose agar or potato gelatin; Leptothrix buccalis, a slender unbranched filament, which may be brought to development on ordinary media, with some difficulty. Even more definitely characteristic mouth bacteria are those which are found in every human mouth (except in very young children) and which are not cultivable in artificial media at all or only under special artificial conditions never met with in nature. Among these - forms may be mentioned the Iodococcus vaginatus, an encapsulated organism which may be stained blue by Lugol’s solution acidified by addition of lactic acid; the Sp. sputigenum, which is found especially at the inflamed margin of the gums; the Spirocheta buccalis, Spirocheta media, Spirocheta microdentium and macrodentium, organisms which are found in the mucus about the teeth, but are especially numerous ‘on denuded areas or in abscess cavities of the gums or in carious teeth. The spirochetes of the mouth have been successfully cultivated by anaerobic methods in serum and in ascitic fluid by several investigators, notably by Noguchi.* The ameeba of the mouth, Entameba (Endameba) buccalis, may be found in nearly every individual in the deposits between the teeth and especially in carious teeth. The cell is 6 to 324 in diameter, actively motile, with few lobose pseudospodia. The nucleus of the living amceba is visible. Its food apparently consists of bacteria and the bodies of * Noguchi, Journ, Exp. Med., 1912, XV, 81. 32 498 MICROBIOLOGY OF SPECIAL SIGNIFICANCE , leukocytes. It does not appear to penetrate living tissue. Other mouth amcebe have been described. Whether they really belong to species distinct from Entameba buccalis is questionable. Recently it has been claimed that the amcebe of the mouth bear a causal rela- tion to pyorrhea alveolaris, but the claim has not been convincingly proven. The various characteristic buccal microérganisms are found in particular parts of the mouth and their numbers vary considerably according to the cleanliness of the mouth and teeth, presence or absence of denuded areas, ulcers, sinuses or carious teeth. The iodine-staining varieties are especially abundant between the teeth and upon starchy . food residues. The spirochetes, on the other hand, are more abundant in the serum exuding from denuded areas and in pus cavities. B. fusiformis (Vincent) is often found in normal buccal mucus but it is especially abundant in the necrotic ulcers of the tonsil in the disease known as Vincent’s angina, in which situation it is always associated with numerous spirochetes. Some of the members of the normal mouth flora occasionally play definite pathogenic réles. There can be little doubt that the starch- fermenting forms produce acid, thus attacking the mineral matter of the teeth and favoring dental caries. The pathogenic réle of Strept. viridans, when it penetrates into carious teeth, causing root abscess and, probably by metastasis from this focus, giving rise to arthritis and endocarditis, is indicated by a mass of circumstantial and experimental evidence which is well nigh convincing. The frequently serious nature of infections with the hemolytic streptococcus are well known. Doubt- less members of this variety of streptococcus in the mouth are ready to acquire virulence whenever lowered resistance of the host presents a favorable opportunity for them to invade the tonsils, the pharyngeal mucous membrane, the Eustachian tube and middle ear, not to mention more distant parts of the body. Certain very specific pathogenic microdrganisms are found in the mouth and pharynx from time to time and they sometimes produce lesions there. Spirocheta pallida is especially abundant in the buccal and pharyngeal lesions of secondary syphilis. The tubercle bacillus is expectorated through the mouth in open pulmonary tuberculosis. The pneumococcus is often found in the mouth and pharynx, even in health and is especially numerous and virulent in cases of lobar pneu- MICROORGANISMS OF THE DIGESTIVE TRACT 499 monia. The influenza bacillus and Bact. diphtherie are also occasion- ally found in the throats of healthy persons as well as of those suffering from the diseases to which they give rise. MICROORGANISMS OF THE STOMACH.—The microbic flora of the healthy stomach consists almost exclusively of organisms swallowed. The gastric juice restrains bacterial multiplication and kills a large majority of the bacteria which enter the stomach. In diseased con- ditions the absence or reduced concentration of the hydrochloric acid may permit the multiplication of yeasts, of large lactic-acid bacilli (Boas-Oppler bacilli), of encapsulated cocci (Sarcina ventriculi), or even of flagellate protozoa, such as Lamblia and Trichomonas. Micro6rGANIsMsS OF INTESTINE.—The duodenum receives from the healthy stomach relatively few living bacteria. The secretions of the liver, pancreas and of the duodenal wall are very free from bacteria and they tend to flush out the duodenum. In health this portion of the intestine is quite free* from living bacteria in the intervals when food is absent and it contains relatively few bacteria during digestion. Among the living microdrganisms most frequently found here are Gram- positive cocci which fail to liquefy gelatin. B. coli is uncommon. ‘In spite of the negative results of culture work upon duodenal juice, it is always possible to see with the microscope abundant bacterial cells in it. These are probably dead. From the upper end of the jejunum to the ileocecal valve, the number of bacteria in the small intestine progressively increases. In the intervals when food is absent, even these portions of the small intestine tend to free themselves from bacteria, in part, probably, because they are continually flushed out by the intestinal secretion, but probably in part also, as has been maintained by Kohlbrugge,t} because of a definite bactericidal property of the intestinal mucous membrane. However this may be, it is certain that living organisms of the B. coli group and various streptococci are commonly found in intestinal contents taken from the jejunum or ileum at operation or at autopsy and that these organisms are quite numerous in the material discharged from the lower end of the small intestine in cases of ileo- cecal fistula.t The relative abundance of the different kinds of bacteria * MacNeal and Chace, Arch. Int. Med., 1913, XII, 178. { Kohlbrugge, Centrabl. f. Bakt. Abt. I, 1901, XXIX, 571; ibid., 1901, XXX, 10; ibid., I901, XXX, 70. t Macfayden, Nencki und Sieber, Arch. f. Exp. Path. und Pharm., 1891, XX XIII, 311. 500 MICROBIOLOGY OF SPECIAL SIGNIFICANCE may be altered by changing the character of the diet, a fact of impor- tance in the treatment of intestinal infections. In the cecum there is a sudden enlargement of the lumen of the intestinal canal and a consequent retardation of the movement of the intestinal contents. The blind pouch also favors stagnation. In this region the whole intestinal contents usually acquire a chemical reaction neutral or alkaline to litmus. All these factors favor the enormous multiplication of bacteria. Indeed, the cecum and the remaining large intestine constitute the great bacterial incubator of the healthy body. Here B. coli multiplies enormously; the strict anaerobes, Bact. welchii and B. edematis flourish under most favorable conditions. Various streptococci, staphylococci and spirochetes multi- ply either in the food residues or in the intestinal secretions. An easily digested mixed diet favors the facultative anaerobes, while excess- ive feeding of starchy foods and of meat leads to an overgrowth of the strict anaerobes, especially those of the Bact. welchii group. Many of these bacteria will then be found to stain blue with iodine, giving the so-called granulose reaction. A milk diet, especially if limited in amount and well digested by the individual, favors the micro-aerophilic B. bifidus of Tissier, the organism which is dominant in the feces of the healthy breast-fed infant and occasionally very abundant even in adults. , In the lower portions of the large intestine, as a result of progressive absorption from the contents of the bowel, there is a concentration and overcrowding of the bacteria which have developed at higher levels. The vast majority of them die and these dead cells, together with the still abundant living microérganisms, make up about a third of the substance of the faces. The feces are composed. of rejected food residues, residues of intestinal secretions, of bile and pancreatic juice and abundant microdrganisms, some of the latter still actively multi- plying, but the majority of them dead and in various stages of disintegration. Tue MicroORGANISMS OF THE Facres.—The microérganisms of the faeces represent the end result of the progressive multiplication or dis- integration, or both, of the organisms originally present in the food together with all those added at various regions of the alimentary canal. The microbic flora of the large intestine is, however, most prominent in the feces. The total quantity and the proportions of the various MICROORGANISMS OF THE DIGESTIVE TRACT 501 kinds of microbes in the feces varies considerably even in health, depend- ing upon various factors, among which age of the individual and character of the food are very important. The first meconium passed after birth may contain few or no micro- érganisms. Within a few hours, however, they appear in the intestinal discharges. The earliest forms are usually large diplococci to which are soon added various bacilli, small diplococci and tetrads. Among the bacilli, a long slender form with a large oval terminal spore, the headlet bacillus of Escherich, is particularly conspicuous. B. coli is also present at this time and several gelatin-liquefying forms of bacilli can be isolated in cultures, among them B. (Proteus) vulgaris and B, subtilis. Anaerobic cultures demonstrate the presence of Bact. welchit, B. edematis and B. bifidus. As the meconium is replaced by the residue of the ingested mother’s milk, the previously variegated bacterial flora suddenly becomes very simple and during the whole period of exclusively breast feeding the stools contain enormous numbers the Gram-positive micro-aerophilic B. bifidus of Tissier, with only small numbers of B. coli and very few cocci. The dominance of B. bifidus may readily be demonstrated by making a series of dilution cultures in tall tubes of glucose agar, according to the method of Veillon, and incubating them for five days or more. When the child begins to take cow’s milk there is a sudden increase in the relative numbers of B. coli and streptococci and with the addition of starchy foods to the diet the fecal flora gradually comes to resemble that of the adult. In the healthy adult taking a mixed diet, the fecal flora consists for the most part of Gram-negative bacilli of the type of B. coli. There are also many diplococci, a few small Gram-positive bacilli (B. bifidus?) a small number of Bact. welchii and its free spores, a few representatives of the’B. edematis group and a variable number of slender spirochetes. Aerobic plate cultures on agar or gelatin often bring to development only B. coli. When a vegetarian diet rich in indigestible residue is consumed, the diplococci are much diminished in numbers; numerous large bacilli, Bact. welchit and B. subtilis, take their place. The con- sumption of excessive quantities of meat and starchy foods may lead to a considerable increase in the numbers of the Bact. welchii group and some of the bacteria of this group may be stained. brown or blue with iodine because of the granulose which *they contain. The bacteria 502 MICROBIOLOGY OF SPECIAL SIGNIFICANCE normally present in the faeces are produced almost altogether by multi- plication within the intestine. It is nevertheless possible for swallowed organisms to appear alive in the feces even though incapable of growth within the digestive tube. , The introduction of foreign organisms capable of multiplication in the gastro-intestinal.canal may lead to a marked alteration in the quantitative relationships of the faecal bacteria or even to the disappear- -ance of certain microbic forms previously present. Thus in cholera, the vibrio of this disease may occupy the intestinal canal so completely that the usual fecal bacteria can no longer be found with the micro- scope. By feeding acid-resisting lactose-fermenting bacteria, such as Bact. bulgaricum along with considerable quantities of milk, it is possible to suppress the putrefactive anaerobes, B. edematis group, which prefer a neutral or alkaline medium. The swallowed bacteria are manifestly, therefore, of some importance in determining the character of the fecal flora, but they are, after all, usually less important in this respect than the chemical composition of the food itself. In every case the original intestinal flora has to be reckoned with as a most essential element. The daily excretion* of bacteria in the feces of healthy men, is, on the average, about 33 million million bacterial cells. The washed and dried substance of these bacteria amounts to about 54 g. per day. From one-sixth to one-fifth of the weight of the dry feeces and probably about a third of the moist feces consists of bacterial substance. The- nitrogen carried away by these fecal bacteria represents a daily loss of 0.5 to 1.0 g. In addition to the bacteria, one often finds in the faeces yeasts and protozoa. Of the latter Entameba coli is probably an almost constant inhabitant of the intestinal tract and its numbers are often augmented in mild chronic digestive disturbances. The flagellates, Lamblia intestinalis and Trichomonas intestinalis are found less frequently. A few other protozoa occur in disease. The physiological effects of the normal intestinal bacteria are not fully understood. Some observers have maintained that continued life and growth would be impossible without the bacteria of the di- gestive tract, ascribing to them an essential part in the nutrition of the body. The experiments of Cohendyt seem now to have disproven * MacNeal, Latzer and Kerr, Journ. Infect. Diseases, 1909, VI, 123. + Cohendy, Annales de l’Institut Pasteur, 1912, XXVI, 106. a oF MICROORGANISMS OF THE DIGESTIVE TRACT 503 this hypothesis. There can be no doubt that the bacteria do enter intimately into intestinal digestion and in some instances bring about changes beneficial to their host, such as the digestion of cellulose, whereas when furnished other food they may exert a harmful influence, as for example in excessive intestinal putrefaction. In diseased conditions of the gastro-intestinal tract one finds more or less well-marked alterations in the fecal flora. These changes include quantitative change in the total bacterial output, change in the proportional relationships of the various normal types and finally the appearance of new or foreign types of organisms, either harmless or pathogenic. In many instances there is furthermore a distinct tendency for some members of the normal intestinal flora to assume pathogenic properties and invade tissues rendered less resistant by disease. Among the intestinal microérganisms which may assume patho- genic réles at times may be mentioned B. coli, B. vulgaris, Ps. pyo- cyanea, B. bifidus, Bact. welchii, the streptococci, micrococci and Trichomonas intestinalis. Among the definitely pathogenic forms are B. typhosus, Msp. comma (Sp. cholere asiatice), B. paratyphosus, B. enteritidis, Bact. dysenterie, Bact. anthracis, Bact. pestis, Bact. tuber- culosis, Entameba dysenterie (histolytica), Coccidium hominis and Lamblia intestinalis. The technical procedures necessary for the recognition of some of these organisms in the feces and for their isolation in pure culture are in some instances highly specific. Thus if one is searching for B. bifidus it is best to employ dilution cultures in tall tubes of glucose agar inoculated with feces of a healthy nursling. The same material plated on gelatin will yield only colonies of B. coli. B. welchii is most readily isolated by pasteurizing a suspension of the feces and introduc- ing it into blood broth or litmus milk in a Smith fermentation tube. The cholera organism is searched for by introducing considerable quantities of faeces into flasks of pepton-salt solution and transplanting from the surface film after six hours to new flasks. On account of its very rapid multiplication in this medium the cholera germ, if present, outstrips the other fecal bacteria. Subsequently it is necessary to apply specific agglutination tests to the spirals thus obtained in order to recognize them with certainty. The typhoid bacillus, on the other hand, is sought by inoculating media containing substances which restrain bacterial growth in general without inhibiting the growth of 504 MICROBIOLOGY OF SPECIAL SIGNIFICANCE B. typhosus. , Broth and agar containing brilliant green are now used for this purpose.* The tubercle bacillus when present, may some- times be separated by digesting the feces in alkali or in antiformin solution, washing the residue and planting it on Petroff’s mediumf or injecting it into guinea-pigs. Entameba coli and Entameba dys- enteri@ should be searched for with the microscope in fresh warm feces obtained after a dose of salts. This brief mention of a few procedures indicates the specialized character of the microbiological technic in this field. The laboratory worker will find it essential to consult the general references below and to study carefully the original papers bearing upon his field of work. GENERAL METHODS OF STUDY CoLLecTION oF MATERIAL.—Material for microbiological study may be: ob- tained from the mouth, fauces or pharynx by means of a sterile cotton swab, by the ordinary platinum loop or other instrument suitable for the special purpose in’ view. This material should be examined promptly, or, if this is impossible, it should at once be spread upon slides for subsequent microscopic study and, if cultures are to be made, it should be suspended in sterile salt solution, or better in sterile ascitic fluid, and refrigerated until the proper media can be inoculated. From the stomach, fluid may be readily obtained through a stomach tube and the contents of the duodenum or of upper portions of the small intestine may be withdrawn through the slender duodenal tube of Einhorn. The contents of the lower part of the small intestine and the upper part of the large intestine can be readily obtained only at surgical operations upon the intestine, at autopsies or from individuals in whom an intestinal fistula has been established. The contents of the lower part of the large intestine are best collected by means of a special glass instrument in the case of . young children. In older children and adults a natural stool or one obtained after salts or other cathartic may be utilized. In every instance, contamination of the material with extraneous organisms is to be strictly avoided by careful sterilization of implements and receptacles and any alteration of the specimen after collection must be reduced to the minimum by examining it promptly, although, for some purposes the use of refrigerated speci- mens may be permitted. The quantity{ of microbic cells present may be ascertained by numerical count of those present in an accurately measured portion of the material, or if they are very abundant they may be physically separated out from a weighed portion by fractional sedimentation in the centrifuge, after which they are dried and weighed (method of Strasburger). * Krumwiede, Pratt and McWilliams, Journ. Infect. Diseases, 1916, XVIII, p. .. + Petroff, Journ. Exp. Med., 1915, XXI, 38. t For detailed directions concerning quantitative methods as applied to the study of fecal bacteria, see MacNeal, Latzer and Kerr. Journ. Infect. Diseases, 1909, VI, 123; ibid., 1909, VI, 572. ’ MICROORGANISMS OF THE DIGESTIVE TRACT 505 A preliminary classification of the recognizably different kinds of microbes should be made by microscopic examination of film preparations stained (1) with Loeffler’s methylene blue, (2) by Gram’s method, (3) by the Ziehl-Neelsen method and (4) simply with Lugol’s solution. It is best to count from s00 to 1,000 microbic cells as they are met with in successive microscopic fields and to classify them according to form, size and structural details brought out by the different stains. Permanent records and, if possible, permanent mounted preparations should be preserved, so that the microbes subsequently brought to development in the cultures may be indentified with some of those present in the microscopic picture of the original material. Cultures are best made upon a quantitative basis, employing for inoculation measured amounts of accur- ately prepared dilutions of the original material. There is no single culture medium or method which can be relied upon to give any approximate conception of the numerical relations of the microbes of the digestive tract. Each cultural method necessarily favors certain species present in the mixture and allows others to develop only poorly or not at all. Adequate information con- cerning the quantitative relationships is obtained only by comparing the results of the culture work with the direct quantitative estimations and by fitting the cul- tural results into the original microscopic picture. A great variety of culture media and culture methods, aerobic, anaerobic and micro-aerophilic, must be em- Fic. 138A.—Two types ployed in making even an incomplete general survey of of instrument for obtain- the microbes from any portion of the digestive tract. 18 feces from infants for For the detection of certain single species, on the other Dackenolonies eae » tion. (After Schmidt and hand, one may sometimes rely upon a single medium, Strasburger.) such as blood-agar for the streptococci of the mouth, Loeffler’s serum for diphtheria bacilli in the pharynx or blood-broth in fermenta- tion tube for spores of B. welchii in the feces. Thus the numerous time-consuming procedures may be very much abridged and many of them may well be omitted when one wishes to ascertain merely the presence or absence of a certain single species of microbe. GENERAL REFERENCES Schmidt und Strasburger, Die Faeces des Menschen, IV “ Auflage, Berlin, 1914. Kuester, Die Flora der normalen Miindhéhle, Kolle und Wassermann, Hand- buch, II Auflage, Jena, 1913, VI, 435-449. Ktiester, Die Bedeutung der normalen Darmbakterien fiir den gesunden Men- schen, Kolle und Wassermann, Handbuch, II Auflage, Jena, 1913, VI, 468-482. CHAPTER VII* THE MICROBIOLOGY OF ALCOHOL PRODUCTS WINE Wine may be defined shortly as the product of the alcoholic fer- mentation of sound, ripe grapes and the usual cellar treatment. The classifications of wines are numerous and the varieties in- numerable. They may be separated, however, into a few main groups, depending on chemical composition and methods of manufacture. _Dry wines are those in which practically all the sugar has been re- moved by fermentation; sweet wines, those in which enough sugar remains or is added to be noticeable to the taste; fortified wines, those that have received an addition of distilled wine spirits; and sparkling wines, those highly charged with carbon dioxide, produced by supple- mentary fermentation in the bottle. Each of these groups includes white wines made from the expressed juice of the grape, and red wines made from both the juice and skins of red grapes. GRAPE JUICE AND WINE AS CULTURE MEDIA Grape juice, known technically as must, is a sugary, acid, organic so- lution very favorable to the growth of yeasts and of many other fungi, but unfavorable to most bacteria. Wine is of a similar composition but contains alcohol instead of sugar and is, therefore, less favorable to the growth of most microérganisms. Both liquids are of highly complex composition. Their character as culture media is indicated by the following table: * Prepared by F. T. Bioletti. 506 THE MICROBIOLOGY OF ALCOHOL PRODUCTS 507 ‘ ; ComposiITIon oF Must anp Dry WINE Must | Wine Specific gravity........... 1.0600 to 1.1090 0.9850 to 1.0000 Fermentable sugar........ I2.0 to 25.0 per cent o to 0.5 per cent Alcohol by volume......... none 8.0 to 15.0 per cent Acidity (as tartaric)....... v.5 to 1.25 per cent 0.35 to 1.0 per cent Nitrogenous matters (soluble)................ 0.2 to o.4 percent variable but small Tannin: soi:daw aatangnkaeare Traces ‘ traces to 0.30 per cent «DEY Extract: cncaows Avis cte da) aide din eteas ev alonae teases as I.4 to4.o percent ASB io 3532 craaieenmiwn ess, 0,20 to 0.70 per cent 0.13 to 0.50 per cent Fortified wines (sweet wines are usually fortified) usually con- tain enough alcohol to make them practically antiseptic to all microérganisms. THE MiIcroORGANISMs FOUND ON GRAPES On the surfaces of grapes, as they are brought to the cellar, may be found any of the bacteria and fungi usually carried by the air and by insects. Many of these are incapable of growing in grape must, and are, therefore, without effect on the wine. Motps.—The spores of the common saprophytic molds, Penicil- lium, Dematium, Aspergillus, Mucor, are always present in abundance, and they find in must excellent conditions for development. Botrytis cinerea, a facultative parasite of the leaves and fruit of the vine, is also nearly always present in larger or smaller quantities. All these molds are harmful to the grapes and the wine. Some of them, such as Penicitl- lium, may give a disagreeable, moldy taste to the wine, sufficient to spoil its commercial value. Others, such as some Mucors and Asper- gilli may injure the wine but slightly except by destroying sugar and diminishing the alcohol. Dematium pullulans may produce a slimy condition in weak white musts and most of them may injure the brightness and flavor to some extent. On sound ripe grapes these molds occur in comparatively small numbers and being in the spore or dormant condition they are unable to develop sufficiently to injure the wine under the conditions of proper wine making. On grapes which are injured by diseases, rain or insects, 508 MICROBIOLOGY OF SPECIAL INDUSTRIES they may be present in sufficient quantities to spoil the grapes before they are gathered. On sound grapes which are gathered and handled carelessly, they may develop sufficiently before fermentation to injure or spoil the wine. An exception to the generally harmful effect of these molds is Botrytis cinerea (Sclerotinia fuckeliana) which under certain circum- stances may have a beneficial action. When the conditions of tem- perature and moisture are favorable, this mold will attack the skin of the grape, facilitating evaporation of water from the pulp. This results in a concentration of the juice. The mycelium then penetrates the pulp, consuming both sugar and acid, principally the latter. The net result is an increase in the percentage of sugar and a decrease in that of acid. This, where grapes ripen with difficulty, is an advantage, as no moldy flavor is produced. Two harmful effects, however, follow:. the growth of the mold results in the destruction of a certain amount of material, and a consequent loss of quantity, which is, in certain circum- stances, more than counterbalanced by an increase in quality (wines of the Rhine, Sauternes); again, an oxidase is produced which tends to destroy the color, brightness and flavor of the wine. This can be counteracted by the judicious use of sulphurous acid. Yeasts.—The true yeasts occur much less abundantly on grapes than the molds. Until the grapes are ripe they are practically absent, as first shown by Pasteur. Later, they gradually increase in number and on very ripe grapes often become abundant. In all cases and at all seasons, however, their numbers are much inferior to those of the molds and pseudo-yeasts. The cause of this seems to be that in the vineyard the common molds find conditions favorable to their development at nearly all seasons of the year, but yeasts only™ during the vintage season... Investigations of Hansen, Wortmann and others show that yeasts _exist in the soil of the vineyard at all times, but in very varying amounts. For a month, or two following the vintage, a particle of soil added toa nutritive solution contains so much yeast that it acts likealeaven. For the next few months, the amount of yeast present decreases until a little before the vintage, when the soil must be carefully examined to find any yeast at all. As soon as the grapes are ripe, however, any rupture of the skin of the fruit will offer a favorable nidus for the’ development and increase of any yeast cells’ which reach it. Where - THE MICROBIOLOGY OF ALCOHOL PRODUCTS 500 these first cells come from has not been determined, but as there are still a few yeast cells in the soil, they may be brought by the wind, or bees and wasps may carry them from other fruits or from their hives and nests. The increase of the amount of yeast present on the ripe grapes is often very rapid and seems to have (according to Wortmann) a direct relation to the abundance of wasps. These insects, passing from vine to vine, crawling over the bunches to feed on the juice of ruptured berries, soon inoculate all exposed juice and pulp. New yeast cultures are thus produced, and the resulting yeast cells quickly disseminated over the skins and other surfaces visited. The more unsound or broken grapes present, and the nrore honey- dew or dust adhering to the skin, the larger the amount of yeast will be. The same is true, however, also of molds and other organisms. In the older wine-making districts, much of the yeast present on the grapes will consist of the true wine yeast, S. ellipsoideus. The race or variety of this yeast will differ, however, in different districts. Usually several varieties will be found in each district. The idea prevalent at one time, that each variety of grapes has its own variety of yeast seems to have been disproved, though there seems to be some basis for the idea that grapes differing very much in composition, varying in acidity and tannin contents, may vary also in the kind of yeast present. Several varieties of S. ellipsoideus may occur on the same grapes. In new grape-growing districts, where wine has never been made, S. ellipsoideus may be completely absent. -Besides the true wine yeast, other yeasts usually occur. The com- monest forms are cylindrical cells grouped as S. pasteurianus. These forms are particularly abundant in the newer districts where they may take a notable part in the fermentation. Their presence in large numbers is always undesirable and results in inferior wine. Many other yeasts may occur occasionally and are all more or less harmful. Some have been noted as producing sliminess in the ‘wine. Many of these yeasts produce little or no alcohol and will grow only in the presence of oxygen. Pseudo-yeasts—Yeast-like organisms producing no endospores always occur on grapes. Their annual life-cycle and distribution are similar to those of the true yeasts, but some of them are much more abundant than the latter. They live at the expense of the food §10 MICROBIOLOGY OF SPECIAL INDUSTRIES materials of the must and when allowed to develop cause cloudiness and various defects in the wine. The most important and abundant is the apiculate yeast, S. api- culatus. According to Lindner this is a true yeast, producing endo- spores. The cells of this organism are much smaller than those of S. ellipsoideus and very distinct in form. In pure culture these cells show various forms, ranging from ellipsoidal to pear-shaped (apiculate at one end) and lemon-shaped (apiculate at both ends). These forms represent simply stages of development. The apiculations are the first stage in the formation of daughter cells, the ellipsoidal cells, the newly separated daughter cells, which later produce apiculations and new cells in turn. Many varieties of this yeast occur, as in the case of S. ellipsoideus. They are widely distributed in nature, occurring on most fruits, and are particularly abundant on acid fruits such as grapes. Apiculate yeast appears on the partially ripe grapes before the true wine yeast and even on ripe grapes is more abundant than the latter. The rate of multiplication of this yeast is very rapid under favoring conditions and much exceeds that of wine yeast. The first part of the fermenta- tion, especially at the beginning of the vintage and with acid grapes, is, therefore, often almost entirely the work of the apiculate yeast. The amount of alcohol produced by this yeast is about 4 per cent, varying with the variety from 2 to 6 per cent. When the fermentation has produced this amount of alcohol the activity of the yeast slackens and finally stops, allowing the more resistant ellipsoideus to multiply and finish the destruction of the sugar. The growth of S. apiculatus, however, has a deterring effect on that of the true wine yeast so that where much of the former has been present during the first stages of fermentation the latter often fails to eliminate all the sugar during the last stages. When the apiculate yeast has had a large part in the fermentation, the wines are apt to retain some unfermented sugar and are open to the attacks of disease-producing organisms. Their taste and color are defective, often suggestive of cider, and they are difficult to clarify. This yeast attacks the fixed acids of the must, the amount of which is, therefore, diminished in the wine, while on the other hand the volatile acids are increased. Many other yeast-like organisms may occur on grapes, but, under ee THE MICROBIOLOGY OF ALCOHOL PRODUCTS Sil ordinary conditions, fail to develop sufficiently in competition with apiculatus to have any appreciable effect on the wine. Most of them are small round cells, classed usually as Torule. They destroy the sugar but produce little or no alcohol. A group of similar forms, known collectively as Mycoderma vini, occurs constantly on the grapes. These, being strongly aerobic, do not develop in the fermenting vat, but under favoring conditions may be harmful to the fermented wine. Bacteria of many kinds occur on grapes as on all surfaces exposed to the air. Most of these are unable to develop in solutions so acid as grape juice or wine. Of the acid-resisting kinds, a number may cause serious defects and even completely destroy the wine. These, the “‘disease-producing bacteria’? of wine, are mostly anaerobic and can develop only after the grapes are crushed and the oxygen of the must exhausted by other organisms. Practically all grape must contains some of these bacteria, which, unless the work of the wine maker is properly done, will seriously interfere with the work of the yeast, thus causing injury to the wine. The only bacteria which may injure the grapes before crushing are the aerobic, acetic bacteria, which may develop on injured or carelessly handled grapes sufficiently to interfere with fermentation and seriously impair the quality of the wine.° THE MicroOrGANnisms FOUND IN WINE Wine microdrganisms may be conveniently divided into two groups: _ those which grow only in the presence of notable supplies of free oxygen, and those which require, or grow better in, the absence of free oxygen. AERopic Orcanisms. Mycoderme.—If a normal wine, especially one strong in alcohol, is left with its surface exposed to the air, it will usually, in a few days, be covered with a whitish film, thin and smooth at first but gradually becoming thicker and finally rough and plicate. This is what is known to wine-makers as “wine flowers.” This film con- sists of yeast-like cells, somewhat longer and more cylindrical than S. ellipsoideus, reproducing by budding and forming large aggregations. Pure cultures show that there are many varieties of this organism differing in the color and texture of the film, in the cloudiness of the liquid and in the character of the deposit. They are called collectively 512 MICROBIOLOGY OF SPECIAL INDUSTRIES Mycoderma vini, though one form which has been found to produce endospores has been called S. anomalus. , These organisms are strongly aerobic and can develop only on the surface in full contact with the air. They are a serious enemy to the wine, rendering it insipid and cloudy. They attack the extract, fixed acids, and alcohol, producing at first volatile acids and finally causing complete combustion of the organic matters to carbon dioxide and water, destroying the wine completely. Acetic Bacteria—The film formed on wines exposed to the air, especially on those of low alcoholic content, will often differ from that due to Mycoderma vini. It will be thinner, smoother and consist of bacteria. These are the vinegar bacteria described on page 539. They grow not only on the wine at the expense of the alcohol, but on crushed grapes and must at the expense of the sugar, producing acetic acid in both cases. Acetic acid in small amounts is produced by the yeast and is a normal constituent of wine. Unless in excess its effect is not injurious. There may be present from 0.12 g. in 100 c.c. in light white wine to 0.14 g. in a heavy red wine without deterioration of quality. In sweet wines, even a somewhat larger amount may be present without causing injury. Much larger amounts are injurious in two ways. When the acetic acid is perceptible to the taste, the wine is spoiled. When an abnormal amount of acetic acid is produced before or during fermentation it stops or interferes with the work of the yeast. In such cases, the wine “sticks,” that is, fails to eliminate all its sugar and becomes especially open to the attacks of other bacteria. Wines high in alcohol are less liable to acetic fermentation than weaker wines. Sound wines containing over 14 per cent by volume of alcohol are almost immune, but such wines may be spoiled during fermentation by the growth of acetic bacteria on the exposed float- ing ‘‘cap” of pomace or on the crushed grapes, especially at high temperatures. ANAEROBIC ORGANISMS (Facultative and Obligate)——Some of the worst, most frequent, and most difficult diseases and defects of wine to treat are due to organisms which develop only in the absence of oxygen. These organisms are all bacteria and appear to include a large number of forms, though, owing to difficulties of isolation and culture, the different forms have not been well studied or described. THE MICROBIOLOGY OF ALCOHOL PRODUCTS 513 Slime-forming Bacteria——Musts and wines become slimy rarely through the action of Dematium pullulans (Wortmann) and wild yeast (Meisner) in the presence of oxygen but more frequently through the action of bacteria. In most cases only young wines after fermentation, and when contained in closed casks or bottles exhibit this defect. A slimy wine has an oily appearance, pours without splashing and in extreme cases, becomes cloudy and will hang from a glass rod in strings. In such wines, the microscope reveals large numbers of almost spherical or more or less elongated bacteria in long chains. Some ob- servers have noticed a diplococcus anda sarcina. Kayser and Manceau % PS eae Ay S SDMA ES oh WT VN 17K one” a ao sa te gt oo ee “> Pe Pc es Fic. 139.—Bacteria of slimy wine. A,B,C, pure cultures of various forms; D, muci- 3 .laginous sheath of slime bacteria. (Afler Kayser and Manceau.) have recently investigated the subject very thoroughly and described a number of forms which are mostly short rods of from tp to 2u by 0.74 to 1.24. One large form, 3 to 4u X 1.6% to 1.7u was also noted. They all form chains, usually of considerable length. They all produce an abundant slimy sheath and stain easily with carbol-fuchsin and other aniline dyes and are Gram-positive (Fig. 139). These bacteria attack the sugar but neither the glycerin nor the alcohol and produce mannit, carbon dioxide, lactic and acetic acids and ethyl alcohol. The disease is usually not serious and disappears under the ordinary cellar treatment. Alcohol above 13 per cent, free tartaric acid, tannin and sulphurous acid in small amounts prevent their growth. ; Propionic and Lactic Bacteria——The most serious and perhaps the commonest disease of wines is characterized by persistent cloudi- ness, disagreeable odors and flavors, increase of volatile acid and injury to the color or its complete destruction. Wines affected are 33 514 MICROBIOLOGY OF SPECIAL INDUSTRIES characterized commonly as mousey, lactic or turned wines (Pousse and Tourne of the French). The disease is due to bacteria. Enormous numbers are readily revealed by the microscope in badly affected wines. There seem to be several or many closely related forms, all short rod-shaped, isolated in the first stages of the disease, but later forming chains or filaments of various lengths. The most noticeable change caused in the com- position of the wine is the decrease of fixed and increase of volatile acidity. The tartaric acid and tartrates are destroyed, and carbonic, acetic, lactic, propionic and other acids formed. Fic. 140.—Bacteria of wine diseases. A, bacteria of “turned wine,” young wine (After Bioletit); B, bacteria of “turned wine,”’ old wine (After Biolettz); C, mannitic bacteria (After Maze and Pacottet); D, bacteria of “bitter wine” (After Pacottet). Light wines of low acidity are most subject to this disease which may be prevented by measures which increase the acidity and alcohol, by rapid and complete defecation and attenuation of the wine with the proper use of sulphurous acid, and finally by timely filtration and pasteurization. Wines noticeably affected can be used only for dis- tilling; those badly affected are valueless. Mannitic Bacteria.—Very sweet grapes of low acidity in hot climates are subject during fermentation to a similar trouble characterized by increase of volatile acids and a persistent cloudiness and a vapid sweet-sour taste. The disease is commonly confused with the preceding but is caused by bacteria of different forms. The form described by Gayon is a very fine short rod which does not unite in filaments. It attacks the sugar, especially the levulose, producing volatile acids and THE MICROBIOLOGY OF ALCOHOL PRODUCTS 515 mannit. The latter may reach over 2 per cent and the former 5 per cent, giving a sweet-sour wine which is completely spoiled. The bacteria grow abundantly only at high temperatures approach- ing 40° and can be controlled by cool fermentation, increase of acidity and proper use of sulphurous acid. Butyric Bacteria.—In the cooler climates, wines, especially old red wines in bottles, often become bitter. This trouble is due to com- paratively large rod-shaped bacteria, first described by Pasteur. The cells remain united in angular filaments, short at first, but be- coming longer and finally thicker with age by incrustations of coloring matter. The tannin, coloring matter, and glycerin of the wine are attacked, acetic and butyric acids being formed. In small amounts the bacteria do little or no harm, in larger amounts they may spoil the wine. Means which increase the alcohol, tannin and acidity diminish the liability to the disease. Prompt attenuation and clarification and in extreme cases pasteurization will cure wines not too badly affected. All the above anaerobic bacteria of wine diseases probably exist in most wines. Which develop most, or whether any develop sufficiently to injure the wine depends on conditions, chiefly the composition of the must and the temperature at which the wine is fermented or stored. Most diseased wines show a mixed infection of several forms. Re-— cently W. V. Cruess has found bacteria in wine containing twenty per cent of alcohol. These bacteria were living and causing cloudi- ness and increasing the volatile acids. CONTROL OF THE MICROORGANISMS Given grapes of suitable composition, the quality of the wine depends on the work of microérganisms. The art of the wine-maker consists almost entirely in the control of these microérganisms. His success in facilitating the work of the useful form (true wine yeast) and in preventing or hindering the work of injurious forms determines the quality of his product. BEFORE FERMENTATION.—On the skins of sound ripe grapes as they hang in the vineyard, the microdrganisms are comparatively few and in an inactive condition. When proper methods are used they cannot injure the wine. On broken or injured grapes, the number is greater f 516 MICROBIOLOGY OF SPECIAL INDUSTRIES and the forms more active. If many such grapes occur they should not be mixed with the sound grapes if the best wine is to be made. Care should be taken to avoid unnecessary bruising of the fruit if it cannot be worked immediately. Molds, wild yeasts and acetic bacteria multiply rapidly on grapes wet with juice. : The sooner the grapes can be crushed and placed in the fermenting vat or pressed the easier it is to obtain a sound fermentation. Cleanliness is essential. Grapes, which are gathered in moldy, vinegar-sour boxes, hauled in dirty wagons or cars, and passed through dirty crushers, conveyors and presses, may be so completely infected with injurious germs that it is impossible to obtain a good fermentation. The most injurious forms of dirt are must, grapes, or wine, which have been allowed to become moldy or vinegar-sour. Dust or soil is less injurious and, if excessive, may often be removed by sprink- ling, especially is this true if the grapes are too sweet and require dilution.* Washing with antiseptics is not permissible. A weak solution of potassium metabisulphite might be used with benefit if it were not for the difficulty of regulating the amount of sulphurous acid entering the fermenting vessel. If the grapes have to be kept for some time before crushing, they should be kept as cool as possible to delay the growth of molds. Gathering in the cool of the morn- ing is desirable and if grapes are gathered when warm they should be left in boxes to cool off during the night whenever possible. If the grapes are cool when they reach the fermenting vat, they will neutralize 4 certain proportion of the heat of fermentation, and the difficulty of avoiding injuriously high temperatures is diminished. However carefully the grapes are handled, a certain amount of dust containing germs and other injurious matters will reach the vats and presses. In the manu- facture of white wines, especially, it is desirable to get rid of these matters before fermentation. ‘This is best accomplished by settling and decantation. As the juice runs from the press, it is pumped into a settling tank or cask. If it is cold, below 15°, and of full normal acidity, the impurities may settle in twenty to forty-eight hours. If the temperature is higher than 15° and the acidity low, molds and yeasts will develop or fermentation will start and prevent settling. A slight sulphuring with the fumes of burning sulphur or with a solution of potassium metabisulphite is therefore usually necessary. The.sulphuring should be as light as possible with acid musts as it tends to preserve the fixed acids. For the same reason it benefits musts of low acidity. In from twelve to twenty-four hours, the must is purged of all its gross impurities including dust, and solid particles de- rived from the skins and the stems and pulp of the grapes. It may be slightly cloudy or nearly clear. It can then be drawn off into clean casks and fermen- tation started with yeast. The microérganisms settle only in part but they are all paralyzed temporarily. This. defecation is of great value, ridding the must of substances that would affect the flavor of the wine in the heat of fermentation and eliminating the excess ‘ * Formerly a decision of the U.S. Dept. of Agriculture forbade the use of the term “pure wine” when water in even the smallest quantities had been used. By the federal law of 1916 dilution with water up to 35 per cent. is allowed. THE MICROBIOLOGY OF ALCOHOL PRODUCTS 517 of protein matters that would serve as food for injurious bacteria. Centrifugal machines have been devised to hasten the process of defecation, but their work is imperfect. Sterilization by heat has been tried for the same purpose but with indifferent success. High heating caramelizes part of the sugar and oxidizes the must, thus injuring the flavor. Discontinuous heating at lower temperatures in an atmosphere of carbon dioxide is preferable but troublesome and expensive. All methods have the defect of extracting undesirable substances from the solid matters which are heated with the must. ' Chemical sterilization is still less practicable. No substance could be used for this purpose except sulphur dioxide; this used in sufficient quantities would seriously injure the flavor of the wine. The effect would be totally different from that of the small quantities used in defecation. All the methods discussed have for their object the diminution or elimination of microérganisms of all kinds. With the injurious forms the true yeast is also re- moved. The more perfect these methods, the more necessary it is to add wine’ yeast. Without this addition, in fact, all these precautions may result in harm, for the wine yeast, being present in much smaller numbers than many of the injurious forms, may be completely removed while enough of other forms are left to spoil the wine. A “starter” of some kind is therefore necessary with defecated must and useful in all other cases. A Starter —One method of producing a starter is to gather a suitable quantity of the cleanest and soundest ripe grapes in the vineyard, crush them carefully and allow them to undergo spontaneous fermentation. Perfectly ripe grapes should be selected and the fermentation allowed to proceed until at least 10 per cent of alcohol is produced. If imperfectly ripened grapes are used or the starter used too soon, the principal yeast present will be S. apiculatus. Toward the end of the fermentation, S. ellipsoideus predominates. From 4 to 12 1. (1 to 3 gallons) of this starter should be used for each 400 I. (100 gallons) of grapes or must to be fermented. Too much starter should not be used in hot weather or with warm grapes, otherwise it may be impossible to control the temperature. This starter is used only for the first vat or cask. Those following are started from the first fermentations, care being taken always to use the must only from a tank at the proper stage of fermentation and to avoid all tanks that show any defect. An improvement on a natural starter of this kind is a pure culture of tested yeast. Such yeasts are being used extensively in most wine-making regions, usually with excellent results. ‘The methods of use would require too ‘much space to describe here, but they are simple and such as could easily be devised by anyone with some knowl- edge of microbiological technic. They do not aim at obtaining an absolutely pure fermentation, which is unnecessary, but endeavor to have an overwhelming pro- portion of a thoroughly tested and suitable yeast which will rapidly and perfectly attenuate the wine before the few injurious microérganisms present have time to do any harm. ? 518 MICROBIOLOGY OF SPECIAL INDUSTRIES DurING FERMENTATION.—However carefully the injurious germs have been excluded and the good yeast increased,.fermentation will not be successful unless conditions as favorable to the latter and unfavorable to the former as possible are maintained. The temperature of the crushed grapes or expressed must is of importance. If it is below 15°, unless the weather is warm, the grapes should be warmed to 20° or 25°. Unless this is done, the molds and S. ‘apiculatus, which require less heat than S. ellipsoideus, will develop more quickly. This is especially true when starters are not used. In the warmer and earlier districts the grapes are practically never too ‘cold. On the other hand, unless there is great carelessness, the grapes are never too warm for the commencement of fermentation. The warmer they are, however, the more artificial cooling will be necessary later, and the sooner it will have to be applied. Thorough crushing is necessary in the case of white wine, to facili- tate the expression of the juice. For red wine, the grapes are also thoroughly crushed and the skin, pulp and juice are fermented together. Imperfectly crushed grapes ferment unevenly and incompletely; thus the growth of mold is much facilitated. The must should be thoroughly saturated with air at the beginning of fermentation to insure the multiplication of the yeast. The aeration received in the processes of stemming, crushing and pressing is usually sufficient for this purpose. More aeration would be harmful by injur- ing the flavor and color of the wine by over-oxidation and promoting the growth of injurious aerobic organisms. An objection to the sterili- zation of must by heat is the expulsion of the air and the difficulty of replacing it in the proper amount. The proper use of sulphurous acid in the regulation of fermentation is one of the most important and necessary but least understood parts of the wine-maker’s art. Only by this proper use can wholesome wine of the highest quality be produced. Improper use will injure or completely spoil the wine. Its beneficial effects are due primarily to its action on microdrganisms, on enzymes and on the color of the wine. In the small quantities properly used in winemaking, it is antiseptic in a degree varying with the amount. All microédrganisms are susceptible to its action in vary- ing degrees. Bacteria are particularly sensitive, molds and psuedo-yeasts less so, while wine yeast is the most resistant of the ordinary forms found in must and wine. The result of the use of the proper amount of sulphurous acid in crushed grapes THE MICROBIOLOGY OF ALCOHOL PRODUCTS 519 and must before fermentation is the almost complete suppression of bacterial action, the discouragement of molds and pseudo-yeasts and the promotion of the growth of wine yeast which is given a clear field unhindered by the deleterious excretions of competitors. Its action as regards enzymes is hardly lessimportant. It would be impossible to make the finest wines of Sauternes and the Rheingau without its use on account of the oxidase produced by the Botrytis cinerea which is abundant and necessary on the best grapes of these regions. In other regions where this mold and others occasionally occur its use is also necessary. In hot climates it is especially useful, not only because bacterial action is more intense in such regions but because of its action in preserving the natural fixed acids of.the grape, which are, there, nearly always deficient. This preservation, according to Wortmann, is due to the sup- pression of acid-consuming bacteria, but experiments of Astruc tend to show that the prevention of the action of unknown acid-destroying enzymes is in part the cause. Its action on the color of wines is also of importance. By the action of oxygen, the color of red wine is gradually made insoluble and precipitated, and the greenish or golden color of white wine is turned to brown. Both these actions are prevented or much diminished by the use of minute quantities of sulphurous acid. The most commonly used source of sulphurous acid is the fumes of burning sulphur. Sulphur is burned in a cask and the must caused to take up the fumes by being pumped into the cask through the upper bung hole. It is almost impracticable to apply sulphurous acid from this source to crushed grapes for red wine. The method is defective in many ways. It is impossible to tell within very wide limits how much sulphur dioxide has been absorbed by the wine. Moreover, the sulphur burns incompletely and the volatilized sulphur acted upon by the yeast may produce sulphuretted hydrogen. Other sulphur compounds are also pro- duced during the burning, to some of which the so-called sulphur taste of wine is said to be due. Several devices have been invented to decrease these defects but none remove them completely; accordingly progressive wine-makers are adopting more reliable sources. An improvement is the use of potassium metabisulphite (K2S.0;) a salt which can be obtained in the requisite purity in commerce containing 50 to 55 per cent by weight of sulphur dioxide. The amount of potash added by this salt in the doses used, is very small, and far within the limits of variation between different wines. By the use of this salt, exact amounts of sulphur dioxide can be applied both to white and red wines. Other sulphites are not permissible. The best source of the acid, recently brought into limited use, is the liquefied gas, which can be manufactured comparatively cheaply in great purity. By its use all the benefits of sulphurous acid are obtained and the defects eliminated. Some grapes, owing to their composition, especially their high acidity, are very resistant to the attacks of injurious bacteria. Others, owing to their low acidity or highly nitrogenous nature, are very susceptible. The addition of tartaric or citric acid to the latter has ‘ 520 MICROBIOLOGY OF SPECIAL INDUSTRIES therefore a deterring effect on some of the most dangerous forms. It is seldom necessary, however, to modify the composition for this pur- pose if the other means of control are used. The addition of acid or its decrease by dilution or neutralization should be solely for the direct improvement of the taste. The quality and character of the wine depends greatly on the tem- perature of fermentation. If too low, the fermentation may be unduly prolonged, the wine yeast may have difficulty in overcoming its com- petitors and the wine may remain inferior and cloudy. With red wine, the desired color, tannin and body may not be secured. On the other hand, if the temperature is too high the results are worse. The growth of bacteria is promoted, injuring the wine by the volatile acid and dis- pleasing flavors produced and preventing the proper action of the yeast. Such wines may remain sweet on account of the failure of the yeast to do its work and become unpleasantly acid owing to the volatile acids produced by the bacteria. ‘Some means of controlling the temperature is therefore always needed. Where heat is deficient it may be supplied by direct heating of the must or part of it, or by heating the cellar. Where the heat is excessive, it may be diminished by crushing only cold grapes, using small fermenting vats to promote radiation and finally by the use of cooling machines applied directly to the fermenting wine. The best temperature for fermentation depends on the kind of wine. For light white wines, the maximum should not exceed 25°, for heavier wines 30°, while for heavy red wines where high extract and tannin are required, it may be allowed to reach 35°. Sound wines can be made at _all these temperatures. As already explained, the ordinary processes of treatment of grapes result in sufficient aeration for the multiplication of the yeast. With grapes containing little sugar, this may suffice to complete fermentation. With sweeter grapes, the fermentation usually slackens when the alcohol reaches 11 or 12 per cent by volume or sooner, unless some supplementary aeration is given. With white wine this is seldom done, with the result that the time of fermentation is prolonged. With red wine, the necessary stirring of the pomace to promote color extraction or the pumping over of the must in the cooling process usually gives a large amount of aeration which is sometimes excessive. Too much aeration results in extremely rapid fermentation and consequent THE MICROBIOLOGY OF ALCOHOL PRODUCTS 521 difficulty in controlling the temperature. It may also have a dele- terious effect on the color, especially if sulphur dioxide has not been used. In any case, the main part of the fermentation should be over in from three to five days in the case of red and in from seven to fourteen days in the case of white wine. With heavy musts, however, there will still remain from 0.5 to 1 or 2 per cent of sugar. With certain special wines such as Sauternes it is desirable to retain the slight sweetness due to this small amount of unfermented sugar. This is accomplished by the judicious use of sulphurous acid, prompt clarification by filtration or fining and when necessary by pasteurization. The pasteurization tends to remove those proteins which are coagulated by heat and which are the preferred food of bacteria. In the case of dry wines, protection from bacteria is best obtained by prompt and complete attenuation. Fermentation should not be al- lowed to cease until all the sugar has disappeared. For this purpose, one, two or more aerations by pumping over are usually necessary im- mediately after the end of the tumultuous fermentation. The tem- perature of the wine should not be allowed to fall sufficiently to check the action of the yeast until all the sugar has disappeared. AFTER FERMENTATION.—As soon as all the sugar has been destroyed in the case of dry wines, or the desired degree of attenuation has been obtained in the case of sweet wines, all the useful work of microérgan- isms has been accomplished. The quality and safety of the wine then depend on freeing it from all organisms present and preventing the entrance and action of all others. As soon as bubbles of carbon dioxide cease to be given off, the yeast and other solid matters will settle to the bottom and the liquid become clear. This often occurs before the fermentation is complete. In this case the yeast should be stimulated by aeration as described above. If the wine is dry, it should be racked (drawn off, decanted) from the sediment into clean casks. The first racking is usually done while the wine is still slightly cloudy during the first month or six weeks to remove the more bulky sediment. If left too long on the yeast the autophagy or degeneration of the latter may produce substances which injure the brightness and flavor of the wine. A second racking is necessary at the end of winter before the spring rise of tem- perature tends to renew the activity of the microérganisms which always remain in the wine. A well made wine at this time should be perfectly bright and all solid 522 MICROBIOLOGY OF SPECIAL INDUSTRIES matters consisting of yeast and bacteria, coagulated proteins and crystals of bi- tartrate should have accumulated in the sediment. Racking should take place when possible only in settled weather, when the baro- metric pressure is high. Low atmospheric pressures diminish the solubility of the carbon doxide with which the wine is saturated. Under these conditions, therefore, bubbles of gas are apt to be given off, bringing up particles of sediment and rendering the wine cloudy. However long wine is kept in wooden casks, it will continue to deposit sediment owing to chemical changes due to the action of oxygen which pene- trates slowly through the wood. Repeated rackings are therefore necessary, oc- curring at least twice a year until the wine is bottled or consumed. Abundant aeration is necessary during fermentation. A moderate supply of oxygen is necessary for the proper aging of wine. Experience has shown that exactly the proper amount of pure filtered air will obtain access to the wine for the latter pur- pose through the wood of ordinary casks of proper size. If the casks are too small the oxidation may be too rapid, if too large the maturing of the wine may be unduly pro- longed. The temperature of the storage cellar is the main modifying factor. The warmer the cellar the larger the casks should be. With sound, completely fermented wines, all aeration, other than that due to the porosity of the wood, should be avoided as much as possible. This is accomplished by keeping the casks tightly bunged and completely filled. Evaporation through the wood continually diminishes the volume of wine and the lack must be supplied by filling up, at first two or three times a month and later every month or two. The drier the air of the cellar, the more frequent the fillings necessary. A light sulphuring of the clean casks into which the wine is racked is usual. This should be practised with great caution. Very little is needed with sound wines, especially if it has been used before or during fermentation and a slight excess will injure the flavor. The amount should not exceed 1.25 g. per hectoliter for red or 2 g. for white wine. One-half to one-third of this is‘sufficient for old wines. The amount can be accurately measured only when using metabisulphite or the liquefied gas. The utility of the sulphur dioxide with perfectly sound wines is to diminish oxidation; with wines liable to disease, to discourage the growth of bacteria. All manipulation of the wine should be conducted with strict attention to cleanliness. This applies especially to empty casks, pumps and hoses. These should be thoroughly cleaned immediately after use and, if of metal or non- absorbent material, kept perfectly dry. Utensils of wood, rubber or other porous material should be preserved from bacterial or mold growth with sulphurous acid. The clarification of a perfectly sound new wine may be facilitated and hastened by thoroughly stirring up the yeast one or two days before racking. The yeast in settling carries down much of the finer suspended matter, thus effecting a rough fining. Materials such as kaolin, pure silica sand, charcoal and filter-paper can be used with the same effect. The fining, however, is never perfect and the flavor of the wine is often injured. A very pure clay, known commercially as Spanish~ clay, is used largely for clearing sweet wines where the flavor is not so delicate. From 75 to 125 mg. per hectoliter are used for this purpose. The best wines are nearly always fined at least once, immediately before bottling. , One or two finings may precede this to hasten aging, defecation and bottle ripeness. THE MICROBIOLOGY OF ALCOHOL PRODUCTS 523 The materials used are soluble gelatinous or albuminous substances which are capable of being coagulated and precipitated by some ingredient of the wine. The best of the commonly used substances are isinglass (ichthyocol) 2 or 3 g. per hecto- liter, for white wines; the white of fresh eggs, 1 or 2 per hectoliter for red; and gelatin, 10 or 12 g. per hectoliter for either. The proper quantity of the finings is dissolved in a little water diluted with wine and stirred into the cask. The tannins and acids of the wine cause a gradual coagula- tion in minute particles throughout the liquid. These particles gradually coalesce, forming larger particles which include all the other floating solid matter of the wine as in a net. These larger particles contracted by the alcohol then settle to the bottom, leaving the wine perfectly bright. The coagulum consists of a combination of the gelatinous matter and the tannin. Some of the latter, therefore, is removed from the wine. With astringent red wines, this may be an improvement. If there is no excess of tannin present, enough must be added to combine with the finings used. With white wines which contain little or no tannin, this addition is always necessary. The amount to use varies with the quality of the finings and of the tannin and with the composition and temperature of the wine. To precipitate commercial gelatin of good quality about an equal quantity of good tannin is necessary; isinglass properly prepared requires only from one-half to one-third this amount. Eggs require only minute quantities. Specially prepared casein of milk is used for fining white wine. Its chief merit is that the acids of the wine alone cause its complete precipitation and no addition of tannin is needed, though a little is sometimes helpful. Many other albuminous substances such as milk, blood and various proprietary preparations are also used, but they are all inferior to the three mentioned and many of them introduce foreign matters such as milk sugar and bacteria which are a source of danger to the wine. Wines containing many disease-producing bacteria may be injured by the intro- duction of finings. The evolution of gases due to the bacterial action may prevent the settling and the protein matters introduced will favor the multiplication of the disease-producing organisms. By the use of 5 to 10 g. of sulphurous acid per hectoliter added to the wine immediately before the addition of the gelatin, the bacteria may be temporarily paralyzed and the finings will then settle and remove the bacteria with the other floating particles. The bright wine should be racked from the finings very soon after the sediment has settled, especially when disease-producing bacteria are numerous. This will be in from ten to twenty days. If the wine is not clear in three weeks it should be filtered. ‘ Filtering is inferior to fining in producing a perfectly bright wine. It is more rapid, however, and is useful in clearing common wine and wines refractory to fining. Filters of innumerable forms are used. They are of two main types. For rough clearing of very cloudy wines some form of bag filter is usually employed in which the wine passes through a cloth tissue. The passage at first is rapid and the filtration imperfect. As the solid matter accumulates on the filtering surface, the filtration im- proves but the passage of the wine is retarded. The first wine is passed a second time 524 MICROBIOLOGY OF SPECIAL INDUSTRIES through the filter and as soon as the rate of filtration becomes too slow, the operation must be stopped and the filtering surface renewed. For wines containing little sediment, the filter must be primed. This is accom- plished by putting some finings in the wine first passed through the filter. The priming is more effective and the output of the filter much increased if a little in- fusorial earth is used with the gelatin. For the more perfect clearing of old wines some form of pulp filter is used. These are various devices by which the wine is forced through a mass of cellulose or as- bestos pulp and freed from all floating matter. Some of the best of these, carefully used, remove nearly all of the bacteria present. BEER Beer is an alcoholic beverage made from certain cereal grains by transformation of the starch to sugar, dilution with water, and fermen- tation with yeast. There is usually an addition of hops and sometimes of materials containing sugar. The liquid before fermentation is called wort. TYPICAL COMPOSITION OF VARIOUS BEERS Lager Ale Porter Weisbier _jomper Water.............. 90.40 88.30 87.30 92.00 Alcohol (by vol.)..... 4.85 8.00 7.00 3-45 2.00 Extracts: s232mex ase 4.20 5.54 6.45 4.63 3-95 Sugars cess avers ws 1.60 1.33 1.83 1.71 1.98 Lactic acid.......... ©.10 °.20 0.22 0.27 0.04 ASD: ieee foie Dau ee 0.23 ©.30 0.40 0.16 Raw MATERIALS AND’ MICROORGANISMS OF BREWING Grains EmMPLovED.—Barley, rice and maize are the grains most commonly used, wheat, rye and oats but rarely. Cane and beet sugars and syrups sometimes form part of the fermentable material. : Yeasts oF BEEr.—The yeast used is usually one of the many forms of S. cerevisig. In some spontaneously fermented beer, other yeasts, Torule and bacteria take part, but in ordinary beers most of these are considered as disease-producing organisms and injurious. Kinps or BeEr.—The principal varieties of beer are: Jager beers, fermented with bottom yeasts; ales, fermented with top yeasts (and Torule); porters, similar to ale but dark in color owing to the use of caramelized malt; weisbiers, in which lactic bacteria are abundant; and certain local ‘types in which bacteria produce con- siderable quantities of lactic and acetic acids. THE MICROBIOLOGY OF ALCOHOL PRODUCTS 525 Process OF BREWING OvutLwwe.—The manufacture of beer takes place in four main stages. First, a portion or all of the grain is soaked in water, allowed to germinate and then dried. This produces the malt which contains the enzymes necessary for the conversion of the starch into sugar and the disintegration of the tissues of the grain. The malt is then crushed (and usually mixed with unmalted cereals or sugar) and heated with water. This constitutes mashing. During this process, the starch changes to maltose and dextrins which with other matters dissolve in the water; then bacteria produce a small amount of lactic acid. The resulting solution con- stitutes the wort. The wort, by the addition of yeast is fermented and changed to beer. The fourth stage includes all manipulation of the fermented beer to prepare it for con- sumption. Mattinc: Propuction or ENzyMES.—The best malt is made from barley, but for special beers may be made from wheat or other grains. Steeping consists in soaking in water to start germination. This requires from thirty-six to seventy-two hours and causes an increase in weight of about 45 per cent. The temperature should be about 12.5°, If higher, injurious molds will develop. If much lower, germination will be retarded. The water should contain little organic matter or chlorides, nitrates or iron salts. A little calcium sulphate is favor- able. If it contains many microérganisms it should be sterilized by boiling. A very little sulphite of lime or of potassium may be used to discourage molds. During germination several enzymes appear, of which the most important to the brewer are diastase which changes insoluble starch into soluble sugar, rendering it available for the growth of the young plant; peptase, which performs a similar function as regards nitrogenous matters; and cytase which helps in the disintegration of the cellulose. All these are necessary to prepare for the work of the yeast. When the plumule has grown to about two-thirds the length of the grain, sufficient enzymes have been formed. This requires from about sixteen to twenty days. The growth of the sprouting seed is at this point stopped by careful drying with artificial heat ina kiln. The kilning must be sufficiently rapid to kill the germinat- "ing seedling quickly, but not too rapid or at too high a temperature, otherwise the enzymes will be weakened or destroyed. The enzymes are more sensitive when moist, consequently the heat may be increased as drying proceeds. The process commencing at a temperature between 30° and 35° is increased gradually to 50° and 55°. In twelve to twenty-four hours, the malt should appear dry. The temperature is again raised gradually for another twelve to twenty-four hours to 80°-100°. The lower the temperature the lighter the color of the malt. Higher temperatures, especially while the malt is moist, produce dark malt. e 7 526 MICROBIOLOGY OF SPECIAL INDUSTRIES As soon as the kilning is finished the radicles are removed by friction and screening in special machines. Work oF ENZYMES AND BACTERIA.—The malt is first crushed by pressing between rollers to facilitate the work of the enzymes and the solvent action of the water. If wnmalted grain is to be used as well, this must be ground and the starch made soluble by heating under prevte with three or four times its weight of water and a little malt to 80°-85° for about an hour. The methods of mashing are very various. They consist in general of mixing the ground malt with warm water, bringing the mass toa temperature of 35° to 45° which is gradually raised to 60°-65° by the addition of hotter water. When the action of the enzymes commences, the heated decoction of unmalted grains is added in va- rious ways, and the temperature controlled by additions of hot water or by heating a portion of the mash. The whole mashing process requires from two to five hours according to the methods used. During the mashing, the starch is transformed partly into maltose and partly into dextrins. The ratio of these products will vary according to the amount of diastase present and especially according to the temperature used. At about 60° the maxi- mum amount of maltose is produced; at higher temperature (65° to 75°) the unfermentable dextrins increase. The amount of alcohol and the amount of extract in the beer therefore depend to a great extent on the method of mashing. During the first part of the mashing, while the temperature is about 45°, lactic bacteria develop. If their action is too intense they will render the beer unpleasantly acid. If moderate, the acidity they com- municate to the wort is useful in preventing the growth of the harmful butyric bacteria which might develop. After mashing, the wort is separated from the solid matters by drawing off, extracting the mash with hot water (sparging), and filtration. It is then boiled‘from one to eight hours according to the result desired. Boiling sterilizes the wort, kills all bacteria and destroys any enzymes which remain. These results are obtained almost instantane- ously owing to the lactic acid present. Coagulation of protein sub- stances is also brought about, effecting a clarification of the wort. This requires one or more hours, according to the nature of the wort. It is necessary also in some cases to concentrate the wort, which is done by prolonged heating in open kettles. This may require several hours. The Hopping of the wort takes place during the boiling. Sometimes the hops are added just at the end of boiling; sometimes in two or three THE MICROBIOLOGY OF ALCOHOL PRODUCTS 527 portions, one of which may be at the beginning and one after boiling. Hops contain an aromatic essential oil, resins and tannin. The essential oil is quickly soluble and volatile. To preserve its aroma in the beer, the hops must not be boiled too long. The resins are anfiseptic and help to preserve the beer. They dissolve with more difficulty and require longer boiling. . FERMENTATION: Work OF YEast.—After boiling, the wort is sepa- rated from the hop débris by straining. It is then cooled by means of refrigerators consisting usually of serpentine tubes through which cold brine or water runs. The hot wort runs or drips over the outside of these tubes in contact with the air. The final temperature of the wort is from 12° to 18° in top fermentation and 4° to 6° in bottom. By this means the wort is thoroughly aerated, which is necessary for the proper work of the yeast. It also effects a partial clarification by oxidation which causes a precipitation of solid matters. The fermentation takes place in two stages, the violent or tumultu- ous fermentation in vats and the secondary or after fermentation in casks. During the violent fermentation, the temperature is allowed to reach a maximum of 7° to 9° with light beers, 8.5° to 10.5° with dark and 12° to 20° in top fermentations. At the end of the first fermentation, the beer is cooled gradually to 3.5° or 5.0° and drawn into fermenting casks where the after-fermentation takes place. The yeasts used in brewing vary very much. Besides the division into top and bottom yeasts, various types of each are recognized. One of the chief characteristics used for this division is expressed by the per- centage of the total extract fermented by the yeast. The Saaz type leaves all the dextrins and some of the maltose untouched and produces beers light in alcohol and high in extract. The Logos type destroys all the maltose and much of the dextrins. The result is high alcohol and low extract. The Frohberg type is intermediate. These differences are probably due to differences in the amount and perhaps in the kinds of enzymes. The yeasts of spontaneously fermenting beers are of various species, S. ellipsoideus, S. pasteurianus and others. To produce fermentation, yeast is taken from previous vats so long as the yeast remains sufficiently uncontaminated with foreign organisms. The condition of the yeast is determined by the character of the fermen- 528 MICROBIOLOGY OF SPECIAL INDUSTRIES tation, the degree of attenuation, and by microscopic examination. In breweries where modern pure culture methods are not used, the yeast present is always of several forms or types. In any case, after a certain number of transfers, the yeast deteriorates and finally may become thoroughly infected with bacteria. The bac- teria are revealed by microscopic examination. Where pure cultures are used, contamination with foreign yeast is shown by a change in the time of spore formation. By this method a contamination of 1:2c0. may be discovered. When the yeast becomes contaminated, a new start must be made with yeast from another brewery, which is uncertain or by a starter of pure yeast, which is the only reliable method. | The new start with pure yeast may be made by employing a kilo- gram of pure pressed yeast or a corresponding amount of liquid yeast and gradually increasing it to the desired amount by repeated small additions of sterile wort. This must be done with special precautions against contamination. Many large breweries use large pure yeast machines which produce directly sufficient yeast to start a fermenting vat. AFTER TREATMENT.—The violent fermentation requires from eight to eighteen days according to the temperature. It takes place in open vats or sometimes, in top fermentation, in barrels. When sufficiently attenuated, the beer is drawn off into large casks where the slow secondary fermentation takes place at a low temperature and the beer clears by depositing yeast and other sediment. The time required for the secondary fermentation is from six to ten weeks or, with certain types of beer, from two to four months or longer. A certain amount of dissolved carbonic acid is necessary for the quality and keeping of the beer. This is obtained by tightly bunging the casks at a suitable stage of the secondary fermentation. The clarification of the beer is sometimes assisted by placing a quantity of chips of beech or other tasteless wood in the casks. Top fermentation beers are often fined by the use of isinglass or animal gelatin. Low fermentation beers are usually filtered. The beer is then ready for delivery to the consumer and is placed in barrels with precautions to retain the dissolved carbonic acid. The clear beer may be put directly into bottles with the same precautions. Bot- tled beers which are to be kept for some time or which are to be shipped to a dis- tance are pasteurized after bottling at 60° to 65°. THE MICROBIOLOGY OF ALCOHOL PRODUCTS 529 DISEASES OF BEER Beer may show defects due to imperfections in the raw material or in the methods of manufacture. These are principally abnormal flavors and lack of clearness. The diseases properly so called are due to wild yeasts or to bacteria. The disease-producing yeasts may be derived from the starter, from the vessels with which the beer comes in contact, or from the air. They develop most commonly during the secondary fermentation or in the bottle. Some may produce a disagreeable bitterness (S. pasteurianus T) or other unpleasant flavor (S. faetidus); many produce a persistent cloudiness (S. ellipsoideus, S. apiculatus, S. exiguus, S. anomalus). They are to be combated by preventing contamination, by proper attenuation and by pasteurizing. Bacterial diseases were more common before effective methods of purifying yeasts were known. s Many forms of lactic bacteria may affect the beer, rendering it acid and cloudy. They occur principally where the temperature is allowed to become too high and where proper care in the cleaning and steril- ization of utensils is not exercised. Acetic bacteria may occur under the same conditions and give a taste of vinegar to the beer. They are more common in top fermented beers. Various forms of Sarcine may cause persistent cloudiness, acid, unpleasant flavors or both. This contamination may be from the air or the water and is relatively common. Their growth is most rapid at 16° to 20° and is retarded by the antiseptic properties of hops. Several kinds of bacteria, bacilli, cocci and sarcine may cause the beer to become slimy or viscid and injure the flavor. This trouble is particularly common in spontaneously fermented beer. Wort and beer, being organic solutions containing very little acidity, are favorable media for the growth of bacteria, many forms of which may cause trouble. With modern methods of using pure yeast, cleanliness and the pasteurization of bottled beer, diseases can be controlled. 34 530 MICROBIOLOGY OF SPECIAL INDUSTRIES MISCELLANEOUS ALCOHOLIC BEVERAGES CIDER AND PERRY These beverages are made by the alcoholic fermentation of the juices of apples and pears respectively and come next to wine and beer in the quantities produced. The composition of the fruit varies very much according to the variety, especially in the matters of acidity, tannin and pectic sub- stances. The following analysis is that of a good cider apple: SURAT je cnc asduon’e Pui sindese ie men aantinin? 4 be sken 167.0 g. per liter. Tannin os ocsdade tanya aeaicanhradenkatin Rag 2.4 g. per liter. Acidity (as sulphuric) ...................0. 0005 1.6 g. per liter. The pectic matters vary from 2 g. to 25 g. per liter but should not be too high. Pears contain usually about the same amount of sugar as apples, more tannin and much less pectic substances. The microérganisms occurring naturally on the surface of the fruit are similar to those occurring on grapes, but special forms of Sac- charomyces are found. Pure cultures of wine yeast are used success- fully in cider making where a perfectly dry cider is wanted. Where a small remnant of unfermented sugar is desired, the difficulties of using pure cultures have not yet been overcome. The wild yeasts occurring -on the fruit in large quantities usually take precedence. Attempts to sterilize the juice by heating have not been successful owing to the production of a persistent cloudiness. Sulphurous acid is even more effective than in grape juice in delaying or preventing the action of the microédrganisms. Its use must therefore always be supplemented by a starter of pure yeast. The principles of the control of the microérganisms, good and bad, are the same as in wine making. The same care in gathering and keeping the fruit and in extracting and handling the juice are necessary. -The fermentation is similar to that of wine, but the cider should be taken off the yeast sooner in order to promote clarification and the retention of a little unfermented sugar. Cider is subject to the same bacterial alterations as wine and requires the same treatment. It is more difficult to keep when made in the ordinary way and is usually consumed during the first year. It is THE MICROBIOLOGY OF ALCOHOL PRODUCTS 531 particularly subject to turning brown, owing to the large amount of oxidase present in apple juice. The use of Sulphurous acid for preliminary defecation, pure yeast in the fermentation, and fining, followed by pasteurization soon after the fermentation, seem to offer the best means of improving present methods. These methods were introduced into a cider-vinegar factory in California by W. V. Cruess with excellent results. FERMENTED BEVERAGES OF VARIOUS FRUITS Many other fruits, especially those rich in sugar and with moderate acidity, are used locally to produce alcoholic beverages. The methods of fermentation are similar to those used in wine making, but additions of sugar and water are usually made to correct defects of composition. Very often distilled alcohol is also added after fermentation to preserve the liquid, which is thus rendered unsuitable for an ordinary beverage. HypDROMEL OR MEAD An alcoholic beverage made by the fermentation of honey and water is much used in eastern Europe. Honey contains from 65 to 74 per cent of reducing sugars and from 2 to 10 per cent of saccharose. It is diluted with water to reduce its concentration to 22° Bal.*-24° Bal. A few yeast cells are usually present in the honey but these are of various kinds and often unsuit- able. The use of a good pure yeast is therefore advisable. As honey contains little mineral or nitrogenous yeast food, an addition of nutritive substances is often necessary. The following formule are recommended by Kayser and Boullanger to be used in one liter: A. Dicalcic phosphate. ............0..0 020-20 eee Ig AMMODIA. focica iawn st ee edeiaeds s258 takes 2 ¢g Bitartrate of potash.................. 20000005 2 g. _ Magnesium sulphate....................0005. O.I g. Be MaltOpéeptone:ncssecccc yee cnaaens le iss wan’ Fe Bitartrate of potash....................000005 I.5¢g. Ammonium phosphate...................2.0. 1.08. * “Balling” refers to the degrees of the special hydrometer for determining the specific | gravity of saccharine solutions such as must or beer wort. Its purpose is to indicate directly the percentage of solids in solution at a temperature of 60°F. 532 MICROBIOLOGY OF SPECIAL INDUSTRIES The same results may be obtained by mixing from 20 to 50 per cent of grape must or apple juice with the diluted honey. MISCELLANEOUS FERMENTED BEVERAGES Fermented beverages of some kind are made in practically every part of the world. They are very numerous and varied but fall natur- ally into three groups; those made from the sweet juices of fruits or’ other plants in which the methods of manufacture resemble those of wine making; those made from starchy materials in which the methods resemble those of brewing; and finally those made from the milk of cows or other mammals which are discussed in Chapter IV, Div. IV. Belonging to the first group are numerous beverages made from the juices of sugar cane, various palms, and tropical fruits. The best known of these is the Mexican PuLquE made by the spontaneous fermentation of the sweet juice of the agave. Little is known about the microflora concerned, but it includes alcohol-forming organisms which produce about 6 per cent of alcohol, and bacteria which cause rapid deterioration and spoiling of the fermented product. The pulque is ready for consumption twenty-fours hours after the commencement of fermentation and cannot be kept more than a day or two. Of the beverages produced fromstarchy materials the Japanese SAKE, RicE BEER, has been most studied. It is made from rice by the diastatic action of Aspergillus oryze and yeast fermentation. The process includes three stages. First the preparation of koji which consists of steamed rice on which the spores of the fungus are sown and allowed to grow at 20° until the whole mass is penetrated with mycelium. The next stage is the preparation of moto which is a thick liquid consisting of steamed rice, water and koji in which the fungus transforms the starch into sugar at 0° to 10° in a few days. Fermen- tation then starts spontaneously, alcohol being produced by the action of several yeasts and lactic acid by bacteria, both present accidentally. In about two weeks the moto is ready. The last stage is the principal fermentation which occurs on mixing together steamed rice, koji, moto and water. This requires two weeks. The liquid is then separated, cleared and stored. It contains a considerable amount of alcohol and and can be kept and aged like wine. Sake is said to average 18 per cent of alcohol and may reach 24 per cent, the highest alcohol content known to be produced by fermentation. = THE MICROBIOLOGY OF ALCOHOL PRODUCTS 533 PomBg is a kind of beer made in Africa from millet seed by sprouting to saccharify the starch and subsequent spontaneous fermentation in water. It is interesting as the source of the genus Schizosaccharomyces which appears to take the main part in the fermentation. _ GINGER BEER is an acid, slightly alcoholic beverage made by the fermentation of a 10 to 20 per cent solution of sugar containing a few pieces of ginger root. The fermentation is induced by adding small pieces, of the so-called ginger-beer plant which consists of Bact. vermi- jorme and S. pyriformis. The bacteria form a thick gelatinous sheath and seem to live symbiotically with the yeast, each developing best in the presence of the other. DISTILLED ALCOHOL INTRODUCTION UsEs AND Sources oF AtcoHoL.—Distilled alcohol is used as a beverage and a medicine or for innumerable purposes in the arts and industries. Certain methods and sources employed for the latter pur- poses are inadmissible for the former. In all cases, it is made by the preparation from saccharine or starchy substances of a sugar solution suitable for the work of yeast, the fermentation of this solution, and, finally, the distillation of the alcoholic liquid. Where the raw materials are sugary, methods similar to those of wine-making, and where starchy, to those of brewing, are employed, modified to suit the conditions of each case. The principal potable alcohols are brandy, made from grapes, rum from sugar cane, and whiskey from rye or other grains. Many other sources are used and any fermented beverage will, by. distillation produce a potable spirit varying in character and quality with the source. Industrial alcohol may be made from any substance capable of undergoing alcoholic fermentation, the limiting factor in practice being, principally, the cost of the raw material per unit of alcohol. METHODS PREPARATION OF THE SUGAR SOLUTION.—Saccharine Raw Materials. —When spirits are to be made from grapes or other fruit, the juice is fer- 534 MICROBIOLOGY OF SPECIAL INDUSTRIES mented in the same way as for the corresponding beverage and then distilled. The juice, however, is diluted to 20° Bal. or less, as it is not necessary or desirable to have too much alcohol in the fermented liquid. The product is consumed directly as brandy or used to fortify sweet wines. The principal fruits used besides grapes are apples, peaches, plums and cherries. Industrial alcohol has been made from inferior or spoiled fruits and from cannery wastes, but the cost per unit of alcohol is usually high. The difficulties of fermentation are great, owing to the presence of large quantities of molds and other injurious organisms, and the extraction of the juice is troublesome. A careful use of sulphites and pure veast much simplifies the process. Sugar cone and its products are used in several ways to produce alcohol. Toa limited extent the juice of the cane is fermented directly and distilled. The product is known as Jamaica rum. Much larger quantities of alcohol are manufactured from the cane-sugar molasses and appear in commerce as rum, taffia, arrack or neutral spirits. : For the making of Jamaica rum the juice is pressed from the crushed canes, and diluted with 20 per cent of vinasses (the residue of a previous distillation) to increase the acidity, and give the required flavor. Cane molasses which contain from 50 to 60 per cent of fermentable sugar are diluted with water or vinasses to15°-18°Bal. and partially neutralized with lime when the acidity is excessive. One of the principal sources of industrial alcohol is the sugar beet. This alcohol is also used for the adulteration or imitation of potable spirits. It may be made by the direct fermentation of the beet juice, extracted by grinding and pressing, by methodical maceration or by diffusion. Sulphuric acid is added during extraction. This facilitates the extraction by setting free organic acids, and represses the growth of injurious microérganisms. The amount used should be such that a minute quantity of sulphuric acid remains free. Most beet alcohol is made from the coarser molasses of the sugar factories. The molasses are diluted to 20°-30° Bal. with water, further diluted and heated with steam and acified with sulphuric acid. The sulphuric acid neutralizes the lime which has been used in the manufacture of the sugar, sets free the volatile acids and breaks up the nitrites producing nitrogen peroxide. The liquid is then boiled for about one quarter of an hour to drive off the volatile acids and the oxides of nitrogen which would prevent yeast fermentation. The liquid after cooling is then fermented with yeast. Starchy Raw Materials —In the preparation of a fermentable solu- tion from starchy materials three methods for the conversion of the starch into sugar may be used, depending respectively on the action of malt, dilute mineral acids, and certain molds. THE MICROBIOLOGY OF ALCOHOL PRODUCTS 535 The malt used in saccharification may be made, in a manner similar to that described for brewing, from barley, oats, rye or maize. As the object in this case is to cause complete conversion of the starch with as little malt as possible, the malt should have the maximum diastatic power. For this reason, germination should be carried further than for brewing and the malt used green. Drying the malt de- stroys half its diastase. The conversion may also be accomplished by boiling one part of grain in four parts of water with hydrochloric or sulphuric acid. With the former acid, 10 per cent of the weight of the grain is used and 5 per cent with the latter. The con- version requires from eight to twelve hours’ boiling. The starch is first converted into dextrinsand theninto glucose. If the boiling is too prolonged some of the glucose may be lost by conversion into caramel. The amount of acid and the time of boil- ing may be much reduced by operating under 2 to 3 kg. pressure. In this case 200 liters of water are heated with 100 kg. of grain and 4 kg. of acid. Conversion occurs in from 40 to 60 minutes. The power of certain molds, especially mucors, to convert starch into sugar has been utilized. Mucor rouxii found in Chinese yeast, Mucor oryze in Ragi, and related forms have been used for this purpose. This is known as the Amylo Process. The grain is first soaked for a few hours, then heated with twice its weight of water under a pressure of three and a half to four atmospheres until soft and the starch rendered soluble. The liquefaction of the starch is facilitated by slightly acidu- lating the water with hydrochloric acid. The mixture is then cooled to 38° and inoculated with a pure culture of the Mucor. A current of filtered air is then passed through the mass for twenty-four hours, by which time the mycelium has permeated the mass. The temperature is then reduced to 33°, pure yeast added and aeration continued for twenty-four hours longer to promote the multiplication of the yeast. Conversion of the starch and fermentation of the sugar then continue together. The mucor is capable of fermenting the sugar and producing alcohol, but the yeast acts more rapidly. The malting process is the most commonly employed. The acid process de- stroys a greater part of the value of the residues of distillation and the amylo process, requiring costly special equipment and large expenditures for fuel, has not come into general use. The starchy substances used being usually neutral or of low acidity the sugar solutions produced would be very liable to bacterial invasion unless means of prevention were used. In the amylo process, the sterilization of the solutions and the use of pure cultures accomplish this end. In the acid process, the minute 536 MICROBIOLOGY OF SPECIAL INDUSTRIES quantity of free mineral acid remaining in the completed solution pre- vents any considerable growth of bacteria. Inthe malting process the injurious bacteria are restrained by lactic acid produced by lactic bacteria, originating in the malt or inthe yeast starter. The requisite bacteria are obtained by keeping the starter or mother yeast .at 50° to 58° for a certain time. This is a favorable temperature for lactic and too high for the development of acetic or other injurious bacteria When the acidity of the solution reaches 3.5 g. to 5 g. per liter the danger- ous butyric bacteria cannot develop. Purelacticacid may beaddedimmediately after saccharification and the loss of sugar, due to the action of the lactic bacteria avoided, but the high cost of the pure acid prevents the practice. Yeast being much less sensitive to the presence of certain antiseptics than bacteria it is possible to control the latter by the addition of suitable amounts of an antiseptic to the sugar solution. In certain cases moreover by gradually increasing the amount, yeast can be accustomed to concentrations of antiseptics which render:the growth of bacteria impossible. In Effront’s method for the preparation of distillation material, hydrofluoric acid is used. This acid is added to the mother yeast at the rate of 10 g. per hectoliter and to the sugar solution in somewhat smaller amounts. This results in the inhibition of lactic, butyric and other bacteria and an increase in the fermentative power of the trained yeast. FERMENTATION.—The sugar solution properly diluted and acetified or sterilized is fermented by the addition of a mother yeast, usually taken from a previous fermentation. The original yeast may be obtained by a spontaneous fermentation as is usual in the manufacture of rum. Such a yeast is always impure, containing various yeasts, molds and bacteria, and is therefore very variable and uncertain in its results. In the fermentation of beet juice and beet molasses, beer yeast of the Frohberg type or special distillers yeasts are used. A starter or mother yeast is prepared for each vat or the process is made continuous by leaving one-third to one-half of the contents of a fermented vat to start a fresh addition of the sugar solution. With the latter method the yeast in time becomes weak and badly contaminated and a new start must be made with fresh yeast. In the fermentation of solutions made from potatoes, corn or other THE MICROBIOLOGY OF ALCOHOL PRODUCTS 537 starchy substances, each vat is started with a mother yeast. The tem- perature should be kept below 30° by means of refrigeration, otherwise alcohol will be lost by the multiplication of bacteria. By the use of pure yeast, the yield in alcohol is greater as no sugar is wasted in the production of lactic acid. The cost, however, is greater owing to the necessity of the use of more heat in sterilization. The fermentation of sugar-cane molasses for the production of arrack is brought about by the use of a mother yeast called tape], prepared from ragi or Java yeast. Tapej is made by mixing powdered ragi with boiled rice. In two days the rice is reduced to a semi-fluid condition and contains bacteria, molds and yeasts. The bacteria seem to have no part in the process but when too numerous are injurious. The mold Mucor oryze converts the rice starch into sugar and the yeast S. vordemanni produces alcohol from the sugar. The other molds present are more or less injurious. CHAPTER VIII* THE MANUFACTURE OF VINEGAR AcETIC FERMENTATION NATURE AND ORIGIN OF VINEGAR.—Vinegar is a condiment made from various surgary or starchy matters by alcoholic and subsequent acetic fermentation. It should contain from 4 to 8 per cent of acetic acid and natural flavoring, coloring and other matters, varying accord- ing to its origin. Acetic acid (CH;COOH) is a monobasic organic acid, the second in the fatty acid series. It is a colorless liquid with a strong suffocating odor, crystalizing when pure at 16.7° and at lower temperatures when diluted with from 1 to 13 per cent of water. Its specific gravity is 1.08 at o° and it boils at 118° under 760 mm. pressure, producing an inflam- mable vapor. It is a solvent of many organic substances and is soluble in water and alcohol in all proportions. The metallic acetates are poisonous and are formed in most cases by simple contact of metal and acid. Certain alloys of tin resist the action of the acid. Acetic acid is formed by the oxidation of ethyl alcohol which takes place in two stages according to the following reactions: C.H,O + O =C.H,O + H.0 Ethyl -+ Oxygen= Acetic + Water. alcoho. aldehyde C:H,0+ O = C.H.02 Acetic + Oxygen= Acetic acid. aldehyde These reactions may be brought about by chemical means, but in practice they are due to the action of certain microérganisms, mainly bacteria. Acetic acid is also made by the distillation of wood but the product is not suitable for consumption. q VINEGAR BActTERIA.—If wine, beer or a similar organic solution con-_ * Prepared by F. T. Bioletti. 538 THE MANUFACTURE OF VINEGAR 539 taining alcohol, is exposed freely to the air, it soon becomes covered with a film, the alcohol disappears, is replaced by acetic acid and the liquid is converted into vinegar. This film, the Mycoderma aceti of Pasteur, consists of bacteria cohering by means of a glutinous sheath surrounding each cell, forming a zooglea. If the film is undisturbed, the liquid remains clear until converted into vinegar, if disturbed, portions may sink, new films form and finally a large gelatinous zoogleic mass, “‘the mother of vinegar,” may form in the liquid. Sometimes, especially on liquids containing sugar and large amounts of alcohol, such as sweet wines, the film formed consists, not of bacteria, but of a yeast-like fungus, Mycoderma vini. Wines which have been sterilized, often remain without acetifying for a considerable time. Those containing free sulphurous acid acetify slowly and with difficulty. Ordinarily at warm temperatures, exposed wines develop a bacterial film very rapidly owing to the almost constant presence of some acetic bacteria in all wines. Hansen was the first to show that the vinegar bacteria included more than one species. He isolated and described three species concerned with the spontaneous souring of beers. Later it was shown by A. J. Brown, Henneberg, and others that several other species commonly occurred in vinegar factories and that many more were capable of pro- ducing acetic acid in small amounts. The species which have been most thoroughly studied and which seem to occur most usually in vinegar factories are Bact. aceti, Bact. pasteurianum, Bact. kiitzingianum, Bact. xylinum, short descriptions of which follow: Bacterium aceti (Kiitzing), Hansen. This speices consists of rods about ry or 2u in length, somewhat constricted in the middle and lying in parallel chains in the surface film. This film is moist, smooth, veined, and forms in about twenty- four hours at 34°. On wort gelatin, it forms gray, waxy, raised colonies which are -usually round, with unbroken edges but somtimes star-shaped and consisting of separate rod-shaped cells. Bacterium pasteurianum, Hansen.—The cells of this species are somewhat larger than those of aceft and more commonly produce thread-like and swollen involution forms. The film is dry and soon becomes wrinkled. Colonies on wort gelatin are smaller than those of acefz, rugose, ‘and the cells retain their arrangement in chains. The mucilaginous sheath is stained blue with iodine-potassium iodide solution (saturated solution of KI colored brown by the addition of a few drops of an alcoholic solution of I), in this differing from Bact. aceti (Fig. 141). 540 MICROBIOLOGY OF SPECIAL INDUSTRIES Bacterium kiitzingianum, Hansen.—The cells resemble those of Bact. aceti, but are usually free or in pairs. The film resembles that of Bact. aceti but has a tend- ency to climb up the sides of the flask above the liquid. The colonies on wort gelatin are smooth and shiny. The mucilage stains blue with the iodine solution. Bacterium xylinum, A. J. Brown.—This species forms a thick tough, leathery film, the gelatinous substance of which stains blue with iodine and sulphuric acid. B. acetigenus, B. oxydans, and B. industrius are motile species. All species are strictly aerobic and grow quickly only when freely supplied with oxygen. This oxygen is necessary for the acetification of the alcohol. Duclaux has calculated that one centigram of the bacterial film is capable of uniting 1.3 g. of oxygen to alcohol, 130 times its own weight. The optimum temperature for most species is about a ee a eo 0.0009 9% Y 9 Or > 0 oO Lat; e220 W227 4 ie c oo Fic. 141.—Vinegar bacteria. A, Bact. aceti; B, Bact. pasteurianum; C, Bact. Pee D, Bact. pasteurianum, showing mucilaginous sheath. (After Hansen. 34° and the range of temperature at which they grow is between 4° and 7° to 42°. They all form acetic acid from ethyl alcohol, propionic acid from propyl alcohol and most of them gluconic acid from dextrose. B. industrius and B. oxydans, according to Henneberg, can form acids from a large number of sugars and related substances, including saccharose, maltose, starch, dextrin, glycerin and mannit. ‘ The presence of too much alcohol prevents the growth of acetic bacteria, the limit being about 14 per cent under manufacturing con- ditions. At 14 per cent and above, the film forms with difficulty, and the oxidation of the alcohol is incomplete, aldehyde and irritating products being formed. Acetic acid in amounts above 10 to 12 per cent is moreover antiseptic to the bacteria. Below 14 per cent of THE MANUFACTURE OF VINEGAR 542 alcohol, the bacteria develop readily and produce in suitable solutions, besides acetic acid, agreeable ethers which are more abundant when the oxidation is slow. Below 1 or 2 per cent of alcohol, the bacteria attack these ethers, and finally the acetic acid itself, causing complete oxidation according to the equation: C.HsO> + 40 = 2CO, + 2H,0. The addition of a new supply of alcohol, however, immediately arrests this reaction. In practice the acetification should be stopped when the alcohol has fallen to 1 or 2 per cent, otherwise there is a loss of flavor and of acetic acid, which may continue until all the acid is destroyed. The length of time during which the acetic bacteria retain their vitality varies with the moisture and the temperature. In nutrient solutions, they live from one to as many as ten years; in the dry state, from three months at ordinary temperatures, to twelve months at 2°. 1 PROCESSES OF MANUFACTURE Raw Materrarts.—Originally vinegar was made from wine, as indicated by the etymology of the word which means “‘acetified wine.” Later, other alcoholic beverages such as cider and beer were used for the same purpose. In these liquids, the acetic bacteria find all the mineral and organic matters necessary for their development, together with alcohol in amounts favorable for acetic fermentation. At present, a large number of materials containing alcohol, or starchy and sugary matters, which, by preliminary yeast fermentation, can be changed into alcohol are used as sources of vinegar. The most important of these are honey, malt, and various fruit juices. All these materials make wholesome vinegar of varying degrees of quality. Those of wine and cider are usually classed as the best, and those of malt and honey next. The great bulk of the vinegar of com- merce, however, at present is made by the acetification of distilled grain, potato and molasses alcohol. This is not vinegar strictly speak- ing but an imitation, consisting of a dilute solution of acetic acid with- out the various flavors which are an essential part of pure vinegar. In order to give it a semblance of the latter, it is often colored with caramel and flavored with various substances. 542 MICROBIOLOGY OF SPECIAL INDUSTRIES Other imitations of vinegar sometimes appear on the market, con- taining wood vinegar, or even mineral acids. These, however, are more or less poisonous and their sale, as food, is usually forbidden by law. FERMENTATION.—If the raw material to be used is starchy or sugary, it must be first changed into an alcoholic liquid containing from 6 to 12 per cent of alcohol by volume. This is accomplished by one of the methods discussed in the preceding chapter. This alcoholic fermentation must be kept rigidly distinct from the acetification and is best carried out in a separate building. The yeast must finish its work before the bacteria commence theirs. The reason for this is that yeasts are very sensitive to acetic acid and a small quantity may paralyze their activity and prevent the change of all the sugar into alcohol, with a consequent loss of strength and quality in the final product. The quality of the vinegar will depend on the quality of the raw material from which it is made. Wine or cider spoiled by bacterial fermentation, moldy casks, etc., will make inferior vinegar. An exception to this may be made of so-called “pricked”’ wines, which are simply wines in which acetic fermentation has started spontaneously. The wine or other alcoholic liquid should be perfectly clear and clean tasting and, if necessary, should be fined, filtered or pasteurized im- mediately before use. It should contain no antiseptic which would interfere with the development of the acetic bacteria. Sulphurous acid, if present in the free state, should be removed or oxidized by thorough aeration. Commerical alcohols made from corn, potatoes, beets, molasses and other products can be used. The special flavors of these alcohols, due to their origin, disappear almost completely in the vinegar. This, however, is not true of denatured alcohol or that containing methyl alcohol or acetone. The alcohol must be diluted to from ro to 12 per cent by volume, and then made suitable for the growth of acetic bacteria by the addition of nutritive substances containing nitrogen and phosphates. This is ac- complished usually by adding 10 per cent of wine, beer, malt-extract, yeast decoction, or similar material to the diluted alcohol. The waste liquids from a brandy distillery may be used instead of water for dilution. After resting a few days, the mixture is filtered and is then ready for acetification. THE MANUFACTURE OF VINEGAR 543 Before starting the acetic fermentation, it is a usual and good practice to add about ro per cent of good vinegar to the liquid, which is thus rendered acid and therefore less liable to alteration by injurious bacteria and other microdrganisms. All the processes of vinegar-making depend on the same principle, which is to expose the liquids prepared as above to the action of acetic bacteria with full access of atmospheric oxygen at a suitable tem- perature. The rapidity of the process depends on the number of! active bacteria present, the nutritive value of the liquid, the tem- perature, and especially on the free access of oxygen. STARTERS AND PurE Cuttures.—The 10 per cent of vinegar added to the liquid to be fermented usually contains sufficient bacteria to in- sure a prompt start. Where this is not the case, a starter may be prepared by exposing a suitable liquid in a shallow vessel to the air of a warm room for several days. Any liquid containing about 4 per cent of alcohol, 2 per cent of acetic acid and a moderate amount of nitrogenous matter is suitable. A decoction made by boiling 50 g. of fresh yeast in 1,000 c.c. of water, filtering and adding the proper amount of vinegar and wine or beer will serve. After thorough aeration, such a liquid in a few days becomes covered with a film of acetic bacteria. This film may be used asa starter by gently submerg- ing the vessel in which it is formed in the liquid to be acetified, or by removing with a clean sliver of wood which is afterward floated in the liquid. In practice, such a starter gives a sufficiently pure fermentation of ~ acetic bacteria. The particular species of acetic bacteria, however, is left to chance. Pure cultures of a particular selected form would in all probability improve the certainty of the production of good vinegar, but the method has not entered into general practice. AppaRATUS.—Most metals of all kinds should be avoided as much as possible. The hoops of barrels and buckets may be protected by a coating of paraffin. Pumps may be of wood or of the special alloys al- ready mentioned, or they may be so constructed that they will not come in contact with the liquids. Domestic MetHop.—A cask of convenient size (40 to 200 liters) is fitted as illustrated in Fig. 142. The wine or cider to be acetified, after filtering, if necessary, is poured into the cask until it is about one-half to two-thirds full, the i 544 MICROBIOLOGY OF SPECIAL INDUSTRIES object being to have as large a surface as possible for the growth of the bacterial film. Free circulation of air is insured by a 5-cm. hole in each head of the cask, one near the surface of the liquid and one near the top of the cask. These holes should be covered with varnished metal netting to prevent the entrance of flies. The top bung hole is then closed with a cork, through which a funnel passes, furnished at its lower end with a glass tube extending to within a few inches of the bottom of the cask. By means of this funnel new liquid can be added without disturbing the surface film. The lower bung-hole is closed with a cork, through which passes an L-shaped glass tube which serves as an indicator of level and which — also can be used to draw off the vinegar. Fic. 142.—Vinegar barrel. L, surface of liquid; O, O, openings for entrance of air; ¢, tube for introducing new supplies of wine without distrubing surface’ films; E, glass tube to show level of liquid and for drawing off vinegar. (Original.) When this apparatus is working well, from one-fifth to one-quarter of the contents may be taken off every three or four weeks. This depends on the temperature, which should be between 10° and 18°. The vinegar drawn off is immediately replaced with wine or cider which, if added slowly, will, owing to its lower specific gravity, remain at the surface in contact with the bacterial film. ; OritEans MetHop.—This is practically the same as the method just described with slight modifications to adapt it to large scale opera- sl THE MANUFACTURE OF VINEGAR 545 tions. It is the oldest commercial method and produces vinegar of the highest quality. Barrels of about two hectoliters are usually employed, fitted essentially like that already described but with the omisson of the funnel and drawing-off tubes. The wine is first cleared in a vinegar filter. This consists of a wooden vat filled with beech chips which have been extracted by soak- ing for several days in cold water. The wine remaining in contact with these chips for three or four days deposits most of its sediment. The cask is first one-third filled with good vinegar and to or 15 1. of the filtered wine added. The same amount of wine is added every week for four weeks by which time the cask is half full. At the end of the fifth week an amount of vinegar equal to the wine added is drawn off and the operation repeated. The vinegar is filtered as soon as it is drawn off, placed in full tightly bunged casks and kept in a cool cellar. PastEuR MetHop.—Pasteur long ago pointed out the defects of the old Orleans method and suggested improvements. The main defects of the old method are that it is cumbersome, laborious, slow and costly. There is a loss of about ro per cent of material by evaporation and the repeated additions of liquid break the bacterial film, which then sinks to the bottom, grows anaerobically and exhausts the nutrients of the solution without producing acetic acid. . These submerged bacteria finally form a large gelatinous mass which interferes with the regular progress of the operations, depreciates the quality and necessitates frequent expensive cleanings of thecasks. Many attempts, more or less successful, to overcome these defects in accordance with Pasteur’s ideas have been made, that of Claudon is one of the best and will serve to exemplify all. It consists essentially of a wide, shallow, covered square vat, furnished with numerous openings near the top by which the entrance of air can be facilitated and regulated. This vat is filled to the bottom of the air vents with a mixture of four parts of good new vinegar and six parts of wine which has been pasteurized at 55° and, when necessary, filtered. On top of this liquid is floated a light wooden grating which helps to support the bacterial film and prevent its breaking and sub- merging during the various operations. When filled, the process is started by placing a small quantity of a good bacterial film on top of 35 546 MICROBIOLOGY OF SPECIAL INDUSTRIES ‘the liquid which soon becomes completely covered when the proper conditions of temperature and aeration are maintained. Each acetifying vat is connected with a small measuring vat from which the proper amount of liquid is added every day after a correspond- ing amount of vinegar has been removed. These two vats constitute a unit, several of which, usually six, are united in a battery. A factory includes several of these batteries. The batteries are fed from a large vat or reservoir, where the mixture of wine and vinegar is prepared and stored. The vinegar drawn from the batteries runs directly to filters, thence to a pasteurizer, and finally to the storage casks. The output of these batteries is from two to five times as great per square meter of acetifying surface as that of the old method; the cost of the operation is considerably less, the loss by evaporation much reduced and the quality equal and much more under control of the manufacturer. GErman Metuop.—In all the methods described, the surface of the liquid exposed to air, where alone acetification occurs, is small compared to the volume of the liquid. In order to hasten and therefore cheapen the process, various devices for increasing the surface in contact with air have been devised. The simplest of these is one sometimes em- ployed in wine-making countries. The pressed pomace of red wine is broken up and placed loosely but uniformly in a tall narrow vat. Ina few days, acetic fermentation commences in all parts of the mass. Wine is then sprinkled periodically on top and trickling down over the pomace, it is changed to vinegar by the bacterial film which encases every particle of the mass. The ‘“‘quick” or German method of vinegar- making is based on this principle. The apparatus used in this method consists of a tall cylindrical or slightly conical wooden vat provided with a perforated false head a few inches from the bottom and another, similar in structure, at the same distance from the top. The space between these two false heads is filled with long thin chips or shavings of beech wood which have been thor- oughly extracted, first with water and then with good strong vinegar (Fig. 143). Various substitutes for beech chips have been used with more or less success. Rattan shavings and wastes are suitable; dried corn-cobs can be used but are not durable; wood charcoal in lumps is used successfully in the manufacture of alcohol vinegar. THE MANUFACTURE OF VINEGAR 547 fe la ; fe boy a Spocoaoodeocedq, Onttsceoseecececoe oe cence O Za | fe] Be : Be a] ey Fic. 143.—Rapid process.inegar apparatus. V, mass of beech chips over which the alcoholic liquids run from H; H, false head with numerous small holes and threads for the slow and equal distribution of the liquid; £, tilting trough for the intermittent supply of liquid; 0, openings for the entrance and exit of air. path of air. 1 path of liquid. (Original.) 548 MICROBIOLOGY OF SPECIAL INDUSTRIES In operation, the liquid to be acetified is distributed over the top false head intermittently in small amounts. This intermittence of supply is accomplished by various automatic devices. If the supply is continuous, the liquid tends to run in streams or currents in certain parts of the vat and much of the acetifying surface is lost; if too rapid, the bacterial film is removed from the upper part of the mass of beech chips and only the lower part is effective. From the false head, the liquid passes through numerous small holes’ to the mass of beech chips, over which it trickles slowly and is acetified by means of the bacterial film which covers them. By the time it reaches the lower false head, the alcohol is in greater or less amount converted into acetic acid. Usually the liquid must pass through from two to five times or through an equal number of vats before it is com- pletely changed into vinegar. The number of passages depends on the amount of alcohol present, the height of the acetifying column, the rapidity of the flow, the temperature, and on the perfection of the apparatus. Oxygen is supplied by the air which, entering holes in the vat below the lower false head, passes through numerous holes in the latter, through the interstices between the chips and out through short tubes fixed in the upper false head and holes in the top. The passage of air is insured by the heating of the interior due to the fermentation. It can be regulated by the number and diameter of the air holes. The temperature, which should be close to 30°, must be carefully regulated. If the temperature rises too high, the loss by evaporation will be much increased; if it remains too low the acetification will be retarded. Many modifications of this method exist, having principally for their objects the more complete regulation of the temperature and air supply, the recuperation of the volatile matters, and the avoidance of the need of repassing the liquid through different acetifying columns. Rotatinc BARRELS.—Several methods are in usé which attempt to combine the rapidity of the German machines with the quality of the Orleans method and which are suitable for use with wine and cider. These liquids cannot be acetified conveniently by the German method on account of the large amounts of solids and extractive matter they contain. This coats the beech chips rapidly and interferes with the perfect working of the machine. THE MANUFACTURE OF VINEGAR 549 These methods make use of a barrel filled partially or wholly with beech chips and half filled with the liquid to be acetified. By rotating the barrel at short intervals the liquid trickles repeatedly over the chips and, with proper aeration, the acetification is rapid and complete. AFTER-TREATMENT.—Alcohol vinegars require little treatment. They should be filtered and are usually colored slightly with caramel. Being little more than dilute solutions of acetic acid without ethers or bouquet, there is no object in aging them. Wine and cider vinegars, for the best results, require aging and careful treatment. They should be filtered and pasteurized as soon as made and stored in clean casks which are well bunged and kept con- stantly full in a cool place of even temperature. If too dark in color, they may be decolorized with pure animal charcoal carefully extracted with acids and water. Before using or bottling, the vinegar should be fined with isinglass (see page 523). DISEASES The most troublesome pest of vinegar factories is a minute nema- tode, the Anguillula aceti or vinegar eel. It often develops around the edges of the surface of the liquid in vinegar barrels and in the acetify- ing columns and, if neglected, may cause putrefaction and spoiling of the vinegar. Frequent and thorough cleaning of all apparatus, pasteur- ization of liquids and light sulphuring of empty casks will prevent its development. The vinegar eels are easily killed by heating the vinegar to 50°. They may be removed by filtration or fining. Microscopic mites are sometimes troublesome in neglected factories. They can be reduced by the methods recommended for vinegar eels and their entrance into the barrels or acetifying columns prevented by painting a ring of turpentine or some viscid substance around each air hole. Vinegar flies (Drosophylla cellaris) are often troublesome, but can be excluded by proper screening of buildings and barrels. Bacteria other than acetic may develop in vinegar and some of them may depreciate its quality. These have been little studied but the most harmful seem to be anaerobic forms which develop in the lower parts of the liquid protected from oxygen by the screening film 550 MICROBIOLOGY OF SPECIAL INDUSTRIES i of the acetic bacteria. They produce butyric acid and putrid odors and, if neglected, may completely spoil the vinegar. Sulphuring, fining, and pasteurization are the remedies. Darkening or persistent cloudiness may be caused by oxidase as in wine and cider and is controlled in the same way. A similar defect may be caused by the tannic extractive matters of new casks or contact with iron. Aeration followed by fining will remove the cause of the trouble. CHAPTER IX* THE MANUFACTURE OF OTHER FERMENTED PRODUCTS PREPARATION AND CONSERVATION OF Foop MATERIAL ComprESSED YEAST.—Yeast in the form of a thick paste is produced in large quantities for the use of bakers. It should be white, vigorous and rich in nitrogen. In the Vienna method, a saccharine solution is made in a manner similar to that used in distilling, by saccharification with malt and acidi- fication by lactic bacteria. Many grains, principally barley—malt, rye and corn, are used. A mixture of the three gives a solution which has the required concentration and the proper degree of viscosity to facilitate the separation of the yeast. In the method by aeration a sweet wort is made from green malt, _ that is, sprouted barley. The malt is crushed, mixed with water and “mashed” for several hours at about 60° to saccharify the starch by diastatic action. The mash is then cooled to 50° and inoculated with a culture of lactic acid bacteria. When the liquid is sufficiently acidified by the action of the bacteria it is sterilized by heating to boiling. It is then filtered and cooled. This sweet wort should contain about 14 per cent of total solids. The cooled wort is inoculated or “pitched” with a large starter of veast and thoroughly aerated during fermentation by means of com- pressed air. Yeast growth and fermentation are complete in twenty- four hours or less. The yeast is separated from most of the liquid by means of a centrifugal machine and is then made drier by means of a filter press. It then forms a paste that can be molded into any desired shape or size. The liquid or “wash” separated from the yeast is distilled and the alcohol used for the manufacture of distilled vinegar. A small number of lactic acid bacteria in the finished product is * Prepared by F. T. Bioletti. 551 552 MICROBIOLOGY OF SPECIAL INDUSTRIES considered desirable. The yeast is sometimes spoiled by bacteria ‘causing sliminess or producing butyric acid, but this does not, occur when the process is properly carried out. BreaD.—The rising of the dough to which the lightness and porosity of bread are due is caused by the production of carbon dioxide by yeast fermentation. The yeasts are always accompanied by bacteria and the character of the bread is determined in great part by the extent of bacterial fermentation. If we make a dough of flour and water and allow it to stand in a warm place it will rise slowly. Yeasts and bacteria, occurring naturally in the flour and water, are the causes. Bread is sometimes made in this way (Graham bread, salt-rising bread). The rising is more or less uncertain and the flavor and acidity of the bread very variable, owing to differences in the kind and degree of bacterial action. Many yeasts and a large number of bacteria have been isolated from the spontaneous fermentation of dough. Among the bacteria are forms producing lactic and acetic acids, others which dissolve gluten and transform starch into sugar and others which produce alcoholic fermentation with evolution of carbon dioxide. Usually the dough is leavened by incorporating more or less im- pure yeast. Bread yeast may be prepared by allowing a culture medium composed of water, sugar, hops and potatoes with a little salt to ferment spontaneously, but the results are uncertain. Usually, in the United States, compressed yeast is employed. In some cases the yeast from breweries is used. In most parts of Europe a leaven made from a piece of dough kept over from the last baking is preferred. ; In a general way, the process consists in making a thick dough or thinner sponge by thoroughly mixing the flour with water, yeast or leaven and a little salt. This mixture is then allowed to stand in a warm place (20°-30°) to promote the growth and multiplication of the microdrganisms. It is then kneaded, usually with more flour and put aside to rise. This kneading with fresh flour and rising may be repeated several times. If a large quantity of a relatively pure yeast is used, the rising will be rapid, there will be little bacterial action and only one kneading is neces- sary. This is the method commonly employed in the bakeries in the United States. Bread made in this way is usually of fine grain, white THE MANUFACTURE OF OTHER FERMENTED PRODUCTS 553 and flavorless. It dries out very rapidly and is palatable only when very fresh. In the common household method, a smaller amount of yeast is mixed in a thin batter or sponge and allowed to multiply for ten to eighteen hours. This batter is then thickened to a stiff dough and allowed to rise until it doubles its volume. A second kneading is then given and a little more flour added. After rising again for a few hours it is baked. Bread made in this way is usually somewhat more open in texture, not so white and with more flavor. It dries less rapidly and remains palatable for two or three days. The difference seems to be due prin- cipally to the action of bacteria on the gluten and other nitrogenous sub- stances in the latter case. The bacterial action, however, is not sufficient to give a perceptible acidity. Where leaven made from old dough is used, as in most parts of Southern Europe, the part of bacteria in the fermentation is much greater. The bread has a thicker and firmer crust, a fuller flavor and a distinct acidity which is often excessive. It holds its moisture well and keeps for a week or longer. The making of so-called French bread, as it is carried out in Paris, is a successful attempt to combine the good qualities of the above ex- tremes and to avoid their defects. It is based on the fact that where a leaven consisting of a mixture of yeast and bacteria is used, the yeast develops more rapidly at the beginning and the bacteria at the end. By successive additions of flour and aeration by repeated kneading, sufficient yeast growth is obtained to make the bread light and the bacteria kept within the limits necessary for flavor and keeping quali- ties without causing undue acidity. YEAST AS Foop.—Enormous quantities of yeast are produced in the various fermentation industries and thrown away as a waste prod- uct after use. It has been calculated that over 60,000 tons of yeast are produced by the breweries of Germany alone and nearly twice as much by the distilleries. Recent tests have shown that this yeast has a highly nutritive composition and can be used as a profitable nitrogenous ration for all farm animals. By careful methods, a human food can be prepared from it which is appetizing and nearly three times as nutritious as beef. 554 MICROBIOLOGY OF SPECIAL INDUSTRIES Large quantities have always been used as food unwittingly, either for animals, which consume it in certain brewery wastes of which it constitutes a large portion, or by human beings who consume it in their bread. It has been estimated that the bread consumed in Germany in a year contains about 147,000 tons of yeast. Most of the yeast produced by the fermentation industries, how- ever, is wasted owing to the difficulties of transportation and con- servation. Recently methods of drying waste yeast have been de- vised which overcome these difficulties and it promises to become a valuable form of cattle food. VEGETABLES.—Various vegetables, cabbage (sauerkraut), string beans, cucumbers, etc., can be preserved by covering them with weak brine and allowing them to undergo spontaneous fermentation out of contact with the air. The vegetables are cleaned, cut into pieces of convenient size, mixed with x to 3 per cent of salt and tightly packed in a fermentation vessel of wood, earthenware or cement. A perforated cover is placed on top and weighted down with stones. The vegetable juices are forced out by the combined action of the salt and pressure and the solid matter re- duced in volume one-third or one-half. A gaseous fermentation commences within twenty-four hours if the temperature is favorable, 18° to 20°, and continues for several weeks. At the end of this time the sugar in the juices has been destroyed and acids, principally lactic, produced to the extent of 0.5 to 1.0 per cent. The liquid is then drawn off and replaced with 4 to 8 per cent of brine in which the vegetables will keep in good condition for a long time if kept from the air. The fermentation is due to a large number of microérganisms originating on the surface of the vegetables and in the water. The yeasts attack the sugar and exhaust the oxygen. The lactic bacteria at the same time produce lactic acid. This is the principal fermentation and produces the acidity to which the conservation of the mass is due. Many other substances are formed by the complex fermentation, the principal products being alcohol, succinic acid, volatile acids, mannit, amid-bodies, carbon dioxide, hydrogen, methane and various aromatic esters. Weiss has isolated 65 different species of bacteria from sauerkraut. Most of these are probably indifferent or harmless, and some harmful. THE MANUFACTURE OF OTHER FERMENTED PRODUCTS 555 When the process is successful the lactic bacteria multiply rapidly from the first and quickly produce enough acidity to restrain growth of the harmful kinds, among the worst of which are the butyric bacteria. StaRcH.—Starch is prepared from potatoes, corn, wheat, flour and other amylaceous substances. The present method of separation is by chemical means. Formerly it was accomplished by a complex fermentation. For the fermentation method, the grain is soaked in water until soft, then ground and made into a paste which is allowed to ferment sponta- neously or started with a leaven taken from a previous fermentation. Alcoholic, lactic and butyric microérganisms attack the sugar while others attack the gluten and cellulose. The fermentation lasts from twelve to twenty-five days according to the temperature and the resistance of the raw materials. During fermentation, lactic and butyric acid, hydrogen sulphide, ammonia and carbon dioxide with traces of alcohol and acetic acid are produced. The process is stopped as soon as gas ceases to be given off and before putrid fermentation sets in. The starch which is set free settles to the bottom and is separated by decantation, washing and screening. The washed starch is then allowed to settle for three or four days in water. The sediment that is formed consists of three layers, the top consisting principally of gluten, the second of gluten and starch and the bottom of comparatively pure starch. The layers are separated and the starch extracted from the two upper layers by repeated washings on inclined planes. The starch, owing to its higher specific gravity, re- mains near the lower parts of. these planes. Sucar.—In the manufacture of sugar, microérganisms have no useful part but many forms may be injurious and cause serious losses. The juices of beets and sugar cane and the saccharine liquids obtained by presses or diffusion batteries form excellent media for the multi- plication of many Saccharomyces and bacteria. They are controlled by cleanliness, rapidity of handling, and sterilization by heat. They are injurious by destroying sugar and thereby diminishing the yield, by inverting a portion of the saccharose and rendering the crystal- lization difficult and by forming gelatinous masses in the liquids. Many of them are very resistant to heat. S. zopfii withstands a temperature of 66° for half an hour. Streptococcus mesenterioides forms 556 MICROBIOLOGY OF SPECIAL INDUSTRIES chains of cocci surrounded by voluminous gelatinous sheaths which unite in zoogleic aggregations sometimes very troublesome in sugar factories. On account of its sheath it is very resistant to adverse conditions. It retains its vitality after. drying for three and a half years. It is not killed by heating to 86° for five minutes and occurs in: the hot liquids of the diffusion batteries. ‘Topacco.—To prepare tobacco leaves for the use of the smoker they must be dried, fermented and aged. Unless all these processes are properly carried out the tobacco does not possess the desired aroma, it contains protein substances that give a bad odor to the smoke, and is usually too rich in nicotin. The processes of preparation are often referred to as “curing,”’ though this term is generally restricted to the preliminary drying. The curing is more than a simple drying, as various important changes of composition take place. When young leaves are removed from the stalk and dried they lose from 12-15 per cent of their dry weight, when dried on the stalk the loss is greater; from 14-18 per cent. Riper leaves lose more, up to 35 or 40 per cent. This loss is due to _ transpiration and to translocation of substances from the leaves to the stalks and is caused by diastatic, proteolytic and other enzymes. The principal changes are the disappearance of starch and reducing sugar; a decrease in proteins, nicotin, pentosans and malic acid and an increase in citric acid. Ammonia is formed and the changes after curing are more rapid with rising temperature up to about 130°F.* After curing the dried leaves are piled in masses, moistened and allowed to undergo a fermentation which raises the temperature to 50°-55°. Sometimes the leaves are then sprinkled with a solution containing sugar, honey, various aromatic substances and sometimes alcohol and passed through another fermentation. The leaves are then tied up in bundles, partially dried, and pressed into boxes where another slow fermentation often takes place. The principal chemical changes which take place in this “curing” of tobacco are a considerable diminution of the nicotin, the destruction of nitrates and the production of ammonia and sometimes of butyric acid. There are three theories as to the cause of these changes. Accord- * Garner, Bacon, and Foubert, Bull. 79, U. S. Dept. of Agr., 1914. THE MANUFACTURE OF OTHER FERMENTED PRODUCTS 557 ing to Suchsland they are due to bacterial activities and by the use of pure cultures he claims to have much improved ordinary tobacco. According to Nessler and Schléssing, the bacteria are useful only in raising the temperature of the mass which is thus made more subject to the action of atmospheric oxygen. This is the immediate cause of the chemical changes. The third theory is that of Loew who ascribes the changes to the action of enzymes, oxidases, peroxidases and catalases existing in the leaves of the tobacco. It seems probable, according to many investigators, that the changes are due in the first place to hydrolyzing, proteolytic and oxidizing enzymes, and that these diastatic transformations are supplemented by the bacteria which destroy nitrates and produce ammonia; variations in these various factors account for variations in the characteristics of tobacco. PREPARATION AND CONSERVATION OF MISCELLANEOUS PRODUCTS Inpico.—This dye was formerly made only from certain species of Indigofera, principally I. tinctoria. This plant contains a glucoside, indican, which by fermentation and oxidation yields indigo. The plants are placed in water at a temperature of 25° to 35° and undergo a spontaneous alkaline fermentation which splits up the indican into a sugar (indiglucin) and indigo white which remain in the solution. This solution is then thoroughly aerated and the indigo white oxidized into indigo blue which is insoluble and forms a sediment. This sedi- ment is dried and constitutes the old indigo of commerce. . Many bacteria are found in the fermenting liquid, but the cause of transformation has been shown to be a specific form, Bacillus indigo- genus, Closely related to Friedlander’s pneumonia bacillus. It is strongly aerobic and surrounded by a gelatinous envelope. Rettinc.—The separation of the fibers of flax, hemp, ramie and similar plants is brought about by a complex spontaneous fermentation. The plants are either left on the surface of grassy meadows exposed to alternate wetting and drying or immersed in water. In either case, the tissues are gradually disintegrated by microbial action, more rapidly in the wet process. The fermentation, principally bacterial, is due to many species. Several have been described as being the principal agent in the process 558 MICROBIOLOGY OF SPECIAL INDUSTRIES © but it is probable that the effects are due to the united action of several, both aerobic and anaerobic. Among the forms to which the retting has been attributed are B. amylobacter of van Tieghem, an anaerobic form which attacks the pectic matters and to some extent the cellulose. Granulobacter pectinovorum of Beyerinck and van Delden, also anaerobic, transforms the pectic matters into sugars which it deconiposes, producing butyric acid. Many other forms have been described and part of the work has been ascribed to Mucor, Penicillium and various molds. Cultures of Granulobacter pectinovorum and other forms have been successfully used to hasten the process. _ Tanninc.—In the manufacture of leather the hides are received salted or dried, or more usually, fresh from the slaughter house— “‘green hides.” The green hides are thrown immediately into a pit containing a lime solution; the salted and dried are first soaked in water before being placed in the lime pits. The combined action of the lime and bacteria loosens the hair. The liquids in old pits contain large numbers of bacteria and are much more effective than new lime solutions. On removal from the lime pits the hair is slipped from the hides by hand scrappers. Wool is removed from sheep skins after allowing them to undergo incipient putrefaction in an air-tight room. Light hides and skins after removal of the hair are placed in a soak- ing pit for a few hours to remove some of the excess of lime. They are then placed in the bating wheel which is a wooden tank furnished with a large, revolving wooden wheel which keeps the hides in motion. The liquor in the bating wheel contains pigeon, chicken or dog excre- ments and is kept at about 40°. Rapid bacterial action takes place and the hides “fall”? or become soft and most of the lime goes into solution. If left too long in the bating liquor the hides lose weight by destruction of their proteins and may finally be completely dissolved. For this reason the bating process must be closely watched and stopped as soon as the hides are well softened. Heavy hides for sole and harness leather do not go through the bating wheel, but are simply soaked and agitated in water to remove the lime. Bating would result in too much softening and loss of hide substance. In some cases a dilute solution of lactic acid is used to de-lime the hides. THE MANUFACTURE OF OTHER FERMENTED PRODUCTS 559 The de-limed or bated hides go next to the “pickle,” a dilute solu- tion of NaCl and H,SO.. Here the remaining Ca(OH). separates as crystals of CaSO, and is removed. The hides are ‘“‘plumped”’ in the pickle and increase in thickness. Formerly a fermenting mixture of - bran and water was used to plump the hides, but this has been almost superseded by the mineral or lactic acid pickle. The plumped hides go progressively through a series of ‘“‘tan-pits” containing each a tannin solution of increasing strength, that of the last being saturated. Tan bark is used for ordinary leather and sumach or other light colored tannin material for light colored leather. A lactic acid fermentation occurs in the tan pits favoring osmotic action which tends to keep the hides plumped and thick. The tannin prob- ably acts as a mild antiseptic, keeping down putrefaction bacteria (W. V. Cruess.). CHAPTER X* MANUFACTURE OF VACCINES INTRODUCTION On July 1, 1902, by Act of Congress, the Secretary of the Treasury, through the Public Health Service, was placed in control of all manu- facture and sale of viruses, serums, toxins, and analogous products for human use. In order to manufacture and place such products upon the interstate market, any individual or corporation must secure a license from the Secretary of the Treasury through the Surgeon General of the United States Public Health Service. All candidates, before securing federal approval for such licenses, must allow federal inspectors the privilege of examining their laboratories, including the details involved in the processes of manufacture. At frequent intervals the Hygienic Laboratory purchases samples of licensed products upon the open market for the purpose of subjecting these to careful examination. If the samples of any products are found to be misrepresented as to potency or kind and amount of preservative, or if contaminating organ- isms are present, the manufacturer is immediately notified to recall such products from the market. July 1, 1913, by a similar Congressional Act, the Secretary of Agri- culture, through the Bureau of Animal Industry, was authorized to regulate the preparation and sale of viruses, serums, toxins and analogous products intended for use in the treatment of domestic animals. The federal control of the manufacture of vaccines, serums, toxins and other biologic products related to specific infectious diseases, has reduced to a minimum the danger formerly involved in the use of such materials. For one who is not a student of microbiology and preventive medicine, or not familiar with the technic involved in preparing biologic *Prepared by W. E. King. 560 MANUFACTURE OF VACCINES 561 materials such as serums, tuberculins and vaccines, it is difficult to realize the various steps necessary in the production of a safe and active product. The manipulations attending the preparation of the materials require large equipment, expensive apparatus and the services of trained laboratory experts. Animals which are used in the work must be quarantined and carefully inspected before being placed under treat- ment. ‘The sanitary conditions of the laboratories, operation rooms and stables must be of the best. Infection of the animal organism is due to absence of natural o1 acquired resistance. The natural resistant forces of the animal body may be such that insusceptibility to specific microbial invasion is present; such a condition is called natural immunity. Acquired im- munity, on the other hand, refers to a condition in which the natural susceptibility of the animal body is replaced by a temporary or permanent resistance toward specific microbial invasions. Acquired ‘immunity may be active or passive, and may be brought about by appli- cation of a vaccine or an antiserum. The application of smallpox vaccine causes a specific reaction in the body, stimulating the develop- ment of natural defences against smallpox virus, and is followed by a condition of active immunity which is relatively permanent in dura- tion. The use of diphtheria antitoxin, which contains the antibodies capable of neutralizing the diphtheria toxin molecules, results in passive immunity and affords temporary protection. ACTIVELY IMMUNIZING SUBSTANCES (VACCINES) Vaccines* are essentially weakened or modified viruses. Such materials as blackleg and anthrax vaccines may be used with safety, as a rule, only on animals which are free from the specific disease in question, because, theoretically, if a specific vaccine were applied to a patient suffering from a given infectious disease, the introduction of the attenuated organisms, or virus, would tend to augment the virulence of the infection. The action of such vaccines is preventive or prophylactic, and not curative. ATTENUATED VirusES.—There are several methods which may be employed in attenuating or modifying viruses. The processes involve * The term “vaccine” is also loosely applied to suspensions containing killed microorgan- isms. 36 562 MICROBIOLOGY OF SPECIAL INDUSTRIES the treatment of viruses in such ways that they may be injected into the normal animal body without danger of producing serious symptoms or lesions, while at the same time sufficient specific infectious qualities must be present to produce mild reactions. The successful vaccine is attenuated or modified to a degree which insures both safety and activity. The following are the more important methods used to modify viruses: Attenuation by growth at a temperature above the optimum. This is illustrated by Pasteur’s method of preparing anthrax vaccine. Attenuation by passage of the virus through some species other than the animal for which the virus is specific. Smallpox vaccine may be regarded as a virus modified by passage through a heifer or other animal. Atter uation of the virus by drying at constant temperature. The Pasteur method of prophylactic treatment for rabies is based upon this method. Attenuation by chemicals. The growth of certain pathogenic bacteria in the presence of weak antiseptics reduces their disease- producing activities. Other methods of modifying viruses for the purpose of active im- munization: The simultaneous method or hypodermic application of the virus together with protective serum, as in hog cholera vaccination. The association or combination of the specific pathogenic bacteria with those of other species as illustrated by the apparent restraining action of yeasts upon pyogenic bacteria and the antagonism which Ps. pyocyanea exerts toward Bact. anthracis. The filtration of liquid cultures of pathogenic organisms, such as Bact. diphtherie or B. tetani, and the consequent separation of the organisms from the toxin. The toxin is used to immunize animals in the production of antitoxin. The destruction of young living cultures of specific bacteria by moist heat at a temperature slightly above their thermal death-point. Heated cultures of B. typhosus and Bact. pestis are used as prophylactics against typhoid fever and bubonic plague. There are many vaccines in practical and experimental use at the present time. Among those which are of recognized value as shown by extensive practical use and reliable clinical statistics, the following MANUFACTURE OF VACCINES 563 are the most important: smallpox vaccine, blackleg vaccine, rabies vaccine, typhoid vaccine and perhaps Pasteur’s anthrax vaccine. The simultaneous method, or injection of hyperimmune serum, to- gether with the specific virus is used in vaccinating against hog- cholera, cattle plague (Rinderpest), anthrax and foot-and-mouth disease. Asiatic cholera, bubonic plague, tuberculosis, acne, pertussis, pneumonia, canine distemper, furunculosis, septicemia hemorrhagica, gonorrhcea and various inflammatory processes are treated, practically and experimentally, by various methods of vaccination, either as prophylactic or curative measures. SMALLPOX VACCINE.—The first experiments relative to vaccination against smallpox date back to 1796. Prior to that time, the only specific preventive method used in warding off this disease depended upon the inoculation of healthy individuals with smallpox virus from a mild case of the disease. The present method of vaccination util- izes cowpox virus as the protective material. It has not been con- clusively determined that cowpox in cattle and smallpox in man possess intimately related causative factors, but notwithstanding, abundant evidence proves the efficacy of cowpox virus as a specific prophylactic against smallpox in man. In the practical preparation of smallpox vaccine, the virus or “seed” is first secured by removing the pulp from the vesicles which appear on infected heifers. Most laboratories which engage in this work use a stock mixture of cowpox virus which originated from spontaneous cases of cowpox, and which is known to produce active smallpox vaccine. Great care is exercised in the selection and preparation of animals used in making the vaccine. Heifers (calves or yearlings) are most frequently used in this work, older cattle being employed in a few European laboratories. When first purchased these animals are placed in a detention stable where they are inspected by a qualified veterinarian and carefully tested for tuberculosis. If, after several weeks’ quarantine, they are passed as healthy in every way, they are admitted to the vaccine laboratory after their bodies have been scrubbed with soap and water and a weak antiseptic solution. The operating room and propagating ward should be constructed with a view to thorough cleanliness. Concrete floors, enameled walls and ceilings and simple sanitary apparatus should characterize the 564 MICROBIOLOGY OF SPECIAL INDUSTRIES appointments. Floors, walls, ceilings and all equipment of these rooms should be carefully cleansed with disinfectant solutions at frequent intervals. After the heifers are prepared for the work, they are inoculated with the seed virus. The animal under treatment is placed on a special operating table, the ventral surface of the body is shaved and cleansed, and, with a sterile instrument, is scarified in parallel straight lines over the greater portion of the abdomen and inner surface of the flanks. The stock virus or “seed” is inoculated in the scarified areas and the animal is released and placed in the propagating room. During the process of propagation of the vaccine all possible precautionary meas- ures should be used to avoid the introduction of contaminating bacteria. It is important that an attendant be constantly present, day and night, whose duty it is to remove instantly all dirt and feces and keep the room as clean and free from microbial contamination as possible. At the expiration of from seven to nine days, characteristic vesicles will have developed on the inoculated areas. These are filled with a thick, sticky purulent material. At this time the animal is removed to the operating table, the field of operatign is washed with sterile water and the contents of the vesicles removed with a sterile curette. Accord- ing to regulations of the Federal Government all animals used in this work must be slaughtered before the vaccine is removed and later submitted to careful autopsy. After removing the pulp, or vaccine, the material is handled under aseptic precautions and mixed with about 50 per cent glycerin, which serves as a preservative. Small portions of the material are then inoculated into guinea-pigs for safety tests and the product is placed in the refrigerator. Under the in- fluence of the glycerin extraneous microbial contamination gradually disappears. Potency tests of the vaccine are conducted by the cutane- ous application of the vaccine on calves, rabbits or on slightly scarified, scrotal surfaces of guinea-pigs. In addition to the safety and potency tests, inoculations are made into culture media which are placed under both aerobic and anaerobic conditions toinsure the absence of harmful bacteria. For the detection of the presence of B. tetani the product is submitted to a special test by transferring 1 c.c. into a quantity of glucose beef bouillon or other special culture media, placing the culture under anaerobic conditions and incubating at body-temperature for about ten days. After the incubation any resulting growth is MANUFACTURE OF VACCINES 565. removed by filtration and the filtrate is injected into guinea-pigs. The absence of symptoms in the treated animals shows that no tetanus toxin has been elaborated in the culture medium and therefore that the vaccine does not contain B. tetani. After the tests are completed, the product is distributed under aseptic conditions, in small, sterile, capillary tubes or upon sterile, ivory points, sealed in sterile, glass containers properly labeled, dated and kept in the refrigerator until placed upon the market. If kept in a cold dark place, smallpox vaccine retains its protective activity for a considerable period. Under the influence of heat and light it rapidly deteriorates. For this reason it is difficult to ship the vaccine to tropical countries. Under suitable conditions the product should remain active for a period of about one year. BLACKLEG VaccinE.—The production of blackleg vaccine depends upon the use of a virulent culture of B. anthracis symptomatici. A heifer is inoculated with a small portion of the virus and rapid, acute symp- toms are usually produced. Death usually supervenes in about three days. The carcass and ward are thoroughly disinfected, the body of the animal is suspended, and, after again carefully disinfecting the out- side of the body, portions of the skin are removed and the muscular tissue is inspected. ‘Those areas of the muscles which show the dark color, gaseous formation and characteristic lesions of blackleg, are removed to the laboratory and examined microscopically for the presence of the specific organisms. After the muscle is freed from the gross connective tissue, it is suspended in strips or finely chopped, and allowed to dry spontaneously. It is then ground and sterile water is added until the mass becomes pasty or putty-like in consistency, after which the material is placed in small shallow pans and attenuated by drying ‘at temperature of 85° to roo° for six or seven hours. In preparing the “single vaccine” most laboratories attenuate the virus by drying at an average temperature of about 90° for six hours. In addition to the aseptic precautions observed in conducting the above processes, microbial contamination is practically eliminated by the devitalization and probable death of any extraneous vegetative forms during the attenuation process. Blackleg vaccine (single) is tested, according to the method recom- mended by the Bureau of Animal Industry, U.S. Dept. of Agriculture, as follows: A series of eight guinea pigs are injected intramuscularly 566 MICROBIOLOGY OF SPECIAL INDUSTRIES with the vaccine under test; three each with three-fourths the dose for cattle, three each with one-half dose and the remaining two with one- third dose. ‘ Temperatures of the test animals should be recorded for three sub- sequent days. Vaccine of proper strength is indicated when thermal, reactions occur in practically all the test animals together with local reactions in some instances. None of the animals in the series should die. As an additional test for potency a heifer may be injected sub- cutaneously with one dose and a few weeks after the vaccination the animal may be exposed to the disease by receiving an injection of the virulent living organisms. If the animal remains normal the activity of the product is indicated. In order to test the vaccine in regard to safety, heifers may be injected with several doses each. The absence of severe disturbances shows that the material may be used without danger. For the purpose of eliminating possible danger from the use of blackleg vaccine a “double vaccine” may be employed. This consists of two vaccines, each possessing different degrees of attentuation, which are controlled by the degree of heat and the period of time used in attenu- ating the organisms in the affected muscle tissue. When the final product, either single or double blackleg vaccine, is ready for use it is usually distributed.in the form of a powder, prepared threads or small pills. The latter, first suggested by Houghton in 1898, are injected hypodermically. Rapies Vaccine.—The successful preventive treatment for rabies, or hydrophobia resulted from the brilliant researches of Pasteur. The method devised by Pasteur in 1885, with some modifications, con- tinues to be the only practical, specific preventive treatment for rabies. This treatment consists of a series of vaccinations, each vaccination involving the application of rabies virus having a known degree of attentuation. In each succeeding application of modified rabies virus the patient receives increasingly more virulent material until finally active immunity is acquired and subsequent attack. from the disease is successfully resisted. The preparation of rabies vaccine begins with the attenuation of a virus having a known degree of virulence. The material may be'secured from an ordinary case of ‘street rabies.” A dog suffering from the MANUFACTURE OF VACCINES 567 disease is killed and a small portion of the brain removed. The brain tissue is emulsified in sterile water or salt solution and a few drops of the material thus suspended in liquid, are injected subdurally into a rabbit. This may easily be accomplished by trephining the skull, after anesthe- tizing the animal, and with a small syringe inoculating a few drops of the suspension just under the exposed dura mater. The inoculation of ordinary rabies virus usually produces symptoms of ‘dumb rabies’ and the death of the animal in fourteen to eighteen days. In order to increase the virulent properties of the same strain of rabid material, it is transmitted from rabbit to rabbit by subdural inoculations until the incubation period is shortened to about six days. Experience has shown that when the virus has reached its maximum degree of virulence for the rabbit, the animal shows symptoms on the sixth or seventh day after inoculation. When the virus attains this degree of virulence it is called ‘‘fixed virus” and may be used in the preparation of the vaccine. The “fixed virus” or spinal cord of the rabbit which has succumbed to the disease in six or seven days, is removed aseptically and placed in a special drying chamber. The cords are suspended over caustic potash and dried at a temperature of 23° for a period of from one to ten or fifteen days. The treatment of the patient consists in the hypodermic applica- tion of the “fixed virus” which has been attenuated by drying. The exact nature of the vaccine used in the initial vaccination and the time consumed in the series of injections depend, to some extent, upon the case in hand. Frequently, the patient is first vaccinated with a sus- pension of a spinal cord which has been attentuated by drying for four- teen or fifteen days. On the succeeding days of the treatment use is made of the suspension of spinal cords, which have been less and less attenuated. The treatment usually lasts about twenty days or until the patient has received an injection of the least attenuated “fixed virus.” It is very important, when one is bitten by a rabid animal, that the Pasteur treatment be begun as early as possible, in order that active immunity may be secured before the expiration of the incubation period. In many of the larger cities of the United States, for some time, labora- tories have been maintained for the purpose of administering the Pasteur treatment. More recently, commercial laboratories have 568 MICROBIOLOGY OF SPECIAL INDUSTRIES developed methods of preparation and distribution, so that any phy- siclan may purchase the vaccine and administer it to his patients. Hogyes* substituted dilutions of the ‘fixed virus” for the dried spinal cords. For the initial treatment, a few cubic centimeters of a r:10,000 dilution was used. In the succeeding injections graduated dilutions were employed. While the work of Hogyes has been con- firmed by other investigators, the method is not generally regarded as possessing the safety of the original Pasteur treatment. - Harris t has devised a simple method of preparing the vaccine by freezing the infected spinal cord of the rabbit with CO. snow, and then drying the material im vacuo over sulphuric acid at a temperature of To° to 15°. The product is kept in the refrigerator in hermetically sealed vigls. It is claimed that the material so prepared maintains its original strength or infectivity several months. Cummings’{ method consists in the dialysis of the rabic material in standard suspensions. Dialysis for twelve to twenty-four hours possesses the advantage of destroying the infectivity of the virus, without disturbing its immunizing properties. Dorset-NILes (Hoc-CHOoLERA) SERUM.§—To prepare the material for this process of immunization it is first necessary to secure a virulent strain of hog cholera virus. This may be obtained from any typical outbreak of the disease. A specimen of blood may be drawn, asep- tically, from the carotid artery of a pig suffering from the disease, and tested for activity. Frequently a given strain of virus may not produce the acute form of hog cholera. In attempting to raise the virulence of a relatively weak virus it may be passed through a series of young pigs until it uniformly produces symptoms after four to six days’ incubation and death in less than fifteen days. None except a virus having this degree of activity should be used in manufacturing the hyperimmune serum. The virulent blood used in the process of hyperimmunization should be obtained from susceptible pigs weighing from 25 to 50 kg. (50 to 100 pounds) each. The animals to be used as the “hyperim- * Hogyes, Acad. des Sciences de Budapest, Oct. 17, 1897. ft Harris, Jour. Infect. Dis., 1912, 10, p. 369. } Proceedings 15th International Congress on Hygiene and Demography, Washington, D. Os 08s. § U. S. Bureau of Animal Industry, Bull. No. 102. MANUFACTURE OF VACCINES 569 munes” should be healthy hogs, each weighing from 100 kg. to 150 kg. (220 to 330 pounds) and possessing either natural or acquired immunity to the disease. The blood is best secured from a diseased pig by suspending the animal with the head down covered with a shroud wet with a disinfectant solution. The neck is shaved and disinfected. A small incision is made on the median line through which a specially devised bleeding knife, properly sterilized, is introduced. The blade of this knife severs the large vessels at the base of the heart and the blood flows through the hollow handle into sterile containers. After the blood is obtained it is defibrinated, the serum separated from the clot, and the clot discarded. The number of pigs necessary to furnish sufficient virus for the hyperimmunization of one hog depends upon the weight of both the virus pigs and the immune hog. The immune hogs may be hyperimmunized either by the “slow” or “quick” method. In the former, now seldom used, the animals receive several injections at intervals of every few days, each succeeding dose being increased in proportion to the weight of the animal. In the “quick” method the virus is injected in one large dose, the amount being determined by the weight of the animal. The virus may be injected intramuscularly, intraperitoneally or intravenously, the latter method now being used almost exclusively. Ten days to two weeks after the hyperimmune hog has received the last injection of virus, the animal is ready for bleeding. When bled from the tail, the end of the appen- dage is severed with a sharp instrument, several hundred cubic centi- meters of blood are collected aseptically, defibrinated, a preservative added and the material placed in the refrigerator. This process is repeated several times, at intervals of one week to ten days, when the animal is ready for final bleeding. By this procedure all the blood is secured from the animal according to the method described for bleeding virus pigs. The “slaughter” method, used in many laboratories, consists of only the final bleeding, thus eliminating tail bleedings. Asa rule the different lots of serum representing the different bleedings from several hyperimmune hogs are mixed and the whole subjected to test. In order to test the potency of the product eight susceptible pigs, each weighing about 23 kg. (so pounds) are inoculated subcutaneously, each with 2 c.c. of virus. Six of these pigs are simultaneously injected with graduated doses (15 to 25 c.c.) of the serum under test. If the hyperimmune §7° MICROBIOLOGY OF SPECIAL INDUSTRIES \ serum possesses potency the test pigs should remain in a normal con- dition throughout the test, except for the presence of thermal reactions and slight clinical symptoms, while the two control pigs should show severe symptoms in five or six days and should die in less than fifteen days. The practical method of treatment in the field consists in the simul- taneous injection of the hyperimmune serum and virus (double treat- ment), into healthy hogs for the purpose of immunization. The amount of hyperimmune serum which should be injected varies from 30 ¢.c. to go c.c., depending upon the weight of the hog to be treated. Thus, a hog weighing 34 kg. to 45 kg. (75 to 100 pounds) usually re- ceives 4o c.c. of serum, together with 1 c.c. of virus. The usual dose of virus for hogs above 34 kg. (75 pounds) weight is 2c.c. For pigs weighing less than 23 kg. (so pounds) ¥ c.c. of virus should be injected. ANTHRAX VACCINE.-—While several methods have been used in vac- cinating against anthrax, probably the most important, at present, is that devised by Pasteur. This method consists in the use of cultures which have been attenuated by growth on artificial culture media at temperatures above the optimum. The inoculation of such attenuated cultures into healthy animals results in active immunization. The stock culture of Bact. anthracis is usually obtained from the blood of a typical case of anthrax. The culture is transferred to agar or broth and incubated. Two vaccines are prepared, the first being less active than the second. Vaccine No. 1, is made by placing in suspension in sterile, physiological salt solution or other liquid, the anthrax organisms which have been grown at a temperature of 42° for a period of fifteen to twenty days. Vaccine No. 2 consists of a similarly treated culture of Bact. anthracis which has grown at a tem- perature of 42° for ten to fifteen days. Tests of both vaccines for activity and safety are made by animal inoculations. Vaccine No. 1 should kill white’mice but should not cause fatal results in guinea-pigs or rabbits. Vaccine No. 2 should prove fatal for both white mice and guinea-pigs, but not for rabbits. Healthy animals are first injected subcutaneously with about 1 c.c. of vaccine No. 1. Several days or a few weeks after, the application of vaccine No. 1, the second vaccine is injected. A severe reaction and sometimes death follows the use of the vaccine. Accidents of this kind MANUFACTURE OF VACCINES 571 have resulted from careless methods employed in standardizing and administering the vaccine. The most important objection to Pasteur’s anthrax vaccine is due to the danger involved in the use of the living, attenuated anthrax organisms. Scalvo* advocates the use of the serum from animals actively im- munized to anthrax. This method may be employed either in the form of the immune serum alone, or the immune serum and anthrax culture simultaneously. Eichhorn} advises the use of antianthrax serum for curative purposes, and the simultaneous treatment with antiserum and a care- fully standardized spore vaccine as a preventive. When vaccine alone is to be employed Eichhorn prefers the spore vaccine rather than the ordinary Pasteur vaccine. TUBERCULOSIS VACCINE.—Among the experimental products for the prevention of animal tuberculosis may be mentioned von Behring’s “bovo-vaccine.” The technic involved in the preparation of this vaccine is not generally known. Romer{ describes the material as being composed of the living tubercle organisms which are dried for a period of thirty days in sealed glass tubes. After this process of attenua- tion the organisms are injected, in carefully graduated doses, into healthy calves. Field tests which have been made upon calves with bovo-vaccine indicated unsatisfactory results. In human practice various tuberculins prepared both from the bouillon culture and from the cellular elements of Bact. tuberculosis are used as therapeutic and diagnostic agents. Products containing the cellular elements are similar in nature to bacterial vaccines. BACTERIAL VACCINES (BACTERINS) Opsonins may be defined as the elements in the blood or body fluids which are capable of modifying invading bacteria in such a way that they become ready prey to the leucocytes. In the presence of opsonins, therefore, phagocytic activity is increased. Opsonins are apparently distinct from agglutinins, lysins, and other analogous substances, because different degrees of heat are necessary for their destruction. Moreover, a given serum may agglutinate, or may exert lytic action, without possessing opsonic activity. * Scalvo, Centralbl. f. Bakt., 1809, 26, p. 425.. f Eichhorn, Bull. No. 340, U. S. Dept. of Agri. t Romer, Beitrage z. Exp. Therapie, 1904, 7. 572 MICROBIOLOGY OF SPECIAL INDUSTRIES Wright and Douglas first advanced the theory of opsonic action, and suggested that the subcutaneous injection of a given species of bacteria, killed by heating, caused the blood of the treated individual to exert greater opsonic activity toward the species of organisms in question. The results of the work of others proved to be confirmatory. To prepare a bacterial vaccine, the specific organism is isolated and after being grown for twenty-four hours or longer at a temperature of 37°, it is emulsified in sterile physiological salt solution, heated at approximately 60°, or killed by the use of chemical agents, standardized as to the number of bacteria present in 1 c.c. of the emulsion, and a preservative added. If the patient and attending physician are conveniently situated in respect to a laboratory, the ‘‘opsonic index” may be taken before and during the treatment. This consists in the determination of the aver- age number of the given species of bacteria ingested by the leucocytes of the patient, as compared to that which the leucocytes of normal blood are capable of destroying. It is usually found that immediately following the injection of specific bacterial vaccine there is a “negative phase” during which the leucocytes of the patient destroy a smaller number of bacteria. This is followed by a “positive phase,” character- ized by more active phagocytosis. For practical purposes the determi- nation of the opsonic index is unnecessary as the clinical reaction fol- lowing the injection of a given vaccine indicates correct dosage and progressive. results of the treatment. The use of bacterial vaccines has yielded excellent results espe- cially in the curative treatment of furunculosis, acne, sycosis, puerperal infection, arthritis and other affections caused by pyogenic organisms, and in chronic infections of the genito-urinary tract. The material may be used in the form of ‘‘autogenous”’ or ‘“‘stock” vaccines. An autogenous (personal) bacterial vaccine is one prepared from a culture of the specific organism isolated from the case under treatment. Bacterial vaccines, prepared from stock cultures of the specific organ- isms, may be manufactured and kept until needed for use. Some of the more common stock bacterial vaccines represent the following organisms alone or in various combinations: Sérept. pyogenes, M. pyogenes var. (albus, aureus and citreus), M. gonorrhea, Bact. pertussis, M. pneumonie, and B. colt communis. The study of bacterial vaccines occupies a position of so much importance in preventive medicine and therapeutics that many new MANUFACTURE OF VACCINES 573 combinations of killed bacteria are being constantly added to the list of experimental products. Some of these have been under ob- servation for a considerable time and are recognized as possessing valuable properties. TypHow FrEvEeR.—The typhoid bacterial vaccine of Wright* is generally accepted as a valuable preventive against infection. For prophylactic treatment, a series of three hypodermic injections (500,000,000, 1,000,000,000 and 1,000,000,000) of killed typhoid organisms are usually given. The curative value of typhoid bacterial vaccine has not yet been determined. Canine DistEMPER.—Ferry,{ corroborated by Torrey{ and Mc- Gowan§ found this disease to be primarily an infection of the upper respiratory tract due to a small motile bacillus, B. bronchisepticus. Ferry and Torrey proved that suspensions of killed cultures of this organism will immunize dogs against experimental inoculations as well as against the ordinary street infection. The bacterial vaccine is being used for prophylactic purposes in graduated doses of 200, 400 and 600 million bacteria per c.c., given at intervals of about five days. AstatIiC CHOLERA.—Two methods of vaccination against this disease have been proposed and statistics which relate to field tests show positive results with both. The method of vaccination resulting from the work of Haffkine** depends upon the use of cultures of the spirilum of Asiatic cholera, attenuated by growth at temperatures above theoptimum. Vaccines of different strengthsareused. Kolle*** has proposed the use of heated (killed) cultures of the organism. StrongTf has developed a vaccine for Asiatic cholera consisting of the filtrates from suspensions of killed and living Msp. comma (Sp. cholere asiatice). This vaccine is standardized in terms of immunity units, one unit “equaling the amount of immune serum which will protect a guinea-pig of 250 g. weight against the intraperitoneal inoculation of ten times the fatal dose of living cholera organisms.” Busonic PLacuE.—Practically the same methods of procedure * Wright, Jour. of Hyg. 2, 1902, p. 385. { Ferry, Am. Vet. Rev., r9ro, Vol. 37, p. 499. Jour. of Infec. Dis., ror1, vol. 8, p. 399. t Torrey, Jour. of Med. Research, 1913, Vol. 27, 291. § McGowan, Jour. of Path. and Bact., rorz, Vol. 15, p. 372. ** Haffkine, Brit. Med. Jour., 1895, 2. pp. 727, 1509. *** Kolle, Deut. med. Woch., 1897, 23. p. 4. TT Strong, Am. Med., 1903, Vol. 6, p. 272. Strong, Philip. Jour. Sci., 1907, Vol. z. p. 155. 574 MICROBIOLOGY OF SPECIAL INDUSTRIES have been followed in the experimental vaccination against bubonic plague as in the case of Asiatic cholera. Cultures of the plague bacillus, killed by heating at a temperature of 60° for one hour, have been used with success. SENSITIZED VACCINE *Besredka has developed modified bacterial vaccines known as sensitized vaccines. In the preparation of these the living micro- érganisms are brought into contact with the homologous antiserums and the mixtures allowed to stand for approximately twenty-four hours at room temperature. The organisms are then removed by centrifugal- ization, washed and placed in suspension. The remaining processes of manufacture are similar to those employed in the preparation of ordinary bacterial vaccines. Besredka and his associates explain the advantage of sensitized vaccines by the fact that in such preparations the microdrganisms, by reason of having been in contact with homologous antiserums, are sensitized with specific amboceptors. Therefore, the sensitized organ- isms are capable of immediately combining with complement, when introduced in the blood of the patient, and prompt immunization should follow. Both living and killed sensitized microdrganisms have been used experimentally, Besredka} having advocated the use of the former as devoid of harmful properties and more certain of successful results. Sensitized vaccines are still in the experimental stage, and their ad- vantage over the ordinary bacterial vaccines is at present a debated question. : Toxin—ANTITOXIN MIxTURE. Babes, t in 1895, first suggested the use of diphtheria toxin and antitoxin mixture as a method of immunization against diphtheria. Through the work of Park and Zingher§ and others who preceded, this method is being adopted in practice, especially asa means of prophylaxis against diphtheria in schools and hospitals. The mixture consists of active diphtheria toxin and antidiphtheric serum in the proportion of 80 per cent. of the L + dose of toxin to one unit of antitoxin. * Besredka, Compt. Rend. de l’Acad. Sci., 1902, 134, p. 1330. + Besredka, Bull. de l’Inst., Pasteur, 1910, 8, p. 241. } Babes, Bul. Acad de Med., Paris, 1895, 34. p. 216. § Park and Zingher, Jour. A.M.A. 1915, 65, p. 2214. CHAPTER XI* THE MANUFACTURE OF ANTISERUMS AND OTHER BI- OLOGICAL PRODUCTS RELATED TO SPECIFIC INFECTIOUS DISEASES. The principles involved in serum therapy are those of passive immunization. Therefore, the employment of an antiserum as a pre- ventive or curative measure is an attempt to supply the patient with certain specific substances which are capable of neutralizing and de- stroying the specific toxic materials and pathogenic microdrganisms. ° Presumably, the patient receives nothing in antiserums which stimulate the development of protective bodies. Active immunity does not follow as in the case of vaccine treatment. As the result of serum treatment, the patient enjoys relatively temporary pro- tection (preventive treatment), or cessation of pathologic processes (curative treatment), because of the application of specific antisub- stances. The substances contained in the serum are developed in the blood of some other species, as the horse, through repeated injections of the animal with the specific organism in question or its toxin. Antiserums are divided into antitoxic and antimicrobial serums. An antitoxic serum is one possessing substances which, in contact with the specific toxin, unite with it, forming chemically stable and physiologically inert compounds. Under the term “‘antitoxic serum,” in addition to antidiphtheritic and antitetanic serums, are grouped anti- serums for the soluble toxins of B. botulinus (specific meat poisoning), abrin, ricin and crotin (plant toxins), snake venom and spider toxin, and the soluble toxin of B. anthracis symptomatici (blackleg in cattle). The antimicrobial serums constitute the majority of serum products. Included among these are antimeningococcic, antistreptococcic, anti- gonococcic, antistaphylococcic, antityphoid, antidysenteric, antirabic, antipneumococcic, antituberculosis, antiplague, anticholera, antihog cholera, antianthrax serums and serums for swine erysipelas, fowl cholera, *Prepared by W. E. King. 575 576 MICROBIOLOGY OF SPECIAL INDUSTRIES white scours of calves, sheeppox, foot-and-mouth disease, canine dis- temper, rinderpest and spotted fever. The action of this group: is directed more especially against the specific microérganisms involved, resulting in dissolution of the cells or lysis due to lytic bodies in the antisera (bacteriolysins). In addition to the presence of lysins in antimicrobial serums, other antisubstances are known to exist, as agglutinins, bacteriotropins (opsonins), and precipitins. The antibody content of antimicrobial serums is comparatively little understood and the clinical interpreta- tion of lysins, agglutinins and precipitins is not clear. ANTITOXIC SERUMS DIPHTHERIA ANTITOXIN.—A culture of the organism may readily be secured from the throat of a patient by transferring some of the diph- theritic exudate, on a sterile cotton swab, to Loeffler’s blood-serum culture medium. After the growth of the bacteria at incubator tem- perature, contaminating organisms may conveniently be eliminated by the inoculation of a guinea-pig and the isolation of the diphtheria organ- isms from the tissues. A pure culture is necessary in the preparation of the antitoxin, but any given culture should not be relied upon until tests have been made of the final product. To produce the diphtheria toxin with which the antitoxin horses are treated, the diphtheria organisms, in pure culture, are transferred to beef broth, contained in large flasks, and incubated at a temperature of 37°. A rapid growth takes place, during which the specific toxin is -elaborated by the organisms. After a period of incubation of about two weeks, the bouillon culture is removed from the incubator, examined microscopically in order to make sure that contamination is not present, a preservative is added, usually carbolic acid, trikresol, or purified cre- sols, and the organisms are removed from the culture by passing the liquid through a Berkefeld filter. The filtrate (diphtheria toxin) is then placed in the refrigerator until used. The horses which are used in the manufacture of antidiphtheritic serum, as well as for the preparation of other antiserums, must be submitted to rigid inspection before being placed on the treatment. These animals, when purchased, are placed in a detention stable for several weeks. During this time they are subjected by a qualified THE MANUFACTURE OF ANTISERUMS 577 veterinarian to a thorough physical examination and to the mallein test for glanders. Finally, only those animals which are pronounced normal in every way are admitted to the antitoxin stables. The stables and the operating rooms with their appointments, which are designed for the antitoxin horses, should be constructed with a view to perfect sanitation and cleanliness. Concrete floors, sanitary stalls, mangers, stocks and apparatus, good water, free ventilation and plenty of light should characterize the quarters. The antitoxin horses are injected subcutaneously with the diphtheria toxin. The initial dose of toxin usually consists of only a fraction of a cubic centimeter, then increasingly larger doses are administered until the animals are finally able to receive 30q¢c.c. or more at a single treat- ment. The intervals between injections and the rate of increase of succeeding doses at any given time depend upon the condition of the animal. During this treatment a constant process of antitoxin forma- tion is taking place in the body of the horse. In order to produce a po- tent serum, the injection of the toxin should be continued throughout the course of treatment as rapidly as the resulting reactions, following each injection of the animal, will allow. After the completion of: the initial toxin treatment, which occupies a period of from six weeks to three months; the horse is allowed a test of about two weeks, during which time all the toxin which has been injected should be absorbéd. During the remainder of the antitoxin- producing period of the animal’s life (approximately two years), treat- ment with diphtheria toxin is continued at regular intervals. When desired, a small sample of blood serum may be secured from the horse for preliminary potency tests. Finally, the animal is bled from the jugular vein, under aseptic conditions. As much blood is secured as the horse can conveniently yield, varying in quantity from 10 to 15 1. The blood may be drawn through a sterile canula and rubber tube into tall, sterile glass cylinders. After the blood has clotted the serum separates and at the end of twenty-four to forty-eight hours, the clear, amber-colored fluid is poured from the cylinders into large, sterile glass containers, a preservative is added and the material is transferred to the laboratory. The serum is then passed through a Berkefeld filter. Each lot of antidiphtheritic serum is submitted to rigid tests relative to potency, safety and microbial contamination. In deter- mining the potency, varying amounts of the serum under test are 37 578 MICROBIOLOGY OF SPECIAL INDUSTRIES mixed with the L-++ dose.of diphtheria toxin and injected into a series of guinea-pigs, each weighing 250g.* TheL-+ dose of toxin is the least amount of toxin, which, when mixed with one unit of standard antitoxin (supplied by the Hygienic Laboratory) and injected into a guinea-pig of 250 g. weight, is sufficient to kill the animal in four days. From the results of this test it is possible to determine the smallest amount of the antitoxin which will protect a guinea-pig of 250 g. weight, when the animal has received simultaneously the L+ dose of toxin. This mini- mum amount of antitoxin represents one unit. Thus, if }é99 c.c. of the given antitoxin represents the smallest amount which is capable of neutralizing the L+ dose of toxin, then the antitoxin would possessa potency of 500 units per c.c. 4 In order that the antitoxin may be tested for safety, each of several guinea-pigs is injected subcutaneously with about 2 c.c. of the serum. These animals are not released until the observer is satisfied that the serum contains no injurious properties. For the purpose of detection of microbial contamination, relatively large amounts of the antitoxin are placed in culture media and incubated under both aerobic and anaerobic conditions. Diphtheria antitoxin is usually distributed in glass syringe containers: ready for immediate use. After the product has been tested relative to potency, safety and microbial contamination, it is put up in sterile glass cylinders. These cylinders are so constructed that accompanying sterilized needles and pistons may be conveniently applied and theanti- toxin injected hypodermically directly from the containers. Each container must bear a label indicating the number of antitoxin units enclosed and the date of preparation. Finally, after the diphtheria antitoxin has been distributed in the glass cylinders, sealed and packed ready for use, sample packages are opened and examined for contamination, usually by two microbiologists. The product is not approved until the independent results of these final tests are compared, and it is assured that microbial contamination is absent. , All antitoxic serums should be kept away from the light and at a temperature of 10° to 15° whenever possible, as the presence of heat and light causes gradual deterioration. Usually an expiration date of from eighteen months to two years is applied to diphtheria antitoxin. * See Bulletin No. 21, Hygienic Laboratory, Washington, D. C. THE MANUFACTURE OF ANTISERUMS 579 It has been demonstrated that the antitoxic content of serum is closely associated with the globulins. Advantage is taken of this fact by most laboratories in reducing the volume of antitoxin, or concentrating the product, by precipitating the globulins with ammonium sulphate, redissolving the precipitate and dialyzing. The concentration of serum by this method increases the unit value per volume and tends to decrease the occurrence of undesirable second- ary effects (‘serum sickness”’). TETANUS ANTITOXIN.—The processes involved in the preparation of antitetanic serum differ but little from those employed in the manufacture of diphtheria antitoxin. The pure culture of B. tetani ’ is inoculated into large flasks of glucose bouillon, placed under an- aerobic conditions and incubated at body temperature. A convenient method of excluding free oxygen, in the presence of which the tetanus organisms will not multiply, consists in boiling the glucose bouillon before the inoculation, to drive off the oxygen, then covering the liquid medium by a layer of oil. These cultures are subjected to a temperature of 37° for several weeks, after which-they are examined microscopically, a preservative is added and they are passed first through a Berkefeld filter, and finally through a Pasteur filter. On account of the presence of spores and the danger attending the con- tamination of any materials or biological products with the tetanus bacillus, it is important that great care should be exercised in the filtration and preparation of the tetanus toxin. Therefore, the filtra- tion process is best accomplished in an isolated room which is used only for the preparation of tetanus toxin.. Tetanus antitoxin is produced by the injection of horses with the specific toxin and the same general methods and precautions are ob- served as in the preparation of diphtheria antitoxin. The anti- tetanic serum is tested relative to potency, safety and freedom from microbial contamination. *The standard unit of tetanus antitoxin is regarded as ten times the least quantity of antitetanic serum necessary to save the life of a 300-g. guinea-pig for ninety-six hours, against the official dose of a standard toxin furnished by the Hygienic Laboratory of the Public Health Service. Tetanus antitoxin is put up for use in the same manner as diph- * See U. S. Treasury Department, Public Health Reports, Vol. XXIV, No. 20, 1904. 580 MICROBIOLOGY OF SPECIAL INDUSTRIES theria antitoxin, being usually distributed in glass syringe containers. The product is used in both human and veterinary practice. 4 ANTIMICROBIAL SERUMS In addition to diphtheria and tetanus antitoxins, certain other antiserums are rapidly attaining practical significance.) At present, however, no methods are in use by which any antiserums other than diphtheria and tetanus antitoxins can be accurately standardized as to potency. Nevertheless, most of the products can be submitted to rigid tests in order to determine the presence of protective qualities. ANTIMENINGOCOCCIC SERUM.—Horses are immunized to cultures of a number of strains of M. intracellularis var. meningitidis, the activity of the resulting serum being determined by agglutination and com- plement fixation tests. Antimeningococcic serum is used in the active treatment of cerebrospinal meningitis and is‘administered by lumbar puncture. The dose depends principally upon the age of the patient and the condition of the blood pressure. ANTISTREPTOCOCCIC SERUM.—Bouillon cultures of Sirept. pyogenes are killed by heating, and injected into horses in increasingly larger doses. Frequently, the killed cultures used in treating the horses are composed of several different strains of the streptococcus. In this case the resulting antistreptococcic serum is designated as “polyvalent,” while the serum obtained after the injection of cultures consisting of but one strain of the organism, is called ‘‘ monovalent” antistreptococcic serum. : “4 In procuring the serum, handling, filtering, preserving and dis- tributing for use, the methods are practically the same as those em- ployed in the preparation of antidiphtheritic serum. Antistreptococcic serum is carefully tested in regard to safety and freedom from microbial contamination. There are no methods avail- able for definitely standardizing the product. The serum is often efficacious in cases of streptococcic infection. Anticonococcic SERUM.—Killed cultures of M. gonorrhee are injected intraperitoneally or intravenously into large, healthy rams, or other animals. The dosage is increased and finally live cultures are applied, the degree of immunity acquired being determined by com- plement fixation and agglutination tests of the serums from the animals. THE MANUFACTURE OF ANTISERUMS 581 Dorset-NitESs (ANTIHOG CHOLERA) SERUM (HyYPERIMMUNE SERUM)*.—This product has been described in the preceding chapter under “hog cholera vaccine” (Double Treatment). When the hyper- immune serum is used unaccompanied by the virus, either among healthy or diseased swine, the process is known as the ‘‘Serum-Alone Method.” Reichel has succeeded in producing an antihog cholera serum which is sterile and free from inert solid matter by precipitat- ing the globulins. Dorset and Henley{ have announced the produc- tion of a clear and sterile serum by employing an extract of common garden beans together with salt to agglutinate the blood corpuscles. AntrraBic SERUM.—Animals which have been immunized to rabies are bled and the immune serum may be used as a preventive and therapeutic agent. While this product is not often employed in prac- tice, yet it has been shown by various investigators that considerable protection is obtained from its use. _ ANTIDYSENTERIC SERUM.—Experimental monovalent and poly- valent antiserums for epidemic dysentery have been developed by Shiga and Flexner, by the injection of horses with the filtrates from bouillon cultures of the dysentery bacillus. THE PRESERVATION OF ANTISERUMS.—The question of a proper .preservative for antiserums has received much attention. The problem of preservation involves several conditions, as the ideal pre- servative, when incorporated in the proper volume of serum in efficient dilutions, must possess marked inhibitive and germicidal power, it must prove inert when injected into the patient, and it must produce no objectionable precipitation of serim proteins. At present, trikresol or purified cresols (0.4 of 1 per cent) is generally employed. BriorocicaAL Propucts OTHER THAN VACCINES AND ANTISERUMS TuBERCULINS.—Koch’s Tuberculin (Old).—Koch’s tuberculin is the concentrated, glycerinated, beef bouillon in which Bact. tuberculosis has been grown. ‘The active substance of the tuberculin is apparently an albuminous body insoluble in alcohol. The product is harmless for the non-infected, but exerts a toxicaction upon tuberculous individuals, the reaction being characterized by a rise in temperature which begins two *See U.S. Bureau of Animal Industry, Bull. No. 102. { Reichel, Proc. 18th Ann. Mtg. U.S.L.S.S. Asso., 1915, p. 127. + t Dorset and Henley, Jour. Agr. Research, Vol.6, May 209, 1916. 582 MICROBIOLOGY OF SPECIAL INDUSTRIES to ten hours after treatment, continues for a few hours and finally subsides. Tuberculin (old) is used as a diagnostic agent in both human and veterinary practice. Tuberculin (old) is prepared from cultures of the human or bovine variety of Bact. tuberculosis. Apparently the active product can be obtained from attenuated as well as from virulent cultures. The organism is inoculated into beef bouillon to which 5 per ‘cent glycerin has been added. The culture medium is usually distributed in flasks and the tubercle organisms, when inoculated, are carefully placed on the surface of the medium. The cultures are incubated at a temperature of 37° to 38° for six to ten weeks or longer, during which time a heavy growth slowly spreads over the surface of the medium and finally falls to the bottom of the flasks. In the successful preparation of tuberculin it is important that the cultures should remain undisturbed, having’ access to plenty of air, that the incubator temperature should be con- stantly maintained without fluctuations, and that the organisms should be allowed to grow until they have completely elaborated the active “tuberculinic’’ substance. After the growth is complete; the cultures are removed from the incubator and sterilized in streaming steam. The killed cultures are then evaporated over a water bath to one-tenth the original volume, the bacteria are removed by passing the cultures through paper and Berkefeld filter and a preservative is added. For cattle the dose of tuberculin concentrated by evaporation to one-tenth the original volume, is 0.25 c.c. too.7 c.c. Because of the fact that the material is thick and syrupy in consistency and the dose is incon- veniently small, it is usually diluted with seven parts of weak carbolic acid golution. During the preparation it may be evaporated to four- fifths the original volume and preserved by the addition of 1 per cent carbolic acid of sufficient volume to dilute properly so that each dose is represented by 2 c.c. Two cubic centimeters of the diluted tuberculin . . is used as the dose for cattle. The product should be tested for activ- ity by injecting known tuberculous animals with the tuberculin under test. The presence of typical reactions in tuberculous animals indi- cates the reliability of the product. _ . In human, as well as in veterinary practice, tuberculin may be applied as a diagnostic agent in various ways. In addition to the hypo- dermic injection of tuberculin (old), as described above, the method of THE MANUFACTURE OF ANTISERUMS 583 Calmette,* von Pirquett and Morof may be used in human practice. Calmette’s ophthalmo test consists in the instillation in the eye of Koch’s purified or refined tuberculin. Purified tuberculin is prepared ‘by treating the original tuberculin with absolute alcohol, washing and drying the precipitate. One drop of a 1 per cent solution of purified tuberculin is placed in the eye. A positive reaction is manifested by a congestion of the palpebral and ocular conjunctiva a few hours after the application of the tuberculin. The method of von Pirquet* depends upon the cutaneous application of the tuberculin. One drop of tuber- culin (old) is placed on the arm, after cleansing the skin, and the small area under the drop is scarified. Two or more small areas may be treated in this way, as well as a control area treated with sterile salt solution or a solution of glycerin and dilute carbolic acid in substitu- tion for the tuberculin. The appearance of a reddish zone in from twelve to twenty-four hours indicates a positive reaction. This area of inflammation gradually increases somewhat in elevation and diameter and finally subsides in a few days. Moro’s modification of von Pir- quet’s method consists in the use of tuberculin ointment prepared by the combination of tuberculin (old) and anhydrous lanolin in equal parts. The ointment is vigorously rubbed on a small portion of the skin of the abdomen. A positive reaction is evidenced by the appearance of a distinct granular or papular eruption at the point of application after about twenty-four hours. For the diagnosis of tuberculosis in cattle, the intradermal test is generally regarded as next in importance to the older subcutaneous test. In conducting this test 0.1 to 0.3 c.c. of a 50 per cent solution of tuber- culin is injected into the cuticle layer of the skin at the base of the tail. A positive reaction is present when, twenty-four to seventy-two hours after the injection of tuberculin, the localized area of skin shows a circumscribed oedematous swelling. Tuberculin (old) is usually distributed in small vials, sealed and labeled. The labels should indicate the amount and dosage and the date of preparation. Under the influence of light and heat the fluid product may slowly deteriorate; therefore, when possible, it should be kept in the refrigerator until needed. * Calmette, Presse Medicale, 1907, 15. ft von Pirquet, Berl. klin. Woch., 1907, 44. fT Moro, Manch. med. Wch., 1908. 584 MICROBIOLOGY OF SPECIAL INDUSTRIES Other Tuberculins—Koch introduced tuberculin “T. R.” (tuber- culin residuum) in 1897 and tuberculin “B. E.” (bacillary emulsion) in t901. The former is prepared by repeatedly centrifugalizing a suspen- sion of the dried and ground tubercle organisms in water. The super- natant fluid “T. O.” after the first centrifugalization is discarded and the final product consists of the constituents of the bacteria which are insoluble in water. One cubic centimeter of the tuberculin ‘'T. R.” should contain the equivalent of 2 mg. of the dry tubercle solids. Tuberculin B. E. is composed of a suspension of crushed or thoroughly ground tubercle organisms in 50 per cent glycerin solution. Each cubic centimeter should contain the equivalent of 5 mg. of tubercle solids. Tuberculin T. R. and tuberculin B. E. are used as therapeutic agents, the latter probably being regarded with more favor by clini- cians. The material is administered by subcutaneous injection, the time intervening between successive treatments varying from three to ten days. The initial dose recommended by most investigators, is 0.0001 mg. or less. Matietn.—Mallein is prepared from cultures of Bact. mallet by practically the same methods as those employed in manufacturing tuberculin from Bact. tuberculosis. The product is used for the diag- nosis of glanders. A few hours after mallein is injected, subcutaneously, into glandered horses a severe local reaction and a rise of temperature usually follow. The thermal reaction is very similar to that produced in tuberculous animals by the injection of tuberculin. The local swell- ing caused by mallein treatment is considered by some to be quite as diagnostic as the temperature reaction. The ophthalmic mallein test, a comparatively recent method, which was first used by Choromansky, appears to be attaining considerable recognition as a valuable aid in diagnosis. The test consists in the application of concentrated mallein to the inner canthus of the eye. A drop of the concentrated mallein in liquid form or a small bit of the same in desiccated condition may be used. In a positive case, hyperemia and swelling of the conjunctiva and a purulent exudate at the inner canthus of the eye will appear from four. to six hours after the instillation of the mallein. ‘ Goodall* advocates the use of the intrapalpebral mallein test which * Goodall, Jour. Comp. Path. and Therap., 1915, Vol. 28, p. 281. é THE MANUFACTURE OF ANTISERUMS 585 involves the injection of a small dose of mallein under the skin of the eyelid. The stock culture of the glanders organism used in the preparation of mallein should be one which possesses known virulent properties. It is grown at a temperature of 37° for several weeks in flasks of glycerin bouillon having a chemical reaction of about three points acid to phenolphthalein. When the cultures are removed from the incubator they are heated in streaming steam, passed through a Berkefeld filter and the filtrate is concentrated, preserved and distributed in labeled vials. SUSPENSIONS FOR THE AGGLUTINATION TESTS Agglutinins are hypothetical bodies existing in the blood and possi- bly other body tissues, of an individual affected with, or convales- cent from, a specific infectious disease. The bodies possess the power of “clumping” and precipitating the specific bacteria which are the cause of the disease in question. Thus, if a dilution of blood serum from a typhoid fever patient is mixed with living typhoid organisms, the specific agglutinins present in the serum. will cause the organisms to cease their motion and agglutinate or-clump together in irregular masses. Normal human blood serum placed under the same conditions will fail to cause the agglutination of the organisms in similar dilutions. The agglutination reaction may, therefore, be used in the diagnosis of certain specific infectious diseases. The serum must be properly diluted in order that the reaction may be of diagnostic value, because undiluted, normal serum will cause a positive agglutination reaction in most cases. The agglutination test is used as a practical aid chiefly in typhoid fever in man and glanders in horses. The test may be conducted either microscopically or macroscopically. In the microscopic method, the diluted serum from the suspected case is placed under the microscope with the live, specific organisms in hanging drop. In the macroscopic method, the serum is added to an emulsion of the killed (heated) bacteria in small test-tubes, and the resulting reaction detected with the naked eye. The emulsion, suspension or “‘test fluid” for the typhoid agglutina- tion test is prepared from a pure culture of B. typhosus. The organism is grown for twenty-four hours upon agar at a temperature of 37°. 586 MICROBIOLOGY OF SPECIAL INDUSTRIES The growth is then removed from the surface of the agar, placed in sterile, physiologic salt solution and the organisms killed by heating on ' a water bath at a temperature of 60° for one-half hour. The emulsion is then roughly standardized by adding sufficient sterile, physiologic salt solution to impart to the fluid the required degree of cloudiness, ’ when compared with control emulsions. To the suspension of dead typhoid organisms or “test fluid” a preservative, usually formalin,. is added and the product is distributed in properly labeled bottles. In conducting the test, the suspected typhoid serum is placed in small tubes, each containing 1 c.c. of the suspension fluid, in such propor- tions that the serum is diluted 1:50, 1:100, and 1:200. A flocculent precipitate of the dead organisms indicates a positive reaction. Suspension fluid for the glanders agglutination test is prepared in practically the same manner as the typhoid test fluid. The glanders organisms are grown on acid agar and the suspension fluid is usually preserved by the addition of carbolic acid. In conducting the glanders . . agglutination test, the suspected serum is usually placed in the follow- ing dilutions: 1:200, 1:500, 1:800, 1:1200, and 1:1,800, The agglutination reaction has been applied experimentally and practically, with more or less success, in the diagnosis of Malta fever, Asiatic cholera, bubonic plague, pneumonia, tuberculosis, contagious abortion (bovine) and other infectious diseases. SUBSTANCES UsEepD ror D1aGnostic TESTS Luertin.—Noguchi* has developed a preparation known as luetin which is used in the diagnosis of syphilis. The material is prepared from a number of strains of Spirocheta pallida grown, under anaerobic conditions, on special ascites agar and bouillon media. After abundant growth of the spirochetes occurs, the agar cultures are ground and mixed into a paste. To this material fluid cultures are added in sufficient proportion to form a liquid emulsion. The organisms are then killed by heating at 60° for one hour and a preservative is added. In applying this diagnostic material to a suspected syphilitic case 0.05 C.c. is very carefully injected into, not beneath, the skin. An area on the antero-internal surface of the upper arm is usually chosen as the site of injection. A positive diagnosis of syphilis is indicated if, after * Noguchi, Jour. Exp. Med., xiv, Vol. 16. , THE MANUFACTURE OF ANTISERUMS 587 _the third day a marked cutaneous eruption appears at the point of inoculation. ANTIGENS.—Certain antigens, such as gonococcus and syphilitic antigen, are of value for the purpose of conducting complement fixation tests in laboratory diagnosis. Gonococcus antigen consists of an ex- tract or filtrate prepared from a suspension of polyvalent gonococci. Syphilitic antigen consists of an extract prepared from either luetic or certain normal tissues such as beef or human heart muscle. Tuber- culosis antigen, as described by Craig,* consists of the filtrates of specially prepared cultures of Bact. Tuberculosis. THE Scuick Trst.—The susceptibility or non-susceptibility of individuals to diphtheria may be determined by the application of the test described by Schick.t For this purpose standardized diphtheria toxin is required. o.1~-o.2 c.c. of a relatively fresh normal saline solu- tion containing 149 minimum lethal dose of diphtheria toxin, for a 250-g. guinea-pig, is injected intracutaneously. The appearance of a circumscribed area of redness at the site of injection after twenty-four to forty-eight hours indicates that the individual possesses practically no immunity against diphtheria. + Craig, Am. Jour. Med. Sci., 1915. 150, p. 781. * Schick, Munch, Med. Woch. 1913, 60, p. 2608. DIVISION VI* MICROBIAL DISEASES OF PLANTS INTRODUCTION Although the earliest study of bacterial diseases in plants antedates the isolation of the tubercle bacterium and the cholera spirillum, this branch of bacteriology has not been marked by the progress which has characterized the investigation of animal diseases. The loss of a human life or of a valuable domestic animal has appealed to the student of disease more strongly than the blighting of a pear tree, or the wilting of a potato vine, and, quite naturally, he has directed his efforts along those lines which have offered the greater inducements, and which have demanded immediate attention. - However, with the introduction of new plants, foreign seeds, and strange nursery stock, many previously unheard-of plant diseases have made their appearance. As the farming communities have become more thickly populated, with less uncultivated land between the fields, these diseases have spread from farm to farm more rapidly than in the earlier days, and the losses from these causes have been so heavy during the past decade that the farmers, gardeners and orchardists have come to the Agricultural Experiment Stations all over the country for advice and assistance in combating their troubles. This has stimulated an increased interest in plant diseases, especially along bacteriological lines, with the result that to-day some forty bacterial diseases af piants have been described. It is a matter of not infrequent observation that closely related species of plants, as well as animals, exhibit a marked difference in their susceptibility to the same disease-producing agents. The Bartlett pear, for example, suffers more severely from blight than the Kieffer, and, among apples, the Toleman Sweet more than the Rome; the small- leaf, stemmy varieties of tobacco seem to be more resistant to the Gran- ville wilt than the large-leaf kinds. Resistance of this sort, which ap- * Prepared by W. G. Sackett, except a protozoal disease ‘‘Fingers.and Toes" by J. L. Todd, revised by E. E. Tyzzer. 588 INTRODUCTION 589 pears to be nothing other than a natural, inborn quality, may be de- signated as natural immunity, and it is immunity of this kind which plant breeding for disease resistance has secured. A good illustration of this is to be found in the wilt-resistant water melon of the Carolinas, which is the result of crossing a naturally susceptible watermelon with a naturally resistant citron. Acquired immunity in the plant world is-a field veh to be explored. Cases have been cited in which active immunity appears to have followed the disease, but these are extremely rare and the evidence is very _ questionable. Passive immunity, at the present time, is unknown. CHAPTER I BLIGHTS Stem BLIGHT OF ALFALFA Pseudomonas medicaginis—Sackett History AND DistRIBUTION.—The disease has been known in Colo- rado since 1904 and was described briefly by Paddock in 1906 and more fully by Sackett in 1910. It is distributed generally over Colorado, and is reported to occur in Utah, New Mexico, Nevada, Nebraska and Kansas. Symptoms.—The disease is primarily a stem infection. In the earliest stages, the stems have a watery, semi-transparent, yellowish to olive green appearance along one side. Soon there oozes from the dis- eased tissue a thick, clear, viscid liquid which spreads over the surface and collects here and there in little bead-like droplets. The exudate al- so dries in a short time with a glistening finish, and gives the stems very much the appearance of having been varnished, and where the liquid has collected in little amber-colored scales and has hardened, it looks as if the varnish had run and dried. Stems in this condition have a dry, slightly rough feel to the touch. The exudate also dries uniformly over the surface or just beneath it, and there produces a dark brown, resinous surface which blackens with age. Such stems are very brittle and easily broken, which fact makes it almost impossible to handle the crop without an immense amount of shattering. The leaves attached to the blighted stems usually show the disease, and sometimes they exhibit the infection independent of the stem. In this case, the petioles become watery and pale yellow, then droop. The malady may be confined to the petiole and base of the leaflet, or it may involve the whole of the blade. Occasionally leaves are found where the inoculation has been made, apparently, in the margin of the leaflet, and the infection has proceeded toward the middle. In such instances, the tender tissue has a watery look, as if it had been bruised. 59° BLIGHTS 5901 One-year-old plants may exhibit blackened areas in the crown, and black streaks which run down into the tap root. As the plant grows older, this blackening increases until the whole crown becomes in- volved, and either the crown buds are destroyed or the root is no longer able to perform its functions, and the plant dies. So far as our present observations go, the disease appears to run Ts course with the first cutting, and those plants which have sufficient vitality throw out a good growth for the second and third cuttings. CAUSE OF THE DISEASE.—If a small piece of the yellowish green, watery tissue from a diseased plant, or a fragment of the dried exudate is placed in a drop of clean water on a glass slide, there will appear on all Fic. 144.—Pseudomonas medicaginis. Twenty-four hour culture on nutrient agar; stained with aqueous fuchsin; X1000. (Original.) sides of it, after half a minute, a dense, milky cloud, which can be seen readily with the naked eye, and which slowly diffuses out into the drop. When this preparation is examined under the low power of the micro- scope, this milky zone easily resolves itself into swarms of motile bacteria. The organism grows readily upon the ordinary culture media and pure cultures of the germ, inoculated into scarified stems of healthy al- falfa plants, produce the disease in seven to nine days with typical symptoms. 592 MICROBIAL DISEASES OF PLANTS , MEtuHop oF InFECTION.—Under field conditions the causal organ- ism which, presumably, lives in the soil, enters the plants early in the growing season with soil through stems which are cracked and split by late freezing. In some instances, inoculation appears to take place by stomatal and water pore infections. CausAaL OrcANniIsm.—The writer has given the name Ps. medicaginis to the causal organism, the characteristics of which are as follows: It is a short rod with rounded ends, about 1.24 to 2.44 by o.5u to 0.84 the majority being 2.14 by 0.7p. It is actively motile by 1 to 4 bi-polar flagella; non-spore forming and non-capsule forming. Filament formation occurs frequently. The organism stains readily with the aqueous stains, but is Gram-negative. It produces a surface pellicle on broth. Shining, grayish white on nutrient agar, becomes fluorescent green after three days. Gelatin stab, surface growth only, and no liquefaction. Potato discolored, moderate growth, cream to light orange yellow, starch not destroyed. No growth in Cohn’s solution. Good growth in Uschinsky’s solution. Plain milk shows no change. Litmus milk becomes bluer after seven days, no curd and no peptonization in thirty days. No indol. No hydrogen sul- phide. Ammonia produced from asparagin solution, Dunham’s solution and nutrient, broth, but not from nitrate broth. Nitrates not reduced. No gas and _ no acid from dentin etc. Obligative aerobe.. Optimum temperature 28°; no growth at 37.5°. Thermal death-point 49.0° to 50.0°. Habitat, soil. Pathogenic for alfalfa (Medicago sativa). Controu.—The only practical way of combating ae controlling the blight is by the introduction of resistant varieties, but no entirely resistant strain has been obtained up to the present time, although the Grimm alfalfa is practically free from it. As a means of control, the writer recommends that the frosted al- falfa be clipped, as soon as there is reasonable certainty that danger from late frosts is past. This will rid the plants of the diseased por- tions, and afford an opportunity for the early growth of a new cutting. If this is done in time, the regular number of cuttings should be secured with little or no loss in tonnage. BACTERIOSIS OF BEANS Pseudomonas phaseoli—Erw. Smith Frequently the foliage, stems, and pods of the common beans, as well as the Lima bean are attacked by a bacterial disease. Symptoms.—The pods and leaves seem to furnish the best food supply for the microdrganism, and it is here that we find the most BLIGHTS 593 typical lesions developing. Small, reddish spots appear which in- crease rapidly in size and develop into watery, amber-colored blisters, surrounded by a pink or reddish border. These blisters are filled with myriads of bacteria, and in time, they dry down, forming a pale yellow or amber-colored crust over the affected areas. Ultimately the dis- eased leaves become brittle, ragged, and are worthless, while the pods curl, shrivel, and rot. METHOD OF INFECTION.—It is believed that the disease is intro- duced with the seed, and when once established, is spread from plant to plant by rain, dew, and leaf-eating insects. Causa Orcanism.—Ps. phaseoli Smith,* is a short, motile rod with rounded ends, which produces a characteristic yellow growth on the different culture media. Gelatin slowly liquefied. Milk becomes slowly alkaline, casein is precipitated by lab ferment and partially redissolved. Very marked diastatic action on potato starch. No gas from glucose, saccharose, etc. Aerobic. Uschinsky’s solution, growth feeble and retarded. Thermal death-point 49.5°. Controi.—Care should be taken to select seed from healthy fields where the disease has never occurred. The disease has been partially controlled by spraying with Bordeaux mixture when the plants were 2 to 3 inches high, again ten days later, and after blossoming. BuicHt oF LETTUCE Ps. viridilividum—Brown The disease has been reported recently from the lettuce-growing sections of Louisiana, and is described as producing a shriveled, dried, burned aspect of the outer leaves, some of which may be in a soft, rotted condition. The deeper leaves exhibit numerous separate or fused spots with a water-soaked appearance; the center of the head is not necessarily involved. Causal OrGANISM.—Miss Nellie A. Brown{ has described the causal organism, Ps. viridilividum, as a short rod with rounded ends, motile by 1-3 polar flagella; stains readily with the ordinary stains; is Gram-negative. No spores have been observed. In young agar cultures, the growth is cream-white mottled with yellow, the mottling disappearing with age. Gelatin is liquefied slowly. Nutrient broth * Smith, Erw., Proc. Am. Asso. Adv. Sci., 46, 228-290, 1897. { Brown, Nellie A., “A Bacterial Disease of Lettuce,” Jour. Agr. Res., Vol. IV., No. 5, P. 475, 1915. 38 594 MICROBIAL DISEASES OF PLANTS is clouded and becomes lime-green in color after ten days. On potato it produces a characteristic transient dark-blue green color which develops promptly and dis- appears on the sixth day or earlier. Growth develops readily in Uschinsky’s and Fermi’s solutions changing them to a pale green color in three to five days; faint growth occurs in Cohn’s solution. Plain milk is cleared without coagulation, the cleared fluid becoming a pale turtle-green color; litmus milk becomes deeper blue. Gas is not produced from the ordinary sugars in Dunham’s solution. Nitrates are not reduced, and some indol is formed. Method of Infection.—Inoculation experiments indicate that infection may take place either through the stomata or through wounds produced by mechanical injury. / : Control.—No control measures have been reported. BuiicHT OF MULBERRY Pseudomonas mori—Boyer and Lambert (Smith) History.—The disease was first studied in 1890 by Cuboni and Garbini in Italy, and later by Boyer and Lambert in France who named the causal organism Bact. mori, but did not describe it. In 1908, Erwin F. Smith* found a similar disease in some of the Southern States, and described the causal organism. Symptoms.—According to Erwin Smith, the blight attacks the leaves and young shoots of the mulberry, producing first water-soaked spots, which later become sunken and black; “‘foliage more or less distorted; shoots soon show sunken black stripes and dead terminal portions. Action of disease rather prompt.” In very young shoots, wood, pith and bark are invaded by bacteria; in older shoots the germs are confined mostly to the xylem. CausaAL OrGANIsM.—The organism is a rod with rounded ends, 3.6u by 1.2u, motile by 1 to 2 polar flagella, attached to one end. No spores observed; pseudo- zoogloea occur. Stains readily with carbol fuchsin; Gram-negative. On agar, spreading, smooth, dull, translucent, shiny, white; medium not stained. On potato, spreading glistening, smooth, white to dirty white, shiny, medium grayed, slight action on starch. Gelatin stab, filiform, no liquefaction. Beef broth, pellicle, strong clouding. Milk, no coagulation, rendered alkaline, becomes clear by solution of fat and casein, litmus not reduced. No growth or scant in Cohn’s solution. Uschinsky’s solution, copious, pellicle, not viscid fluid, bluish-fluorescent color. No gas from dextrose, saccharose, etc. Aerobic. No indol or slight. Nitrates not reduced. Thermal death-point 51.5°; does not grow at 37°. * Erwin F. Smith, Bacterial Blight of Mulberry, Science N S., Vol, XXXI, 803. x BLIGHTS 505 BLADE BLIGHT OF OATS Pseudomonas avene—Manns and Bacillus avene—Manns History AND Distrisution.—A specific bacterial disease of oats has been described by Manns in 1909. What appears to have been a similar trouble, extending from the Atlantic coast west to Indiana, and from the Great Lakes to the Gulf States, was observed as early as 1890 by Galloway and Southworth. Its appearance was noted for the first time in Colorado in 1915. Symptoms.—In the early stages of the disease there is ‘‘a yellowing, beginning either as small round lesions on the blade, or as long, streak lesions extending throughout the blade or even the whole length of the culm and blades. In the advanced stages, the affected blades take on a mottled to almost red color, which has been called ‘rust’ and ‘blight.’” CAUSE OF THE DISEASE.—The disease is produced by the symbiotic growth of two bacteria whose activity is favored by rainy, humid, and cloudy weather. One of these organisms, Ps. avena, alone, is said to be capable of effecting the blight in a mild form, while the other, B. avene, is nonpathogenic; but a mixture of the two germs results in an aggravated attack. MeEtuHop oF InFEctTIoN.—Infection takes place through the stomata, the organisms being spattered on the leaves from the soil by rains. Grain insects are also responsible for spreading the disease. ControL.—It is believed that the control of the disease lies in the selection of resistant strains. STEM BLIGHT OF FIELD AND GARDEN PEAS Pseudomonas pisi—Sackett History AND DistTRrBuTION.—The disease occurs in several of the Western States, particularly in the mountain valleys of the higher altitudes. It was first observed in Colorado in 1915, where it caused a loss of approximately one-third of the field peas in the San Luis Valley, while in other parts of the State where garden peas are grown for can- ning purposes, the crop was materially affected. Symptoms.—The plants usually show the infection before they are 8 inches high, and many succumb before they reach that size. Both * Manns, ‘‘The Blade Blight of Oats, A Bacterial Disease,” Bull. 210, Ohio Exp. Sta., 1909. 596 MICROBIAL DISEASES OF PLANTS field and garden peas are affected alike, and the symptoms simulate the bacterial stem blight of alfalfa. The stems have a watery, olive- green appearance which soon becomes olive-brown, and in the last stages dark brown. The leaves and stipules appear watery at first, as if bruised, and later turn ocher yellow in color; this is often accompanied by wilting. In young plants, the discoloration of the stems is followed by a shrivelling, and ultimately the plants dry up and die; in the older ones, where the infection has taken place later, the same condition may result, but on the whole, the disease appears to be less serious, and in some cases the plants seem to outgrow the blight. Frequently when the first and earliest shoots are destroyed, the plant throws up new shoots from below ground, and a good late crop is obtained, in spite of the trouble. CAUSAL ORGANISMS.—Pseudomonas pisi, n. sp., as described by Sackett,* is a short rod with rounded ends, motile by means of a single polar flagellum; neither spores nor capsules observed; filaments formed commonly; stains readily , with aqueous stains, and is Gram-negative. It produces a flaky surface scum with heavy clouding in broth. On nutrient agar the growth is smooth, glistening, grayish white, and the medium is not dis- colored. Gelatin is liquefied rather rapidly. On potato, smooth, glistening, cream to orange-yellow; medium becomes grayish brown. No growth in Cohn’s or Uschinsky’s solutions. Heavy clouding with white surface pellicle in Fermi’s solution; clouding with surface scum in Fraenkel’s solution; slight, transient cloud- ing in Naegli’s solution. Plain milk is coagulated, and the coagulum is slowly peptonized, the supernatant liquid becoming yellowish green. Litmus milk becomes bluer, and the litmus is reduced, the liquid becoming greenish-gray. Neither indol nor hydrogen sulphide is produced. Ammonia is produced from asparagin and peptone. Nitrates are not reduced. No gas is formed from sugars, but acid is produced from dextrose, saccharose and galactose. Obligative aerobe. Optimum temperature 25° to 28°. Thermal death-point 50°. Habitat, soil. Pathogenic for field pea and garden pea (Pisum sativum var. arvense and Pisum sativum). : Metuop or InrectT1ion.—Experimental inoculations indicate that infections take place either through the stomata or through wounds produced by mechanical injuries. Controt.—There seems to be a close relation between the preva- lence of the disease and a late, cold spring. The low temperatures appear to make the plants more susceptible, and as a result the early * Sackett, Walter G., “Stem Blight of Field and Garden Peas—A Bacterial Disease,’ Bull. 218, Colorado Exp. Sta,, April. 1916. BLIGHTS 597 plantings are the worst affected. As a control measure, planting from two to three weeks later is suggested. PEAR BLIGHT Bacillus amylovorus—(Burrill) De Toni History AND DIsTRIBUTION.—As early as 1780, William Denning, a fruit grower, who lived on the Highlands of the Hudson River, ob- served pear blight in the trees of his neighborhood. It is very probable Fic. 145.—Two pear twigs. The upper one affected with Fire Blight, the lower one healthy. (After Sackett, Mich. Agr. Exp. Sta.) that blight existed many years before this in eastern North America on some of our native wild crabs, hawthorns, and wild plums, and with the introduction of cultivated varieties, it found a new field for attack. As the farming communities became more thickly populated, and the orchards more numerous, it has spread gradually westward over the , ~ 598 MICROBIAL DISEASES OF PLANTS Allegheny Mountains into the Mississippi Valley, across the Great Plains, and over the Rocky Mountains to the Pacific Coast. So gen- erally is it distributed over the United States and Canada that a blight- free orchard is, indeed, a rare sight. The disease has progressed with such severity that, to-day, commercial pear growing in Colorado has been practically abandoned, and the industry in California is being threatened with destruction. So far as our present knowledge goes, the blight is of American origin and is confined to North America. OccuRRENCE.—While the ravages of the disease are worst upon the pear, from which fact the disease derives its name, many varieties of the apple, quince, apricot and plum, together with the mountain ash, service berry, wild crabs and several species of hawthorn, have suffered severly from the same cause, and are capable of transmitting the disease from one to the other. ' Symproms.—The disease is most isily recognized during the grow- ing season, when it attacks the blossom clusters and the tips of the growing twigs. In this formitisknownas blossomand twig blight. The leaves attached to these parts usually turn brown or black, either wholly or in part, the petioles blacken, and the young twigs show a blackened; shriveled bark, having much the appearance of green brush which has been burned only partially. It is from these symptoms that we get the name Fire Blight, so appropriately applied to pear blight. The blackened, withered leaves cling tenaciously to their blighted twigs long after the other leaves have fallen in the fall, and in this \ way afford the orchardist an easy way of recognizing the blighted areas. Frequently the disease finds its way into the larger limbs and even the trunk of the tree, where it produces body blight. This form is characterized in the early stages by a cracking of the bark and the oozing of a thick, dirty white or brown, sticky liquid which collects here and there in drops over the injured surface. As the disease progresses, the splitting of the bark increases and the area involved becomes rough, giving rise toa canker. This is not to be confused with sun scald, in which the bark dries down and adheres firmly to the wood beneath, and which is due to an entirely different cause. The immature fruit manifests the blight by turning black, shriveling and taking on a dried, mummified appearance. Accompanying these changes, drops of a thick, sticky exudate usually appear on the surface. If a cross section is made of a diseased twig or limb, one invariably | Se BLIGHTS 599 finds a blackened ring in the region of the cambium layer. This phenomenon, the significance of which will be explained later, serves as a reasonably reliable means of diagnosis. CausE.—A microscopic examination of either the blackened cam- bium or a drop of the exudate shows swarms of motile rods, B. amylo- vorus, which Burrill of the University of Illinois, as early as 1878, credited with being the cause of pear blight. By inoculating healthy trees with this gummy material, he was able later to demonstrate his point experimentally, and with his work and that of a Dutch Botanist, Wakker, we have the begining of the study of bacterial diseases of plants. MEeEtHops oF InFECTION.—The more careful observers believe that insects, especially bees, plant lice and twig borers are responsible for the initial infection and subsequent spread of the disease. It has been found that the bacteria find protection from the adverse conditions of winter in the margins of the old cankers next to the sound bark, and also in some of the blighted shoots and twigs.* These hold-over bacteria become active with the increased flow of sap and the higher temperature of spring, and soon spread into the adjacent healthy bark. Here they multiply so rapidly that at about the timet the trees are in blossom, they begin to ooze from the cracks in the diseased bark as drops of a thick, sticky material, dirty white or brown in color. Insects are attracted to this ooze, apparently feed upon it, smear their feet, bodies and mouth parts, and then fly away to the opening blossoms. Here they feed upon the nectar and while so doinginfect the flowers. The germs increase rapidly in this sweet liquid, and each bee that visits the flower subsequently carries away millions of germs to’ infect other blossoms. From the flowers, the bacteria find their way into the cambium and softer tissues of the bark, where the disease is confined almost entirely. After about ten days the progress of the germs can be noted by the blackening of the flower clusters, and the wilting and blackening of the leaves of the fruit spurs. Following the collapse of the fruit spurs, the disease may move down the twig an inch or more a day, causing it to appear watery, turn black and shrivel. The blackening may be 10 to 12 inches behind the advancing infection. * The writer examined a number of blighted rear twigs Apr. 14, 1911, collected from different orchards in Colorado and found B. emylovorus alive in 23.53 per cent. The germs occurred in the 2 cm, adjacent to the healthy part of the twigs. ' "4 Whetzel, Bull. 272, Cornell Exp. Station, 1909, 600 MICROBIAL DISEASES OF PLANTS This may continue until the whole limb becomes involved, but as a rule it is only the smaller twigs which are the worst affected. From this it will be seen that the external blackening cannot be relied upon, early in the season at least, as a guide to the exact location of the disease; however, as the season advances, the plant tissues harden, conditions for germ life become less favorable, and as a result, by the middle of summer, the active progress of the blight is checked by natural causes, and the blackening overtakes the advancing infection. Blight which appears on the water sprouts of large limbs later can usually be accounted for by inoculation by plant lice and the pear twig borer. CausaL Orcanism.—According to Jones* Bacillus amylovorus possesses the following characteristics: Short motile bacillus, rounded ends, ry-1.84 by o.su— 0.94; stains readily with the aqueous stains; Gram-negative. No spores observed. Agar slant and potato, growth moderate, filiform, glistening, smooth, grayish white, semi-opaque, butyrous. Gelatin stab, growth rather slow, filiform, slight crateriform liquefaction after twenty days. Nutrient broth, moderate clouding, uniform; if left undisturbed, a delicate pellicle or ring may form which breaks up and sinks with the slightest jar; scant finely granular sediment after ten days. Litmus milk, light blue in four days, pinkish in six days, light blue again in twelve days, upper layer blue in eighteen days; soft gelatinous curd six to ten days, with whey on the surface. Cohn’s solution, no growth. Uschinsky’s solution, no growth. Nitrates not reduced. No indol. Thermal death-point 50°. Optimum tem- perature 23° to 25°. Slight acid production but no gas from dextrose, etc. Starch. is not fermented. ConTROL.—It is obvious that spraying is useless for a disease of this character, where the germs are located beneath the surface. A systematic cutting out of the diseased limbs and twigs wherever and whenever they appear is the only practical method of controlling the blight. It is almost impossible to get all of the diseased material in the summer time when the heavy foliage hides it, but in the fall and winter the blighted branches cqn be recognized very readily by the tufts of dead leaves clinging to them. It is necessary in removing the dead wood to cut well below the discolored part, 10 to 1s inches, for the bacteria may be considerably in advance of the discolored area. Clean out all old cankers by cutting well into the healthy part and by removing the dried, diseased material. Disinfect the freshly cut sur- faces of this wound as well as the exposed ends of twigs and limbs with * Jones, D. H., The Bacterial Blight of Apple, Pear and Quince Trees. Bu!l. 176, Oatario Agr. College. BLIGHTS 601 1: ooo solution of mercuricchlorid. All diseased wood must be collected and burned. STREAK DISEASE OF SWEET PEAS AND CLOVERS Bacillus lathyri—Manns and Taubenhaus History.—The first recorded observations of this disease were made by Diggs on sweet peas in Dublin, Ireland in 1904. The trouble was known locally as “Streak” disease of the sweet pea, and various parasitic fungi were assigned as the cause. One investigator even ventured the assertion that the malady was of a physiological nature. In 1912, Taubenhaus isolated a bacillus from clovers and sweet peas collected in the vicinity of Newark, Delaware, and which bore lesions similar to those described for “Streak.” Subsequent inoculations with pure cultures proved the disease to be of bacterial origin and identical with that observed in England and Ireland. SymptToms.—The disease makes its appearance during the season of heavy dew and is characterized by light reddish-brown to dark brown spots and streaks, almost purple when old, along the stems. They usually originate near the ground, which seems to indicate distri- bution by spattering rain and infection through the stomata. The disease is quickly distributed over the more mature stems, and ulti- mately the cambium and deeper structures are destroyed in con- tinuous areas resulting in the premature death of the plant. Occa- sionally the petioles and leaves show the infection; the latter exhibit the water-soaked areas common to bacterial stomatal infections such as are met with in alfalfa blight and bacteriosis of beans. CausaL OrGANIsM.—Manns and Taubenhaus have described the organism which is responsible for the disease as a new species under the name Bacillus lathyri. It is a small rod, motile by means of 8-12 short, peritrichiate flagella; it grows lux- uriently upon all of the common nutrient media, especially if sugars are present, producing a yellow pigment; on glucose agar, colonies appear in twenty-four to thirty-six hours, showing a tendency to become stellate or auriculate. PaTHOGENESIS.—Bacillus lathyri, n. sp. has been isolated from specific lesions on the following hosts: Sweet pea, Lathyrus spp., red, alsike and mammoth clovers, soy beans, garden beans, cow peas and alfalfa. MeErtuHop oF InFectTion.—Infection appears to take place through the stomata, the organism being spattered on the plants from the soil during rains. 602 MICROBIAL DISEASES OF PLANTS ConTrot.—On small areas, heavy mulching of straw along either side of the row is suggested as a possible means of preventing the distribution of the disease. Tomato BLIGHT Bacterium (?) michiganense—Erw. Smith* The disease is distinct from the wilt, caused by B. solanacearum, in that there is not the sudden collapse of the whole plant, but rather a slow yellowing or wilting of the leaves, one at a time. The causal organism produces cavities in the pith and bark as well as in the vascular system. ' WaLnut BLIGHT OR BACTERIOSIS Pseudomonas juglandis—Pierce History AND DisTRiBuTION.—Attention was first called to this dis- ease as it occurred in California by Piercet in 1893 although it had been observed in Los Angeles County in about 1891. Outside of California, it is known to occur in Oregon, Texas and midway down the Pacific coast of Mexico. What appears to be a similar trouble has been reported from New Zealand and France. Symptoms.{—All of the new, tender, growing parts of the tree, such as young nuts and branches, petioles of leaves, midveins, fine lateral veins and adjoining parenchyma are subject to the attacks. On the branches, the disease always starts in the young succulent growth and manifests its presence by small, discolored areas which under favorable conditions may extend 2 to 3 inches along the green shoot. As the infection progresses, the central portion of the lesion turns black and is surrounded by a water-soaked margin. In the later stages, the whole diseased area becomes blackened and in many instances has a somewhat shrunken, dried-out, deformed, cracked appearance due to the drying out of the tissue. In severe cases the tissue is killed inwardly to the pith, while in the milder attacks only the bark and wood are diseased. As the wood hardens, the infection is checked, and the vitality of the tree is not affected to any extent, the * Smith, Erw., Science, N. S., Vol. XXXI, 803, p. 794, 1910. _t Pierce, N. B., Bot. Gaz., 31; 272-273, I90I. } Smith, C. O., ‘Walnut Blight,” Bull. 231, Calif. Exp. Sta., 320, 1912. BLIGHTS 603 crop suffering rather than the tree. The leaves sometimes exhibit a blackening or browning of the petioles and veins, while the intermediate tissue may develop brownish, circular or angular spots. The disease does not cause serious defoliation of the tree. The catkins are probably not affected. It is upon the young nuts that bacteriosis is especially Fic. 146.—Walunuts affected by Bacteriosis, mostly stigma or blossom-end infection. (After C. O. Smith, Calif. Bull. 231.) destructive, and it would be of little economic importance did it not attack these. Many of the nuts may become infected and fall when they are }¢ to 14 inch in diameter and continue to drop throughout the summer. A conservative estimate of the loss places it at 50 per cent in badly diseased groves. .The most common point of infection is at the blossom end, although, it may start at any place on the nut. In 604 MICROBIAL DISEASES OF PLANTS ‘the early stage, the lesions appear as small, circular, raised, discolored, water-soaked areas; later, these spots-increase in size and turn black. Under favorable conditions, the disease may extend through the hull and shell-forming tissues into the kernel which at length becomes blackened and finally destroyed. CausaL OrGANISM.—According to Smith, C. O., Pseudomonas juglandis, Pierce, isa rod with rounded ends; single or in pairs, rarely in chains; measures 1.54 to 3.014 X 0.3 to 0.51; stains readily with the ordinary aniline dyes; Gram-positive; spores and capsules not observed; motile by means of a single polar flagellum; agar colonies nucleated, circular, moist, shining, pale yellow with regular margins; startiform liquefaction in gelatin; potato, abundant, moist, shining, slimy, raised, white chang- ing to yellow; uniform turbidity and ring in bouillon, slight flocculent precipitate; indol produced; nitrates not reduced; enzymes, diastasic, cytohydrolytic, rennet, proteolytic; milk coagulated, curd digested; litmus milk wine colored; viability, nine and one-half months on potato; methylene-blue milk reduced. Metuop oF Inrection.—It has been shown that the causal organisms live over winter in the old lesions of the wood and bark and that in the spring they exude to the surface and are carried to the new growth, to which they gain entrance through the stomata. The disease is most severe during seasons when the fogs and rainfall are heaviest, and in those localities where rain and fogs are abundant. ‘During one of these fogs the trees become saturated, water dripping from one portion of the tree to another which could easily carry the disease organisms to healthy tissue.” Distribution by this means is thought to be one of the most important, if not the most important, methods of spreading the trouble. Insects probably play some part in the dis- semination of blight. PATHOGENESIS.—Pathogenic for Juglans regia (English) under natural conditions; pure culture inoculations give positive lesions on Juglans nigra (eastern black), Juglans hindsii (nothern Cal. black), Juglans californica (southern Cal. black), Juglans cinerea (butternut). ConTROL.—Systematic spraying experiments with Bordeaux mix- ture, lime-sulphur, and a sulphur spray have demonstrated that spray- ing is impracticable and has little value as a means of control. Applica- tions of lime to the soil have resulted in no benefit. It has been observed that individual trees exhibit great differences in their natural resistance to the blight, and at the present time the selection and propagation of varieties which are more or less immune promises the most practical solution to the problem. CHAPTER II GALLS AND TUMORS Crown GALL Pseudomonas tumefaciens—Erw. Smith and Townsend Crown gall is one of the most recent plant diseases to be traced to bacterial origin. Its occurrence is so common in nursery stock that in a certain Western State, 75 per cent of the young trees and shrubs condemned by nursery inspectors are condemned for crown-gall, and Toumey places the annual loss to orchardists at $500,000 to $1,000,000. Fic. 147.—Crown gall with hairy root on nursery stock. Northern Spy apple. (After Paddock.) History.—Smith and Townsend* working with the gall of the Paris daisy observed bacteria in these outgrowths in 1904, but it was not until 1906 that they succeeded in isolating the causal organism and * Smith, Erw. F., and Townsend, C. O., ‘A Plant Tumor of Bacterial Origin,’’ Science, N. S. Vol. XXV, No. 643, p. 671-673, 1907; “The Etiology of Plant Tumors,”’ Science, N. S. Vol. XXX, No. 763, p. 233, 1909. Townsend, C. O., ““A Bacterial Gall of the Daisy and Its Relation to Gall Formations on Other Plants,’”’ Science, N. S. Vol. XXIX, p. 273 (Abstract), 1909. 605 606 MICROBIAL DISEASES OF PLANTS in securing satisfactory re-inoculations. Subsequent studies* have shown that this same microdrganism is responsible for the pathological condition that we recognize as crown gall in its various forms on the different hosts. One of the remarkable things about this disease is the large number of families which are subject to the infection. PaTHoGENEsIS.—A partial list of the plants upon which crown gall occurs naturally or upon which it has been produced by laboratory inoculation includes the daisy, tomato, tobacco, potato, carnation, peach, rose, cabbage, grape, hop, sugar-beet, turnip, red beet, carrot, radish, chrysanthemum, oleander, marigold, pyrethrum, almond, clover, white poplar, Persian walnut, Pterocarya, gray poplar, cotton, alfalfa, raspberry, geranium, apple, willow, quince. Symptoms.—The swellings or galls, small at first, usually appear just below the ground line (crown), at or near the juncture of the stock and scion. These may be either hard or soft galls; the former are smooth, soft, spongy, white to flesh-colored outgrowths which may reach a very appreciable size during one season and then be entirely decomposed and disappear by the following spring; the latter increase in size more slowly, persist year after year, harden and become rough and warty on the surface with age. Both are crown galls and both are produced by bacteria. According to Smith,f ‘Whether a crown gall shall develop as a hard gall or a soft gall would seem to depend chiefly if not altogether on which meristem cells receive the initial impulse. If the cells first infected are principally the mother cells of medullary rays, we may assume that the gall will be a ‘soft gall,’ and readily inclined to decay. If, on the contrary, the needle or other carrier of infection wounds principally those meristem cells which give rise to tracheids and wood fibers, the gall will be a ‘hard gall,’ of slow growth and long duration.” The structure of the galls is unlike that of club- root of cabbage in that the latter is an hypertrophy while the former is an hyperplasia. Frequently this disease assumes a form known as “hairy root” characterized by the presence of bunches or tufts of closely matted rootlets with enlargements at their bases. As the gallst enlarge, the function of the adjacent conducting tissue is interfered with, and . * Smith, Erw. F., Brown, Nellie A., Townsend, C. O., “‘Crown-gall of Plants: Its Cause and Remedy,” Bull. 213, Bur. Plant Ind., U. S. Dept. Agr., 1911. { Smith, C. O., “Further Proof of the Cause and Infectiousness of Crown Gall,” Bull. 238) Calif. Exp. Sta., 1912. © tT Very hairy roots often accompany these. ome GALLS AND TUMORS 607 . the circulation is impaired, as is shown by the poor growth and dwarfed appearance of the trees. The development of this disease is looked upon by Smith* and his associates as paralleling closely what takes place in cancer in man and animals. The primary tumors have been observed to send out “‘roots” or tumor-strands for some distance into the normal tissue, and from these tumor-strands, secondary tumors may arise which tend to take on the structure of the primary tumor, ¢.g., “if the latter is in the stem and the former in a leaf, the secondary tumor shows a stem structure.” “There are no metastases in crown gall * * * for whether a cancer shall be propagated by floating islands of tissue, or only by tumor- strands, appears to be a secondary matter depending upon the char- acter of the host tissues rather than the nature of the disease.” The salient point is the internal stimulus to cell division which arises from the presence of the microdrganisms within certain cells. MeEtuop oF InFrection.—Little is known about the natural channels of infection, but inoculation through wounds induced by poor grafting careless cultivation, and by borers, nematodes, etc., is undoubtedly responsible for many crown galls. CAUSAL ORGANISM.—Pseudomonas tumefaciens is a short rod with rounded ends, motile by 1-3 polar flagella; measures 1.2 to 2.54 by 0.5 to o.8u; neither spores nor capsules demonstrated; pseudozoogloeee occur; involution forms present; stains with the usual anilin stains; Gram-negative; on agar, slow, four to six days at 25°; filiform, raised, white, glistening, somewhat slimy; potato, growth rapid, white, smooth, wet-glistening; gelatin stab, filiform, no liquefaction; moderate, flat, filiform, white, smooth, glistening, no liquefaction; blood serum, moderate; broth, ring or pellicle, clouding absent or inconspicuous; milk, coagulation delayed, curd not peptonized, litmus gradually blued then reduced; silicate jelly, slow white growth; Cohn’s solution, scanty or absent; Uschinsky’s solution, scanty, not viscid; NaCl bouillon, 4 per cent inhibits, 3 per cent retards; bouillon over chloroform, growth unrestrained; no gas from sugars; ammonia is produced; nitrates not reduced; indol production small; thermal death-point 51°; optimum reaction between +14 and +24 Fuller’s scale; opt. temp. 25° to 28°, max. 37°, min. positive at 0°; killed readily by drying; moderately sensitive to sunlight; invertase and rennet thought to be produced. ControL.—Thorough inspection of nursery stock and care in the cultivation of orchards not to wound the crowns are important factors. * Smith, Erw. F., Brown Nellie A., McCulloch Lucia, “The Structure and Development of Crown Gall; A Plant Cancer. Bull. 255, Bur. Plant Ind. U. S. Dept. Agric., ro12. 608 MICROBIAL DISEASES OF PLANTS ¢ Plant’ on uninfected land and avoid heeling in healthy stock into soil that has previously borne diseased plants. Remvoing the galls results in no practical benefit. OtivE Knot. Bacterium savastanoi—Smith* ft The olive knot has been known for many years, and is even des- cribed by the early Roman writers; its bacterial nature, however, has been recognized only since 1886. It is most prevalent in those countries which border on the Mediterranean Sea, but it also occurs in the olive growing sections of California. So far as is known, the causal organism enters the ena and leaves of the olive through wounds, and there produces roughened, wart-like swellings. The growth of the knots usually begins in the spring, and later in the season, if the trees are badly diseased, they show scant ‘foliage, limited growth, and occasionally dead branches, especially where the galls have entirely encircled the twigs. “FINGERS AND ToES” or “Stump Root” oF CABBAGESt Plasmodiophora brassice—Woronin This organism which is classified as a rhizopod by many is the cause of a common disease of the roots of cabbages and of other. crucif- erous plants. The disease is sometimes called “fingers and toes” and it may cause much damage in market gardens. In it the roots are greatly hypertrophied appearing distorted and lumpy, like fingers bent and swollen with rheumatism. The disease may be controlled to some extent through the destruction, by burning, of all infected material as soon as the disease is recognized. It is usually considered well to rely on the rotation of crops or, in case the soil has become generally infested, to plant crops of another type for several years in order to prevent losses from this infection. The plants attacked are recognized by their stunted appearance and * Smith, Erw., Bull. 131, Part IV, Bur. of Plant Industry, U. S. Dept. of Agriculture, 1908- + Savastano, L. Les maladies de l’olivier et la tuberculose en particulier. Comp. Rend. 103 1144, 1116. II bacillo della tuberculosi dell’olivo, nota suppletiva. Rend. Lincei 5:92-94 1889. ¥ t Prepared by J. L. Todd and revised by E. E. Tyzzer. GALLS AND TUMORS 609 the tendency of the leaves to wilt or turn yellow when an examination of the roots will at once reveal the distinctive features of the disease. The spores are liberated with the disintegration of the diseased roots and become disseminated in the soil during cultivation. Under ap- propriate conditions the spore is ruptured and a small flagellated, amoeboid organism emerges. It is in this form that the parasites Fic. 148.—Roots of Cabbage plant showing characteristic hypertrophy due to Plasmodiophora brassice. (Woronin.) penetrate the roots of the young plants in which they complete their development. The youngest forms seen within the vegetable cells possess two nuclei each with a central mass of chromatin or karyosome. Several organisms frequently invade a single cell. As they grow there is a multiplication of nuclei and the associated organisms tend to fuse together to form plasmodia. Subsequently there occurs a series of changes, certain stages of which are readily distinguished but also 39 610 MICROBIAL DISEASES OF PLANTS others which are more difficult to follow. The nuclei first lose the greater part of their chromatin and appear pale and indistinct, while attraction spheres appear at the opposite poles of each. The nuclei then divide twice by karyokinesis and a small amount of cytoplasm is separated off, constituting a gamete. The gametes now unite in pairs and each pair becomes encysted to form a spore. Fic. 149.—Plasmodiophora brassice. A, a plant cell filled with parasites the nuclei of which are undergoing mitotic division (at the top,is the nucleus of the plant cell). 3B, two plant cells with developed and partly developed spores. (After Prowazek, from Doflein.) Whether during the multiplication of these organisms in the plant, they are able to migrate to other cells and thus spread the infection has been questioned. A number of investigators believe that the number of infested cells is only increased by the division of the infected plant GALLS AND TUMORS 611 cells which not only are greatly enlarged but also show evidence of proliferation in the presence of dividing cells. The hypertrophy of the plant cell is associated with hypertrophy of its nucleus and it is evident that the growth and increase of the parasite is favored by the reaction which its presence excites. TUBERCULOSIS OF SUGAR-BEET ‘Pseudomonas beticola—Smith History.—This new disease of the sugar-beet, resembling somewhat crown gall on the surface, but distinct from it, was first observed in the autumn of rgro on beets from Colorado and Kansas. Symptoms.—Affected beets bear numerous wart-like outgrowths or tubercles on the upper portion of the root. On section these show small, water-soaked, brownish areas with more or less necrotic tissue in their interiors; such areas may develop small central cavities, and the softening may extend into the ungalled part of the beet; the dist eased parts appear mucilaginous and stringy when touched, and under the microscope this broken-down tissue is found to be swarming with bacteria. CausAL Orcanism.—According to Smith* Pseudomonas beticola, n. sp., is a motile rod with rounded ends, single or in pairs, chains or clumps; measures 0.6 to 0.8 by 1.5 to 2.0u; flagella polar; no spores observed; capsule present; liquefies gelatin, but not blood serum; grows in beef bouillon containing 9 per cent NaCl; uniform clouding and copious pellicle which falls easily in bouillon; thermal death- point 51°; grows at 37° but best at 20°; grows slowly at 1°; produces a yellow rim and pellicle in plain milk which is slowly coagulated; whey separates slowly; litmus milk is blued and later reduced; grows readily in Uschinsky’s solution, viscid; no growth in Cohn’s solution; moderate growth on potato; does: not produce gas from dextrose, lactose, saccharose, maltose, mannite or glycerin; agar colonies, circular, smooth or wrinkled; indol is produced; grows in bouillon over chloroform; resists drying; stains by Gram; is yellow or: becomes yellow on all ordinary media. * Smith, Erwin F., ‘‘Crown Gallof Plants: Its Cause and Remedy.” Bull. 213, Bur. Plant Ind., U. S. Dept., Agr., p. 194, IOII. , CHAPTER III LEAF SPOTS Cirrus CANKER Pseudomonas citri—Hasse The disease was‘ probably introduced into the United States on nursery stock from Japan, and since 1912, has occurred in Florida, ~ Alabama, Mississippi, Louisiana, and Texas. : Symproms.—According to Stevens,* citrus canker attacks all varieties of citrus trees of any commercial value in Florida, but it is most severe on the grape fruit. Under field conditions a characteristic spotting of the fruit, foliage and twigs is produced which appears as small light-brown spots, 1.5 mm.—6 mm. (4/¢ to }4 inch) in diameter. These spots may occur singly or several may coalesce to form an irregular area; they are raised above the adjoining tissue and are made up of a spongy mass of dead‘cells, covered by a thin white or grayish membrane, which ultimately ruptures forming a ragged margin around the spot. The fruit is especially susceptible to the infection, and drops soon after it is attacked: The disease is spread rapidly from one part of the tree to another by insects, rains and heavy dews, so that when once infected, a tree frequently becomes worthless in two or three months. CausaAL OrGANISM.—Miss Clara H. Hasse has described the causal organism, Ps. citri, as a short rod with rounded ends, motile by a single polar flagellum. ' On nutrient agar, the growth is filiform, shining, dull yellow in color; on potato, bright yellow, shining, viscid. In nutrient broth, a yellow ring is formed at the surface in old cultures. Litmus milk becomes deeper blue, and the casein is pre- cipitated. Gelatin is liquefied. Indol is not produced. No gas is formed from sugars in Dunham’s solution. Growth is slight in Uschinsky’s solution, and nitrates are not reduced in starch nitrate solution. The organism grows best under aerobic conditions. * Stevens, H. E., “Citrus Canker, I, II, III,’ Bulls. 122, 124, 128, Fla. Exp. Sta., 1914, 1915. T Hasse, Clara H., “Pseudomonas citri, the Cause of Citrus Canker," Jour. Agr. Res., Vol. IV, No. 4, p. 97, 1915. 612 LEAF SPOTS 613 MEtHop oF INFECTION.—Experimental evidence goes to show that infection takes place through stomata as well as through wounds produced by insects, or by other mechanical injuries. PaTHOGENESIs.—According to Berger, the following citrus varieties are subject to citrus canker: Pomelo, citrus trifoliata, wild lime, Navel, sweet seedlings, Satsuma, tangerine, King orange and lemon. ContTRoL.—Removal of the affected parts of the tree by pruning has proven a complete failure as a control measure, and the only practical means of handling the disease appears to be the prompt and complete destruction, by burning, of all stock that shows the slightest trace of infection. : ANGULAR LEAF-SPOT OF CUCUMBERS Pseudomonas lachrymans—Exw. Smith and Bryan HIsTORY AND DIsTRIBUTION.—The angular leaf-spot of cucumbers is a widespread disease occurring in many of the Eastern and Middle Western States. It has been recognized in the field for more than twenty years, but it was not until 1914 that the causal organism was isolated. Symptoms.—The disease is characterized by the “numerous, often confluent, angular, dry, brown spots which tear or drop out when dry, giving to the leaves a ragged appearance. In the early stages a bac- terial exudate collects in drops on the lower surface during the night and dries whitish,”* and because of these tear-like drops of exudate the specific name lachrymans has been suggested for the causal organism. The young stems and petioles may become soft-rotted and crack open, but there is little evidence that the fruit itself suffers from the disease, other than indirectly from lack of nourishment resulting from the destruction of the active leaf surface. CausAL OrcAnism.—Pseudomonas lachrymans is a short rod with rounded ends, motile by means of 1-5 polar flagella. No spores have been observed; capsules are formed on agar and in milk. It is Gram-negative and is not acid fast. On agar, the growth is smooth, shining, transparent, white; agar colonies, two to four days old, exhibit opaque white centers which spread in radiating lines into the thin margin. Gelatin is liquefied slowly, and as the liquefaction progresses the upper part becomes stratiform, the lower part bluntly funnel-shaped. In * Erw. F. Smith and Mary Katherine Bryan, ‘!Angular Leaf-spot of Cucumbers,” Jour. Agr. Res., Vol. V, No. 11, pp. 465, 475, 1915. . 614 ' MICROBIAL DISEASES OF PLANTS nutrient broth moderate clouding occurs, and a membranous pellicle is formed which breaks readily on shaking. On potato, the growth is slimy, shining, creamy- white. Plain milk clears slowly without coagulation, becoming translucent and tawny-olive with age. Lavender-colored litmus milk is completely blued in three _days, and a creamy-white pellicle is formed at the surface; clearing is complete in twenty days, and later the blue color bleaches out leaving the fluid a translucent » brown, The organism grows in Uschinsky’s, Fermi’s, and Cohn’s solutions producing a green coloration in the first two. No gas is formed from the ordinary sugars; acid is produced from saccharose and dextrose. Nitrates are not reduced. Hydrogen sulphid is not formed. A small amount of indol is produced in 2 per cent peptone water and peptonized Uschinsky’s solution. Methylene blue in milk is rapidly reduced, The organism is an obligate aerobe. Optimum temperature is 25° to 27°.; no growth at 36°. MetHop oF INFECTION.—The causal organism enters the leaves through the stomata, no wounds being necessary. Controu.—Laboratory experiments upon the germicidal action of copper sulphate on Ps. lachrymans suggest that Bordeaux mixture, properly applied, may be a remedy for the disease. ’ SPOT OF THE LARKSPUR Bacillus delphini—Erw. Smith So far as is known, this disease occurs only on the larkspurs of Massachusetts. Infection takes place through the stomata, resulting in numerous black spots on the leaves and stems. CausAL OrGANisM.—Smith* describes the organism as a motile, gray-white, non-liquefying, nitrate reducing bacillus. Agar colony has characteristic wrinkled structure. Grows in Uschinsky’s solution. No growth at 37°; thermal death- point 48° to 49.1°. BACTERIAL Spot oF PLuM AND PEACH Pseudomonas pruni—Erw. Smith The first occurrence of the bacterial spot was reported on the Japan- ese plum in Michigan.t Later, what appeared to be the same disease was observed on the peach in Georgiat and Connecticut, and more recently it has been found throughout the South and Middle West. * Smith, Erw. F., Science, N. S., Vol. XIX, No. 480, p. 418, 1904. { Smith, Erw., Science, N. S., Vol. XVIII, 429, p. 456, 1903. f Rorer, J. B., Science, N. S., Vol. XXIX, 753. p. oF4, N909. LEAF SPOTS 615 Symproms.—On the plum, the leaves and green fruit exhibit numer- ous small, water-soaked spots; later the diseased tissue of the leaves falls out, giving a shot-hole appearance, and the plums show black, sunken aréas and deep cracks. The spots may reach a diameter of one-fourth to one-half inch. On the peach leaves, angular, purplish-brown spots one-eighth to one-fourth inch in diameter are formed, which drop out giving the shot- hole effect. The organism also attacks the young twigs and fruit. It destroys the bark of the former, producing black, sunken areas, while on the latter it causes small purplish spots over which the skin cracks. Tn both the plum and the peach, infection is believed to take place through the stomata. It is primarily a disease of the parenchyma, but the vascular system is invaded ultimately. Causa OrcANIsM.—Ps. pruni Smith, is a small rod, motile by one to several polar flagella. It grows readily upon the ordinary culture media. On agar, it resembles Ps. campestris, producing a distinctly yellow pigment, but is distinguished by its feeble growth on potato and by its growth in Uschinsky’s solution, which is converted into a viscid material like egg albumin. Gelatin liquefied slowly. Casein of milk precipitated slowly and redissolved; litmus reduced but color restored later. No gas produced. Thermal death-point 51°. DISEASE OF SUGAR-BEET AND NASTURTIUM LEAVES Pseudomonas aptatum—Brown and Jamieson History.—The bacterial leaf spot of sugar-beet and nasturtium leaves was first observed in the summer and spring of 1908 on nastur- tium leaves growing near Richmond, Va., and on sugar-beet leaves - obtained from Garland, Utah; more recently the trouble has been noted in California and Oregon on the sugar-beet. Symptoms.—Affected nasturtium leaves exhibit water-soaked and brownish spots from 2 to 5 mm. in diameter. The sugar-beet leaves disclose “‘dark-brown, often black, irregular spots and streaks from 3 mm. to 15 mm. in diameter. They occur on the petiole, midrib, and _ larger veins.” Occasionally the discoloration extends along the veins, and the tissue on either side is brown and dry; sometimes cork-like protuberances occur at the central point of the spots. In badly dis- eased petioles the tissue softens as though affected with a soft rot, but where the infection is mild there is no indication of this condition. 616 MICROBIAL DISEASES OF PLANTS Microscopic examination of the diseased spots and adjacent area shows the tissue to be filled with a large number of active bacteria. Sections cut from the central portions of the diseased areas show the cell walls to be ruptured or collapsed, while the cells bordering the ruptured places show that the bacteria are in the cells. The disease is repro- duced readily with typical symptoms by means of needle prick inocula- - tions with pure cultures. So far as has been observed, the causal organism does not attack the beet root, but is confined strictly to the beet leaf. Causa Orcanism.—According to Brown and Jamieson,* Pseudomonas aptatum, n. sp., is a short, motile rod with rounded ends; flagella, bi-polar; involution forms rare; no spores or capsules observed; pseudozodgloez occur; aerobic; smooth whitish colonies on agar plate with fish scale-like markings; clouds beef bouillon in eighteen to twenty-four hours; produces alkaline reaction in litmus milk, with a gradual separation of whey from curd; liquefies gelatin; produces ammonia; no reduction of nitrates; fluorescence greenish; no diastasic action on potato starch; grows in Uschinsky’s and Fermi’s solutions; indol produced after ten days; optimum temperature 27° to 28°; maximum 34° to 35°; minimum 1°; thermal death- point 47.5° to 48°; vitality four to ten months in beef agar, ten to twelve months in beef bouillon, depending on temperature; growth good on litmus-lactose agar; growth much retarded on gentian violet agar; stains readily with basic anilin dyes; not acid fast; not stained by Gram; tolerates acids; oxalic 0.1 per cent; tartaric 0.2 per cent; hydrochloric 0.1 per cent; tolerates sodium hydroxide in beef bouillon, —18 Fuller’s scale; no growth in Cohn’s solution; killed readily by drying; not very sensitive to sunlight; retains its virulence two to three years. PATHOGENESIS.—Pathogenic to nasturtium and sugar-beet leaves; spots have been produced by artificial inoculations on leaves of pepper, lettuce, egg plant, and upon the leaves and pods of the bean plant. MetuHop or InFection.—It is believed that infection takes place only in bruised or wounded tissue, due to insects or to-mechanical injury. ControL.—No practical methods of control have been undertaken. * Brown, Nellie A., Jamieson, Clara O., “A Bacterium Causing a Disease of Sugar-beet and Nasturtium Leaves,’’ Jour. Agr. Res., Vol. I, No. 3, p. 189, 1913. CHAPTER IV ROTS Biack Rot oF CABBAGE Pseudomonas campestris—Pammel (Erw. Smith) This disease is widely distributed in the United States and Europe and has become so serious on many truck farms that gardeners dread its appearance as much as orchardists do pear blight. It is not confined to cabbage, but it attacks other cruciferous plants such as cauliflower kohlrabi, kale, rape, turnips, mangels, rutabagas and mustards. Symptoms.—The first symptom is the withered, yellow margin of the leaf, giving the impression of a ‘burned edge.” The progress of the disease is inward and downward through the vascular system, as is indicated by the brown or black color of the veins and midrib. The tissue of the vascular bundles is destroyed and the cell walls of the adjacent tissue are dissolved, presumably by a cytolytic enzyme.* In this way practically all of the tissues are softened, disorganized, anda general infection of the whole plant may follow. Diseased leaves fall prematurely, leaving a long naked stalk with a tuft of leaves at the top. The dwarfed, one-sided growth of the heads, and in some cases the failure to produce heads is characteristic. METHOD oF InFECTION.—Water poref infection along the margin of the leaf is believed to be the most common method of entrance, although root inoculation at the time of transplanting undoubtedly takes place also. It has been shown, further, that the germ is intro- duced on the seed.t CAUSAL ORGANISMS. §—Pseudomonas campestris Pammel, is a short rod, with rounded ends, relatively shorter in the host tissue than on culture media, 0.74 to * Smith, Bull. 25, Bur. Plant Industry, U. S. Dept. Agr., 1903. + Russell, Bull. 65, Wisconsin Exp. Station, 1898. Smith, Farmers’ Bull. 63, U.S. Dept. Agriculture, 1898. }¢ Harding, Bull. 251, N. Y. Experiment Station, 1904. § For a means of distinguishing Ps. campestris, Ps. phaseoli, Ps. hyacinthi and Ps. stewarti, the student is referred to Bull. 28, p. 149, Div. Veg. Phys. and Path., U. S. Dept. Agr., rgor. 617 618 MICROBIAL DISEASES OF PLANTS 3.0n by 0.4n to 0.54; motile when young by one polar flagellum; no capsule dem- ' onstrated and no spores observed; zoogloez in liquid cultures. Stains readily with aqueous stains. Gram-negative. It grows readily in the ordinary culture media. Upon potato, growth is charac- teristic; at first light yellow, and in old cultures a golden brown, abundant, moist, shining, slimy. Gelatin liquefied slowly. Litmus milk becomes slightly alkaline, casein separated and gradually redissolved. On nutrient agar, translucent, yellow slime. No gas from dextrose, lactose, etc. Uschinsky’s solution, growth retarded and feeble. Aerobic. Indol produced. Nitrates notreduced. Diastase produced. Optimum temperature, 25° to 30°; thermal death-point, 51.5°. ! Controt.—The removal of diseased leaves in the early stages has © been practised by some growers with success, but care must be taken not to remove so many that growth will be checked. Manure con- taining diseased cabbage refuse must not be used. Seed disinfection with 1:1,000 mercuric chloride, fifteen minutes, or formalin 1: 200, ' \twenty minutes, is recommended. Rotation of crops, and planting on new land should be practised whenever possible. If practicable, the seed bed should be made in sterilized soil, so that the plants will be healthy when set in the field. ! WaAKKER’s HyYAcINnTtTH DISEASE Pseudomonas hyacinthi—Wakker Histrory.—One of the earliest landmarks in the study of bacterial diseases of plants is the excellent contribution of Dr. J. H. Wakker,* a ‘Dutch botanist, who between 1883 and 1888 published five papers on a disease of the hyacinth, caused by Ps. hyacinthi. Erwin F. Smithf has carried the investigation farther and has described the causal organism more fully. The disease was first observed in the Netherlands where it frequently causes serious losses in the hyacinth gardens. It is not known to occur in any other part of the world. Symptoms.—The disease is characterized by a yellow siping of the green leaves and the bright yellow slime produced in the vascular bundles of the bulb. The infection in the leaf spreads slowly to the bulb by the multiplication of bacteria in the vascular system, filling the * Wakker, Bot. Centralbl., 1883, 14, p. 315; Archives neerlandaises des sci. ex..et naturelles, ‘Tome XXIII, pp. 18-20. + Smith, Erwin F., ''Wakker’s Hyacinth Germ,” Bull. No. 26, U. S. Dept. Agr., Div. Veg. Phys. and_Path., root. ROTS ' 619 vessels, especially those of the bulb, with a bright yellow bacterial slime. In time, the walls of the vessels.are destroyed and large cavities are formed in the fibro-vascular bundles.’ The disease does not spread rapidly from bundle to bundle in the bulb, but is confined for a long time to the vessels first involved, a year or more being required for the destruction of the host plant. This is due, largely, to the resistance offered by the cells of the parenchyma to bacterial invasion. MetHop oF INFECTION.—The causal organism enters through wounds in the leaves and through the blossoms, and when the disease is once established, it is probably spread by insects which visit the blos- soms or eat the leaves. Daughter bulbs contract the infection from mother bulbs. Wakker believed the disease to be transmitted often by knives used around sick plants. CausaL Orcanism.—Pseudomonas hyacinthi Wakker, according to Erwin F. Smith, is a medium-sized rod with rounded ends, 1.0 to 2.04 by'o.5u to 0.74, motile by one polar flagellum; non-spore forming. It grows well upon the ordinary culture media, on most of which, as' well as in the host plant, it produces a bright, chrome-yellow pigment. Gelatin and blood serum are liquefied slowly (six to seven days). Milk is rendered alkaline, and the casein is slowly precipitated. On nutrient agar, growth is copious, yellow, smooth, wet-shining, translucent, spreading. On 20 per cent cane agar, the zoogloea formed gives the growth a papillose, verrucose appearance. Acid but no gas is formed in dextrose and saccharose broth; indol produced slowly. Nitrates not reduced. Feeble growth in Uschinsky’s solution. Does not grow at 37°; optimum tem- perature 28° to 30°; thermal death-point 47.5°. The hyacinth is the only known host plant. \ ContTROL.—Diseased bulbs should be removed from the fields and destroyed; land on which the disease is present should be used for other crops; the use of infected tools without thorough disinfection should be avoided. The-selection and breeding of disease resistant varieties, as advised by Wakker, suggests the most practical way of ae the trouble. Brack Lec or Basat STEM Rot oF Potato Bacillus phytophthorus—Appel* _ The disease is prevalent in the United States and Europe. It. appears to originate in the seed tubers from which it extends upward * Appel, Otto, “Untersuchungen u. d. Schwarzbeinigkeit. ‘Arb. Bio. K. G. Amt., Berlin, 1903. % \ 620 MICROBIAL DISEASES OF PLANTS into the base of the stem causing it to turn black and rot. The vines grow spindling, turn yellow and die prematurely. The diseased tubers may rot in the soil or later when in storage cause a soft rot of the crop. CausaL OrcaNnisM.—Erwin Smith describes the causal organism as a non- spore forming bacillus, motile by means of peritrichiate flagella. It stains with the ordinary stains, but is Gram-negative. The growth is grayish-white on agar and on gelatin plates large, round, white colonies develop promptly. Gelatin is liquefied with funnel-shaped liquefaction. On cooked potato, white to yellowish growth. Raw potato, white growth and black stain. There is a slow acid coagulation of milk with precipitation of casein and reduction of litmus. Thick pellicle and heavy precipitate in potato juice. No growth in Cohn’s solution. Moderate _ production of hydrogen sulphide. Nitrates reduced. No indol. Acid from dextrose, saccharose, lactose, maltose and galactose. Some gas from inosite, lactose and mannite. Facultative anaerobe. Optimum temperature, 28° to 30°.. Thermal death-point, 47°. Closely related organisms are B. solanisaprus Harrison, and B. atro- seplicus van Hall. Controi.—In view of the fact that the germs are introduced with the seed potatoes, thorough disinfection of the seed with formalin is recommended. BUD-ROT OF THE COCOANUT | Bacillus coli (Escherich) Migula History AND DistrrBuTIoN.—The bud-rot of the cocoanut has been known for more than thirty years in Cuba and is to be found dis- tributed more or less generally throughout tropical America and the eastern tropics. Symptoms.—Johnston* states that in the acute stages of the disease, the bud, or the growing point in the center of the crown, is affected by a vile-smelling soft rot which destroys all the younger tissues. Most of the nuts fall, the lower leaves turn yellow and the middle folded and undeveloped leaves die and hang down between the still green sur- rounding ones. The rot gradually spreads from the base of one spike to another until all are involved and shed their nuts; the leaf stalks become so rotten at their bases that they are no longer able to maintain their _ natural position and droop or else fall off. From a central diseased bud, the infection may spread downward and into the trunk of the tree for * Johnston, John R., “The History and Cause of Cacoanut Bud -rot,” Bull. 228, Bur. Plant, Ind., U.S. Dept. Agr., 1912. ROTS 4 621 a short distance, rotting out the fundamental tissues and leaving only the fibers which are too hard to be disintegrated. It has been estimated that in some cocoanut groves from 75 to 9o per cent of the trees have been destroyed by the rot. Causat OrcanisM.—B. ¢oli (Escherich) Migula. MEeEtTHOD oF INFECTION.—It is believed that the causal organism enters the host through insect bites or other mechanical injuries to the soft tissue. Insects, birds or some form of animal life are held respon- sible for spreading the trouble. , Controt.—The removal of the diseased parts of a tree as well as spraying have proved of no benefit in controlling the disease. “The absolute destruction of diseased trees, a careful watch for the newly infected cases, and their immediate removal has done much to prevent greater loss in the various regions.” Brown Ror, A LEar-pISEASE OF TROPICAL ORCHIDS Bacillus cypripedii—S. Hori History AND DistRiBuTION.—The brown rot of orchids was first observed by Hori* in 1906 on orchids growing in the greenhouses in Tokyo, Japan. Since then the disease has been noted on orchids from Formosa grown in their natural habitat out of doors. In 1898 v. Peg- lionf described a similar trouble in Italy which may be identical with the above. Symproms.—The rot is characterized by dirty cinnamon or light umber colored, depressed spots on the leaf-blade; these become darker with age and may increase in size so rapidly that the entire green leaf is discolored (yellowish) in a few days and dies. The rotting also spreads downward into the stem, and if the diseased leaves are not removed ‘early, the entire stalk will be destroyed. CausaL OrcanisM.—Bacillus cypripedii is a medium-sized rod with rounded ends; single or in short chains; measures 1.5 to 24 X 0.5 to 0.7; stains readily with aniline dyes; Gram-positive; motile by '4 peritrichiate flagella; non-spore forming; smooth, light grayish white colony, with pearl luster on agar; dirty cream, colony *Hori, S., “A Bacterial Leaf-disease of Tropical Orchids," Cent. f. Bakt., Abt. II, Bd. at; “p. 85, I9II. ; tv. Peglion,’ “Bacteriosi delle folie di Oncidium spec,” Cent. f. Bakt., Abt. II, Bd. s. P. 33, 1899+ 4 7 622 MICROBIAL DISEASES OF PLANTS on potato; surface film on bouillon; liquefies gelatin rapidly; coagulates milk; ferments glucose with production of H and CO; in the ratio 1 : 3; indol positive after forty days; methylene blue reduced; ammonia and H.S produced from bouillon; enzymes: amylase, oxidase, peroxidase; facultative anaerobe. PATHOGENESIS.—Pathogenic to orchids grown in the hothouse and also in their habitat. MeEtHop or InFEcTION.—The germs enter the leaf tissue ehieny through wounds caused by careless washing. ConTROL.—Use only a soft sponge soaked in a 1: 1000 solution of mercuric chloride for wiping the leaves, and avoid excessive watering as this favors the disease. \ Rot oF CAULIFLOWER AND ALLIED PLANTS Bacillus oleracee—Harrison _ History anp Symptoms.—This rot of cauliflower and allied plants was first reported in 1901 from truck gardens in the vicinity of Guelph, Ontario. It is characterized by a soft rot of the roots and a blackening of the stems and leaves. Harrison* has found this condition to be traceable to an actively motile bacillus which invades the intercellular spaces of the plant and destroys the middle lamelle. CausaL Orcanism.—Batillus oleracee—Harrison is a rod with rounded ends; occurs single or in short chains; measures 2 X 0.64; motile by means of 7 to 13 peritrichiate flagella; stains with the ordinary aniline dyes; Gram-negative; in broth heavy turbidity and sediment, no pellicle; stratiform liquefaction of gelatin; on agar spreading, thin, whitish, moist, slightly opalescent; neutral red agar no change in color; litmus milk coagulated, soft curd slowly peptonized; blood serum slightly liquefied; growth positive in Uschinsky’s and Fermi’s solutions; potato waxy, straw-colored to moist, shining; opt. temp. 30°, max. 42°, min. 5°; thermal death- point 55°; facultative anaerobe; slight reduction of nitrates; indol slight; H.S positive; slight gas from glucose and lactose, none from saccharose; acid from. sugars; enzymes: proteolytic, diastase, cytase (pectinase). Metuop oF InFection.—Infection takes place chiefly through wounds due either to mechanical or insect injuries. Warm weather combined with excessive moisture appears to favor the spread of the sScaBE, _ * Harrison, F. C., “‘A Bacterial Disease of Cauliflower (Brassica oleracea) and Allied Plants,” Cent. f. Bakt., Abt. II, Bd. 13, pp. 46, 185, 1904. ROTS 623 PaTHOGENESIS.—Pathogenic for cauliflower, cabbage, and turnips; a soft rot can be produced in a large variety of vegetables under labora~ tory conditions by pure culture inoculations. Controt.—Complete destruction of diseased crops by burning and crop rotation are to be recommended. Harding and Morse,* from their extensive comparative studies of micro6érganisms producing soft rots of vegetables make Bacillus oleracee of Harrison identical with B. carotovorus of Jones. Sort Rot or Catia Lity Bacillus aroidee—Townsendt A soft rot of the calla lily, distinct from other soft rots, is scattered over the calla-growing sections of the United States. The disease starts at the top of the corm and causes a rotting of the plant at or just below “the surface of the ground. Asa result the leaves and flower stalk turn brown and fall over. The healthy corms are white, but the infected ones are brown, soft and watery. It is believed that the causal organism lives in the soil and enters the plants through wounds. The disease is undoubtedly spread from one locality to another by shipping slightly diseased corms. As a means of control, only sound corms should be used, and the soil in the calla beds should be changed every three to four years. Sort Rot oF CARROT AND OTHER VEGETABLES Bacillus carotovorus—Jones A number of the cultivated plants of the north temperate zone, notably those grown for their root crops, suffer, at times, from a bac- terial rot caused by a liquefying bacillus. Although probably as widely distributed as any microérganism parasitic upon plants, it was not de- scribed until 1901. Bacillus .carotovorus is a wound parasite which invades the inter- cellular spaces, dissolving the middle lamellz and portions of the inner * See footnote, p. 624. } Townsend, C. O., Bull. 60 Bur. Plant Ind., U. S. Dept. Agr., 1904. fT Jones, L. R., “A Soft Rot of Carrot and Other Vegetables,” 13th Report Vermont Exp. Station, p. 299, 1901. 624 MICROBIAL DISEASES OF PLANTS lamellz, thereby establishing a condition which is known as a soft rot. Jones* has shown this solution to be due to a bacterial enzyme which he has named pectinase. ' CausaL OrcANismM.—The organism is a variable rod, majority 2.0% by o.8y, rounded ends, motile by 2 to ro peritrichiate flagella; no endospores; no capsules; slight pseudozoogleee. Stains readily with aqueous stains. Gram-negative. _ On agar, growth abundant, filiform to spreading, glistening, smooth, white, opaque to opalescent. Potato—glistening, white, decided odor, smooth, butyrous, medium grayed. Gelatin stab—filiform, liquefaction crateriform to infundi- buliform, liquefaction begins second day and complete in six days.’ Broth—thin pellicle, clouding, abundant sediment. Milk—coagulated, slowly peptonized, rendered acid, litmus reduced. Cohn’s solution—no growth. Uschinsky’s solu- tion—abundant growth. Quick tests; soft rot of uncooked carrots, turnips, cab- bages. Slight gas produced from dextrose, lactose, saccharose, but not glycerin. Acid from dextrose, lactose, saccharose and glycerin. Nitrates reduced. Slight indol. Thermal death-point, 48° to 50°; grows at 37°. Optimum temperature 25° to 30°. Pathogenic to the roots of carrot, turnip, rutabaga, radish, salsify, parsnip, , bulb of onion, leaf stalk of celery, leaves and scapes of hyacinth, cabbage, cauli- flower, lettuce, lrish potato, fruit of tomato, eggplant and pepper. B. oleracee@ Harrison, and B. omnivorus van Hall, formerly described as bacterial species capable of producing soft rots, have been reported by Harding and Morse} as identical with B. carotovorus and therefore to be recognized no longer as distinct species. ConTROL.—Jones believes that the soft rots can be practically held in check by rotation of crops; by not using manure into which garden refuse has been thrown; by drying the surface of the roots thoroughly and exposing them to bright sunshine before storage; by maintaining a constant low temperature (4°) during storage. Sort Rot oF HyacintH Bacillus hyacinthi septicus—Heinzt A very active soft rot of the hyacinth bulb, producing a bad smelling, slimy condition in a few days, has been described by Heinz as caused ‘by an unpigmented, motile bacillus, * Jones, L. R., ‘“‘Pectinase, the cytolytic enzyme produced by Bacillus carotovorus and certain other soft rot organisms.” Tech. Bull. 11, New York Agr. Exp. Sta., 1909. { Harding and Morse, Tech. Bull. 11, New York Exp. Sta., 1909. ft Heinz, Cent. f. Bakt., 5, p. 535, 1899. ROTS 625 Sort Rot oF MUSKMELON Bacillus melonis—Giddings History.—Toward the close of the season of 1907 the muskmelons in certain sections of Vermont were attacked by a soft rot. An investigation of the cause of the trouble by Giddings* showed it to be due to a microérganism which he has called B. melonis. Symptoms.—The decay usually begins on that part of the melon next to the soil as shown by the shrunken but generally unbroken skin over the soft diseased area. There is a complete collapse of the melons accompanied by some frothing and a disagreeable odor in the last stages. A microscopic examination of the diseased tissue, both fresh and killed, shows that the bacterial invasion is purely intercellular, and the patho- logical condition of the tissue manifested as a soft rot is due to the solution of the middle lamelle. Infection in the field appears to take place through wounds in the skin, and especially through cracks in the skin and flesh. CausaL Orcanism—According to Giddings, Bacillus melonis possesses the following characteristics: A bacillus r.ou to 1.72 by 0.6u/to 0.9u actively motile by 4 to 6 peritrichiate flagella. Endospores not produced. Gram-negative. Stains readily with aqueous stains. In nutrient broth, strong clouding twenty-four hours, neither pellicle nor ring, slight sediment. Agar stroke, abundant, contoured, shiny, glistening, without color, opalescent growth having umbilicate elevation. Gelatin stab, infundibuliform liquefaction in two days. Cooked potato, abundant, spreading, glistening, odor of decaying potatoes. Litmus milk, coagulated and reddened in three days, no di- gestion. No growthin Cohn’ssolution. Abundant growth in Uschinsky’s solution, ring, pellicle and heavy sediment, odor of hydrogen sulphide. Vegetables rotted— muskmelon, citron, carrot, potato, beett and turnip. Growth and some acid but no gas from lactose, etc. Slight gas production from asparagin broth, abundant in fermentation tubes of milk, this gas being 99 per cent carbon dioxide. Hydrogen sulphide from nutrient broth and potato. Nitrates reduced. Slight indol. Am- monia from asparagin broth; none from broth, gelatin, milk or urea. Thermal death-point, 49° to 50°. Optimum temperature, 30°. Controt.—Spraying with Bordeaux mixture or other fungicides is recommended as a preventive measure. * Giddings, Bull. 148, Vermont Exp. Station, 1910. f B. carotovorus, Jones, associated with several soft rots, does not rot the beet. 40 £ 626 MICROBIAL DISEASES OF PLANTS 1 The melons should be supported by some means to keep them from coming in direct contact with the soil, and should be supplied with adequate water during a dry season to keep them from cracking. : Sorr Rot oF THE SUGAR BEET \ Bacterium teutlium—Metcalf ‘History.—A soft rot of the sugar beet, occurring in Nebraska, has been described by Metcalf and Hedgcock.* Symptoms.—Beets affected with the rot show the lower half badly decayed and honeycombed with “pockets” or cavities filled with a slimy, stringy fluid, colorless, sour-smelling, and alive with bacteria. The vascular bundles remain intact, while the tissue surrounding them is usually consumed. Above ground the beets appear normal. MerHop oF InFEcTIOoN.—The germs gain entrance to the beet through wounds and abrasions in the skin, and there is good reason for believing that nematodes are responsible for many of the inoculations. CausaL OrcANisM.—Bacterium teutlium,' according to Metcalf, possesses the following characteristics: It is a short, non-motile rod, rounded ends, 1.54 by 0.8; neither capsules nor endospores have been observed; the organism stains readily with the aqueous ‘stains. Gram-positive. On nutrient agar, slow, scant,'translucent, porcelain white, non-viscid, and pene- trates the agar. On cane-sugar agar growth more rapid, viscid, watery, vitreous to translucent, colorless. Gelatin stab—scant, filiform to beaded, dirty white, no liquefaction. Cane sugar gelatin—characteristic cumulus cloud appearance in stab, no liquefaction. Nutrient broth—slight clouding and sediment, acid pro- duced. No evidence of growth in milk. No visible growth on potato. Oncarrot, clear, viscid and acid. On sugar beet, viscid, clear, ‘spreading, copious, acid, parenchyma destroyed leaving vascular tissue. No growth in Uschinsky’s, Fermi’s, Pasteur’s, Fraenkel’s or Dunham’s solution, No gas from dextrose, saccharose, etc. Facultative anaerobe. No growth at 37°. Optimum temperature, 17°. Thermal death-point, 45°. Controi.—The rot is less apt to be serious if the beets are grown on relatively dry soil and if rotation of crops is practiced. The selection of resistant varieties seems to be the most practical solution of the problem. * Metcalf and Hedgcock, ‘A Soft Rot of the Sugar Beet,” 17th Annual Report, Nebraska Agr. Exp. Sta., pp. 69-112, 1904. CHAPTER V WILTS WILT oF CUCURBITS Bacillus tracheiphilus—Erw. Smith History AND DistripuTIoN.—The bacterial wilt of the muskmelon, cucumber, squash and pumpkin was first reported by Erwin Smith* in 1893. Itis widely distributed over the United States east of the Rocky Mountains and seems to have different host preferences in different localities. Symptoms.—The disease is characterized by a wilting of the vine, pure and simple, without any visible external cause such as mildew, rust or leaf spot. The leaves and runners wilt suddenly as if from lack of water or too hot sun, the runner becoming prostrate on the ground. From two to three days usually elapse before the wilting of the whole vine is complete, and it may remain in this wilted condition for several days, after which the leaves begin to dry up, but retain their green color for considerable time. One runner may die at a time, beginning - at the tip and working back toward the root, after which a general infection is to be expected. If inoculation takes place upon the main stem, several or all of the runners may show the wilt at the same time. The disease is caused by a bacillus whose growth fills the water ducts or trachee with a white, viscid material which prevents the rise of water, and wilting follows. If the severed ends of a diseased vine are rubbed together gently and separated slowly, this sticky liquid will string out in fine threads 2 to 3 cm. in length. METHOD oF InFECTION.—Under field conditions, the disease is spread principally by insects, especially the striped cucumber beetle and the common squash bug. * Erwin Smith, Cent. f. Bakt., Bd. I, IL., Abt., pp. 364-373, 1895. 627 628 MICROBIAL DISEASES OF PLANTS Causat Orcanism.—Erwin F. Smith describes Bacillus tracheiphilus as a rod 1.24 to 2.54 by o.5u to 0.74, actively motile when young. Growth occurs on the ordinary media. Upon agar, the growth is milk-white and extremely viscid. Upon potato, a gray film is produced, much like that of B. typhosus; the potato is unchanged. Gelatin is liquefied and no change occurs in milk. Acid but no gas is produced in saccharose and dextrose broths. The organ- ism is aerobic and possibly facultatively anaerobic. Optimum temperature is between 20° and 30°. No growth at 37°. Thermal death-point, 43°. Controt.—The same precautions and preventative measures are to be recommended for the wilt-of cucurbits as are given for tomato blight. WILT oF SWEET CoRN Pseudomonas stewarti—Smith The early varieties of sweet corn grown in the truck gardens of Long Island* are subject to a bacterial disease which manifests itself by a wilting and drying up of the leaves. It also occurs in Iowa, and it’ has been reported from certain parts of New Jersey. The wilting may occur at any stage of growth, but the plants seem to be more susceptible at the time of flowering. As a rule the leaves succumb one at a time, although on the younger plants they may all wilt simultaneously. There is no external evidence which_ would indicate the cause of the trouble, but if a diseased stalk is cut length- wise, the fibro-vascular bundles appear as yellow strands in the white pith. A cross-section of such a stalk will show drops of a yellow viscid substance, composed largely of bacteria, exuding from the cut ends of the bundles. The infection is not confined to the stalks but can be found in the vascular system of the leaves, husks and cobs as well. The vessels are the principal structures invaded, but in time small cavities filled with the bright yellow slime are formed in the surrounding - parenchyma. Metuop or InFEcTION.—The germ may enter its host through -— either the roots, stomata or water pores and when once inside the vas- cular system, it multiplies very rapidly and fills the water tubes with a yellow slime and wilting follows. A CausAL OrGANISM.—The organism was first described by Stewart and later named Pseudomonas stewarti by Erwin Smith. * Stewart, F. C., “‘A Bacterial Disease of Sweet Corn,” Bull. 130, N. Y. Agr. Exp. Sta., 1897: WILTS 629 It is a short, relatively thick,.motile rod with rounded ends; occurs usually in pairs. No endospores observed. Stains readily with the aqueous stains. It grows well upon the ordinary culture media. On agar, smooth, shining, yellowish-white to deep yellow, lobate. On potato, spreading, deep yellow be- coming slightly iridescent, smooth; potato is browned. Broth—thin film, slight clouding and slight flocculent white precipitate. Milk—slight peptonization with- out coagulation; litmus reduced. No gas is produced from dextrose, etc. Good growth in Uschinsky’s solution. Facultative anaerobe. Pathogenic for sweet corn. ControL.—lIt is believed that the germ is disseminated on dis- eased seed and therefore disinfection of the seed before planting is recommended. The disease is also spread by the use of manure which contains diseased stalks. \ Varieties differ considerably in their susceptibility, and by the selection of the more resistant kinds some relief can be secured. Rotation of crops and planting on new land, when available, should be practised. Field corn and pop-corn are not affected by the wilt. Witt oF Tomato, EccpLant, IrtsH PoTATo AND ToBACCO Pseudomonas solanacearum—Erwin Smith History.—A bacterial wilt affecting a number -of plants of the potato family has been described by Erwin Smith.* The disease was first observed in the Atlantic coast and southern states. In 1903 Stevenst and Sackett described a wilt of tobacco in Granville County, N .C. and this, too, Smitht has shown to be due to the tomato wilt organism, Ps.. solanacearum. Quite recently Miss Bryan§ has shown the same organism to be the cause of nasturtium wilt. Symetoms.—The disease usually manifests itself by a sudden wilting of the foliage, and, as a rule, with little or no yellowing. This may be indicated at first by the collapse of a single leaf, but in time the whole plant will succumb.. Following the wilting, the parts affected shrivel, * Smith, Erwin F., ‘A Bacterial Disease of the Tomato, Eggplant and Irish Potato,” Bull. 12, U. S. Dept. Agr.,. Div. Veg. Phys. and Path.,.1896. + Stevens and Sackett, “Granville Tobacco Wilt,’ Bull. 188 N. Car. Exp. Sta., 1903. t “Granville Tobacco Wilt,” Bull. r41, U. S. Dept. Agr., Bur. Plant Industry, 1908. § Bryan, Mary K., “A Nasturtium Wilt Caused by Bact. Solanacearum,"’ Journ. of Agr. Research, Vol. IV, No. 5, p- 451, I915. 630 MICROBIAL DISEASES OF PLANTS. turn yellow, then brown, and finally black. If a diseased stem is split lengthwise, black streaks, following the fibro-vascular bundles, can be traced the whole length of the stem and often out into the corresponding leaves. The vessels are packed with bacteria which ooze out on the cut surface as little drops of a dirty white, slightly viscid liquid. The _bacillus destroys the parenchyma of the pith and bark and mechanically ‘plugs the water tubes so that the water supply from the soil is shut off and wilting follows. In the tubers of the potato, the rot begins in the blackened vascular ring and spreads in all directions, producing well- defined cavities next to the ring. MertuHop oF INFEcTION.—Insect enemies are largely responsible for the spread of the wilt, especially above ground, while beneath the surface inoculated soil enters the roots through wounds made either by transplanting, cultivating, or nematodes. In the case of the nastur- tium, stomatal infections have been demonstrated. CausaL Orcanism.—According to Smith, Pseudomonas solanacearum is a medium-sized rod, rounded ends; 1.54 by 0.54; motile by a single polar flagellum, zoogloez formed in liquid media; stains readily with aqueous stains. Zoogloee produced at the surface in beef broth, copious dirty white sediment, reaction made alkaline. Casein of milk dissolved without precipitation and medium becomes alkaline. On nutrient agar, growth is smooth, wet shining, slightly viscid, at first dirty white becoming yellowish, then brown; agar browned. Gelatin stab—growth best at surface, pure white, smooth, wet shining, no liquefaction or very feeble after six weeks. Potato—wet shining, not wrinkled, copious, dirty white and later brown to black; medium browned. Neither acid nor gas produced in any of the culture media or from glucose, etc. Obligate aerobe; ammonia pro- duced in nutrient broth and potato tubes; pigment formation aided by glucose, fructose and sacchasose. Grows well at 37°. Thermal death-point, 52°. rd PATHOGENESIS.—Pathogenic for tomato, potato, eggplant, tobacco, Jamestown weed, black nightshade, physilis, petunia and ‘nasturtium. ContTROL.—If the disease is not too general, it is possible to control its spread by removing the dead plants and burning them; the early and. complete destruction of all insect pests is important; if available and practical, new land or land which has not been planted to any of the potato family for a period of years, should be used; only those seeds and tubers which have come from plants grown in localities free from the | : disease should be planted; the use of infected manure or soil should be avoided. (te vs eee aS WILTS 631 ADDITIONAL BACTERIAL DISEASES Angular Leaf Spot of Cotton, Pseudomonas malvacearum Smith.* um Disease of Sugar Cane, Pseudomonas vascularum Cobb,t Smith. t Leaf Spot of Broom Corn, Burrill.§ Bacteriosis of Tomatoes, Bacillus briosit Pavarino.|| Wilt of Banana and Plantins, Bacillus muse Rorer.** Bacteriosis of Ixia miaculata, Bacillus ixie Severini.tt Bacteriosis of Gladiolus colvilli, Pseudomonas gladioli Severini. t+ Bacteriosis of Orchard grass, Bacterium rathayi Smith.{{ Rot of Potatoes, Bacillus solanisaprus Harrison. §§ A Bacterial Disease of the Mango, B. mangifere||||_ Doidge. * Smith, Erw., Bacteria in Relation to Plant Diseases, I, p. 95, 126. + Cobb, N. A., Rept. New So. Wales Dept. Agr., 1893, pp. I-21. t Smith, Erw., Cent. f. Bakt., II Abt., Bd. XIII, 22-23, pp. 726-729, 1904. § Burrill, Bull. 6, Ill. Exp. Sta., pp. 165-176, 1889. Smith and Hedges, Science, N. S., Vol. XXI, 535, p. 502, 1905. ll Pavarino, G. L., Atti R. Accad. Lincei. Rend. Cl. Sci. Fis., Mat. e Nat., 5, ser., 20 (1911), I, No. §, pp. 355-358. ** Rorer, J. B., Phytopathology, I, (1911), No. 2, pp. 45-49. TT Severini, G., Ann. Bot. (Rome), 11 (1913), No. 3, pp. 413-424. {f Smith, Erw. F., “A New Type of Bacterial Disease.” Science, N.S., Vol. XX XVIII, No. 991, p. 926, 1913; Sitz. Ber. Weiner Akad., 1 Abt., Bd. CVIII, p. 597. § Harrison, F. C., ‘A Bacterial Rot of the Potato Caused by Bacillus solanisaprus." Cent. f. Bakt., Abt. II, Bd. 17, p. 34, 1907. |||] Doidge, Ethel M., Annals of Applied Biology, Vol. II, No. 1, May, 1915, pp. 1145. DIVISION VII MICROBIAL DISEASES OF INSECTS INTRODUCTION* Microbial diseases are of interest to the layman from two economic standpoints: I. At certain stages of their existence, certain insects have an economic value; for this reason their breeding is desirable and any plague which devastates their numbers should be combated. Pas- teur was the pioneer in this line, not only being the discoverer of the first known bacillary insect disease, flacherie of the silk-worm, but he worked out an efficient method for its scientific control. His work is the more notable since he was handicapped by the lack of suitable methods of isolation and study of the organisms discovered. TI. Certain insects or their larve are at times veritable plagues laying waste valuable crops and causing serious hardships, even famine and epidemic disease resulting in many cases. Not infrequently these insects naturally become subject to microbial enemies which make heavy inroads on their numbers, thus checking the insect plague. Such an epizootic occurred among the white grubs in Michigan in 1912. The artificial employment of these microbial enemies naturally suggests itself as a means of voluntary control, and such experiments have been carried out successfully on a practical scale. One of the best examples of this is seen in the arrest of the locust epizootic in Mexico and the Argentine Republic by the use of cultures of B. acridiorum. Another thing worthy of note which has been mentioned many times by those working with microbial insect diseases, is the fact that these diseases seem to be almost explosive in character; an epizootic among * Prepared by Zae Northrup, except paragraphs on “‘ Miscellaneous Fungis Diseases” by C. Thom. 632 MICROBIOLOGY OF DISEASES OF INSECTS 633 insects caused by a fungus disease is after a comparatively short time entirely wiped out and another disease takes its place; in many places bacterial diseases seem to have almost entirely supplanted the fungus diseases. This succession of diseases among insects takes place with such periodicity that those who are most intimately connected with their study, can predict very closely both the duration of the epizootic in progress and the time intervening before the onset of the next one, This same periodicity takes place more or less among the more highly organized animals but the “explosive” character is greatly modified by the length of the life cycle. MISCELLANEOUS INSECT DISEASES Bacillus erausquinii n. sp. was isolated from locusts of the species Romalea miles in Argentina by Cullen and Maggio. It is said to have many characteristics which distinguish it from B. acridiorum. A disease of the caterpillars of Gortyna ochracea, and artichoke pest, is recorded in the Department of Var, France. Bacillus gortyne was isolated as the causal factor. Bacillus pyrameis I and II were isolated from the blood and tissues of the caterpillars of Pyrameis cardui, another artichoke pest. These may be distinct or merely varieties of a single species; they may repre- sent one or more saprophytic species widespread in nature which are readily adaptable to a parasitic life (Paillot). Two associated microérganisms, one a motile rod and the other a coccus were the cause of epizootics destroying nearly all of the cater- pillars of Galleria melonella, the bee moth, which were being raised for experimental purposes (Metalnikov). The rod form was the more viru- lent on injection. The manner in which infection takes place was not determined. SAPROLEGNIACEE AND ENTOMOPHTHORACEZ.*—Some of the Sapro- legnia (all water fungi) form conspicuous masses of mycelium around dead insects in stagnant water. The Entomophthoracee are parasites of insects on land. One of these, Empusa musce, destroys the common house-fly, which, after death from this disease, is found attached by its mouth parts to windows or woodwork. * Prepared by Charles Thom. 634 MICROBIAL DISEASES OF INSECTS , ENTOMOGENOUS FuNcI.*—The practical usefulness of some of these species, notably Sporotrichum globuliferum, as a chinch-bug disease, has been studied carefully.' While the work was markedly successful in causing an epidemic disease when conditions favored it, dependence upon particular conditions was so complete that the production of the disease as an effective destroyer of pests failed. -Similar results have attended the effort to use other fungi as insect-destroyers. The condi- tions which make possible their development in epidemic form only occur occasionally. These conditions in thémselves are, as a rule, very unfavorable to insects. Under other climatic conditions, these diseases appear only as isolated cases, negligible in their effect upon the insect population, no matter how carefully the inoculating material is spread by man. BACTERIAL DISEASE OF JUNE BEETLE Larva, Lachnosterna spp. Micrococcus nigrofaciens—Northrupt History AnD DistrisuTion.—The characteristics of this disease were noted in 1893 by Krassilstschik, Russia, but he did not consider it a disease. It is common everywhere in the United States that white grubs of this and related species are found; infected specimens have also been received from Porto Rico. Symptoms.—The normal larva is white, quite firm, covered with conspicuous hairs; the head is brown as are also the spiracles or breath- ing pores along either side. The diseased larva has black shiny spots, sharply circumscribed, located mainly along the joints of the legs, spiracles, and upon the dorsal or ventral segments of the white portion. Badly diseased larve are almost entirely black or brownish black in color; the whole body often seems to be in a state of advanced putre-_ faction, yet the larva still shows life. ee The progressive destructiveness of the disease is most marked in the affections of the legs. In some cases the infection begins at the tip of the leg and as it progresses, the leg, segment by segment, blackens and drops off, leaving the stumps shiny, black, and sometimes swollen in appearance; in other cases the infection occurs at one of the inter- mediate joints or at the joint nearest the body of the grub, the leg in * Prepared by Charles Thom. + Northrup, Z. A bacterial disease of June beetle larve, Lachnosterna spp. Tech. Bul. 18, Mich. Exp. Sta., 1914. MICROBIOLOGY OF DISEASES OF INSECTS 635 time loosening and breaking off at the point of infection. Within certain limits neither the size nor the number of infected areas seems to affect the activity of the grub. Most grubs are very active unless badly infected with the disease. CausaL OrGanisM.—Pure cultures of M. nigrofaciens show micrococci of vary- ing sizes, o.gu and 1.2 to 1.44 diameter with dividing forms; occur singly, in pairs, threes (triangular), fours (tetrads or diamond shape), and clumps of more or less regular groupings, the individuals in a group arranged in a honeycomb-like order; chains of more than three never observed. Gram-positive; stains well with anilin dyes. Growth on agar abundant, beaded, flat, glistening, opaque, pale orange yellow, butyrous consistency, no odor. Cultures newly isolated are not pigmented and give only a moderate growth. Turbidity in broth, no ring or pellicle, no gas. Litmus milk is slightly reduced and acidified, no curd, yellowish deposit of bacterial cells. Gelatin is liquefied; dextrose, lactose and saccharose not fermented. Moderate indol production; nitrates reduced. Mersops oF INFECTION.—Sterilized soil was inoculated with a broth culture of the micrococcus and in the soil were placed apparently uninfected larve, which were incised to imitate accidental abrasion. Characteristic lesions developed in two days at these points. Under natural conditions these larve bite one another, especially if they are very numerous in any one place; this may account for the rapid spread. of the disease. M. nigrofaciens must be a common soil organism, espe- cially where this disease is common. Parasitic insects or fungi may also aid in making infection possible. Excessively wet soil favors the progress of the disease. Larve of Allorhina nitida, the southern June beetle, are susceptible to this infec- tion but less so than the Lachnosterna spp. The American cockroach, Periplaneta americana, is also slightly susceptible, the infection limiting itself to the legs. : M. nigrofaciens does not lend itself readily to the control of the white grubs on account of the limiting environmental conditions. FLACHERIE, AN INFECTIOUS DISEASE OF SILK-wORMS Streptococcus bombycis—Cohn History AND DistRIBuTION.—Flacherie appeared in the silk indus- try as an epidemic at the end of the sixteenth century. It was again a serious epidemic about the year 1869 in the silk nurseries of southern 636 MICROBIAL DISEASES OF INSECTS France. Later it was found in Italy and other neighboring countries devoted to sericulture. In 1870 Pasteur recognized flacherie in silk- worm as a disease of the silk-worm distinct from pébrine. SymptToms.—Diseased worms refuse to eat, become languid; after the fourth molt when they ordinarily climb up twigs and branches for the purpose of pupating, instead of spinning their cocoons they stretch out and remain motionless until death, or they may fall pendant, hang- ing by their pseudofeet. Worms when dead appear so very life-like that it is necessary to touch them in order to make sure that they are not living. From this appearance comes one of the names of this disease “ morts-blancs.” After death they become soft in a short time and assume a blackish color in twenty-four to forty-eight hours. The body is then filled with a brownish fluid swarming with bacteria. Hundreds of worms in this condition show no polyhedral bodies (characteristic of pébrine). A glance is all that is necessary to distinguish worms dead of flacherie. Causa Orcanism.—In the silk-worms as well as in culture Streptococcus bomby- cis forms short chains of small cocci, 0.894 in diameter; the chains are from s. om to 11.99p long; stain well with anilin dyes and are Gram-positive. In gelatin plate cultures, colonies are small, round, yellowish-gray, sharply con- toured, finely granulated interior, gelatin not liquefied. Subsurface colonies have the same characteristics. Gelatin stab cultures are dull white, not liquefied.. Agar colonies are small, round with a slightly undulating contour, deep brown in color, finely granulated, moist. It is opalescent on glycerin agar and on ordinary agar when first isolated. In _broth at 37° a marked turbidity is manifested after twelve hours without flocculence; after long standing the broth becomes clear. Potato cultures show an iridescence which later becomes a light gray. Strept. bombycis develops in milk without curdling it. It is a facultative anaerobe; the temperature optimum is 37° but it develops well at 20° also. The streptococcus retains its vitality for a long time in culture. It is destroyed at 65°-70° in fifteen minutes. MetxHops or Inrection.—Infection of the silk-worm takes place by means of food infected either with the excrement of sick individuals or with the dust of infected silk-worm nurseries of the year preceding. When silk-worms show all the symptoms of flacherie, if they develop~ into moths the eggs laid by these moths are always infected. If any of the forms in which. the silk-worm exists during its life-cycle becomes infected it is sure to die before the cycle is completed. Certain environmental conditions favor the rapid development of ? MICROBIOLOGY OF DISEASES OF INSECTS 637 flacherie; high humidity due to an approaching storm or to keeping the worms enclosed in a practically air-tight cage prevents the transpira- tion which is so necessary to the worm after the fourth molt. Too many worms together often favors the progress of the disease. / ContTroL.—Pasteur instituted the following means of producing healthy strains of the silk-worm; a small portion of the digestive cavity of a moth was abstracted with a scalpel, mixed with a little water and examined microscopically. If the moths did not contain the character- istic microérganism, the strain they came from might unhesitatingly ~ be considered as suitable for seeding. The flacherie organism was as easily recognized as the pébrine corpuscle, but the infection was more difficult to prevent on account of the environmental conditions above mentioned. BACTERIAL DISEASE oF Locusts Bacillus acridiorum—d Herelle History AND DistrIBu1I0N.—Tropical and subtropical countries covering more than half the earth’s surface suffer periodically from plagues of locusts of different species. Famine and its attendant, epi- demic disease, follow in their wake and decimate the regions invaded. A bacterial epizootic has become a natural means of control. Bacillus acridiorum, the cause of the locust epizootic, was discovered in Mexico in the state of Yucatan by F. d’Herelle. In 1909 a certain mortality was noted among the swarms which arrived from the south of the state where they winter; the following year the epizootic was generalized and raged among a large number of bands; finally in 1911, all of the swarms which appeared were attacked, and in 1912, the locust invasion ceased. These particular locusts were the Schistocerca pallens, Symptoms.—The locusts which are attacked by the natural disease, present symptoms which are identical with those which are experimen- tally inoculated or contaminated per os. After a time of incubation, which varies from one to forty-eight hours according to the virulence of the bacillus, the age and individual resistance of the insect, and the environment (temperature especially), at first the contents of the chyli- fic stomach become liquefied and assume a dark color resembling coagulated blood. The locust ceases to eat, becomes flabby, jumps awkwardly and hides itself under tufts of-herbage. The intestinal con- 638 MICROBIAL DISEASES OF INSECTS tents next become liquefied; they are at first a clear yellow, later dark- ening little by little until they are blackish in color. At this stage a slight pressure upon the abdomen causes the liquefied intestinal con- tents to issue from the anus and the characteristic diarrhoea reveals itself on the vegetation which is fouled with the dejecta of the sick locusts. Some hours afterward the locust falls upon its side and the legs move spasmodically; the locust remains in this comatose state several min- utes to several hours until death occurs. When the virulence of the coccobacillus is very high, very often the chylific stomach only presents the characteristic blackening; death occurs before the intestinal con- tents have undergone a modification. After death the insect putrefies very rapidly and the tegument becomes dark. The intestinal content of locusts attacked with this disease shows microscopically practically a pure culture of this bacillus. The intes- tinal contents of healthy locusts are poor in bacteria, sometimes seem- ingly aseptic. Among the saprophytes found, the most common is a motile, Gram-positive coccobacillus which causes death of locusts by injection but never by ingestion. It is distinguishable from the specific coccobacillus by the disagreeable odor which it produces in the locusts or in culture media. Sometimes staphylococci are found, rarely B. subtilis; only one saprophyte per hundred of the specific bacillus rend- ers the isolation of the latter very simple. All of the tissues of the locust are invaded by this bacillus as has been proved microscopically. A pure culture can be obtained from the blood at the same time that the intestinal contents are attacked, thus B. acridtorum produces a veritable septicemia. CausaL Orcanism.—B. acridiorum, the causal organism of the Mexican locust epizootic is a short, slightly ovoid bacillus, decidedly polymorphous; in the same culture coccus forms of about 0.6 are found beside of forms plainly bacilli, 0.4u to 0.64 X0.9m to 1.54; actively motile possessing peritrichic flagella; Gram-negative but stains readily with anilin dyes; the bacillus takes the stain most deeply at the poles, especially if Ziehl’s carbol-fuchsin is used for one to two seconds. Facultative anaerobe; cultures grow readily from 16° to 43° in all ordinary media, even in Raulin’s medium. It develops very rapidly at 37° in broth, turbidity appear- ing at about the fourth hour. A delicate membrane is formed on broth which clears only after three weeks, leaving a heavy sediment. Young agar colonies are cir- cular and have a waxy appearance; they grow rapidly, being plainly visible after twelve hours; after eighteen hours, they are 2-3 mm. in diameter. Subsurface colonies are small, spherical, whitish and opaque. Gelatin is not liquefied. Milk is coagulated and rendered strongly alkaline. Grows abundantly on potato MICROBIOLOGY OF DISEASES OF INSECTS 639 having a creamy appearance; the culture in the water at the bottom of the tube is so dense that the liquid becomes sirupy and has a strong alkaline reaction. Dextrose, levulose, maltose and galactose are fermented; the inoculated medium containing one of these sugars becomes acid at first, then alkaline. This alkalinity is due to the formation of ammonia. B. acridiorum has lived over two years in sealed tubes. Meruops oF Inrection.—Natural. There are several natural methods by which the epizootic is spread. Sick locusts or nymphs leave their infectious liquid dejecta on the vegetation, the other locusts eat, the contaminated herbage, contract the disease and in turn infect new plants thus continuing the cycle. With certain species of locusts, the Schistocerca for example, another very important mode of contagion exists: when one of their number becomes weak or where vegetation is scarce both the nymphs and the adults eat one another. At the time of depositing the eggs, if the female or even the male is diseased, the eggs will be forcibly soiled with the liquid of the diar- rhoea and the bacillus will be conserved up to the time of hatching upon the eggs or in the mucilaginous matter surrounding the eggs which the locust has provided for their protection. A certain number of locusts among every swarm act as healthy “carriers.” Carriers among nymphs have never been found. The period of life of the insect affects its resistance. The adult locust is individually much more susceptible than the nymph. The habits of life of each, however, have a great influence. The nymphs are continually in contact with the vegetation and with each other as they march in very dense columns; they are endowed with a voracious appetite and undergo in the short period of their larval life, five molts, which are the periods of their least resistance. The winged locusts, to the contrary, passing a large part of their life in the air, are only rarely pressed one against the other, except, for example, when the weather is cold; they also eat much less than the nymph, thus the epizootic will have a greater tendency to become generalized among the bands of nymphs than among the swarms of adult locusts. The age of the nymph or locust influences its resistance; the young nymphs have a maximum resistance, but this decreases gradually, reaching its minimum at the time of the last molt; the adult locust has its minimum of resistance at the egg-laying period. The period of the molt is not a means of protection against this type of disease, which is a generalized septicemia. 640 MICROBIAL DISEASES OF INSECTS ARTIFICIAL.—The virulence of B. acridiorum decreases very rapidly in culture and in order to obtain the desired destruction of locusts it is absolutely necessary to employ cultures of the highest possible virulence as an attenuated virus immunizes the locust and renders it refractory to a culture of the highest virulence when applied later. The virulence is increased by successive passages through locusts or nymphs; twelve series of passages are made using twelve locusts in each series. The cul- ture to be rejuvenated is mixed with a few cubic centimeters of sterile water or broth. Injections are made with a syringe having a very fine sharp-pointed needle. The insect is seized with the left hand, the ventral portion toward the operator, and the needle of the syringe inserted between the second and third anterior abdominal segments at the point of intersection with one of the longitudinal ridges, horizon- tally in the direction of the head to a depth of about 3 mm. for an adult insect, a little less fora nymph. The point of the needle should enter the abdominal cavity, not merely pierce the tegument as in the latter case the effect would be nil. Tf the needle is inserted too deep the internal organs will be injured. A very fine- pointed bent pipette could be employed equally well. One or two drops of the emul- sion of the old culture are injected. As soon as the locusts in the first series become sick or preferably are nearly dead, press the abdomen between the fingers and collect in a watch glass the blackish liquid which issues from the anus.’ Inject a drop of this liquid into the abdominal cavity of the second series of locusts, following the same technic and observing the same precautions as for those of the first series. These-insects will die in a shorter period of time. Obtain as previously, in a watch glass the intestinal liquid of three or four of the first dead locusts of the second series, dilute half with water and sterile broth and inoculate the third series. To inoculate the fourth series, use the intes- tinal liquid of the first dead of the third series diluted to a third; a fifth series with the liquid diluted to a fourth and continue with the series in this way. It is excep- tional that it will be necessary to proceed further than the twelve series. The viru- lence of B. acridiorum is increased sufficiently if death occurs eight hours after injec- tion. One-hundredth of a cubic centimeter of virus at its maximum virulence in- jected into a locust will cause the characteristic diarrhoea in two hours and death an hour later. This method of increasing the virulence takes five to six days and this period of time has to be taken into consideration when it is necessary to employ the culture on a practical scale. When the acridian to be infected belongs to a different species than that for which | the virulence of B. acridiorum has been previously augmented, a large number of passages: may be necessary as a culture virulent for one species may not be able to infect another species. In one case fifty-two passages were necessary in order to kill Stawronautus maroccanas (Algeria) in eight hours while for the same insect at Cyprus only twelve passages were necessary. It is also desirable that the first few series consist of a large number of insects, as there will be apt to be some which will be more sensitive to the virus. Their natural resistance can be weakened ‘ by fasting for several days before inoculation.’ The intestinal contents should not ~be diluted until the virus will kill within fifteen hours. MICROBIOLOGY OF DISEASES OF INSECTS 641 When the virulence is sufficiently increased, the specific bacillus is isolated by means of an agar slant or plate and cultivated for twenty-four to thirty-six hours at room temperature; it may then be isolated if the virus is to be conserved; if desired for direct infection experiments, it may be placed directly in broth. The broth used is made as follows: Water: sc.223% 1,000 c.c. } Peptone...... 40 gt. | Boil, alkalinize slightly and filter; place in bottles, plug witb cotton, Sal Os eisdesaece 5 er. cover mouth and neck with parchment paper cap, and sterilize at Gelatin....... 30 gr. \ 120° for thirty minutes. Dextrose..... 5 ger. The gelatin serves to fix the organisms in place when the culture is sprayed on the vegetation, and on account of the dextrose the plants are greedily devoured by the locusts. It is necessary to remember that the virulence of B. acridiorum lowers very rapidly in culture and is attenuated likewise by re-transplantations, so that a broth culture so prepared should be used within two or three days at the utmost. If the campaign against the locusts lasts for several months or the regions invaded are extensive, it will be necessary to continue the series of passages during the campaign in order to have on hand a virus of maximum efficiency. It is best to make two or three more passages than necessary, rather than too few, for in the latter case the results of a whole campaign may be nullified. The material necessary for a campaign against the locusts consists of a new spray pump, preferably tinned inside, such as is used in spraying fruit trees, and the bottles containing the pure broth culture of B. acridiorum at a maximum virulence. A used spray pump should never be employed as it is practically impossible to free it from the antiseptic contained. The pure culture should be used as soon as it shows turbidity. Broth cultures should never be used which have a putrefactive odor. In practice it is generally necessary to infect the greatest number of locusts in the largest possible number of bands in order to exterminate them with certainty in as short a time as possible. The quantity of broth culture to be sprayed varies with the area covered by the nymphs or locusts. One liter per hectare is sufficient in all cases. For large areas, ¢.g., 100-200 hectares, spray over twenty dif- ferent places, using one-half liter each time, taking in all ten liters. It is better to spray over a large area rather than all in one place, choosing places where the nymphs or locusts are in largest numbers, and always spraying the type of plants preferred and in advance of where they are eating. Spraying should be done in the early morning or preferably in the evening towards sunset. The heat and especially the bright light of day rapidly attenuate the virulence of the bacillus. If necessary to spray in the middle of the day, shady spots should be chosen. The virulence and vitality of B. acridiorum has been conserved in the dejecta and dried cadavers for seven months while in culture the virulence especially, is lost rapidly. A very hard rain will inhibit the progress of an epizootic for several days. The rain washes the dejecta of the locusts from the contaminated vegetation, hindering 41 642 MICROBIAL DISEASES OF INSECTS this mode of contagion; the epizootic little by little regains its normal activity. A rain of short duration, to the contrary, seems to favor the progress of the disease. A curious phenomenon takes place when a band of infected nymphs meet a river in the course of their route. On the near bank is found an actual heap of cadavers, on the opposite bank likewise but they are very much less in number. The epi- zootic seems to be completely checked; it recommences only after several days when it takes its normal course. This is explained by the fact that all the nymphs al- ready badly diseased are not strong enough to make the necessary effort to cross the stream and die without surmounting the obstacle; those which were only slightly diseased could pass it but were so enfeebled by their effort that they died on the opposite bank. Thus the colony which pursues its march is composed only of healthy insects and of several nymphs in which the infection has hardly begun. The duration of an epizootic is impossible to predict for all species of insects and under all conditions; as a general rule it will last several days, most often several weeks, rarely several months. The duration of an epizootic, however, is of little importance; the object is to cause such a reduction in the number of the locusts that these insects will cease to be a plague. To spread the epizootic to great distances, care should be taken to infect the winged adults. Some species of locusts are more sedentary than others, it follows that the more sedentary a species is, the more necessary to multiply the foci of infection. In order to ascertain whether the epizootic is progressing, gather one hundred locusts from different parts of the swarm and by pressing their abdomens, see how many show the characteristic diarrheea. Those insects showing diarrhoea one day will be dead the next. Certain peculiarities were observed during the course of an epizootic. In swarms infected a little while before egg-laying, numerous females lay eggs which never reach maturity; others never reach the laying stage and the eggs are transformed , within the body to a blackish mass. Such bands were annihilated several days afterward. In bands of nymphs infected several days before the last molt are found numerous abnormal adults with poorly developed wings only half their ordinary length which prevent them from flying, and further a microscopic examination of the genital organs shows complete atrophy. SuscEPTipte InsEcts.—I. Acridians—B. acridiorum should be pathogenic for all acridians. The following species are susceptible: Schistocerca americana (or pallens)—Natural epizootic in Yucatan in 1908-1911, induced in the Argentine Republic in 1912. Caloptenus sp?.—Epizootic induced in December 1912 in the region of Rio Negro, Argentine Republic. Stauronautus maroccanus.—Epizootic -induced in 1913 in Algeria in the province of Oran, and in the isle of Cyprus. Schistocerca paranensis is killed by B. acridiorum. (Argentine Re- public.) MICROBIOLOGY OF DISEASES OF INSECTS 643 Zonocercus elegans, the so-called “elegant grasshopper” of South Africa, a non-migratory species, was used in inoculation experiments with this bacillus. It was concluded that this disease at best could be employed only as a supplementary measure in dealing with the inva- sion of these insects under conditions that prevail in South Africa. The Philippine locust, Pachytylus migratoroides, has given negative results with B. acridiorum. (Mackie). II. Ants.—A species of small ant near Paris was annihilated in 1gi1 by B. acridiorum. Selenopsis gemminata, near Buenos Aires was annihilated in 1912. Several drops of the culture were placed in each ant hill. , Alta sexdens, a veritable plague in the tropical and sub-tropical countries was annihilated at Chaco and Tucuman after the virulence had been increased for this species of ant by many passages. III. Caterrrttars.—A field of cotton which was being ravaged by caterpillars was treated with B. acridiorum. Four days afterward alh the caterpillars were dead while a neighboring field, treated simul- taneously with Paris green, still contained many living caterpillars. B. acridiorum does not attack the silk-worm, Bombycis mori; it kills the cockchafer, Melolontha vulgaris, by injection but not by ingestion. Birds and mammals in general are immune to this bacillus. One notable exception is the sewer rat which dies from generalized septicemia a few hours after injection. The rat was immune to cul- tures ingested. BACILLARY SEPTICEMIA OF THE CATERPILLARS OF Arctia caja L. Bacillus caje—Picard and Blanc* History AND DIsTRIBUTION.—In 1913 the vineyards of central France were almost completely destroyed by two diseases; one of these was a fungus disease caused by Empusa aulice, the other was a septi- cemia of bacillary origin. Symptoms.—The caterpillars become flaccid and emit a nauseating odor; their digestive tube contains only a clear liquid free from all organisms. The blood contains a pure culture of a bacillus with which the disease has been produced artificially. * Picard, F. and Blanc, G. R. On a bacillary septicemia of caterpillars of Arctia caja L. Compt. rend. acad. sci. 156, 1913, PP. 1334-1336. 644 MICROBIAL DISEASES OF INSECTS CausaL Orcanism.—B. caj@ is a slightly oval bacillus, about 1.54 in length; motile; Gram negative; stains deeply with crystal violet; treated by Pappenheim’s method it shows a characteristic bi-polar stain. Broth cultures develop in twelve hours at 15°-35° with an optimum of 25°; from these the odor of H.S is perceptible; in twenty-four hours broth cultures have a green fluorescence which is more marked at 25° than at 15’ or at 35°. Grows rapidly on both gelatin and agar showing a green fluorescence, the former is liquefied. Growth on potato is meager, showing only after forty-eight hours and producing no pigment. Metuops or Inrecrion.—Artificial—Caterpillars of Arctia caja inoculated in one of their feet by means of a fine needle dipped in viru- lent blood or in a broth culture, die regularly in three days at 15°, manifesting in their blood swarms of the specific bacteria. If kept at 25° they die in twelve to twenty-four hours. The blood of the cater- pillars kept at the latter temperature appears to be the more virulent. Caterpillars receiving several drops of culture by means of a pip- ette introduced into the pharynx, die in twelve hours at 25° with their blood invaded by the bacteria. This suggests a possible practical application. SUSCEPTIBLE INSECTS AND OTHER ANIMALS.—Caterpillars of Por- thesia chrysorrhea are very sensitive to B. caje and die on inoculation in twenty-four to forty-eight hours. The following Coleoptera: Hydrophilus pistaceus, Dyticus pisanus, Cybister laterimarginalis, Colymbetes fuscus are not killed by inocula- tion; nor are the following Hemiptera: Notonecta glauca, Nipa cinerea, Ranatra linearis. The white rat is not sensitive to intraperitoneal injection of 1 c.c. of a twenty-four-hour broth culture. The tree frog, Hila arborea, dies by inoculation, with the same culture, into the lymphatic sacs in twenty-four to forty-eight hours with the blood invaded by numerous organisms. The blood of dying caterpillars is more virulent for the tree frog than broth cultures; 0.5 c.c. injected into the lymphatic sacs causes the death of the batrachian in twelve hours with an intense bacillary septicemia. B. caj@ seems to belong to the same group as d’Herelle’s B. acridio- yum. It is distinguished from it however by several characteristics, both biological and pathological, being a parasite of the blood of the caterpillars whereas, according to d’Herelle, the site of affection in the diseased locusts is in the digestive tract. MICROBIOLOGY OF DISEASES OF INSECTS 645 GRAPHITOSIS* Bacillus tracheitis or graphitosis—Krassilstschik History AND DIsTRIBUTION.—This disease together with a bac- terial septicemia was noted among the Lamellicornia in 1893 in the southeast of Russia by Krassilstschik. He states that the larve from the Lamellicornia which formerly died en masse of muscardine,f die of this disease very seldom in recent years. Bacterial parasites seem to have replaced it in this part of Russia, and this is distinctly advan- tageous since the bacterial diseases are much more destructive than any of the species of muscardine. Symptoms.—At first the larva is entirely pure white, then several legs change to a bright yellow color, next to a yellow brown. Little by little this coloration extends over all the legs. Later on both sides of the larva, characteristically in the region of the spiracles and around them, the skin takes on a grayish hue which gradually deepens. The larva is generally living at this stage but appears to be diseased. This grayish coloration extends toward the back, and the anterior part of the larva then gradually becomes gray also. At this stage the larva is generally dead. After death, the gray color, spreading characteris- tically from the spiracles, deepens considerably, extending all over the skin, finally acquiring a tint resembling that of polished graphite, whence its name “‘graphitosis.”” This coloration is very characteristic for the larva which die of this disease; it is only very rarely that the cadaver is of a brownish shade. When the infection first shows in the legs, the movements are not inhibited in any way, but when the graying around the spiracles sets in, the larva becomes comatose yet still responding to exterior excita- ~ tions. It soon dies, retaining its characteristic curve but gradually becoming limp and soft and decreasing in size, length, etc. CausaL OrcanisM.—The bacillus of graphitosis is from 2 to 2.24 long having a diameter of more than half its length; spores are produced; very motile, movements quick and rapid; occurs generally in pairs from 3.6u to 4.6u long; long filaments not produced; the longest do not exceed 7y to gu which corresponds to two pairs of bacilli *Bacillary Diseases of Lamellicornia.—In 1893 Krassilstschik described two bacterial diseases attacking the larve of the following insects: Rhizotrogus solstitialis, Melolontha vulgaris, Anisoplia austriaca, crucifera, and fruticola, Cetonia sp. and a larva belonging to the Geotrupini (Lethrus? sp.). + Krassilstschik, I. Graphitosis and Septicemia of Insects. Memoires Soc. Zool. en France, t. vi, pp. 245-285, 1893. 646 MICROBIAL DISEASES OF INSECTS ' end to end. Aerobic; shrinks perceptibly when treated with Gram’s stain, almost to half its size. Stained bacilli show unstained spots (spores). In plate cultures B. tracheitis develops into small circular colonies 0.25 to 0.75 mm. in diameter, which are covered with tubercles when the colony grows in gela- tin. Gelatin colonies are finely granular of a deep brownish-yellow color, opaque center surrounded by a transparent ring. Gelatin is liquefied in twenty-four to forty-eight hours. Gelatin stab cultures are typical, having a cup-shaped hollow funnel at the surface, a short empty stem; then the culture grows in the depth of the gelatin, liquefying it in the shape of a carrot, later becoming the shape of an inverted bottle; the culture is seen along the original path of the stab as a zigzag line which later forms a compact cream-colored ‘deposit as the gelatin becomes entirely liquefied; these cultures have the odor of the white of an egg. Broth cultures are clear the first twenty-four hours; after that they become turbid and a pellicle forms which thickens with age, sediment compact; cultures become wholly transparent in four to six months. MeEtuHops oF INFECTION.—Washing the larve of the Lamellicornie with the natural virus of graphitosis kills 16.6 per cent to 100 per cent but an augmented virus gives 100 per cent mortality. The injection under the skin of a very small drop of graphitosis blood is always fatal for the larva even if the virus is weak. B. tracheitis multiplies first in the blood system, then fills the Malpighian tubes, next characteristically in the trachea and then in the fatty bodies; the trachea becomes typically filled with black amorphous granules from whence the name of this organism; the fat cells are at- tacked. The intima of the muscles, trachea and other organs is covered with bacilli, which however do not penetrate the organs themselves. AMERICAN FouLt Broop Bacillus larve—White* History anD DistrisuTion.—American foul brood is the prevalent disease among bees in America and is distributed through all parts of the United States, in Ontario, Canada, Switzerland, New Zealand, Germany, England, and France and it is probable that it has a much wider geographical distribution. Symptoms.—American foul brood or simply “foul brood” usually shows itself in the larva just about the time that the larva fills the cell and after it has ceased feeding and has begun pupation. * White, G. F. The cause of American foul brood. Cir. 94. Bur. of Ent. U. 8. Dept. of Agr. 1907. MICROBIOLOGY OF DISEASES OF INSECTS 647 At this time it is sealed over in the comb. The first indication of the infection is a slight brownish discoloration and the loss of the well- rounded appearance of the normal] larva. At this stage the disease is not usually recognized by the beekeeper. The larva gradually sinks down in the cell and becomes darker in color, and the posterior end lies against the bottom of the cell. Frequently the segmentation of the larva is clearly marked. By the time it has partially dried down and become quite dark brown (coffee colored) the most typical character- istic of this disease manifests itself. Ifa match stick or tooth-pick is inserted into the decaying mass and withdrawn the larval remains ad- here to it and are drawn out into a thread, which sometimes extends for several inches before breaking. , This ropiness is the chief characteristic used by the beekeeper in diagnosing this disease. The larvacontinues to dry down and gradually loses its ropiness until it finally becomes a mere scale on the lower side wall and base of the cell. The scale formed by the dried-down larve adheres tightly to the cell and can be removed with difficulty from the cell wall. The scales can best be observed when the comb is held with the top inclined to- ward the observer so that a bright light strikes the lower side wall. A very characteristic and usually penetrating odor is often noticeable in the decaying larve. This can perhaps best be likened to the odor of heated glue. The majority of the larve which die of this disease are attacked after being sealed in the cells. The cappings are often entirely removed by the bees, but when they are left they usually become sunken and frequently perforated. As the healthy brood emerges the comb shows the scattered sunken cappings covering dead larve, giving it a char- acteristic appearance. Pupz also may die of this disease, in which case they too, dry down, become ropy, and have the characteristic odor and color. The tongue frequently adheres to the upper side wall and often remains there even after the pupa has dried down to a scale. Younger unsealed larve are sometimes affected. Usually the disease attacks only worker broods, but occasional cases are found in which queen and drone broods are diseased. It is not certain that race of bees, season, or climate have any effect on the virulence of this disease, except that in warmer 648 ' MICROBIAL DISEASES OF INSECTS climates where the breeding season is prolonged, the rapidity of devas- tation is more marked. CausaL Orcanism.—Bacillus larve, a motile, spore-producing bacillus is the etiological factor in American foul brood. It is cultivated with difficulty, growing best on media made as the ordinary laboratory media, substituting bee larve for meat. On these media it produces a large number of giant whips which persist for a long time. These giant whips are also found in decaying larve which are dead from American foul brood experimentally produced by feeding pure cultures of B. larve. B. larve and its spores are killed at go°-100° in ten minutes, and at 100° in less than five minutes. MeEtuops oF InFection.—Artificial American foul brood can be communicated by feeding to a healthy colony the scales from combs which had contained brood affected with American foul brood; likewise when these scales are placed in ordinary meat broth, incubated twenty- four hours and then heated to 65° for twenty minutes; infection in this case is due to the presence of spores. Pure cultures of B. larve mixed with sterile sugar sirup and fed to healthy colonies produces the disease within three weeks. B. larve can be obtained in pure culture from such diseased larve. B. larve may be obtained in large quantities suitable for experi- mental inoculation by diluting and filtering the crushed bodies of bee larve through a Berkefeld or other fine filter. ControLt.—The treatment of an infectious bee disease consists primarily in the elimination or the removal of the cause of the disease. Effort is not made to save the larve already dead or dying, but to stop further devastation by removing all material capable of transmitting the cause of the trouble. The swarm is transferred from the infected hive to a clean disinfected hive; the infected combs from the old hive should either be burned, melted, or boiled thoroughly before the wax is fit for use again. The honey taken from the infected hive should be buried or at least removed so that no bees can use it for food. This treatment may have to be repeated before the disease is under control. Brood from badly diseased colonies should be burned, buried or other- wise destroyed at once. Combs even if they appear white and clean should be melted. Infected hives should be burned over inside with a gasoline or oil torch. MICROBIOLOGY OF DISEASES OF INSECTS 649 SEPTICEMIA OF THE CocKCHAFER, Melolontha vulgaris Bacillus melolonthe—Chatton* History.—In May 1912, while studying the effect of d’Herelle’s B. acridiorum on the cockchafer, Chatton noticed that the cockchafers were dying from a spontaneous septicemia; this he found later was due to a coccobacillus which he named B. melolonthe. Symptoms.—No symptoms are noted. CausAL OrcAnism.—B. melolonthe resembles B. acridiorum of d’Herelle with the exception of the following characteristics: the bacillus is longer, and in agar culture produces a green fluorescence in five to six days. It is distinguished from the bacillus of d’Herelle in addition by its pathogenic action on the silk-worm, Bombycis mort. MeEtHops oF InFEcTION.—Injected into the general cavity, B. melolonthe kills the cockchafer in twelve to thirty-six hours, and where its virulence has been augmented by several passages through this insect, always in less than twenty-four hours, but per os it is as inactive as B. acridiorum. Seventy-five per cent. of healthy cock- chafers show the presence of B. melolonthe in their digestive tube, sometimes in massive culture. This is always the case with cock- chafers affected with septicemia. This blood disease seems to be of intestinal origin however, as with the locust. B. melolonthe, a common parasite of the intestine of the cockchafer passes into the general cavity only under special conditions yet unknown. When this organism is removed from the intestine and injected into the general cavity, septicemia is produced. It is as virulent for the silk-worm by injection as for the cockchafer, and as inactive by ingestion. EvuRoOPEAN Fou Broop Bacillus pluton—Whitet History AND DistRIBUTION.—This type of foul brood, sometimes known as “black brood,” or ‘‘New York bee disease” is not nearly as wide spread in the United States as is American foul brood, but in cer- * Chatton, E. Spontaneous septicemia in the cockchafer and the silk worm due to cocco- bacilli. Compt. rend., acad. sci. 156, 1913, pp. 1707-1709. { White G. FP. The cause of European foul brood. Cir. 157, B. of Ent. U.S. Dept. of Agr. 1912. 650 ‘ MICROBIAL DISEASES OF INSECTS tain. parts of the country it has caused enormous losses. It is spread over England, Germany, Switzerland and other parts of Europe and has been noted many times during the last decade. Symptoms.—The presence of disease can usually be detected in an experimental colony during the week that feeding is begun. The first indication of it may be that only a portion of a larva is seen in a cell, the remaining portion having been removed by the bees. Aside from an observation of this kind, the earliest indication one gets from the macroscopic examination is that sick larve are found among the un- capped brood. Sick larvae manifest certain symptoms during the course of the disease by which its presence can be diagnosed while the larve are still alive. The length of time that a developing bee is sick of European foul brood is variable. In general, the three days just preceding the time when a larva would ordinarily be capped, is the most favorable period for making a diagnosis from the gross examination alone. Healthy larvae at a certain age when slightly magnified show a peristalsis-like motion of their bodies, but larve of this same age when sick frequently exhibit a marked peristalsis which can easily be seen with the unaided eye. Diseased larve may show a yellowish tint or appear transparent instead of the glistening white or bluish white of healthy larve. Another symptom often serves for diagnosis. In a healthy larva a pollen-colored mass is frequently plainly seen through the transparent area along the dorsal median line. If this intestinal mass appears white or yellowish white, the presence of European foul brood is al- most certain. This may be often more plainly observed if the larva is removed from the cell with forceps. European foul brood may be positively diagnosed in living larve of a favorable age and condition by the following method; Remove the larva to be tested from the cell and place it upon glass, preferably with a dark background; with a dissecting needle in each hand and with their points near together, pierce with both needles so as to tear the body wall crosswise, and continue to separate the two portions of the larva. If the larva is diseased, and one is successful, it will be found that the intestinal content will be stripped from and pulled out of the posterior and blind end of the canal. The intestinal content of healthy living larve cannot be removed in this way. The force which is ap- MICROBIOLOGY OF DISEASES OF INSECTS 651 plied in pulling the mass from the intestine frequently causes the typ- ical transparent, mucus-like substance surrounding the central mass to stretch and the enclosed whitish substance to break into segments; this appearance is very characteristic. Tf the disease is more advanced, a portion of the intestinal content may flow out in the form of a sac, the wall of which is very easily bro- ken. When broken the content of this sac-like structure will flow out as a rather thin whitish or yellowish white fluid containing small whitish granules that vary in size. If the disease is far advanced and the larva probably dead, the enveloping substance of the intestinal content is so easily broken that often only the whitish or yellowish-white fluid flows from the ruptured wall of the larva. Dying larve diseased with European foul brood frequently show the segments of the body marked off less distinctly than living healthy larve. Causal OrGANISsM.—B. pluton, the organism of European foul brood, is a small, non-spore forming organism, sharply pointed at one or both ends, about rp long and less than 0.54 in breadth, on the average; occurs frequently in pairs; single individuals vary very markedly in size and shape. This organism has never been cultivated, but sections of larve in various stages of the disease show B. pluton to be the first invader of healthy larve. B. pluton is easily killed between 60° and 65° in ten minutes. MEtHOoDs OF INFECTION AND CONTROL.—These are essentially the same as those for American foulbrood. BACTERIAL SEPTICEMIA OF LARV& OF THE LAMELLICORNE Bacillus septicus insectorum—Krassilstschik History AND Distrisution.—This disease occurred separately and together with graphitosis previously described. Symptoms.—The septicemia produced in larve of the Lamelli- corne by B. septicus insectorum is characterized by a uniform browning of the body of the larve. As death approaches, the larva shrivels up and when dead is about half its natural size and is of a deep brown color. During the progress of the disease, the larva ejects a very black, abund- ant, viscous, semifluid substance from the anus which soils the ex- tremity of the abdomen. 652 MICROBIAL DISEASES OF INSECTS \ Causa OrGANISM.—This bacillus is 1.24 to 1.84 long and from 0.6y to 0. in di- ameter; often in pairs; long filaments not formed. Spores are characteristically dip- lospores although isolated ones occur not infrequently. It shrinks very little when stained by Gram’s method. - Gelatin is liquefied; subsurface colonies are decidedly lemon shaped, yellowish brown, finely granular, surface of colony typically curled. Surface colonies are con- centrically three-ringed, the interior opaque, the second ring more transparent, the third very thin and finely granular. Saccate liquefaction in gelatin stab; the gelatin is blackened and has a very disagreeable odor. ‘Spores are found in the sediment at the bottom of the tube. Broth is made turbid in eighteen to twenty hours, no pellicle, bad odor. MeEtHops oF InFEcTION.—Healthy larve inoculated with B. sep- ticus insectorum by placing cotton saturated with a broth culture on a wound, died in most cases with typical symptoms. The sole habitat of these microbes before death is in the blood system. BACTERIAL DISEASE OF THE GUT-EPITHELIUM OF Arenicola ecaudata, the Lug-Worm Bacterium arenicole.—Fantham and Porter.* History AND DistRIBUTION.—This bacillus was found in the lumen of the gut and within the intestinal epithelium of specimens of Areni- cola ecaudata obtained from Plymouth, England. This disease is not of frequent occurrence. Symptoms.—No external symptoms are noted. Lesions are pro- duced in the epithelium, the cells undergoing degeneration, perhaps shortening the life of the lug-worm. Bact. arenicole seems to be con- fined chiefly to the ciliated tracts, as was determined by microscopical examination of sections. Meruops oF Inrection.—No methods of infection are noted. From the type of the disease, however, infection per os is suggested. CausaL OrGANisM.—Bacterium arenicole* averages about 11m long and 1p broad. Extreme individuals measure 7u to 17p long by 0.7 to, 1.34 broad; some of the larger forms are slightly sinuous in outline; no flagella; chromatophile granules determined by staining with iron-hematoxylin, are present, often in considerable numbers, scattered through the cell; these granules are often concentrated into transverse bars both of which in some specimens are refractile. The cytoplasm stains with difficulty with plasma stains. Division is transverse. One terminal * Pantham, F. B.and Porter, A. Bacillus arenicole, n. sp., a pathogenic bacterium from the gut-epithelium of Arenicola ecaudata. Cent. f. Bakt. I., Orig. §2, 1909, 329-334. MICROBIOLOGY OF DISEASES OF INSECTS 653 spore is formed which does not cause the enlargement of the rod to any extent. No cultural characteristics are given. ImportANce.—This disease is of no special economic importance. Sac Broop, A DISEASE OF BEES Filterable virus—White* History AND DistTripuTion.—A disease which was similar to, but was not foul brood was noted in 1881 by Doolittle in America, by Jones of Canada in 1883, and by Simmins of England in 1887. The larve were found to die here and there throughout the brood comb; the disease would disappear entirely or it would reappear the next season; the bees would frequently remove the dead brood and no further trouble would ensue. Simmins found no microscopic evidence of disease in these larve. In 1892 an editorial in one of the bee jour- nals stated that dead brood had been encountered which did not seem to be infectious and which lacked two decisive symptoms of the real foul brood, 7.e., the ropiness and the “glue-pot” odor. In 1902 G. F. White of the U. S. Dept. of Agriculture began the study of this diseased brood. This disease was described in Switzerland in 1906 and later in 1910. The name “sac brood” comes from the fact that many larve dead of this disease can be removed from the cell without rupturing their body wall. When thus removed they have the appearance of a small enclosed sac. Symptoms.—The strength of a colony in which sac brood is present is frequently not noticeably diminished. When the brood is badly infected, however, the colony naturally becomes appreciably weakened thereby. The brood dies after the time of capping. The dead larve are, therefore, almost always found extended lengthwise in the cell and lying with the dorsal side against the lower wall. It is not unusual to find many larve dead of this disease in uncapped cells. Such brood, however, had been uncapped by the bees after it died. In this disease the cappings are frequently punctured by the bees. Occasionally a capping has a hole through it, indicating that the capping itself had never been completed. A larva dead of this disease loses its normal * White, G. F. Sacbrood. Cir. 169, B. of Ent., U.S. Dept. Agr., 1913. 654 MICROBIAL DISEASES OF INSECTS color and assumes at first a slightly yellowish tint. “Brown” is the most characteristic appearance assumed by the larva during its decay. Various shades are observed. The term “gray” might sometimes appropriately be used to designate it. The form of the larva dead of this disease changes much less than it does in foul brood. The body wall is not easily broken, asarule. On this account, often the entire larva can be removed from the cell intact. The content of this saclike larva is more or less watery. The head end is usually turned markedly upward. The dried larva or scale is easily removed from the lower side wall. There is practically no odor to the brood combs. CausAL Orcanism.—No microérganisms have been found either culturally or microscopically. However, experimental evidence shows that the etiological factor is a filterable virus. | MEtHopDs oF INFECTION.—Larve, sick and dead of sac brood were picked from the combs, crushed and diluted with sterile water. The suspension was filtered by means of the Berkefeld filter. This filtrate was fed in sirup to healthy colonies with the result that the typical symptoms of the disease were produced in all cases. The virus is killed by heating at 60° for ten minutes. Witt DISEASE OR FLACHERIE OF THE Gipsy MoTH CATERPILLAR, Porthetria dispar L. Filterable virus—Glaser* HisTory AND DistRIBUTION.—There is no account of the occurrence of wilt in America prior to 1900. This disease may have been intro- duced on trees or shrubs imported from Europe, in which country “ Wipfelkrankheit,” a wilt disease of the European nun-moth cater- pillars, Psilura monacha, exists. In Europe flacherie has become the “guardian angel” of the central European forests. In the United States there is every reason to suppose that the wilt is distributed over the entire territory infested by the gipsy moth, a territory of about 4,850 square miles (1915) extending over various parts of Maine, New Hampshire, Massachusetts and Rhode Island. Symptoms.—The symptoms of the wilt disease of the gipsy moth * Glaser, R. W. and Chapman, J. W. The Wilt Disease of the Gipsy Moth Caterpillar. Jour. Econ. Entomol., 6, 1913, pp. 479-488. Reiff, W. The Wilt Disease, or Flacherie, of the Gypsy Moth, torr. MICROBIOLOGY OF DISEASES OF INSECTS 655 caterpillars are those of flacherie of the silk-worm. They soon stop eating, become languid, usually crawl up on some object where they remain motionless. In a few hours there drops from the mouth and anus a dirty, blackish, foul-smelling liquid; they become more and more flaccid, one leg after another looses its support and finally the cater- pillar reduced to a black skin is found hanging limply to tree trunks and limbs, still holding on with one or two of its false feet or with the anal claspers. After death their body tissues become degenerated so rapidly that it is impossible to handle them; a slight touch breaks the skin and a thin dark, offensive-smelling liquid flows out, consequently they can never be used for histological work. Causa Orcanism.—A filterable virus seems to be responsible for the death of these caterpillars. It is filtered with difficulty, however. Bacteria are not responsible for this disease. Minute dancing granules are observed in the Berkefeld filtrate, which may be etiologically sig- nificant. No bacteria or polyhedral bodies are observed in the filtrate. MeEtHops oF InFECTION.—Infection naturally takes place through the mouth by means of the food. Predisposition to the disease is se- cured by giving the caterpillars food which has been placed in water and renewed only every three or four days. This causes an increase in the acidity of the leaves which in turn decreases the alkalinity of the caterpillar’s digestive fluid. Before the visible outbreak of flacherie, as an early symptom, a characteristic sweet odor is recognized in the breeding cages which resembles that of withered lilac blossoms some- what. Whenever this odor is noticeable, flacherie soon makes its appearance, and as it progresses the odor increases proportionately. (Fischer). Lack of food, which is necessarily brought about by the cater- pillars themselves, causes them to lose their vitality, thus producing a greater susceptibility to the disease. Defoliation also exposes them to the sun’s rays which have the effect of converting the chronic into the acute form of wilt. In lightly infested woodland this does not hap- pen as the caterpillars can always find shade. Flacherie, however, seems to be influenced by climate and weather conditions less than any other caterpillar disease. Wilt is always prevalent among the older caterpillars; young cater- pillars often live several days before succumbing to the disease. Female ‘caterpillars always succumb more readily to the wilt disease than the 656 MICROBIAL DISEASES OF INSECTS male; this may perhaps be due to the fact that they require a longer time to mature than the male. Diseased females deposit egg clusters reduced in size, which contain usually, embryos, incompletely or not at all developed. In this case there are always found undeposited eggs within the body of the female, which never occurs with healthy moths. Genetic immunity of certain individuals is probable. Sub-, lethal doses of the virulent filtrate may produce active immunization. | Although probable, there is yet no definite evidence that wilt is trans- mitted from one generation to another. PaTHOLOGY oF WitT.—When a caterpillar dies of wilt, all of its tissues are in a state of disintegration. ‘The intestine is the last in- ternal organ to disintegrate. A smear of the brown liquid from a dead caterpillar examined microscopically with a high power lens will be found to contain, besides the elements of disorganized tissues, myriads of highly refractive polyhedral bodies of various sizes. The average polyhedron measures from 1p to 6y in diameter and is never regular as are the silk-worm polyhedra. The significance of these bodies is not known. However, they are believed to be reaction bodies belonging to the highly differentiated albumins, the nucleoproteids. They may be stages of the filterable virus but no evidence has been brought forward . to substantiate this view. It has been determined however, that no diagnosis of wilt is valid unless polyhedra are demonstrated microscopically. The “ Wipfelkrankheit” of the nun-moth in Germany is essentially the same disease as that of the gipsy moth in the United States (Es- cherich and Miyajima.) P£BRINE, AN INFECTIOUS DISEASE OF THE SILK-woRM, Bombycis mort Nosema bombycis History AND DistripuTIon.—About the year 1853, anxious atten- tion began to be given in the southern part of France to the ravages of a disease among silk-worms which from its alarming progress,threatened to issue in national disaster. Symptoms of this disease had been noted as early as 1845. It finally became necessary to import seed (technical term for eggs) for continuing the culture of the silk-worms. This was procured first from Lombardy, but after one successful year the same MICROBIOLOGY OF DISEASES OF INSECTS 657 disappointments occurred. Then Italy was attacked, also Spain and Austria; later seed was procured from Greece, Turkey, the Caucasus, but to no avail; China itself was attacked and in 1864, healthy seed could be obtained only from Japan. This disease, characterized by dark spots on the silk-worms, was called pébrine, from the patois word pébré (pepper), the name given to it by de Quatrefages on account of the resemblance of these spots to pepper grains. Symptoms:—As above mentioned, one of the symptoms of pébrine is the manifestation of dark spots in the skin of the larve; some worms languish on the frames in their earliest days, others in the second stage only, some pass through the third and fourth molts, climb the twig and spin their cocoons. The chrysalis becomes a moth, but the moth shows signs of disease in its deformed antenne and withered legs; the wings seemed singed. Eggs from these moths were inevitably un- successful the following year. Thus, in the same nursery in the course of the two months that it takes a larva to become a moth, the pébrine disease was alternately sudden or insidious; it burst out or disappeared, it hid itself within the chrysalis and reappeared in the moth or the eggs of a moth which had seemed sound. CausaL OrGaANISM.—The causal organism for this disease is micro- scopic but nothing further of its nature is known. In the worms suffering from pébrine, corpuscles or polyhedral bodies, first noted by Pasteur, are found in all tissues and all fluids of the body, even in the material from which the silk is made; naturally they are also found in the dejecta of the worms. These same bodies are found in and on the infected eggs, pupe and moths and in innumer- able quantities in the dust of the infected nurseries; they are easily recognized microscopically. Although these polyhedral bodies are neither known to be the etio- logical factor, nor the effect of the disease, elimination of all eggs, containing these bodies results in its suppression. It is generally accepted, however, that Nosema bombycis is the cause of pébrine. The spores find their way from the caterpillar by means of the dejecta or through the . disintegration of dead forms to other silk-worms. Some of the parasites find their way into the ovary, produce spores, pass through the pupal and imaginal stages of - ‘the host into the next generation of silk-worms. The spores are often regarded as _ pébrine-corpuscles. 42 658 " MICROBIAL DISEASES OF INSECTS , MerHops or INFECTION.—A very common method of infection is due to the habits of the silk-worms crawling over one another. When a worm moves across a diseased worm, its claws cut through the tegu- ment and become contaminated; in its progressit inoculates other worms by means of its soiled fangs. The greatest source of contagion, however, is the excreta which fall on the food of the worms. Luckily this infec- tious material on being exposed to light and air, becomes rapidly at- tenuated. However, the causal organism is not so attenuated when within the egg; it passes the winter in a latent state and develops along with the worm, multiplying within its body and altering more or less profoundly the conditions of existence. Controt.—If moths are not seriously diseased, their eggs will always furnish several healthy larve and if these are isolated as soon as they hatch out and are kept and bred under sanitary conditions, a race of worms free from corpuscles can soon be obtained. This has been found to be the most effective method of combating pébrine. Excessive heat saps the vitality of the silk-worm and makes it ready prey to disease. Open air cages resulted in much hardier, moreactive worms. DIVISION VIII MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS CHAPTER I* METHODS AND CHANNELS OF INFECTION INFECTION DEFINED The term infection implies the entrance of animal or vegetable organisms into the body of another animal or plant, their multiplica- tion and their injury to that body. In most instances the organisms enter the tissues of the animal or plant body, although this is not true in every case of infection. It is possible in certain instances to produce the symptoms of an infection by introducing into the body the chemical products elaborated by some pathogenic organisms. For example, the injection of tetanus toxin into the body causes the typical symptoms of tetanus to result. Tetanus toxin is made by growing B. tetani in beef broth under anaerobic conditions and filtering out the bacteria by passing through porcelain filters. These chemical prod- ucts do not occur naturally unassociated with the pathogenic . organisms and therefore they do not produce infections when artifi- cially injected in the usual sense. The disease-producing organisms with which we will especially concern ourselves in the subsequent discussion are those which are very minute in size and are of three kinds: first, bacteria; second, protozoa; and third, ultramicroscopic microérganisms or viruses. It is essential to have clearly in mind what is meant by an infectious disease and a contagious disease before entering into any detailed dis- cussion, although some authorities attempt to make no distinction. An infectious disease is any disease produced in the plant or animal body which is due to a foreign animal or plant organism. The name is applied to the nature of the cause of the disease. A contagious * Prepared by E. F. McCampbell. 659 660 MICROBIOLOGY OF DISEASES OF MAN’ AND DOMESTIC ANIMALS disease is an infectious disease which is transmitted from one individual to another by contact. The term refers to the method of transmission rather than to the cause of the disease. It is possible that certain conta- * gious diseases may be transmitted by indirect contact or by the agency of fomites but many authorities now hold the view that these factors are non-essential and that most contagious diseases are transmitted by direct contact. MiIcROORGANISMS OF DISEASE CONSIDERED AND CLASSIFIED PATHOGENIC BAcTERIA.—Bacteria which produce disease are known as pathogenic bacteria. Of the many thousand species of bacteria only a comparatively few species have anything to do with the diseased processes in the plant or animal body. Those bacteria which are capable of gtowing in the body of animal or plant may be designated as parasitic bacteria. Some bacteria can grow only in the animal or plant body and do net exist for any period of time outside of it. They are known as obligate parasites. There are others which may produce disease in the animal or plant body which can grow and reproduce outside the body. They are known as facultative saprophytes. There are still other bacteria which ordinarily live outside the animal and plant body and which exist largely upon dead organic material, which when taken into the body occasionally produces disease processes. They are called facultative parasites. As an example of an obligate parasite the Bact. lepre of leprosy may be cited, although in this instance certain observers have claimed to have cultivated the bacillus _ in pure culture. However, the results are not in any sense uniform. Improved bacteriological technic has made possible the cultivation of a large number of bacteria which heretofore were regarded as obligate parasites. As examples of facultative saprophytes the B. typhosus of typhoid fever and the Msp. comma of cholera may be mentioned. As examples of facultative parasites B. tetani of tetanus and Bact. welchit of gaseous gangrene may be mentioned. PaTHOGENIC Protozoa.—There are several infectious diseases in man and animals which are caused by pathogenic protozoa. Among the common diseases due to protozoa there may be mentioned malaria, syphilis, rabies (the nature of the organisms involved in syphilis, and rabies is not well understood however), amoebic dysentery, Texas fever, » » METHODS AND CHANNELS OF INFECTION 661 infectious jaundice of dogs, and the various trypanosome infections, such as sleeping sickness, nagana, dourine, and mal de caderas. It is difficult to artificially cultivate the pathogenic protozoa outside the animal body in pure culture. The Trypanosoma brucei of nagana and the Trypanosoma lewist of the rat have been cultivated. The Entameba coli and the Entameba tetragena of dysentery, the various types of the Plasmodium malarie, and the Treponema pallidum of syphilis have also been cultivated, and it is stated that under certain conditions the Piroplasma bigeminum of Texas fever may be artificially grown. ULTRAMICROSCOPIC MICROORGANISMS OR VIRUSES.—There are some infectious diseases the causes of which have never been discovered. The infectious agents in most instances cannot be cultivated and cannot be stained by the ordinary bacteriological methods. The presence of ultramicroscopic organisms has been demonstrated in several ways. For example, when the ordinary bacterial culture is run through a fine porcelain filter, the filtrate contains no microérgan- isms and consequently when inoculated into animals is non-infectious; although if soluble toxins be present there may be evidences of an intoxication. When the viruses or the infected body fluids of men or animals suffering from the diseases mentioned below are passed through a fine porcelain filter the filtrate remains infectious, therefore demon- strating that the viruses or microdrganisms are filterable and are prob- ably so small that they cannot be seen. Examples of diseases due to agents belonging to this class are as follows: hog cholera, yellow fever, foot-and-mouth disease, rinderpest, epithelioma contagiosum of fowls, chicken typhus, horse sickness, acute poliomyelitis, etc. There are several infectious diseases of unknown cause, the viruses of which are not filterable; for example, smallpox, cowpox and vaccinia, typhus fever and Rocky Mountain spotted fever. There are still other diseases of unknown cause about which nothing is known regarding the filterability of the etiological agents of the disease. Scarlet fever, chickenpox and measles belong to this class. These diseases can be inoculated into animals only with great difficulty and the virus cannot be cultivated or secured in sufficient quantities from the experimental animals for study. A possible explanation of some of these diseases of unknown cause may be found in the proposition that two microérganisms may each produce non-toxic substances, and that when these non-toxic 662 MICROBIOLOGY OF DISEASES OF MAN AND. DOMESTIC ANIMALS substances come together, a toxic substance may be produced. This condition of affairs might explain certain infectious diseases in which microédrganisms are known to occur, and in which they cannot be directly connected with the disease as causative factors. For example, the Strept. pyogenes very frequently occurs in both scarlet fever and smallpox. It has been shown absolutely that this organism is not the cause of these diseases, but theré is a remote possibility that it may act in the so-called associative relation with some other microérganism or virus, as mentioned above, and produce the typical symptoms of these diseases. It has been recently stated that scarlet fever is due to a filterable virus but there is every reason to believe that the occurrence of the Strept. pyogenes materially changes the character of the infection and makes it more severe. The associative relationship of infectious organisms is probably not the logical explanation for all infections of this character. It might be mentioned in this connection that the view is held by some investigators that some of the infectious diseases of unknown etiology are due to enzymes and that a so-called autocatalysis explains the seeming reproduction in the body of the viruses. This theory is, however, without substantial proof. Tue DISTRIBUTION OF PATHOGENIC Micropic AGENTS IN NATURE The causal microérganisms of most of our infectious diseases are found principally in the bodies of diseased man and animals. There are some exceptions to their being found only in the bodies of the diseased. Notable examples are found among certain of the wild animals such as the brush-buck, wildebeast and others which serve as reservoirs for the microérganisms of some of the most fatal of protozoal diseases. These animals seem to be naturally immune. Various insects which are factors in the transmission of certain infectious diseases do not suffer from these diseases in any form and are naturally immune. The most common source, however, is the diseased animal or human body. There is no doubt, for example, that the natural habitat of the Bact. , diphtherie is in the throat and nasal passages of persons suffering from or convalescing from diphtheria. Occasionally these bacteria are also found in the nasal passages and throats of persons who have never had diphtheria. The B. typhosus of typhoid fever also has its natural abode in the intestinal tract of persons suffering from or convalescing from the fever. The same is true with the majority of the causal microdrgan- 7 METHODS AND CHANNELS OF INFECTION 663 isms. There are some microbic agents, however, which exist in the soil but probably do not undergo multiplication such as the B. tetani of tetanus or lockjaw, Bact. welchii of emphysematous or gaseous gangrene, and the B. botulinus of meat poisoning. These bacteria sometimes exist in the intestinal tracts of animals such as the horse and in all prob- ability their occurrence in the soil is due to their deposition in manure. THE OCCURRENCE OF PATHOGENIC Micronic AGENTS Upon AND IN THE Bopres oF HEALTHY ANIMALS AND MAN The exposure to the air of the external surfaces of the body, of course, makes it especially easy for microérganisms to collect upon them. The large percentage’ of the microérganisms which collect on the external surfaces are non-pathogenic but there are frequently dis- ease-producing ones among them. The various varieties of the M. pyogenes are almost universally present on the skin and also on the exposed mucous membranes. Strept. pyogenes, Bact. influenza, Bact. tuberculosis, M. intracellularis var. meningitidis, Strept. pneumonia, Bact. diphtheri@ and many other species may be present. The mouth and nose are excellent places for microdrganisms to collect and excellent for their growth as the requisite conditions such as food, heat and moisture are present. It has been stated on competent.authority that all the species of bacteria which have been described as occurring in various parts of the body have also been found in the mouth. These bacteria do not necessarily produce disease or injure the body unless the vitality is lowered and they enter into the tissues. They feed upon the desquamating cells and the excretions. It is exceedingly interest- ing to note that Bact. tuberculosis and Bact. diphtheria, as before stated, have been found in the nose of persons who have never had these dis- eases. These bacteria have also been shown to be virulent and undoubtedly such persons are extremely dangerous to other more susceptible persons. It is also frequently noted that pathogens are found in the bodies of persons after they have recovered from the dis- ease and that these individuals disseminate the microérganisms and infect non-immune individuals. This may be the case in diphtheria, typhoid, Asiatic cholera and dysentery “bacillus carriers.” In regard to the occurrence of microbic agents in the internal organs of the body the following may be said. For a long time it was claimed that the internal organs of man and animals were sterile. Neisser is . 664 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS one authority for the statement that the internal organs of healthy animals are sterile. This has been shown not to be the case universally. Experiments have shown that fifty per cent of the internal organs of rabbits, guinea-pigs, cats, dogs, mice, horses and cattle are not sterile. Bact. tuberculosis has been found in absolutely normal human and bovine lymph glands. The various pus-producing micrococci have been frequently found in the spleen, kidney, liver, etc. Perhaps the commonest group of bacteria to be isolated from the internal organs are the intestinal forms. It has been demonstrated that intestinal micro- ‘Organisms invade the tissues with surprising rapidity when for any reason the resistance of the body is lowered. It has been noted also that there are more bacteria in the internal organs of animals which have been fasted than in those which have been fed. Peristaltic action and the diffusion of food through the intestinal wall may be influencing factors. The fact.that the internal organs are not sterile in every case ‘is important as it may account for the so-called autogenic infections. THE MANNER INFECTIOUS AGENTS ENTER THE BODY AND THEIR SOURCE Air-borne Infections—The causal microérganisms of infectious diseases are frequently excreted from the body of the diseased individual © and are deposited on the clothing, furnishings, on the floors and walls, or on the ground. These microérganisms probably do not proliferate except in rare instances, but frequently remain virulent for a short period of time and are capable of being carried through the air for short distances, producing in certain instances disease in other individuals. There is no doubt that in diseases such as smallpox, measles, scarlet fever and other acute exanthematous diseases together with such diseases as plague and diphtheria, that the infectious agents may be carried through the air after having been deposited on clothing and furnishings. However, recent investigations have shown that this ° method of transferring infection is comparatively rare and that most infections are transmitted by direct contact. In the beginning it was supposed that the only way that bacteria could be carried in the air was after having been dried on particles of dust and carried by currents of air. This, however, has been shown not always to be the case and we now know that infectious micro- METHODS AND CHANNELS OF INFECTION 665 ¢ érganisms may be carried on small particles or droplets of sputum or moisture. These two types of aerial infection are known, respectively, as dust and droplet infection. Dust Infection Infectious microérganisms to remain virulent and be able to produce infection must be able to successfully resist drying after being affixed to particles of dust. After being dried the particles are frequently moved and whirled about by air currents. The larger particles of material quickly settle down but the small, almost invisible pieces of dried material may remain suspended for three or four hours. It is these small particles which are usually inhaled or deposited on the . skin and mucous membranes of normal individuals that produce in- fections providing the microérganisms have not been killed by drying or exposure to sunlight. Bact. tuberculosis is sometimes carried in this way as well as certain other pathogens. The fact that small-pox virus remains active after drying indicates, at least, that dust containing it may be infectious. The extent of such dissemination is quite limited. Droplet Infections—It has been demonstrated that during the pro- cesses of talking, coughing, and sneezing, small bubbles or droplets of sputum are thrown out into the air. These particles remain suspended ‘for some time and may be inhaled or deposited elsewhere. It is sur- prising the distance that these small particles may be carried. It is stated that they are frequently thrown out thirty feet ormore. It has been shown that Bact. tuberculosis is rarely thrown out over four or five feet by the cough of the tuberculous individual. It should be re- membered that these bacteria will remain alive two to three weeks when in the dark but that they live only a few hours when exposed to the sunlight. The pathogenic microdrganisms or viruses which are commonly disseminated by droplets of moisture are those of whooping cough, mumps, measles, influenza, epidemic meningitis, pneumonia, and pulmonic plague. Air-borne infections rarely occur, as previously stated, and are not of great importance in the open air where sunlight has free access to the disease germs but this type of infection sometimes occurs in crowded quarters such as dark shops, schools, tenements and railway trains. However, the factor of direct contact must be given its due weight in such instances. Water-borne Infections.—Pure infections of this type occur in prac- tically only five diseases, namely, Asiatic cholera, typhoid fever, 666 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS paratyphoid fever, and in dysentery of the amcebic and bacillary forms. The length of time that these microdrganisms will remain alive in the water depends on the quantity and quality of organic matter present. Only under rare circumstances do bacteria proliferate in water of very high organic matter content. Ordinarily microérganisms will live only a few days if the water is absolutely pure. They have, how- ever, been known to live for several weeks in ordinary river water. The drinking of water or of fluids or material contaminated by water is the common but not the only way these diseases are acquired. Infections from Soil.—The soil as a source of infectious micro- drganisms is of importance in only a few diseases, namely, anthrax, tetanus, symptomatic anthrax, malignant edema, emphysematous gangrene, Asiatic cholera, and typhoid fever. In the first five men- tioned infection always takes place through some wound usually in the skin and in the last two diseases mentioned infection is usually through the intestinal tract but may also occur by means of wounds. The microérganisms of anthrax, tetanus and emphysematous gangrene, or more specifically the spores, will remain in soil for long periods of time. They are sometimes found in the active vegetative stage but it is probable that they do not proliferate to any extent in the soil. They exist as ordinary saprophytes. The microdrganisms of typhoid and cholera have been known to remain alive for a year or more in soils containing large quantities of organic matter. The various pyogenic micrococci are also occasionally found in the soil and may enter the body of man and animals through wounds. These last- mentioned organisms may live for indefinite periods of time on the skin and enter the body only when the resistance of some tissue is lowered. Infection from Food.—Quite a large variety of pathogenic micro- érganisms have been found in the various food products. Milk is’ perhaps the most common food product to be infected. The causal agents of diphtheria, scarlet fever and some other diseases have been disseminated by means of milk. Milk contaminated by water con- taining B. typhosus may be the means of conveying typhoid fever, and the dissemination of Malta fever is accomplished by the drinking of the milk of infected goats. Typhoid fever has also been known to have been acquired from the eating of vegetables which have been washed in water containing the pathogens. Oysters and various shell fish have nae METHODS AND CHANNELS OF INFECTION 667 ‘been known to carry the microbic agents of typhoid fever and Asiatic. cholera. Three infections coming from meat sometimes occur, namely, botulism, enteritis and occasionally paratyphoid fever. In these instances the causal microdrganisms are in the meat. Another type of infection known as ptomain poisoning also occurs from the eating of meat or fish which has. been acted upon by saprophytic bacteria and the proteins split up into toxic substances. Animal Carriers of Infection—Animals may communicate disease microérganisms to one another and to man in three ways, namely, first, by direct or indirect contact; second, by serving as mechanical carriers from one individual to another; and third, by serving as inter- mediate hosts for the microbic agent and then subsequently com- municating it’to another. As examples of the first proposition, the fact that tuberculosis has been communicated from cattle to man, that glanders has been communicated from horses to man, and that anthrax has been communicated from sheep to man by contact may be mentioned. It has also been stated that the cat, while not suffering from true diphtheria, seems to be able to transmit this infection and the dog may also transmit rabies to the man. In the second method of transfer, the mechanical carrying of an infection, the insects are principally concerned. It is well demonstrated that common flies frequently carry B. typhosus on their feet from the infected patient or the excreta and deposit them on the food materials thus causing infection when the food is eaten. The various suctorial insects also may suck up the blood of one individual and carry the infectious agent to the normal individual. Notable examples of this are found in the transmission of the various trypanosomiases by the tsetse and other tropical flies, and of Rocky Mountain spotted fever by the wood tick. The same is true of Bact. pestis of plague which is carried by the flea, of Texas fever by the cattle tick, and it has been shown recently that the louse may be one of the agents in the transmission of typhus fever. As an example of the third method, the serving as an inter- mediate host and the carrying of the causal agent, the mosquitoes which serve as the only means of transmission of the causal micro- érganisms of malaria and yellow fever and in which these parasites pass a certain cycle of their existence, may be mentioned. Human Carriers of Infection—It has been mentioned previously that man is capable of carrying infectious agents when he himself is 668 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS not infected. For example, in the’ case of diphtheria it has been repeatedly shown that convalescents from diphtheria, persons who have had the disease, and persons who have never had the disease, frequently: carry the etiological microdrganisms of this disease in a virulent form and are accordingly exceedingly dangerous as dis- seminators. Not uncommonly persons who have had typhoid fever carry large numbers of virulent B. typhosus in their bodies, particularly in the gall-bladder, and disseminate them through the intestinal excreta thus causing many infections when this excreta comes into con- tact with water used for drinking purposes or food supplies. B. typhosus may be carried for many years, one case of twenty years being on record. Asiatic cholera is occasionally carried in the same way. It has also been shown’ that well individuals may carry the etiological agents of epidemic cerebrospinal meningitis and acute poliomyelitis or infantile paralysis. Individuals who carry infectious organisms are: popularly known as “bacilli carriers” and should always be kept under rigid quarantine or observation. Contact Infection —It is only necessary to emphasize certain points in addition to what has been said in the foregoing. It has been stated that animals may communicate an infectious agent to other animals - of the same or different, but susceptible, species by direct contact. Probably the commonest diseases to be communicated by animals to each other are tuberculosis and glanders. This is commonly accom- plished by the rubbing of the mouths and noses together although the disease may be acquired in other ways. Among the human race the diseases which are usually communicated by the contact of one indi- vidual with another are diphtheria, scarlet fever, smallpox, chicken- pox, mumps, measles, gonorrhea, chancroids and syphilis. In the. six first mentioned diseases it seems that the expirations and possibly in rare instances the desquamations of the skin in those which have an eruption carry the causal microérganism. The infectious agent is inhaled into the nose or throat. Some of these diseases may be in rare instances transmitted by intermediate agents, clothing, etc. (fomites). In the last three diseases mentioned, which are known as the venereal diseases, an abrasion of the integument is a prerequisite and the infectious agent must enter by this route. Infection is usually brought about by absolute contact of one individual with another. METHODS AND CHANNELS OF INFECTION 669 In leprosy also almost direct contact is necessary for a transfer of the infectious agent. e THE Routes By Wuicu INFECTIOUS MICROORGANISMS ENTER THE Bopy Microérganisms enter the body through either the external or internal surfaces. It has been shown that the absolutely healthy and intact skin furnishes an efficient barrier to the entrance of infectious agents. Pathogenic bacteria, for example, the streptococcus and the various varieties of the staphylococci are present on the skin almost continuously yet do not often produce infections. When there is an abrasion of the skin or a diseased duct or hair follicle the bacteria frequently pass down into the skin and an infection results. When the pyogenic bacteria, such as mentioned above, are vigorously rubbed into the skin infection sometimes takes place but in this instance also there has been some mechanical injury to the skin. Minute unobserved abrasions of the skin also serve commonly as points of entrance for the Bact. pestis of the plague. The microdérganisms of tetanus, anthrax, symptomatic anthrax and malignant edema always enter the skin through wounds. Sometimes the infectious agents remain local and at other: times are carried from the point of the original entrance. This may take place in different lengths of time. For example, in tetanus the bacteria remain localized in most instances at the point of the original wound and their toxin diffuses from this point. In the various pyogenic infections the bacteria usually remain localized. However, in anthrax the bacteria are carried into the circu- lation in a very few minutes after they enter the wound. In the new- born infection very frequently enters the body through the umbilicus or navel. Tetanus is one of the common diseases acquired in this way in certain localities. Mlicroérganisms may also enter the skin through the wounds made by insects such as mosquitoes, flies, ticks, and fleas. The larger and the clearer cut the wound the less the danger of infec- tion because of the mechanical and bactericidal barrier of the fibrin and the bactericidal action of the blood serum. A free flow of. blood also washes the microérganisms out of the wound. Crushing wounds are especially dangerous inasmuch as there is not a free flow of blood and also there is a good chance for the growth of anaerobic bacteria such 670 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS as those of tetanus, malignant edema, symptomatic anthrax and emphysematous gangrene. The mucous membranes of the nose, throat and mouth are quite resistant to infection. The epithelial coat, the mechanical action of the mucus and saliva and possibly the slight bactericidal action of the saliva are the barriers. Infections of the thin non-resistant mucous membranes of the new-born do occur and necrosis sometimes results (noma). The mucous membrane of the mouth and throat is frequently the seat of primary infection when it is injured. The actinomycotic fungus usually enters a lesion in the mucous membrane made by straws and other substances. The ducts of the salivary glands also serve as . points of entrance for certain infectious agents. The tonsils are very commonly the seat of infections especially with the Strept. pyogenes and Sirept. pneumonia. Septicemias, as for example those occurring in diphtheria, and especially in scarlet fever, frequently arise from infec- tion of the tonsils with Sirept. pyogenes. These structures are also the primary point of invasion in cases of acute rheumatism and possibly in certain cases of pulmonary tuberculosis. The nasal mucous membrane is undoubtedly more permeable to infectious agents than that of the oral cavity. The microdrganisms of acute epidemic meningitis, acute poliomyelitis, measles, leprosy and glanders undoubtedly most fre- quently enter the body through lesions in the membranes of the nose. Infection may be carried into the nose directly or pass from the conjunctiva through the naso-lachrymal duct. The flora found in the eye is quite extensive. The conjunctiva is frequently the seat of primary infections. The pyogenic cocci and the M. gonorrhoee are among the common infecting agents. It is possible that certain points of infection are provided by the conjunctiva being injured by dust particles. The tears are not bactericidal and only serve to mechanically wash the eye. Infections of the con- junctiva are frequently very severe. There is no doubt also that other pathogens are caught in the eye and washed into the nose where they set up infections or are carried through the membranes to set up infec- tion elsewhere. M. intracellularis var. meningitidis of epidemic menin- gitis is known to pass in this way and possibly Bact. pestis of the plague in certain instances. Infectious microdrganisms after being taken into the body through the nose or the mouth may either pass to the lungs through the trachea METHODS AND CHANNELS OF INFECTION 671 or down the oesophagus to the stomach and intestines. During the ordinary inspiratory part of a respiration it is probable that micro- érganisms cannot pass directly into the alveoli of the lung as the tortuous passage, the mucus and the cilia are fairly efficient barriers. Bacteria may be inhaled directly into the finer bronchi and the alveoli during forced inspiration such as that attendant upon hiccoughing and sneezing. Infections of this kind occur in pneumonia, tuberculosis, and influenza. Microérganisms frequently lodge on the membrane of the trachea and are here taken up by the leucocytes and carried to the lungs, bronchial lymph glands, and occasionally to other parts of the body by the blood and lymph. It is probable that such a form of infection occurs sometimes in pneumonia, tuberculosis and plague. Infectious microdrganisms very frequently pass down to the stomach and intestines. The mucous membrane of the stomach is normally very resistant to infection due to the hydrochloric acid which is normally present in the gastric juice and which in normal amount is distinctly antiseptic. It should be remembered that in instances where the acidity of the stomach is lowered that micro- érganisms will develop. All toxins with the exception of that of B. botulinus of meat poisoning are destroyed by the gastric juice. The intestines are less resistant to infection. It is here that the causal microdérganisms of typhoid fever, Asiatic cholera, chicken cholera and dysentery and the various hemorrhagic septicemias find their particular affinities. These bacteria enter or attach themselves to the intestinal wall and in the case of cholera and dysentery this is the only point of infection. The B. typhosus has occasionally been known to enter at other places. This bacterium, however, com- monly localizes in the lymphatic patches (Peyers) of the intestine, and may in certain instances enter the blood from this point. It should be noted that some bacteria can pass through the wall of the intestine when it is seemingly intact. This point has been repeatedly demonstrated in the case of Bact. tuberculosis. The genital organs of the male and female are susceptible to in- fection with microérganisms in certain instances. The M. gonorrhee of gonorrhoea and the Treponema pallidum of syphilis find their usual portals of entry in the genital tract. They have, however, been known to infect other parts of the body as the mouth, rectum, and the con- junctiva. The etiological bacteria of gonorrhcea can penetrate the a 672 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS seemingly intact male urethra but not the vagina of the female on account'of the bactericidal secretion and the character of its squamous epithelium. Other bacteria, as for example, the Sirept. pyogenes, M. pyogenes var. aureus and B. coli are sometimes found infecting the genital tract in cases of chronic urethritis. The kidneys, ureters and bladder are sometimes infected but usually the infecting agent is carried in the circulation although it occasionally ascends through the urethra from without. ‘ In conclusion, the proposition of germinal and antenatal infection must be mentioned. By true germinal infection is meant the carrying of the infectious organisms of a disease by the ovum or the spermatozoa and its incorporation in the development of the embryo and fetus. It is doubtful if this ever occurs. Some authorities claim that it is possible and that it has been demonstrated that the spermatozoa may carry the Treponema pallidum of syphilis, but this is not generally accepted. Antenatal infection or infection of the fetus before birth does occur. Infectious organisms enter the fetus only as a result of intimate contact with the mother and,it has been repeatedly shown that tuberculosis and syphilis may be acquired in this way. It is essential, however, that the mother be infected and in most instances this infection is localized in the placenta. Smallpox, scarlet fever, measles, dysentery, various pyogenic infections, and in rare instances pneumonia, have also been acquired by placental infection. In rare cases in animals, anthrax, symptomatic anthrax, chicken cholera, and glanders have been acquired by antenatal infection from the mother. VARIATION IN INFECTION It should be noted that there may be a variation in the infection depending upon the route by which the infectious microdrganism enters the body. For example, in the case of Bact. tuberculosis, if the bacterium enters the skin a usually non-fatal infection called lupus vulgaris results; if it enters the lymph glands or joints and localizes there an inflammation of not necessarily a fatal character results; if it enters the lungs pulmonary tuberculosis or consumption occurs which usu- ally, after being well established, runs a fatal course; if it enters the intestine, intestinal tuberculosis may result which is nearly always fatal; and if it enters the meninges, tubercular meningitis results which ’ s METHODS AND CHANNELS OF INFECTION 673 is rapidly fatal. Just so with the Strept. pyogenes, depending on whether it enters the circulation, the lymphatic vessels of the skin or the connective tissues there results septicemia, erysipelas, or abscesses which obviously differ in their severity. The same is true of practically all the pathogenic bacteria which invade the plant and animal body, the variation in the route produces a great variation in the type and the results of the infection. It was mentioned in the beginning of this discussion that infection included certain things such as the entrance of bacteria into the body tissues, their increase and their injury to the body. There is some variation in what constitutes an infection depending upon the infectious microédrganism and the tissue it attacks. For example, Msp. comma of Asiatic cholera does not produce an infection unless it comes into contact with the intestinal mucosa and in this case it does not enter the tissues but sets up an inflammatory process on the surface. If this same bacterium comes into contact with tissues such as those of the nose, throat, lungs, no infection results. In the case of B. typhosus of typhoid fever the bacterium not only attacks the intestinal mucosa, but in addition it enters the tissue of the lymphatic patches and sets up an inflammation. This microérganism may also invade the circulatory system directly. In order for such an organism as the Strept.. pneumonie to produce pneumonia it is only necessary for the bacteria to come into contact with the thin, single-celled, alveolar wall through the blood or air passages. In case this bacterium produces an abscess it is necessary for it to first enter into the tissues. In the pneumonic form of plague, although infection is supposed to be ac- complished generally by the inhalation of bacilli, the Bact. pestis may be carried to the alveolus through the circulation and thus enters the tissues of the lung before actually- invading the alveoli. This some- times occurs in case of Sirept. pneumonia. It also gives rise to abscesses occasionally but only when it invades lymphatic glands. The same is true of the large number of infectious microbic agents; there is a varia- tion in the infection due to the variation in the microérganism and the point where this agent attacks the body. The severity of an infection, as for example, a pneumonia due to Strept. pneumonie or to Strept. pyogenes, or to Bact. pneumoniae (Friedlander) or to Bact. pestis, would vary with the infectious agent, its virulence and number, and with the resistance of the individual infected, i . 674 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS THE Factors wich INFLUENCE THE RESULTS OF AN INFECTION There are four principal factors which influence the results of an infection. ‘They are as follows: The virulence of the infecting micro- érganism; the number of the infecting microdrganisms; the avenue by which the infectious microérganism enters the body; and the resistance of the plant, animal or individual infected. VIRULENCE.—-It is a self-evident fact that the more virulent a micro- drganism is the more serious will be the infection which results from its invasion of the body. There is a great difference in the virulence. For example, Sirept. pyogenes may infect the skin of mucous membranes and produce only an abscess of varying proportions. Again, it may be more virulent. The resistance of the infected individual may be lowered somewhat and the streptococcus may enter the lymphatics of the skin and produce erysipelas or the blood stream and produce a fatal septicemia. Furthermore, one strain of the streptococci in the blood may produce a very virulent infection and another a less severe one. Virulent streptococci are not phagocytized by the leu- cocytes. The same variation in virulence is noted in all the pathogenic bacteria and the infections are modified thereby... The fact that an organism is virulent for an animal is not evidence that it is virulent fot man. ‘The virulence of an organism depends upon its ability to form toxins or other poisonous substances. NumBer.—The number of infecting microérganisms which are introduced into an animal body is of importance. In anthrax, for example, it has been shown that one bacterium is capable of multiply- ing and setting up an infection. In tuberculosis and typhoid fever and most of the infectious diseases it requires a rather large number before an infection will take place. The leucocytes, bactericidal substances in the blood, and the body cells in general are capable of destroying many infectious agents. Furthermore, it can be readily understood how a few bacteria might be able to cause a mild infection and an increasing number be able to so overcome the bodily resistance as to cause a more or less severe infection. AVENUE.—It has been pointed out previously how the avenue of infection modifies the infection. A very virulent microérganism may occasionally produce a very mild infection when introduced in a certain locality while in another place the same organism may. produce METHODS AND CHANNELS OF INFECTION 675 a very severe type. The results of the infection will be materially modified depending on the avenue of entrance which the virulent micro- érganism takes. For example, in addition to those mentioned previ- ously, suppose Bact. pestis, the causal agent of plague, enters the blood through the skin, or the lymphatics through the tonsils, it is carried to the lungs and there produces a very severe and usually fatal pneumonia; if bacteria enter the lymphatic system in large numbers they frequently localize in the lymph glands producing buboes or glandular enlargements which are not always fatal. These bacteria may also enter the blood current and produce a rapidly fatal septicemia. It has not been established in man that plague can be produced by the © ingestion of Bact. pestis, but in some susceptible animals such as rats, the disease in a very fatal form is rapidly acquired when the bacteria enter the intestines. RESISTANCE.—This factor is one of the prominent ones which modify the results of an infection. It is a familiar fact that two or more individuals may be infected with the same microérganism, as for example, B. typhosus and one will not become infected or have a very mild form of the disease, while the other will have the severest and most fatal form of typhoid fever known. Again, the age of the indi- vidual infected is important in determining the resistance. The adult resists infection such as diphtheria, scarlet fever, and measles more than the child. The very young child resists pneumonia and tuber- culosis more than the adult. The resistance of the body depends on the presence in that body of natural or acquired antibodies. It is, therefore, obvious that the higher resistance or immunity of the indi- vidual infected, the less severe will be the results of the infection on that individual. Tue Exact Cause oF INFECTIONS We are familiar with the fact that all of our infectious diseases are due to microdrganisms or viruses of some form or other. The causal agents of many of these diseases are known but in the case of those that. are not known there is reasonable certainty as to the types of the in- fecting agents. The exact substances which are produced by the micro- érganisms and which are responsible for symptoms of the various diseases will be briefly considered in the following paragraphs. 676 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS SOLUBLE Toxins.—It is a known fact that there are some patho- genic bacteria which secrete through their cell walls poisons which diffuse into the surrounding media. To these poisons or toxins the disease symptoms are due. Bact. diphtherie of diphtheria, B. tetani of lock-jaw or tetanus, Bact. dysenterie of bacillary dysentery, B. botulinus of meat poisoning, and Ps. pyocyanea, the causal organism. of blue-green pus, are about the only bacteria of this character. Some bacteria, such as Sirept. pyogenes and M. pyogenes var. aureus produce hemolytic toxins. There are certain protozoa as, for example, cer- tain entamoebe and the various trypanosomes which secrete soluble poisons. Among the animals, the venoms of the poisonous snakes, the poison of the centipedes and spiders, the serum of the eel, and the excretion of the dermal glands of the toad are examples of secreted toxins (zootoxins). Again, among the plants are abrin from the jequerity bean, ricin from the castor oil bean, and others, are examples of soluble toxins the product of plant cells (phytotoxins). The cells producing these toxic substanees, therefore, are only indirectly re- sponsible for the infections for it is the toxins themselves which produce the pathogenic effect on the body. Enpotoxins.—Many of the pathogenic bacteria and some of the protozoa do not secrete their toxins outside the cell wall but hold them within the wall in combination with the protoplasm. They do not liberate these substances until the microédrganisms die and are disintegrated. Such toxic substances are called endotoxins to dis- tinguish them from those secreted from the cell, namely, the soluble toxins. Two of the best examples of pathogenic bacteria of this type are the Msp. comma of Asiatic cholera and B. typhosus of typhoid fever. Toxic Bacrer1aAL Proreins.—There are some bacteria and other parasitic cells which produce a small amount of endotoxin and in certain instances some soluble toxin but not enough of either of these substances to account for the toxicity of the organism. It has been found that when organisms of this character are ground up and washed to free them of their endotoxin and are washed free of all soluble toxins, they are still toxic. It has been shown that this toxicity is due to the protein substances of the cell. The Bact. tuberculosis and the Bact. mailei of glanders are two notable examples of microdérganisms of this character. When, for example, the proteins of Bact. tuberculosis t ‘ METHODS AND CHANNELS OF INFECTION 677 are injected into the circulation of susceptible animals, tubercle formation occurs showing that these proteins are poisonous. OTHER PossrBte Exact CavsEs.—In certain infectious diseases it is also claimed by certain writers that enzymes are responsible. This lacks substantiation. It is also stated that in such infections as anthrax that the mechanical effect of the bacteria plugging up the capillaries and producing mycotic emboli is a factor. Thismay betrue but in addition other factors are concerned as previously mentioned. In mixed infections of two or more organisms, which frequently occurs, the infected individual may have within the body soluble toxins, endotoxins, and toxic bacterial proteins and in such a case it is - difficult to differentiate their action. THE METHODS BY WHICH INFECTIOUS MICROORGANISMS ARE DISSEMINATED The microérganisms of some of the infectious diseases such as diph- theria and Asiatic cholera and usually tetanus remain local and seldom enter the body generally. From the locus of the infection they dis- seminate their toxic or poisonous products. In the case of tetanus the toxin is carried over the body along the sheaths of the motor nerves; in diphtheria the toxin is usually carried by the lymph, occasionally by the blood; and in the case of cholera the blood and lymph both serve to carry the toxic agents. In diphtheria and cholera the microdrgan- isms very frequently extend along the mucous membranes from the original point of infection. There are other infections in which the causal microdrganisms extend only from the point of original invasion into the surrounding areas. Such is the case with Strept. pyogenes in the infection of the lymphatics of the skin in erysipelas and of Bact. influenze in all infection through the respiratory tract. Many of infec- tious agents are carried by the blood and occasionally by the lymph, as for example, in tuberculosis, syphilis, glanders, plague, leprosy, pneumonia, and the septicemias due to the pyogenic cocci. It is pos- sible in certain. cases that the leucocytes acting as phagocytes may carry virulent infectious agents through the blood and lymph from one part of the body to the other. \ ' 678 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS Tue METHODS BY WHICH INFECTIOUS AGENTS ARE ELIMINATED FROM THE Bopy The etiological microérganisms of the various infectious diseases may be eliminated from the body in two general ways, namely, by a direct method and by an indirect method. For a microérganism to be directly eliminated from the body it is necessary for the focus of the infection to communicate with the outside of the body in some way or other. In the case of infections of the mucous membranes and the skin there is, of course, direct communication with the outside. In diseases of the respiratory organs and the intestines the infectious agents are discharged into the lumen of the air passages and the intestines and then thrown out from these passages. Examples of the partial direct elimi- nation from the skin may be found in such diseases as smallpox, measles, syphilis, scarlet fever, lupus vulgaris, and in suppurative conditions such as carbuncles and furuncles. From the present evidence little significance perhaps is to be attached to the elimination of the infectious agents mentioned directly from the skin. It is probable that the micro- érganisms which are eliminated remain alive for only ashort time and are not factors of consequence in the transmission of these infections. As examples of diseases in which direct elimination from the various mucous membranes occurs infections such as typhoid fever, tubercu- losis, cholera and dysentery from the membranes of the intestines; influenza, pneumonia and tuberculosis from the bronchial membranes; diphtheria, leprosy, glanders and scarlet fever from the membranes of the nose, throat, and tonsils; and gonorrhea, syphilis and tuberculosis from the membranes of the genito-urinary tract may be mentioned. In elimination from the various internal membranes sometimes re- infections occur such as in the case of the elimination of Bact. tubercu- losis from the respiratory tract, the swallowing of the sputum, and the subsequent infection of the intestines. In the second, or indirect method of elimination, two distinct prop- ositions present themselves; first, the infectious microdrganism must enter the lymphatic or blood circulation; and, secondly, in order to get out of the body they must pass through the cells of some of the organs, the mucous membranes or skin. It is a common occurrence for bac- teria and other microdrganisms to get into the circulation in some of the infectious diseases such as typhoid fever, pneumonia, plague,. METHODS AND CHANNELS OF INFECTION 679 and in the various septicemias. They may pass through the epithelium ‘ of the kidney and be eliminated in the urine; they may pass through the liver and be eliminated in the bile, finally passing out through the in- testines; and they may pass through the mucous membranes of the intestine and possibly pass through the glandular epithelium of the sebaceous and sweat glands and be eliminated through the skin. They have also been known to pass through the glandular epithelium of the milk glands when these glands are not grossly diseased and through the salivary glands. It has been recently well demonstrated that there must be some form of lesion in the liver and kidney in order for the microdrganisms to pass through. Infectious microédrganisms are some- times destroyed by the lysins in the blood, carried to and deposited in the spleen and bone marrow and gradually disintegrated and dissolved. In certain infections in which a recovery seems to have occurred all the infectious microédrganisms are not always eliminated from the body. As mentioned previously, B. typhosus and Bact. diphtherie are frequently carried by persons fully recovered from these diseases. Sometimes, however, inflammatory infections are set up by these bac- teria. It has been suggested on seemingly good evidence that inflam- mations of the gall-bladder and gall-stone formation may be due to the toxic action of the bacteria of typhoid fever which have been retained in the gall-bladder for a considerable time following an attack of typhoid fever. It is known that frequently repeated attacks of malaria are due to the retention of some of these protozoan parasites for a time in the quiescent stage. Repeated attacks of erysipelas caused by the Strept. pyogenes may also be due to the same condition. It is also claimed by some (Von Behring) that Bact. tuberculosis is taken into the body in infancy, that it is not eliminated, and that it sets up infection in later life. In conclusion should be mentioned one other indirect way in which infectious agents are eliminated from the body, namely, by being taken up by suctorial insects from the blood. It is necessary that this be done in order to perpetuate the parasite and completeits life cyclein certain instances, as with the mosquitoes in yellow fever and malaria. In other instances the parasites are only taken up by the insect and subsequently injected into another individual or digested as the case may be. This occurs with the ticks in the transmission of certain of 680 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS ’ the piroplasmoses and with the tsetse flies in the transmission of certain of the trypanosomiases.. THE EFFECT OF INFECTIOUS MICROORGANISMS ON THE Bopy It becomes necessary to consider briefly the effect of the various infectious microérganisms and their toxic substances on the body. Tue Periop or Incusation.—This period is that elapsing after the entrance of the infecting organism into the body until the symp- toms of the disease develop. This period is variable in most diseases and depends upon the same factors which modify the results of an infection, namely, the virulence of the infecting organism, the number growing in the body or its tissues, the avenue, and the resistance of the individual. The period can be in a measure controlled and shortened in experimental animals by inoculations into the circulatory system and in other regions depending on the organisms used. In some of _,, the human diseases, particularly those of unknown cause, the period of incubation is quite constant, as for example, in smallpox and measles. Locat Reactions.—The local effects of the toxic substances of microérganisms are usually first inflammatory and later possibly ne- crotic, that is, they produce a death of the tissue involved. The inflam- matory changes may be confined, to those of an acute character as, for example, in the various serous, hemorrhagic, suppurative and fibrinous: inflammations, or be chronic and proliferative in nature. There is always a variation in the type of inflammation depending on the loca- tion of the infection and also a variation in two different individuals of the ‘same species infected at the same point with the same agent. In some diseases such as tetanus the local point of infection may entirely heal and still the bacteria be localized at this point and disseminate their toxin. In some cases of tuberculosis and glanders the bacteria may become localized at the points of infection and after an acute inflammatory stage the point may become the seat of a chronic process and proliferative changes occur in the tissues. GENERAL REActTioNs.—Metabolism.—The general metabolism of the body is affected by the changes produced in the amount and the chemical constitution of the food substances which are taken into the body. By changing in the same way the substances which natu- rally are eliminated from the body and by setting up new and abnormal METHODS AND CHANNELS OF INFECTION 681 changes in the functional activity of the tissues, the general metabolism may be disturbed. Muscular weakness, delirium, pain and loss of appetite, together with vomiting, diarrhea, disturbance of intestinal absorption and the digestive juices are often noted in cases of altered metabolism. The fats, carbohydrates, and then the proteins are in the order named rapidly used up, producing certain changes in the respired air and in the urine and feces. Infectious microérganisms may also reduce the power of the hemoglobin to carry oxygen and perhaps cause a narrowing of the respiratory passages thus preventing the necessary amount of oxygen reaching the lungs and subsequently the tissues. Infecting microédrganisms may alter the composition of the food substances and after being taken into the body produce abnormal compounds which have little or no nutritive value on absorption or they may produce toxic substances related to ptomains. Substances which normally should be eliminated from the body are often retained and abnormal losses of such substances, as water and various albumi- nous compounds, occur. Attendant upon the changes in metabolism usually there occurs _fever in all infectious diseases. It is probable that the fever is the result of the effect of the toxic protein compounds of the infecting microdrganisms on the tissues, or the disintegration of the protein compounds of the body due to the action of toxins. It is evident that the fever-producing substances, in certain infectious diseases, act in a very characteristic manner as is demonstrated by the so-called typical fever curves. It seems to have been demonstrated that fever is a good sign and that it is indicative of the reaction of the body to the toxic substances of the infecting agents. It has also been shown that the fall of fever in certain infections is attendant upon the formation and saturation of the body fluids with antibodies. Blood-forming Organs.—There are usually changes in the blood- forming organs in most all types of infection. The spleen frequently shows enlargement and contains numbers of myelocytes which are also found in the blood in increased numbers. This is probably dueto the disintegration and deposition there of red blood corpuscles and also to the action of toxic substances. The endothelial cells and leucocytes of the spleen are actively phagocytic. The bone marrow, particularly the fatty marrow, shows large numbers.of myelocytes of 4 682 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS the neutrophile type and it eventually becomes lymphoid in nature in a large number of infections. The lymph glands also frequently show endothelial proliferation. : Parenchymatous Tissues.—All sorts of degenerations of the kidneys, heart, liver and some of the other organs frequently occur in infections. Amyloid formation and necrosis of tissue sometimes occurs. Many of the toxins have special affinities for tissues, such as the tetanus toxin for nerve tissue and this produces organic changes. It is possible also that the fever is responsible for a certain portion of the changes in the parenchymatous organs in infections. Epithelial and Endothelial Tissues—In certain infections, as for example, diphtheria, the epithelial tissues are subject to inflammation; and in other infections, as for example, syphilis, the endothelial tissues of the blood-vessels undergo inflammation and sometimes proliferation. The epithelial and endothelial cells are frequently actively phagocytic ' and large numbers of the infecting microérganisms are taken up and ‘destroyed. Some of the infectious microdrganisms produce no effect whatever on these tissues, while others produce pronounced destructive changes. : Erythrocytes and Leucocytes——Lytic or dissolving substances for the red blood corpuscles are frequently produced in infections (hemo- lysins). Strept. pyogenes, M. pyogenes var. aureus, and Ps. pyocyanea areamong the bacteria which produce hemolysins. Anemia is, therefore, not an uncommon finding in many infections. Normal human blood and that of some animals contains an antilysin for the staphylolysin and it is sometimes produced in large amounts. Agglutinating sub- stances for red corpuscles are produced by some pathogenic micro- °° érganisms and it is possible that these are the cause of the so-called agglutination thrombi which occur in infections like typhoid fever. The most marked changes seen in the leucocytes in infections is their rather constant increase in number; in most cases a leucocytosis. In uncomplicated tuberculosis and typhoid fever, in measles and German measles, in malaria, and in dengue, there is no increase in number. In acute inflammations it is the polymorphonuclear leuco- cytes that undergo an increase. This increase is sometimes preceded by a decrease (leucopenia). The leucocytes act as the principal phago- cytes of the body and are attracted (positive chemotaxis) to the bacteria or other microérganisms after they have been sensitized by the opsonins METHODS AND CHANNELS OF INFECTION 683 in the body fluids. Besides acting as phagocytes they may, according to Metchnikoff, produce antitoxins and bactericidal substances. It has been suggested that the initial leucopenia in some cases is due to the production of negative chemotactic substances. Some virulent bacteria cannot be phagocytized probably because they produce very strong negative chemotactic substances. Antibody Formation.—One of the general effects of infectious micro- érganisms in certain infections is the production of antibodies of various kinds. These may be antitoxins as in the case of tetanus and diph- theria, or bactericidal substances as in typhoid fever and cholera, or opsonic substances as in the pyogenic infections. Agglutinins, pre- cipitins, and other bodies are also sometimes produced in conjunction with the other immune substances mentioned. CHAPTER II* IMMUNITY AND SUSCEPTIBILITY GENERAL Derinition.—A clear understanding as to what is meant by the terms immunity and susceptibility is of fundamental importance. By immunity we mean the resistance which an animal or plant body possesses to the etiological microdrganisms of an infectious disease and to the disease itself. The name has been adapted from the Latin immunis which meant a person who was free or exempt from public duties and later, one who was exempt from the action of poisons. Briefly stated, immunity is resistance to disease. It results commonly as a natural termination of the process of self-healing in many infectious diseases. The absence of such resistance, which may be total or partial, characterizes what is known as susceptibility. Throughout thé animal kingdom and also among the plants there is a great variation in the immunity and susceptibility in the different species to the various dis- eases. Immunity bears no relation to the contagiousness of a disease and the term is only applied as a rule to strictly infectious diseases and not to metabolic diseases. _ HYPERSUSCEPTIBILITY OR ANAPHVLAXIS.—It has been shown that animals and. man are occasionally hypersusceptible to certain proteins. For example, there are individuals who are always seriously poisoned by the ingestion of eggs, pineapples, and strawberries. Certain indi- viduals when injected with diphtheria or tetanus antitoxin which is carried in horse serum are seriously intoxicated and occasionally die. In such instances the proteins of the horse serum and not the antitoxin are responsible. It has been demonstrated that an animal may be sensitized or rendered hypersusceptible to almost any protein by first injecting a minute amount of the protein and then, after a period of at least eight to thirteen days, may be seriously intoxicated, if not killed, by the injection of a slightly increasing dose of the same protein. The *Prepared by E. F. McCampbell. 684 IMMUNITY AND SUSCEPTIBILITY 685 proteins of the bacterial cells have been shown to act in thesame way. Animals injected, as described above, may be rendered hypersusceptible to all bacterial proteins. Furthermore, as referred to above, individuals may be naturally hypersusceptible to bacterial and other proteins. The manner of the original sensitization in these cases is not known. Sensitization which has been either naturally or artificially acquired is transferred in most instances in utero to the first generation; that is, a mother may be sensitized, convey the sensitizing substances to her young while in the uterus, and when these offsprings are subsequently injected after birth with the same protein they may be intoxicated or killed. In this connection it should be stated that the so-called in- herited tendency to specific diseases may be something more definite than we are ordinarily accustomed to regard it. Suppose a mother becomes tuberculous and is, therefore, sensitized to the proteins of Bact. tuberculosis; it is quite possible for her to convey to the offspring this susceptibility to the particular proteins of Bact. tuberculosis as has been demonstrated artificially. After birth, or in later life when the causal microérganisms of tuberculosis are taken into the body, as they are in about ninety-five per cent of all persons in civilized countries, the bacteria may find & more than ordinarily susceptible individual and may develop with comparatively little hinderance. If this condition be true naturally as it is when produced experimentally in suscep- tible animals a very interesting and scientific explanation of the so-called inherited tendency to tuberculosis is at hand. PREDISPOSITION AND NON-INHERITANCE OF InFEcTIOUS DISEASES. —There is probably no such thing as a truly inherited infectious disease. This point has been debated and discussed for a great many years and the above conclusion has been reached by the majority of investigators. By inheritance is meant the transference of a property, or in this in- stance, a pathogenic microérganism by the nuclear substance of either the spermatozoén or the ovum. It is only the nuclear substances which. combine to form the new individual. It is true among certain of the lower animals, such as the fowls and some insects, that microérganisms are carried within the egg but the eggs are quite different in structure from the human or mammalian ovum. The egg of the above-mentioned animals is composed largely of yolk-furnishing food and there is ample opportunity for microbic growth, while the mammalian ovum contains - no yolk. Such instances should be referred to as germ-cell transmission, 686 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS not inheritance. If the microdrganisms were present they would be immediately incorporated within the new developing embryo. If the microérganism ever did find its way into the human or mammalian ‘germ cells it would be a mechanical impossibility for the cells of the embryo to divide and multiply in proper manner. Such pathogens would rapidly destroy the developing cells in the embryo. It is true that the offspring of certain individuals are born diseased. For ex- ample, children are not infrequently born with syphilis and tuberculosis. At first thought it might seem that this is inheritance but on careful analysis it will be found that the mother is either syphilitic or tubercu- lous. Furthermore, the locus of the infection is most frequently in the uterus and the microérganisms are transferred to the unborn offspring by means of the fetal circulation. This condition is what is known as antenatal acquirement; it isnot heredity. It is absolutely impossible for the male to communicate any disease to the offspring unless the female is first infected. Colle years ago formulated a law which bears his name in which he stated that a father could communicate syphilis to his child without the mother being infected. This law has been disproved since the introduction of the new serum tests for syphilis and it can be posi- tively demonstrated in all such cases that the mother isinfected. Ante- or prenatal acquirement may then be recognized. What can be said in regard to the predisposition to a definite infectious disease? There is a, question as to whether true predisposition does exist. Many cases are on record to show that disease seems to run in families and in localities. For example, tuberculosis and cancer are frequently said to be subject to inheritance or to predisposition in certain cases. It can be easily seen that if one parent is diseased the germ cell of the parent will be less healthy and when combined with a normal healthy germ cell of the other parent will not give rise to as healthy an individual as when both cells are from healthy individuals. Again, the result when -the germ cells of both parents are unhealthy due to the parents being unhealthy, is evident. Predisposition seems to resolve itself into the inheritance of a weakened constitution, a constitution which will not withstand the ordinary infections easily. It-seems not to be a predis- position to any particular disease but a predisposition to all diseases, infectious and metabolic. Diseases such as tuberculosis are so \ prevalent that it is very possible that infection may take place and it be interpreted as inherited because the parent died of the same cause. IMMUNITY AND SUSCEPTIBILITY 687 As mentioned above, it may be that the true explanation of the phenomena of predisposition is found in anaphylaxis or the sensitiza- tion to various proteins of microdrganisms. Further work is necessary along these lines. IMMUNITY Immunity and susceptibility to disease are always relative and never absolute; that is, it is always possible to produce some sort of an infection in a supposedly immune animal by modifying the conditions under which the animal is accustomed to live. For example, the chicken is immune to tetanus but by keeping this animal for some time at a temperature higher than its normal it may be infected. The cow cannot ordinarily be infected with typhoid but when large numbers of the B. typhosus are injected under the skin an abscess may be produced. These and many other examples might be mentioned. Our standard of immunity in a particular animal is based upon the conditions as they exist naturally and on the average resistance of animals of the same species. Immunity to disease may be of two kinds, natural and acquired. Natural immunity is that resistance which is possessed normally by an individual. Acquired immunity is that resistance which is acquired by having an infection, or by being vaccinated, or immunized against an infection with the specific etiological microérganism or its antiserum. NatTuRAL IMMUNITY AND SUSCEPTIBILITY.—Attention should be directed to certain forms of natural immunity and susceptibility. Racial Immunity and Susceptibility.—It is a familiar fact that certain species of animals and certain races of man differ in their resistance and their susceptibility to infectious diseases. As examples of racial im- munity among animals the native cattle of Austria-Hungary and of Japan which are relatively immune to bovine tuberculosis, a disease which causes great loss among other races, may be mentioned. Again, the sheep of Algeria are relatively immune to anthrax while all other sheep are extremely susceptible. Field mice are immune to glanders while the common house mouse is susceptible. The negro is more re- sistant to the infectious microbic agent of yellow fever than other races, but is without doubt more susceptible to tuberculosis. The Japanese are said to be more resistant to scarlet fever than other races. The 688 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS Melanesians are very susceptible to measles and the Malaysians to beri- beri, while otHer races are relatively immune. Familial Immunity and Susceptibility—It is true that certain families vary in their immunity and susceptibility when compared with other families in the same community. For example, tuberculosis undoubtedly shows a tendency to run in families. In determining a case of this kind it is, of course, necessary to take cognizance of the environment’ of the individual and the association with other diseased persons. The so-called tuberculous diathesis does exist and perhaps we have an explanation of it in anaphylactic phenomena as mentioned previously. Measles and scarlet fever also in certain instances seem to run in families. Individual Immunity and Susceptibility—vVariation among indi- viduals associated together is noted in regard to their resistance and susceptibility to disease. It is well known, for example, that in a herd of cattle, which are in the main tuberculous, there are certain individuals who never contract the disease. These animals may be of the same breed and be fed and handled the same as the rest of the herd, still they never become infected. Again, in the human race, with the acute exan- thematous diseases such as scarlet fever and measles, there are children, _ for example, in the same family and of nearly the same age and living under exactly the same conditions, who contract the disease and others who do not. The exact cause of the individual, familial and racial immunity cannot be satisfactorily explained at the present time. ‘There is also a variation in the individual’s resistance at different times dependent upon food, sleep, work and general hygienic conditions. ~ Factors oF Narurat Immunity.—The natural immunity of any individual to an infection may be dependent upon several things as follows: The Protection A fforded the Body by the Surfaces.—The body surfaces may be conveniently divided into those which are external and those which are internal. Skin and Cutaneous Orifices—The first protective mechanism that we wish to call attention to is the skin. It is a well-known fact that virulent bacteria are frequently present on the skin of seemingly normal and healthy individuals. Perhaps the most common of these is the Strept. pyogenes and the M. pyogenes vars. aureus and albus. These microdrganisms and others live largely as saprophytes, feeding upon 44d IMMUNITY AND SUSCEPTIBILITY 689 the dead and desquamating epithelial cells. The skin is impermeable to these microérganisms when it is unbroken in its normal state. Experiments have been performed to determine whether the skin is normally permeable to bacteria. Bacteria have been rubbed into the skin and have produced infection but in these instances the skin has been abraded by the mechanical irritation. Bacteria may infect the sudoriferous and sebaceous glands and their ducts, in case the metabolic activity of these structures is disturbed. The ducts and the glands of the skin are protected ordinarily by a flow of the secretions. In case the flow of the secretions is decreased and the orifices of the ducts contracted as in cold weather, while bacteria find it more difficult to pass down than before, they occasionally do produce an infection. When a hair follicle is diseased and the shaft contracted or perhaps dropped out, bacteria may pass down and produce an infection. B. tetanit of tetanus or lockjaw frequently passes through the skin by means of deep penetrating wounds. The same is true of some other pathogens. In case bacteria are successful in permeating the skin either directly -or by means of cutaneous orifices, they are usually able to set up a marked inflammation of these structures and produce necrosis of the epithelium. It is in this way that pustules, boils, carbuncles, and various forms of cellulitis are produced. The secretions of the sebace- ous glands are not germicidal but are perhaps slightly antiseptic due to the salts which are contained therein. Furthermore, as soon as the serum from the blood is extravasated there may be slight germicidal action on the bacteria infecting the skin. The soluble toxins of bacteria cannot be absorbed through the unbroken skin. The Subcutaneous Tissue—In case the bacteria are successful in permeating the skin and penetrating the subcutaneous connective tissue, again various protective mechanisms show themselves. ‘This resistance is due to a very rapid production of new connective tissue which serves to mechanically limit the infection. It is due, further- more, to the germicidal action of the serum, the mechanical and germi- cidal action of the fibrin and the phagocytic activity of the leucocytes. These various factors will be discussed subsequently in connection with the phenomena and the protective mechanisms of inflammation. The Exposed Mucous Membranes of the Body—The exposed mucous membranes of the body usually are covered with a variety of bacteria, 44 690 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS some of which are pathogenic. Their moist condition favors the growth of microdrganisms, but the mucus which is secreted upon them forms a mechanical bearer to the bacteria and serves to wash them away. This mucus is not germicidal but is perhaps slightly antiseptic. The only mucous membranes of the body that are really exposed are those of the eyelids, lips, anterior nares, genito-urinary apparatus and theanus. It is perhaps more convenient to discuss these membranes in detail in connection with the cavities which are connected with them. Nasal Cavity.—Microérganisms find a barrier to the entrance of the nasal cavity in the hairs which protect the anterior nares and serve to keep out the dust from the inhaled air. The membranes of the nasal tract, besides being covered with mucus, which acts as above mentioned, are also covered with ciliated epithelial cells which move from within out and serves to wash the mucus containing the bacteria from the surface. Infections of the nasal mucous membranes are, however, not uncommon. Bact. influenze, Strept. pyogenes, M. pyogenes vars. aureus et albus, Bact. diphtheria, M. intracellularis var. meningitidis, and occasionally Bact. mallei produce infection through this membrane. The Mouth—The mouth probably contains the largest variety of bacteria to be found anywhere in the body. A large number of these bacteria are non-pathogenic, although pathogenic microérganisms do occasionally occur. All the requisite conditions for bacterial growth are provided in the mouth, namely, temperature, moisture and food. The food supply is largely derived from materials which have been de- posited during the process of mastication between the teeth and in the various depressions of the mucous membrane. The microérganisms also feed upon the desquamated squamous epithelial cells. They are being constantly washed off the membrane by the saliva which contains a certain portion of mucus. The saliva is not germicidal, and in all probability only very slightly antiseptic. The most permeable part of the mouth is in all probability the tonsils which separates this cavity from the pahrynx or throat. These lymphatic structures have many deep crypts, and bacteria once entering the tissues of the tonsils may gain access to the lymphatic circulation through these structures. In case bacteria are successful in passing the obstacles of protection afforded in the nose and in the mouth and pass into the throat, there are two routes for their entrance into the internal body, namely, through IMMUNITY AND SUSCEPTIBILITY 691 the trachea and bronchi into the lungs and through the cesophagus into the stomach and intestines. The Lungs.—In case infectious microdrganisms pass down the trachea and bronchi they meet first with the obstruction of the mucus which is secreted upon the surfaces of these tubes. In addition, ciliated epithelial cells are present and serve to cleanse the surfaces from microédrganisms as in the nose. Occasionally microérganisms lodge along the trachea and the bronchi and produce slight irritations which if left undisturbed may immediately produce serious infections. How- ever, the leucocytes from the neighboring bronchial and mediastinal lymph glands pass through the walls of the trachea and bronchi, ingest the microdrganisms, carry them back to the glands and in a majority of instances destroy them. Occasionally, however, leucocytes contain- ing virulent microérganisms get into the lymphatic circulation and these are carried by the diffusion currents in the lymph vessels down to the alveoli of the lungs and here may cause inflammations of a more or less serious character. It is probable that the Sirept. pneumonie is very frequently carried to the alveoli of the lungs in this way. Without doubt, microédrganisms cannot be directly inhaled through the air passages into the alveoli of the lungs during an ordinary inspiration, but it has been shown that in forced inspirations, such as those attend- ing upon coughing, hiccoughing, sneezing and sighing that they may be so carried. The Stomach.—In case the microérganisms pass down the cesophagus into the stomach, they immediately come into contact in the normal organ, with the gastric juice, which contains the hydrochloric acid in such concentration that it is at least antiseptic if not germicidal. In case the functional activity of the stomach is disturbed and the hydrochloric acid is diminished in amount, microérganisms may grow in the stomach to a limited extent. Furthermore, in case all the particles of food are not thoroughly broken up in the stomach, bacteria which may be contained within these particles may pass through the stomach into the intestine. The Intestines——In the intestines the microérganisms come into contact with the alkaline pancreatic juice which is slightly antiseptic and with the bile which is antiseptic and in certain instances bacteri- cidal. They find no particularly favorable conditions for growth in the upper part of the small intestines under normal conditions. Here 692 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS also mucus covers the surfaces. However, if the functional activity | of the small intestines is disturbed, bacteria may enter the lymphatic structures (Peyer’s patches, solitary follicles) low down in the small intestines and produce infection. Such is the case with B. typhosus of typhoid fever and with the Msp. comma of Asiatic cholera. Bacteria which have been prevented from development in the small intestines frequently find the opportunity in the large intestine. Here the concentration of the various digestive juices is lowered and the requisite condition for maximum bacterial growth is provided. Nevertheless, infections of the large intestine with bacteria are not common but may occur, colitis of various forms resulting. The various dysentery amcebe very frequently develop in the large intestine. The Genito-urinary Tract—The mucous membranes of the genito- urinary tract, varying in male and female, present the same features as those of other mucous membranes. Besides the secretion of mucus, various other acid-containing secretions are often present. In addi- tion, in the urinary tract the mechanical factor of irrigation removes the microérganisms. Not infrequently, however, microdrganisms do enter these mucous membranes and produce serious infections, such as the Treponema pallidum of syphilis, the M. gonorrhee and the B. chancroide mollis. Sometimes these membranes are infected with ordinary pyogenic bacteria. The Conjunctiva.—The conjunctiva is protected against infection in several ways. First, the eyebrows with their hairs and the eye- lashes prevent microdrganisms and particles of dust and dirt carry- ing microdrganisms from entering the eye. Again, the lacrymal secretion or the tears flowing across the eye from the outside in serve to wash this membrane. Bacteria are frequently washed off the conjunctiva and pass down through the lacrymal duct into the nose where they meet the obstructions which have been previously dis- cussed. Jn all probability the tears are only slightly antiseptic and not germicidal at all. The conjunctiva is sometimes infected with microdrganisms and furthermore serves as a point of entrance into the body when it itself is not infected. There is no doubt that the Bact. influenze, the Srept. pneumonia, and other microérganisms may enter the body and get into the lymphatic and blood circulation in this way. IMMUNITY AND SUSCEPTIBILITY 693 It is evident, therefore, that the protection afforded an individual by the body surfaces is a decided factor in the natural immunity of that individual. The Protective Nature of Inflammatory Processes—It has been mentioned in a previous discussion that when bacteria successfully enter a tissue and develop in that tissue a complex local change results which is designated as inflammation. In the majority of instances inflammation is of a beneficial nature. Fundamentally, it is always beneficial. Few examples of the pernicious results from inflammation can be given. In this connection may be mentioned the thickening of the cerebral blood-vessels in syphilis and the increase of connective tissue in cirrhosis of the liver. In these instances the inflammatory processes are brought about by the reaction of the various tissues to the irritation of the infecting microérganisms. Unluckily these reac- tions are not on the whole beneficial to the body, but, as before stated, inflammation is usually beneficial and may be characterized as the re- action of tissues to injury. The exact processes of inflammation may be traced in case an infecting microdrganism succeeds in entering the tissues of the body. The organism having produced its toxic substance first causes a congestion of the blood-vessels in the region (hyper- emia). Following this localized congestion there is an extravasation of plasma from the blood-vessels. This plasma immediately on leav- ing the vessels coagulates or clots, producing throughout the infected area fibrin and blood serum. This fibrin serves in a mechanical way to limit the infection, and it has been recently demonstrated that the fibrin possesses germicidal properties in addition. Furthermore, the serum in a large number of instances exerts a bactericidal effect upon the microérganisms. Following the extravasation of blood plasma from the capillaries, the leucocytes pass out and gather around the infected area. These leucocytes are attracted to the area due to the presence of various chemical substances (chemotaxis). They will come as close to the microérganisms as possible, depending upon the effect of the toxins which have been produced. In certain in- stances they will ingest the bacteria and destroy them. In such cases, the bacteria having been removed, the inflammation rapidly subsides and the infection is, therefore, checked. Such are the char- acteristics of an acute inflammation. Inflammations, however not infrequently depending especially upon the microérganism producing 694 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS the infection, may become chronic, and in such a case the inflamma- tion after passing through the acute stage, as indicated above, stimu- lates a proliferation of the connective tissue in the part infected. In such cases, around the outside of the ring of leucocytes, which have been unable to ingest the bacteria, young.embryonic connective- tissue cells which are known as round cells are found. In case the inflammation progresses, the leucocytes are destroyed and the round cells next to the infected area assume more of an elliptical shape and are known as epithelioid cells. On the outside of this layer of epi- thelioid cells will be found newly produced round cells, and on the outside of the round cells an area of recently migrated leucocytes, those passing out in the beginning having been destroyed by the toxic action of the infecting microédrganisms. Frequently the newly produced connective tissue passes on to the adult type and in this instance completely walls off the area of infection and the infecting micro- érganisms. In such cases the inflammation and the infection are checked. Among the diseases caused by microérganisms which have a tendency to produce chronic inflammation may be mentioned those of tuberculosis, leprosy, syphilis, actinomycosis and glanders. It is not an uncommon observation in man to note in the lungs and in other parts of the body healed areas of tubercular infection; areas that have been completely walled off by the development of adult fibrous tissue. It is probable that about ninety-five per cent of all individuals living in civilized communities are infected with Bact. tuberculosis some time during their lives. The inflammation produced by this microdrganism passes through the acute stage and into the chronic before being suc- cessfully combated and thoroughly walled off. Such an area is known as a tubercle, and in the other diseases mentioned, similar areas of like structure are produced. It depends entirely upon the virulence of the infecting microérganisms and the resistance of the connective tissue of the individual infected as to whether healing will result. Natural Antitoxins—It is an observed fact that certain animals resist the action of toxins produced by bacterial and other plant and animal cells. The question arises as to whether these animals are immune to the toxins on account of the presence in their bodies of natural antitoxins or other substances. If antitoxin is present, it can be detected by experiments made by drawing off the blood serum of the animal and combining it in varying proportions with the toxin in IMMUNITY AND SUSCEPTIBILITY 695 question. These experiments may be made im vitro. When toxins and antitoxins are combined in proper proportion and incubated together a non-toxic molecule is produced which when injected into a susceptible animal will produce no effect. It is, of course, necessary in this connection to inject the animal with a minimum lethal dose of the toxin in question as a control. If no natural antitoxins are present in the serum of the animal in question, the animal experimentally injected with the combined toxin and serum will die as a result of the non-combination of the toxin. In this way natural antitoxins may be tested. Natural antitoxins for diphtheria have been detected in the blood serum of about fifty per cent of normal humans and in about thirty per cent of horses. However, their occurrence in other animals for this specific bacterium and for other species is comparatively rare, and the explanation of the fact that certain animals are immune to toxins must be found elsewhere. It has been shown for example that the frog is immune to tetanus toxin, and that, when this animal is injected with this toxin that a large part of the toxin remains unchanged in the circulation for a variable period of time and may be later drawn off in the blood serum and will produce a toxic effect when injected into a susceptible animal. There are no natural antitoxins present in the blood serum of the frog and it has been found that the immunity of this animal is due to the fact that there are no cells in the body possessing the necessary side-chains (open valencies) for chemical combination with the toxin and the subsequent intoxication of the cells does not result. It seems that the best explanation of the fact that certain animals are immune to toxins is found in the fact that there are no chemical substances in the cells with which toxin can combine. It is probably not true that natural antitoxins explain all the phenomena in this connection. Natural Antibacterial Substances.—Natural antibacterial substances are present in the blood serum and body fluids of a large number of animals. In order to demonstrate the presence of the natural anti- bacterial substances it is necessary to inject the experimental animal with a carefully washed culture of the bacteria in question. If the animal remains uninfected, two possibilities present themselves: First, the presence of natural antitoxins; and second, the presence of anti- bacterial substances. It is necessary, of course, to have excluded the possibility of natural antitoxins, its having been demonstrated that 696 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS the organism injected produces its diseased effects by endotoxins held within the bacterial cell rather than by toxins. There is no evidence indicating the presence of natural antiendotoxins in any animal. The antibacterial action of the blood may be due to two constituents, namely, cellular substances (leucocytes) and chemical substances in the serum. The rat and the dog are both immune to anthrax but the immunity of the dog is not due to antibacterial substances but to the phagocytic activity of the leucocytes, while in the rat the immunity is not due to the leucocytes but to the antibacterial substances. In order to demonstrate the fact that the leucocytes are not responsible for the immunity in the given animal it is necessary to combine the bacteria in question with the leucocytés and serum im vitro and after incubation make a careful examination with these cells to see if they have taken up any bacteria. Antibacterial action is due to two substances in the serum: First, the thermostable substance which combines with the bacteria called an amboceptor; and second, a thermolabile substance called a complement, which combines with the amboceptor after this substance has combined with the bacterial cell. It is sufficient to say at this time that these substances occur in normal sera and that the result of their combination with the bacterial cell causes the death of the bacteria and in some cases a lysis (solution) of the bacteria in addition. There may be present in the blood of animals antibacterial sub- stances of three kinds: First, those just killing bacteria (bactericidal); second, those killing the bacteria and dissolving them (bacteriolytic); and third, the leucocytes which are active in the ingestion of the specific microérganisms. In all probability the overactivity of leucocytes in every case of natural phagocytic immunity is due to the presence of normal opsonins—substances which sensitize the bacteria and render them susceptible to phagocytosis. Normal Hemolysins~—Normal hemolysins (hemoglobin-liberating substances) are present in the serums of certain animals for the red blood corpuscles of other animals of different species, and for the same species, but never for the red corpuscles of the animal from which the serum was obtained. Such substances known respectively as heterolysins and isolysins and if the latter occurred the name aufolysin would be applied. Normal A gglutinins.—Normal agglutinins for various bacteria, such as B. typhosus, Msp. comma, Bact. dysenteri@, B. coli, and Ps. pyocyanea IMMUNITY AND SUSCEPTIBILITY 697 are present in the blood serum of some animals. It is necessary, of course, to exclude normal agglutinins when testing the serum of the infected case for the purposes of diagnosis as will be mentioned later. Normal Precipitins —No normal precipitins for bacteria occur in the serums of animals. Precipitins for various blood sera, however, do occur. For example, human serum will precipitate the monkey serum, etc. These substances will be discussed in detail under acquired immunity. Acgurrep Immunity.—Acquired immunity is that resistance which is acquired after-having an infection or from artificial inoculation with the etiological microdrganism of an infection or from inocula- tion with the products remaining in the body after infection, whether natural or artificial. Acquired immunity may be divided into two classes, namely, active and passive. Active immunity is that immunity resulting from an infection or vaccination. In it the body cells react and give rise to the formation of antibodies. When antibodies pro- duced in active immunity are inoculated into other animals the im- munity conferred is referred to as passive immunity. Active Immunity.—Active immunity may be produced artificially in the following ways: By the injection of living bacteria; by the injec- tion of bacteria of reduced virulence; by the injection of dead bacteria; by the injection of the secretory and excretory products of bacteria (toxins, etc.); by the injection of the disintegration products of bacteria liberated after the death of the cells (endotoxins); and by the injection of bacteria or bacterial products which in no way are related to the bac- terium against which immunity is conferred. As a result of the injection of living bacteria in small amounts or of bacteria of reduced virulence the body cells react and produce bacteri- cidal substances (lysins, etc.). Asa result of the injection of dead bac- teria, the opsonins are increased in the blood. Asa result of the injec- tion of the secretory and excretory products of the bacteria, namely, toxins, antitoxins are produced. As a result of the injection of the dis- integration products of bacteria, namely, endotoxins, bactericidal sub- stances are produced. In cases where bacteria or bacterial products, which are in no way related to the bacterium against which immunity is conferred are injected, it is probable that bactericidal substances are produced. This condition only occurs in rare instances. Passive Immunity.—Passive immunity may be conferred by the injection of antitoxins, and by the injection of bactericidal substances. 698 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS In this type of artificially produced acquired immunity the body cells do not react to any great extent and the injected antibodies remain practically unaltered. Various other antibodies may be injected into other animals and confer upon them passive immunity. The principal antibodies produced in active immunity will be subsequently discussed. THE ORIGIN AND OCCURRENCE OF ANTIBODIES The toxic and some of the non-toxic substances of bacteria and cells from other sources when introduced into the body of a susceptible animal usually have the power to produce antibodies. Substances having the power of producing antibodies are known as antigens. Among the antibodies produced are antitoxins, bactericidal and lytic substances, opsonins, antiferments, agglutinins and precipitins. The antigenic substances for these antibodies will be discussed later. The mechanism of action of the antigen is of interest. It is supposed that the antigen can combine only with the cell which has the proper com- bining groups or receptors. The antigen combines in the same way that , food products combine with the tissue cells. In case there is no group in the tissue cell with which the antigen can combine that tissue is naturally immune to the antigenous substances in question. If all the tissue cells in the body are in this condition then the individual may be said to be naturally immune. It occasionally occurs that certain cells of the body are not susceptible to the action of antigens at one time while at another they are susceptible. For example, the red blood corpuscles of the young chick are not affected by the lysin-toxin in spider poison while those of the adult are readily hemolyzed (hemo- globin liberated). It also occurs in rare cases that the antigen when injected into an animal whose tissue cells show no affinity for it or no proper receptors, will remain in the circulation for days and weeks without combining and producing any effect. The antigen, for ex- ample, a toxin, can be isolated from the blood in such a case in the same concentration and form as when it was injected. Some antigens have special affinities for certain tissues, as for example, tetanus toxin and nerve cells. In this case, however, the larger part of the anti- toxin is produced by cells other than those of the nervous system. The production of antibodies for antigens probably occurs in the fol- IMMUNITY AND SUSCEPTIBILITY 699 lowing way; the antigenous substances combine with the cells utilizing all the available receptors, leaving none open for food and thus pervert- ing the general metabolism of the cell. In such cases there is a regener- ation of these chemical receptors by the tissue cells which more than compensates for those with which the antigen has combined and as a result the cell discharges them (chemical substances) free into he body fluids. The various antibodies are usually produced with more avidity by certain tissues than by others. Antibody formation may be of a strictly local character depending upon the point where the antigen is injected. For example, when abrin is placed in the eye, antiabrin is produced, but only in the eye so injected. In the majority of cases the antibodies are produced in some special tissue or tissues at a distance from the point of injection. : Following the injection of an antigen into the body of an animal there is always a decrease in the resistance of that body and a decrease in the antibodies produced followed in a short time by a marked increase in their formation. The former condition is spoken of as the “negative phase” and the latter as the “positive phase.” Antibodies may be transferred from mother to young before birth, but only after fetal circulation is established. It has been positively demonstrated that antibodies are not transferred by the ovum or the spermatozoén directly. They are only carried from the blood of the mother and diffused through the placenta into the blood of the fetus. It has, however, been shown that the eggs of immunized chickens con- tain antibodies occasionally. This is “‘germ-cell transmission” and not true hereditary transmission. The transferred immunity or anti- bodies do not remain over two or three months in the bodies of the offspring after birth. ANTITOXINS.—Antitoxins are so called because they combine with and render inert the soluble toxins. Antitoxins are produced for all the bacteria producing soluble toxins and for the toxic substances of a large number of other plant and animal cells. Antitoxins are the free chemical receptors of ¢ertain of the cells of the body. That is, they are chemical substances which have been thrown off from the cells of the body and in all probability were normally used for the purpose of taking up food substances. These chemical substances are produced in excess of those actually needed by the cell due to a stimulation of the cells by 700 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS the toxin. The antitoxins are labile substances which cannot be analyzed. They may be similar to euglobulins. They are composed. of molecules of large size. Antitoxins when present in the body of an animal are protective and in many cases curative. According to Ehr- lich, toxins are assumed to possess two chemical combining groups, one known as the haptophore group which combines with the cells and another known as the toxophore group which combines with the cell after the haptophore group has combined and this produces an intoxication of the cell. The haptophore group of the toxin molecule is thermostable (heat resistant) and the toxophore group is thermolabile (heat suscep= tible). When a toxin is injgcted into the body of an animal, or is pro- duced during the process of an infection, the haptophore group combines with the cells with which it has an especial affinity and with the receptors (chemical substances which are unsaturated and open to combination with other chemical substances) of these cells. The chemical receptors of the cells with which the toxin-haptophore group combines are designated as haptophile receptors. tis probable also that the toxophore group of the toxin combines with other chemical receptors in the cell after the haptophore and haptophile groups have combined. These are designated as toxophile receptors. The haptophore receptors of the toxin having combined with the haptophile receptors of the cells, the toxophore group of the toxin then combines and intoxicates, stimu- lates or sometimes kills the cells depending on the affinity for the cells and the concentration of this group. In case the cell is not killed, it is stimulated and begins after a time to return to its normal functions. All the available receptors of the cells having been occupied and com- bined with, the cell sets about to generate new chemical receptors in order that food substances and other chemical substances may be taken up. The cells produce these haptophile receptors in excess, that is, there is over-compensation, and they are subsequently excreted into the lymph and blood. These haptophile receptors are in fact the chemical substances which we know as antitoxins. It is not only the cells with which both the haptophore and toxophore groups of the toxin combine because of special affinity, which make all the antitoxin, but cells which are widely separated from those which have an especial affinity for the toxin, also produce antitoxin. For example, tetanus toxin has an especial affinity for nerve tissue but this tissue produces little of the antitoxin. In this case most of the antitoxin seems to be produced in IMMUNITY AND SUSCEPTIBILITY 7OI the ‘spleen, lymph glands and bone marrow. The haptophore groups of the toxin have at least combined with these cells and stimulated them to the overproduction of haptophile receptors. It has been mentioned that the antitoxins are protective to the body infected. The haptophile receptors (antitoxins) before they are thrown off combine with the toxin-haptophore and often the toxophore group does not have the opportunity for combining and killing the cells. This is in case there is no special affinity for the cells, as in the above-men- tioned chief antitoxin-producing cells in tetanus. In such cases fre- quently all the available toxin is bound and very little is left to combine with the tissue with which it has an especial affinity, as is the case with tetanus toxin and nerve tissue. The antitoxins serve in this instance as protective substances. Furthermore, in case the antitoxin is ex- creted into the blood and lymph it serves in addition as a curative agent, all the toxin which is produced combining with all the available anti- , toxin in the circulation and none is left to combine with the cells of the body. The maximum affinity is always between toxin and anti- toxin rather than between toxin and cell, if there is any antitoxin present. Antitoxins are prepared artificially and used for both pro- phylactic and curative purposes in the treatment and prevention of certain of the infectious diseases such as tetanus and diphtheria. Antitoxins are also produced in the bodies of animals which are to all appearances immune to the toxins concerned. For example, the alligator is immune to tetanus but when tetanus toxin is injected into this animal tetanus antitoxin will be produced. In this case the hapto- phore group of the toxin has combined with certain of the cells of the body, but with such cells as give no opportunity for the toxophore group to combine, or have no affinity for this group. In the case of the alligator the nerve tissue seems to possess no chemical receptors for the toxin. There are certain animals which are very susceptible to the action of certain toxins and which will not produce antitoxin when the toxin is injected. For example, the guinea-pig and the rabbit will not produce tetanus or diphtheria antitoxin when injected with small and gradually increasing doses of tetanus or diphtheria toxin. If the toxin is modified chemically by the addition of chemicals such as terchloride of iodine or by heat these animals may be immunized and will produce antitoxin. In this instance the virulence of the toxophore group is reduced and it 702 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS is possible to inject the animals with more toxin, thus combining with more cells and finally liberating more antitoxin. It should also be noted that animals of the same species vary in their power to produce antitoxin. The production of the product varies with the age and general condition of the animal and with the duration and the degree of toxicity of the toxin used. On account of this condition it is necessary to establish units or standards for determining the strength of antitoxins. As stated in the discussion of natural immunity to toxins, there are some animals which when injected with toxins do not possess cells which have receptors open for chemical combination and as a result the toxin remains free in the circulation for varying periods of time. For example, as before stated, the frog is immune to tetanus and an injection of toxin will not produce any antitoxin. If tetanus toxin is injected into this animal it will remain in the circulation in the same form as injected and can be withdrawn after a few weeks or a month. The Mechanism of the Neutralization of Toxin by Antitoxin.— At one time it was supposed that the antitoxin was but a toxin in a little different form but this has been absolutely disproven. The amount of antitoxin produced is much greater than the amount of toxin which is injected or produced during an infection. The union between toxin and antitoxin is of a definite chemical nature. After these two substances unite the resulting compound is absolutely harmless and differs from both the toxin and the antitoxin in that it is much more stable. In the beginning all experiments dealing with the union of toxin and antitoxin were performed in the body of an experimental animal (in vivo) but finally Ehrlich showed that they would act and combine equally well in the test-tube (2m vitro) and could be studied much more easily. The various toxins are neutralized by their antitoxins with varying rapidity. The concentration of these chemical substances, the tem- perature, the character of the medium in which they are placed, and the » amount of electrolytic salts present, are accountable for the differences in length of time of combination. In the main these substances act like most chemicals and some of them show evidences of following the laws of multiple proportions. As a matter of fact the same laws which IMMUNITY AND SUSCEPTIBILITY 703 govern the union of toxin and antitoxin govern other antibodies and their antigens. As before stated, toxins have a greater affinity for the free hapto- phile receptors of cells (free antitoxin) than for those still associated with the cells. Toxin and antitoxin will always combine, if the opportunity presents itself, before toxin and body cells will enter into chemical union. Furthermore, in certain instances, such as in diph- theria, when the toxin has been partially bound by the body cells and antitoxin is produced in sufficient quantities or is injected, the toxin- cell chemical union will be broken up and the toxin and antitoxin will combine. Obviously, antitoxins of this kind are very valuable in effecting a cure in certain infections. In the above-mentioned cases, the union between the toxin and the cell is comparatively unstable but this is not true in every case, as for example, in tetanus or lockjaw. In this case when once the toxin is combined with the cells of the nervous system and other body cells it is very difficult to break up the chemical combination by the addition of antitoxin. It requires exceedingly large doses and these rarely act efficiently. The union between toxin and body cells in this instance is very stable. We have here an ex- planation why tetanus antitoxin is of so little use for therapeutic purposes. It is, however, of use as a prophylactic when free toxin is being produced in the body. Diphtheria antitoxin is efficient both as a curative and prophylactic agent for the reasons which have been discussed above. : Antitoxin like toxin is fairly unstable and such agents as heat, light, and chemicals, affect it and reduce the toxicity. It may, how- ever, be dried and kept for long periods of time in the dark. It is necessary in the commercial preparation of antitoxin and in its ex- perimental study to have a unit or standard of measurement. Units of Antitoxin.—In order to arrive at a standard it is necessary to accurately test a given antitoxin to determine the number of so- ealled antitoxic units it contains. In the accurate study of the neutralization of the toxin by the antitoxin it is noted that adding fractional amounts of the antitoxin to the L° dose of the toxin and injecting the resulting mixture into test animals (guinea-pigs), there is not a corresponding decrease in the toxicity as would be expected. The toxin is made up of various parts. The part just mentioned has a great affinity for the antitoxin but is not 7°04 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS really toxic. Such parts of the toxin molecule are called protoxoids. The protoxoids compose about one-fourth of the amount of toxin necessary to saturate one immunity unit. After the one-fourth antitoxin is added to the L° dose of toxin the mixtures of toxin-anti- toxin become less toxic for the experimental animals down to the point where approximately three-fourths of the amount of toxin necessary to saturate one unit of antitoxin has been used (three-fourths of L° dose). This fraction is true toxin. The toxicity of the mixture does not decline from this point when antitoxin is added up to one im- munity unit, and it has been demonstrated by Ehrlich and others that this is due to another part of the toxin molecule which has a lesser affinity for the antitoxin than the true toxin itself and the protoxoid. This part of the molecule is called an epitoxoid, true toxoid, or toxon. The toxin molecule necessary to saturate one unit of antitoxin is, therefore, made up of one-fourth protoxoid, one-half true toxin, and one- fourth epitoxoid, true toxoid or toxon. The toxon is in certain instances slightly toxic and is supposed by some to be a secondary toxin and in certain diseases such as diphtheria, this substance has a weak affinity for antitoxin and is a possible cause of diphtheritic paralysis. Antitoxins may be prepared for all the bacteria producing soluble toxins, such as Bact. diphtheria, B. tetani, B. botulinus and Ps. pyo- cyanea. Antitoxic substances may also be made for some of the products of other bacteria such as the Sirept. pyogenes but these differ from true antitoxins. Antitoxins may also be prepared for the toxins of certain plant cells, such as abrin, recin, crotin, and for the toxins of animals, such as snake venom and spider poison. These substances are in the main similar to those produced by bacteria, although in certain characteristics they differ materially. LysIns AND BACTERICIDAL SUBSTANCES.—Under the lysins will be discussed those substances occurring in normal and immune sera which have the power of destroying and disintegrating bacteria, those disintegrating and liberating the hemoglobin of erythrocytes (red blood corpuscles) and those substances which have a lytic action on various body cells. The substances which act on the bacteria are called bacteriolysins, those acting on erythrocytes are called hemolysins, and those acting on the other body cells are called cytolysins. The mechanism of these lytic processes is quite complex. It should also be noted in this connection that there are certain substances which IMMUNITY AND SUSCEPTIBILITY 75 kill or seriously injure bacteria and body cells and do not actually disintegrate them. Such chemical bodies are designated respectively as bactericidal substances and cytotoxins. The first observations in regard to bactericidal and bacteriolytic substances were made by Nuttall and later by Biichner. Biichner noted these substances in normal serums and other body fluids and named them alexins (Gr. to guard). He assumed that they were concerned in the immunity of the body. This is not necessarily true as certain blood serums are frequently highly bactericidal when the in- dividual is relatively susceptible. This is true of human blood serum and B. typhosus. Furthermore, in certain instances the animal is immune to the disease and the serum is not in any sense bactericidal. This is the case with the dog and Bact. anthracis. Pfeiffer a number of years ago observed that when Msp. comma of Asiatic cholera was introduced into the peritoneal cavity of the normal guinea-pig that the bacteria underwent lysis. He also noted that the process was much more rapid in the immune guinea-pig. Pfeiffer had the idea in the beginning that lysis did not take place anywhere but in the body of the animal but later it was demonstrated by a number of men, among them Metchnikoff, that the lytic action would also take place in the test-tube (in vitro). Bordét and others later showed that some normal serums possess the power of liberating the hemoglobin in red blood corpuscles. It was also shown that these hemolytic substances could be developed in the body of an animal if that animal were injected or immunized with a suspension of erythrocytes. The phenomenon of hemolysis is easily observed and studied and the amount of the hemolytic agent can be accurately determined as the amount of hemoglobin liberated varies accordingly. The mechanism of hemolysis and bacteriolysis corre- spond exactly and accordingly much about the latter process was first worked out by experimentation with hemolysins. Lytic substances can be prepared for a large number of bacteria and for many body cells, as before stated. These substances may be markedly increased by the usual processes of immunization. Those substances which have the power to produce lysins are called Lysinogens and are distinct antigens. The lysins are antibodies. Thelysins may be prepared by injecting the experimental animal with the live cells, the 45 706 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS dead cells, the disintegration products of cells and in some cases with the metabolic products of; cells. The Structure of Lysins—Lysins and bactericidal substances have been shown to be composed of two distinct parts: one a thermolabile part known as the complement which is destroyed at a temperature of 55° to 60° for thirty minutes; and another part which is thermo- stable, known, on account of its double combining ability, as an ambocep- tor. This amboceptor will withstand heating to 60° for twenty-four hours but if the temperature is raised to 70° it is readily destroyed. If kept at ordinary room temperature or in the ice box amboceptors will remain active for years. According to Ehrlich, amboceptors are the free chemical receptors of the body cells. They are produced in the same way as antitoxins but differ from these bodies in that they have two combining groups, one known as the cytophile group with which the amboceptor combines with the bacteria or other cells, and the other known as the complementophile group, with which it combines with the complement. The complement seems to be a normal constituent of the blood serum and other body fluids. It is undoubtedly produced by the various body cells (leucocytes et. al) and during the immuniza- tion of animals with certain antigens it is probably increased only slightly, if any, in amount. The complement is supposed to be com- posed of two groups also, one a haptophore with which it combines with the amboceptor, and another a zymophore which readily produces the lytic action after the haptophore has combined with the ambocep- tor. On heating the complement the zymophore group is destroyed and a complementoid isproduced. This substance is similar to a toxiod and will combine with amboceptor but no lysis will result. It is, however, the amboceptor, or so-called immune body, that undergoes the decided increase during the processes of immunization. It can be accurately demonstrated that the amboceptor must combine with the cell in question before the complement can combine. Cells, such as bacteria or erythrocytes, may be saturated with amboceptor and washed and when the complement is added and combined, lysis takes place. The complement will not combine with the cells under any circumstances unless amboceptor is present and has first combined with the cells. It is probable in a given serum or body fluid that there are several complements which may activate a variety of amboceptors. IMMUNITY AND SUSCEPTIBILITY 7°7 However, it has been-shown that the same complement will activate a variety of amboceptors of certain kinds. While the majority of lytic serums are thermolabile some have been noted which are thermostable to a certain degree. Hamilton has de- scribed such a serum resulting after immunizing animals to Bact. pseudodiphtherie and Horton’ has noted thermostable substances in normal rat serum which are lytic for Bact. anthracis. Various serums have been noted which possess amboceptors for certain cells but are not lytic because they do not possess the necessary complement. For example, the serum of the dog contains amboceptors for Bact. anthracis but no complement. If in this instance a foreign complement such as that in guinea-pig or rabbit serum is added there will-be lysis of the bacterial cells. Occasionally the absence of complement is of benefit to the animal in question and may account for the seeming natural immunity. For example, the venoms of the poisonous snakes are nothing more than amboceptors and when these substances are injected into an animal body such as a hog, which does not possess the required complement, no lysis of the body cells takes place. On the other hand, should the animal, such as a rabbit or man, possess the necessary complement, as they do, lysis will take place. Substances are sometimes present normally in serums which have the power of combining with the amboceptors which may be present, and prevent the latter from combining with the cells so that when the complement is added there will be no lysis. Such substances must be designated as antiamboceptors. These antiamboceptors (antiantibodies) may be developed in an animal by immunization with amboceptors of definite kinds. There are other substances which may also engage the amboceptors which cannot be called amboceptors in the true sense but they accomplish the same purpose and are, therefore, classed with these bodies. The Deviation of the Complement—The complement may be deviated in several ways and as a result lysis of the cells in question may be prevented. Occasionally there is noted in serums normally substances which may combine with the complement and prevent this body from com- bining with the amboceptor. Such substances are called anticomple- ments and may be produced artificially by the immunization of animals 708 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS with complement. Occasionally complement is absorbed by tissue cells and prevented from combining with amboceptor. In case there is an excess of amboceptors in a serum and only a small amount of com- plement, it may be deviated. In this case the cells will have taken up all the possible amboceptor and there will be an abundance of ambo- ceptor free in the serum. It has been demonstrated that complement will combine with free amboceptor before it will combine with the amboceptor which has been bound to thecells. In this case all the avail- able complement will be taken up by the amboceptor which is free and consequently there will be nolysis. This fact is of importance in certain infections where the development of bacteriolytic substances are of importance and necessary in effecting a recovery. The infectious ‘microdrganisms may not be destroyed for the above reason. The Deflection of the Complement as a Test for Antibodies.—A very in- genious procedure has been devised for the testing of serums for unknown antibodies similar to bactericidal substances and lysins. The method of demonstrating the fixation of the complement was first worked out by Bodét and Gengou. The reaction is made use of in the test for syphilis which is briefly stated as follows: when the syphilitic antigen is combined with the supposed amboceptor in the blood serum of the suspected case of syphilis and a foreign complement, which has been accurately standardized, is added, this complement is bound and is, therefore, prevented from combining with red blood corpuscles, and a hemolytic amboceptor which may be added later. Hemolysis is, therefore, prevented. The technic of the test is as follows: the syphi- litic antigen is prepared by making an aqueous or alcoholic extract of the liver of syphilitic fetus or in several other ways. This antigen is - supposed to contain the protein products of the Treponema pallidum, the etiological microérganism of syphilis. The blood serum of the suspected case of syphilis is heated to 56° for thirty minutes in order that the normal or immune serum complement may be destroyed. The new complement is supplied from normal guinea-pig serum. Before beginning the test it is necessary to have a rabbit immunized with some hemolytic antigen, such as sheep erythrocytes. There is developed in the serum of the rabbit the hemolysin for sheep corpuscles which when combined with these corpuscles will cause a liberation of hemoglobin. In the rabbit serum there are both hemolytic amboceptors and comple- ment. It is necessary to heat this hemolytic rabbit serum to 56° for IMMUNITY AND SUSCEPTIBILITY 7°9 thirty minutes‘in order to destroy its complement and also it is necessary to accurately find out the amount of guinea-pig serum which will com- plement the resulting hemolytic amboceptor. This definite amount of complement having been determined, it is mixed with syphilitic antigen plus the syphilitic amboceptor, mentioned above, and allowed to incu- bate for one hour and thirty minutes at 37°. If the serum is from a case of syphilis the antibodies (amboceptors) will be present and com- bine with the antigen, and alsothe guinea-pigserumcomplement. The next step in the technic is to add to the above-mentioned mixture the hemolytic amboceptor and its antigen, sheep corpuscles. If the com- plement has been bound there will be none left to combine with the hemolytic amboceptor and no hemolysis of the sheep corpuscles will result. If the patient’s serum does not contain syphilitic amboceptors or antibodies, the complement will not be bound and hemolysis will result. This test has been designated as the Wassermann test on account of the man first working it out in the case of syphilis, and has shown itself to be very efficient in the diagnosis of this disease in suspected cases. Many modifications of this test have been devised, some of which are very accurate. The fixation of the complement may be made use of in the detection of any bacterial antibody, the procedure being approximately the same as above indicated and the hemolytic system used as an indicator as in the case of syphilis. Theantigen, however, is different. When working with specific bacteria a suspension of bacterial cells in 0.85 per cent sodium chloride solution constitutes the antigen. Cytotoxins and Cytolysins —The names cytotoxin and cytolysin are used synonymously and are applied to those substances in serums and other body fluids which have the power of destroying cells other than erythrocytes. In a broad sense any substance destroying a cell would be cytotoxic but the terms are usually applied in the more limited manner, as above indicated. Cytotoxins are produced in the same manner as other antibodies. The immunization of an animal, for example, with renal (kidney) cells, produces in the blood serum of that animal a cytotoxin for the paren- chymatous cells of the kidney. Cytotoxins can be produced for prac- tically all the parenchymatous cells of the body. These immune bodies are not very specific and even careful experimentation leads to confusing results. For example, when an animal is immunized to kidney cells there is produced in the body of the immune animal cytotoxins for 710 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS kidney cells and also cytotoxins in smaller amounts for other paren- chymatous cells such as those of the liver. In the beginning it was sup- posed that the cytotoxins would be of value in the study of the physio- logical’ functions of organs and tissues. For example, a cytotoxin having been produced for the thyroid gland or adrenal gland it would be possible to inject this into another animal, destroy the gland, and then note the effect on the body. It was thought it might be possible to produce anticytotoxins which would be able to counteract the action of those cytotoxic substances which are produced in the body during the course of infections. However, the lack of specificity of the cytotoxin renders these procedures only theoretically possible. The fact that cytotoxins are produced for cells other than those used in the process of immunization indicates that there are similar chemical substances in the various cells. There are autocytotoxins produced in the body. These probably result from the absorption of the products of disintegrated tissue cells. If no anticytotoxins for these autocytotoxins are produced, or they are not destroyed in some way, a very “vicious cycle” would result in that more of the specific cells of the organ or tissue used would be de- stroyed. Cytotoxins have been prepared for leucocytes and these substances are sometimes developed during the progress of an in- fection. The leucocytotoxins have perhaps been studied more than any one of others. When ova are used for the purpose of producing cytotoxins, besides producing these substances in the serum of the immune animal, cytotoxins for spermatozoa of the same species are also produced, showing that these cells have some chemical substances in common. Metschnikoff, following his idea that old age is due to a destruction of tissue by the mononuclear leucocytes, hope that it would be possible to produce a cytotoxin for these cells. It is claimed by some that there are specific substances produced by the exhaustion of certain cells, that is, a toxin of fatigue. Weichardt has produced an antibody for this toxic substance which must in reality be an anticytotoxin. It has been suggested that the cardiac hypertrophy in nephritis is due to the effect of a nephrocytotoxin on the peripheral blood-vessels causing increased diastolic pressure on the heart. Another interesting substance has been produced and this is called syncytiolysin. It is prepared by immunizing animals with placental IMMUNITY AND SUSCEPTIBILITY Vit cells. It is claimed that this cytotoxin produces on injection symptoms similar to those noted in eclampsia and it has been suggested that the production of such a body in the pregnant woman from the placental cells may be the cause of this serious condition. Liepmann claims to have demonstrated placental constituents in the blood of pregnant women by means of the precipitin test. These bodies must be the antigen of cytotoxins. He states that when the blood of the pregnant woman is mixed with the specific syncytiolysin produced by im- munizing an animal with human placenta that a precipitate occurs. He suggestst he possibility ofaserum test for pregnancy. Abderhalden has reported some interesting results with the serum test for preg- nancy and cancer. His findings cannot, however, be regarded as conclusive. Cytotoxins are similar to bacteriolysins and hemolysins. They consist of amboceptors which are activated by the complement which is normally present in the serum or other body fluids. THE OpsonINsS AND PHAGOCyYTOSIS.—It was shown a number of years ago that certain types of leucocytes and other body cells were capable of ingesting bacteria and other plant and animal cells. The mechanism of this process was not known until Wright and Douglas demonstrated certain substances in the blood serum and other body fluids which have the power of rendering the bacteria susceptible to phagocytosis. These substances are known as opsonins (Gr. I pre- pare food for). The phenomena of the phagocytosis depend almost wholly on these special opsonins. Leucocytes which have been washed free from all serum will not take up bacteria except a few in rare in- stances. Bacteria which have been placed in contact with blood serum or other body fluids may be thoroughly washed, and then when they are placed in contact with the leucocytes, they will be taken up. The opsonin reacts chemically with certain substances within the bacteria, and so to speak, sensitizes them. Opsonins are present in many normal serums for the various bacteria. They may be produced in animals not containing them by the process of immunization with various antigenous microérganisms. Opsonins are destroyed at about 60° for thirty minutes, but there is some variation among them. When kept at o° opsonins will remain active for several days, but at a temperature of the body, 37°, after the serum has been withdrawn, they rapidly deteriorate. Many opsonins have the structure of 712 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS agglutinins and precipitins, although they bear some points of re- semblance to antitoxins and complements. They possess two so- called chemical groups, a “combining group” by which they enter into chemical union with the bacteria and a “functional group” which really sensitizes the microérganism and makes it phagocytable. It has been shown that the opsonins may be increased in the serum of the animal or injected individual by the injection of heated (60°) cultures of the specific etiological microérganisms. Such sub- stances are called opsonogens or vaccines (bacterins). Vaccines are used to a certain extent in the treatment of the various pus infections due to the staphylococci and also in tuberculosis and pneumonia. It is supposed that the opsonins are produced in the subcutaneous tissues and in the muscles. The Opsonic Index.—The concentration of the opsonins may be recorded in an individual in the following ways. Suppose the leu- cocytes of the infected individual take up a certain number of bacteria, say an average of 5, after counting 50 to 100 polymorphonuclear leu- cocytes. In this case the phagocytic index is said to be 5. Again, suppose the leucocytes of the normal individual take up 15 of the bac- teria in question, the average after counting 50 to 100 leucocytes being always taken. The phagocytic index in this case would be 15. In order to determine the opsonic index of an infected individual the phagocytic index of the normal individual is taken as a denominator of a fraction and the phagocytic index of the infected individual as the numerator of the fraction. In the above illustration this would be 545, 14 or reduced to decimals 0.33+. The opsonic index, it can be seen, is somewhat of an indication of the resistance of the particular individual to the infecting microdrganism in question. By the use of vaccines the opsonic index may be raised to at least 1.0 or even more, showing that the leucocytes are actively phagocytic and the opsonins increased in concentration in the blood serum. In such a case recovery would be indicated. When vaccines are injected in the treatment of infections the opsonic index has been shown to vary from time to time. Within a few hours after the injection the opsonic index falls below what it was at the time of the injection. This lower- ing of the index is known as the “negative phase.” Following the fall in the index there is a continuous rise to a point equal to what it was in the beginning and above this point. This rise in the opsonic IMMUNITY AND SUSCEPTIBILITY 713 index is known as the “‘ positive phase.” The individual receiving the vaccine usually shows an increase in the symptoms during the “‘ega- tive phase.” Obviously, it is necessary not to give a subsequent in- jection of vaccine until the patient is at the height of the “positive phase.” This can be best determined by determining the opsonic index. Occasionally counts are made of the number of leucocytes which are actually taking up bacteria, disregarding the number of bacteria within the cells. The determination is always made on the basis of 100 and the per cent of leucocytes which are phagocytic is taken as the so-called percentage index. The percentage index also gives an idea of the re- sistance of the individual. It has been shown that in the practical work of treating infections with vaccines it is not absolutely necessary to determine the opsonic index or percentage index. The positive and negative phase may be determined fairly well by general clinical observations on the infected individual. Virulent bacteria are not readily phagocytized. For example, virulent streptococci and pneu- mococci are not phagocytized as easily as non-virulent forms. It seems in this instance that there is some toxic or poisonous substance produced by the bacteria that is antagonistic to the opsonins or perhaps an antiopsonin is formed. The presence of opsonins in the body fluids of an animal is not absolute proof that such animal is highly resistant to infections. The resistance really depends on the activity of the phagocytes and in certain cases where the opsonins are high in concentration the phago- cytes are not active. In other cases the reverse is true and in these cases opsonins and phagocytosis are of the utmost importance in the immunity of individuals. For example, in anthrax the immunity of the dog is due to opsonins and phagocytosis, while in the rat,-although opsonins are present, there is no phagocytosis and immunity is due to antibacterial substances in the blood serum. In certain infections, such as typhoid fever, influenza, and uncomplicated miliary tubercu- losis, there is a deficiency in leucocytes (leucopenia) and consequently even if the opsonins were concentrated and the bacteria sensitized there would be very little increase in the immunity from these causes. Hemoépsonins.—It has been demonstrated that very frequently opsonins for red corpuscles are present in the sera and body fluids of animals. Such bodies sensitize the red blood corpuscles and render 714 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS them susceptible to phagocytosis by the polymorphonuclear leucocytes and the epithelial and other body cells. They are designated as hemo- Gpsonins. Occasionally iso- and autohemodpsonins are present in normal sera. For example, in human blood serum, it is probable that the process of red blood corpuscle destruction which takes place in the spleen may be referred to the action of these types of opsonins and various phagocytic cells. AGGLUTININS.—Agglutinins are substances, present in the blood serums and body fluids of normal and immune animals, which have the power of producing a clumping and sedimentation of the microérganisms causing the specific infection or used in artificial im- munization. The relationship of the agglutinins to the phenomena of immunity and the other antibodies which are produced during the process of infection and experimental inoculation is not known. One of the first agglutinins to be observed was that occurring in the blood serum in cases of typhoid fever and. the agglutination reaction is now made use of in the diagnosis of this disease (Widal test). Agglutinins are specific substances and at high dilutions only cause a clumping of the microdrganisms which give rise to their formation (antigens). Normal A gglutinins —Agglutinating substances, as above stated, are frequently found in normal serums. In this case no direct connection between their formation and specific microérganisms can be established. Normal human serum frequently contains agglutinins for B. typhosus, B. coli, Bact. dysenterig, and occasionally M. pyogenes var. aureus and Msp. comma in certain rare cases. Agglutinins for B. typhosus which are present normally in the serum may give rise to confusion when this test is'used for the diagnosis of typhoid fever. It is, there- fore, necessary to dilute the serum of a suspected case of typhoid fever at least one to forty or one to fifty times in order to exclude the normal agglutinins and the so-called coagglutinins. The Production of A gglutinins—Agglutinins may be produced arti- ficially by the injection of bacteria, dead or alive, into the veins, sub- cutaneous tissues or peritoneal cavity. In rare cases they may be pro- duced by feeding the bacteria, injecting them into the air passages of the lungs or by rubbing them into the skin. It is probable that the highest concentration of agglutinins results from the injection of dead bacteria. It is, however, necessary that these bacteria be not subjected to a temperature above 62°. Many pathogenic and non-pathogenic IMMUNITY AND SUSCEPTIBILITY 715 bacteria form agglutinins when injected into the body. The concen- tration of the agglutinins produced varies greatly. Very high agglu- tinating serums are noted, such as, for example, one in one million when B. typhosus is used and one in two million when Msp. comma of Asiatic cholera is used. Often two strains of the same organism will vary greatly in their power to produce agglutinins. Again, the concentra- tion of the agglutinins in an infected animal varies from day to day, and in order to make an accurate observation it is necessary to make repeated examinations on subsequent days. For example, in typhoid fever the agglutinins one day may be thirty times as strong as on a subsequent day. The Distribution of Agglutinins in the Blood.—As before stated, these antibodies are found in practically all the body fluids. They reach their highest concentration in all probability in the blood serum. In certain cases they are in high concentration in the milk. Agglutinins are also present at times in the sputum, tears, and the humors of the eye. Inherited A gglutinins—Agglutinating substances may be transferred from the mother to the offspring im utero. It has been frequently demonstrated, for example, that the offspring of mothers who have recently recovered from typhoid fever or who are infected at the time of the birth, have agglutinins in the body fluids. The same is true of the offspring of glandered horses. Notwithstanding the fact that the milk is frequently rich in agglutinins, these substances are not trans- ferred to the offspring to any great extent by this means.. The Substances Concerned in A gglutination—There are two distinct substances concerned in this reaction, one substance which is present in the serum or body fluids of the infected or immune individual, and other substances which are present in the microdrganisms which are agglutinated. The substance in the serum, as before stated, isknown as the agglutinin; the substance (antigen) in the bacteria or other micro- érganisms is known as the agglutinogen. When agglutinins and agglu- tinogens are combined together a new substance is formed which is designated as an agglutinate. As to the location within the bacterial cell of this agglutinogen (agglutinum) there is some dispute. Various authorities have stated that it is present in the cell wall or on the cell wall. Others have held the view that it is located within the cell proto- plasm and in certain instances in the flagella. Without doubt, in certain cases this substance is excreted from the cell into the surround- 716 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS ing medium, as is shown by the fact that when filtrates of bacterial cul- tures are injected they frequently give rise to the formation of agglu- tinins. This agglutinogenic substance is specific and varies with the species. There are, however, very closely related substances of this character among some groups of bacteria. When these agglutinogenic substances are injected into the animal they frequently give rise to agglutinins which when combined with other members of this group will produce agglutination in low dilutions. Such a reaction and prop- erty is known as “group agglutination,” and the agglutinins produced in such a case are known as coagglutinins. For example, the serum of the patient suffering from typhoid fever or of an animal immunized with B. typhosus will produce an agglutination first of B. typhosus, but in addition, an agglutination of B. coli, B. paracoli, B. paratyphosus, and B. enteriditis. The agglutination of these last-named organisms, of course, will not be active except in low dilutions, and in order to exclude them satisfactorily it is necessary to dilute the serum to a higher point. This phenomena of, coagglutination is due to the fact that there are some chemical substances (agglutinogenic) within these bacteria which are common to all and which give rise to the formation of agglutinins, which are chemically similar to each other in certain . respects. Structure of Agglutinins and Agglutinogens According to Ehrlich’s conception the agglutinins are composed of two chemical groups, a haptophile or combining group with which it combines with the hapto- phore group of the agglutinogen and a zymophorous or agglutinophor- ous group which actually produces the agglutination. The agglutino- gen is also composed of a combining group known as the haptophore group with which it combines with haptophile of the agglutinin. It is probable that this same haptophore group will combine also with vari- ous tissue cells and give rise to formations of agglutinins which are really free haptophile receptors of the tissue cells which have been acted upon by the agglutinogenic substance contained in the bacteria. Agglutinoids.—It is possible by means of heat and chemicals to destroy the zymophorous group of the agglutinin leaving only the hapto- phileigroup. Such a substance is known as an agglutinoid, being similar to a toxoid. A temperature of not to exceed 60° to 70° is necessary to produce this substance. Agglutinoids will combine with the agglu- tinogen of the bacteria but they will not produce a clumping or an agglu- IMMUNITY AND SUSCEPTIBILITY 717 tinate. Occasionally in some fresh serums substances are found which have a greater affinity for the agglutinogen of the bacteria than the agglutinins have. Such substances are designated as proagglutinoids and are in this respect similar to protoxoids. The Stages of Agglutination—There are two distinct stages of the agglutination reaction. Neither of these stages can take place unless some salts or electrolytes are present. Sodium chloride is the common salt present. The first phase of the agglutination reaction is a union between the agglutinin and the agglutinogen of the bacteria. The second phase is the actual clumping of the bacteria. It is supposed that in this last phase the zymogenic group of the agglutinin is acting. In the first phase the haptophile group of the agglutinin is combined with the haptophore group of the agglutinogen. There are some bacteria that cannot be agglutinated, as for example, Bact. pneumonie of Friedlander, and in rare instances B. typhosus cannot be agglutinated. It is possible, for example, to grow B. typhosus at a temperature of 42° and cause it to lose its power of producing agglutinins. Bacteria may also be modified chemically so that they will lose the power to produce agglutinins. Agglutinins bear no relationship to bactericidal substances, anti- toxins, opsonins or any of the other antibodies. They are both of use in the determination of species of bacteria when a known agglutinating serum is used, and they are also of use in determining the cause of infections where a known culture or agglutinogenic substance is used. The agglutination reaction is used in the diagnosis of typhoid fever, paratyphoid fever, glanders and dysentery. Hemoagglutinins.—Agglutinating substances are sometimes pro- duced for red blood corpuscles when these cells are used in the immu- nization of an animal. Such agglutinins when combined with the corpuscles produce a clumping which is known as hemoagglutina- tion. The mechanism of the reaction is the same as that of bacterial agglutinins. It is possible that hemoagglutination is one impor- tant factor in the production of agglutination thrombi in' certain infectious diseases such as typhoid fever. Precipitins.—Another group of substances, which are antibodies, is produced through the processes of immunization which have not been definitely connected with the phenomena of immunity. These substances are known as the precipitins. Precipitins may be produced 718 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS for the protein substances of most bacterial cells and a large variety of other plant and animal cells, such as blood serum, milk and grains. They were first demonstrated in 1897 by Kraus, who noted that the bouillon filtrates of cultures of B. typhosus, Bact. pestis, and Msp. comma would cause precipitates when mixed with the blood serum taken from cases of these diseases. The precipitin reaction is definite and specific. The protein substance used in immunization or concerned in the infection is the only one which is precipitated when the anti- serum is added. To the protein substance which produces the precipi- tins the name precipitinogen is applied. To that substance in the blood serum and body fluids of the immunized or infected animal or person the name precipitin is applied. The combination between the precipi-. tinogen and the precipitin forms a new chemical substance known as a precipitate. Precipitin may be formed in various parts of the body, for example, in the parenchymatous cells of the organs and by the leucocytes. Bact. diphtherie will not act as a precipitinogen and will not produce precipitins. This is practically the only bacterium which will not yield these antibodies. Normal Precipitins—Precipitins for alien blood serums have been found in the organs and blood of seemingly normal animals. Normal precipitins for bacterial proteins have not been demonstrated to a certainty. Mechanism of the Formation of Precipitins—-The mechanism of the formation of precipitins is similar to that of other antibodies. When the precipitinogen is injected into the body of an animal, it combines with certain of the body cells, occupying chemical receptors which otherwise would be used for the taking up of food products. As a result the cells produce new receptors and the number of these more than compensate for the ones already utilized. The chemical. receptors are finally thrown off into the body fluids and form the pre-. cipitins. It is supposed that the precipitinogen contains haptophore receptors which combine with the haptophile receptors of the cells... When these haptophile receptors are regenerated and produced in excess, as before stated, they are thrown off into the body fluids and are really what we know as precipitins. Precipitins are produced most commonly for widely different or heterologous substances or serums (heteroprecipitins). - Autoprecipitins and Isoprecipitins—It has been demonstrated IMMUNITY AND SUSCEPTIBILITY 719 that animals will not produce precipitins for their own protein sub- “stances. For example, if an animal is bled and injected with its own blood serum an antibody will not be produced. Therefore, autopre- cipitins do not occur. Again it has been shown that only in rare instances do animals produce precipitins for members of the same spe- cies. For example, if an animal, such as a goat, is bled and the blood serum injected into another goat, it is only in rare cases that the second goat will produce an antibody which is capable of producing precipi- tation of the proteins in the first goat’s blood serum. Such precipitins are known as isoprecipitins and occur only in a very small per cent of - cases and with no regularity. The Phenomena of Specific Inhibition —When precipitins are heated to low temperature (50° to 60°) or are subjected to the action of light or certain chemicals, their power to produce a precipitate when combined with a precipitinogen is destroyed. The precipitin which has been heated becomes a precipitoid similar to an agglutinoid or a toxoid. Their ability to combine with the precipitinogen still remains. It is possible, therefore, for precipitoids to combine with all the avail- able precipitinogen so that when fresh precipitin is added no precipitate will occur. This is known as specific inhibition and sometimes leads to very confusing findings in the study of these immune bodies. Antiprecipitins——When an animal has produced a precipitin in its blood serum due to the injection of the antigenous substance which in this instance is known as the precipitinogen, this precipitin, which is a definite antibody, may be used for the immunization of another animal and an antiprecipitin produced; that is, a body which will combine with the precipitin in such a way as to prevent precipitation when this substance is combined with the precipitinogen. This is then, in fact, an antiantibody and is practically the only example we have in immune reactions of such a substance. The antiantibody is the limit for antibody formation. The Precipitmogen.—As before stated, the precipitinogen is any protein substance which will cause the formation of precipitins. Cer- tain of the precipitinogens are composed of two groups, one which is thermostable and another which is thermolabile. Therefore, when these precipitinogens are heated and this thermolabile substance de- stroyed there results a substance which is exactly similar to the precipi- toid produced by heating the precipitin. Such bodies are known as 720 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS the precipitoids of the precipitinogen in distinction from the precipitoids of the precipitin. These precipitoids retain their power to combine with precipitin, but no precipitate results on such combination. The Precipitate—When precipitin and precipitinogen combine it requires some little time before precipitation occurs. This is dependent upon the temperature (37° best) and certain other factors. The presence of the trace of organic acids materially facilitates this reaction. Furthermore, the reaction will not take place without the presence of certain electrolytes or salts. Coprecipitins —The phenomena of “group precipitation” does not occur as often as does “group agglutination.” The bacterial precipitins are very markedly specific but some of the blood precipitins are not so specific. For example, in a case where two rabbits have been im- munized, one with the blood serum of man and the other with the blood serum of the monkey, it,is found that the serum of the rabbit immunized to human blood serum will precipitate monkey blood serum to aless. degree, of course, than human serum. This is due to the fact that there are certain chemical substances in common in the blood sera of the monkey and man. There are other rare instances of coprecipitins which will not be discussed. The Forensic Use of Precipitins—The precipitins are of use on ac- count of their great specificity in the identification of various albumi- nous substances. They have been used, for example, in the identifica- tion of bloods. Before the knowledge of the precipitins was available, the only means of determining one blood from another was by means of the microscopic examination of the corpuscles. If the corpuscles were in a good condition, it was possible, for example, to differentiate between a mammalian and fowl blood, on account of the nucleation of the cor- puscles of the latter. By the use of the spectroscope it was also possible to determine whether a particular stain was blood or not. When it ‘came to determining the exact species from which the blood came it was impossible. By means of the precipitins this can be done. For example, a stain which is supposed to be blood is carefully dissolved out in 0.85 per cent sodium chloride solution and placed in a sterile test-tube. A series of animals, such as rabbits, have been immunized to the various known blood sera and after immunization their sera are drawn off. These sera contain the precipitins for the various sera and corpuscles used in immunization. These precipitins are combined IMMUNITY AND SUSCEPTIBILITY 721 separately in small test-tubes with the salt solution preparation of the blood in question. A precipitate occurs when the corresponding pre- cipitating serum is-added. It is necessary, of course, to place these preparations in the incubator at 37°. By this method all types of mammalian blood may be separated from each other with the possible exception, as before stated, of monkey and human blood. In this instance it is necessary to make careful comparisons in order to deter- mine the concentration of the precipitins. The precipitins may also be used in the identification of various meats and other albuminous substances such as eggs. In some ways the precipitins resemble colloids and it has been shown that organic colloidal substances such as ferric hydroxide, etc., when in aqueous solution, may be precipitated by the addition of certain elec- trolytic salts. The precipitation occurs in this instance in a very similar manner to that of the organic precipitins. THE THEORIES OF IMMUNITY Various theories have been proposed which attempt to account for the resistance naturally present in animals, and the resistance which may be artificially produced. One of the first theories proposed was the so-called noxious retention theory which held the view that in natural immunity there were natural noxious substances present in the body which prevented the growth of the infectious microdrganisms. In acquired immunity it was supposed that, as the result of an infection, specific noxious substances were produced and consequently new infect- ing microdrganisms of the same species as those producing the original infection were unable to grow. This theory has long been discarded. Another theory, for a time prominent, was known as the exhaustion theory. It was conceived that natural immunity was due to the fact that the body tissues did not possess the necessary food products for the invading microérganisms and that in acquired immunity these necessary food products were exhausted completely so that when a sec- ond infection was attempted none could possibly occur. This theory has also been discarded. One of the most prominent theories is the one which has been held more recently, with some modifications, namely, the chemical side-chain theory of Ehrlich. It is claimed that tissue cells are made 46 722 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS up of definite chemical substances which possess chemical side-chains which are open for chemical combination with other substances. It is by means of these chemical side-chains that food products are absorbed and assimilated by the cells. Furthermore, it is by means of these chemical side-chains that toxins and various poisons are absorbed by the cells. It seems to have been clearly demonstrated that as a result of the absorption by certain cells of the body of toxic substances, particularly bacterial toxins, that the cells are stimulated and produce or open up an excess of these chemical side-chains for combination with various substances. It is conceived that if enough toxin (not enough to kill the cells), is assimilated by the cells the chemical side-chains which are definite chemical substances will be split off from the original cell compound and escape into the circulation. It is these escaped chemical side-chains which constitute the antitoxin or bactericidal substances. In the case of antitoxins, they possess a maximum affinity for the toxin and will combine with the toxin much more readily than the toxin will combine with the remaining chemical side-chains of the original cell compound. In the case of bacteri- cidal substances they will combine with the bacteria and destroy them and liberate in this way the endotoxins which may subsequently combine. with antiendotoxin (?) or tissue cells. Inasmuch as no antiendo- toxins are ever produced, the presence of bactericidal substances in a large percent of instances is a detrimental factor. The production of antiendotoxins by.some method or other is extremely desirable. Since the majority of our diseases are due to bacteria-producing endo- toxins, such a product would be of immense value in combating these infections. The chemical theory of Ehrlich explains many features of the phenomena of immunity. This theory has been the basis of nearly all of the preceding discussions on the various antibodies. Metschnikoff suggested what may be called a phagocytic theory of immunity. According to his ideas and those belonging to his school, the phagocytes, and principally the mononuclear and poly- morphonuclear leucocytes, are concerned in immunity. He explains natural immunity to toxins on the basis of an increased toxin-absorp- tive power on the part of these cells for toxins. He explains natural antibacterial immunity to an increased power of phagocytosis for the invading microdrganism by the leucocytes. He conceived that in acquired immunity to toxins these cells develop as the result of an in- TMMUNITY AND* SUSCEPTIBILITY 723 fection or artificial injection of microérganisms, an increased power of absorption of toxin and the power of producing antitoxin, and that acquired immunity to bacteria-producing endotoxins is due to the in- creased power of the phagocytes to ingest and digest invading micro- organisms. We find the best explanation for the phenomena of immunity in both the theories of Ehrlich and Metschnikoff. Undoubtedly certain forms or types of immunity are due to definite chemical substances known as antitoxins or bactericidal substances, while other types are due to the activity of the phagocytes. CHAPTER III MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS Diseases CausED By Morps* AND YEASTS The diseases produced by fungi in higher animals are mostly localized infections of the skin (dermatomycoses), of the mouth and throat (thrush), and of the lungs and air passages (pneumomycoses). In insects, one large series of forms, the Laboulbeniales, produce local affections only; many other forms from widely different groups are destructive to particular insects. PNEUMOMYCcosISf ASPERGILLOSIS.—The fungus disease of the lungs and air cells of birds is quite uniformly attributed to Aspergillus fumigatus which is widely distributed upon feed and grains as well. The agency of this species in causing disease is well established. It grows best at blood- heat. Inoculation experiments have reproduced the disease. Iso- lated cases are recorded where the same organism is regarded as the cause of disease in horses or cattle and even man, but not proved. Other species of Aspergillus, A. flavus, A. nidulans, A. niger, have been listed among pathogenic forms from their presence at times in diseased tissue. Whether these species are ever a primary cause of disease is doubtful. Secondary Infections Spores of any species of fungus found in the locality may find lodgment in wounds, orifices open to the outside, . such as the external ear or the air passages. Many of these spores will germinate in such situations. If favored by dirt, pus, mucus, or existing pathological condition the resulting growth in some species develops into a secondary infection; most species lack entirely the power to produce disease. The appearance of molds, especially species of * Arranged generically as far as possible. + Prepared by Charles Thom. 724 MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 725 Mucor, Penicillium, and Aspergillus, in such situations has been fre- quently reported in literature. In very large measure at least such presence may be regarded as evidence of lack of care, cleanliness, of even ordinary precautions when the infection involves man. THRUSH* The parasite of thrush, Oidium albicans Robin, (Saccharomyces albi- cans, Reiss), in culture produces a scanty mycelium, submerged in the i Fic. 150.—Oidium albicans, from a culture obtained from Kral. substratum, which branches monopodially. The tendency to budding and to the entire suppression of the mycelium leads some to regard this form as a yeast (Fig. rs0). It attacks the mucous membrane of the Fic. 151.—Oidium albicans. (Kohle and Wassermann.) mouth and throat in young animals only, producing vesicles, then white membranous patches composed of the mycelium of the fungus (Fig. 151). It is to be recognized in such cases by microscopical examination. The same disease affects children and is found in fowls, calves, and colts. * Prepared by Charles Thom. 726 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS DERMATOMYCOSES* The molds which cause skin diseases form a small group, with re- lationships to the commoner forms of fungi very ill-defined. They pro- duce a vegetative mycelium within the tissues of the host with fertile branches which bear conidia but indicate little as to their group rela- tionships among fungi. Certain of these diseases have been carefully studied, mostly from the pathological side. BaRBER’s ItcH, RINcworM, Herpes TONSURANS, ‘TRICHOMYCOSIS. —The disease due to Trichophyton tonsurans (Fig. 152), Malm, has re- ceived many names in different languages. It attacks man and dom- estic animals, the ox, horse, dog, cat, sheep, hog, probably other animals as well. It is characterized by the formation of circular patches from which eventually the hairs fall. These patches enlarge radially and fuse into large areas covered with crusts with more or less discharge in the center. The fungus is recognized microscopically by examination of hairs pulled from the growing edge of the infection. The hyphe penetrate the layers of the skin and especially surround the roots of the hairs which, when first affected, stand stiff and straight. * Prepared by Charles Thom. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 727 The appearances of the disease differ in the various species of in- fected animals, as also does the length of time it continues. The disease does not affect the general health greatly, since it primarily attacks the drier and more horn-like portions of the skin, but becomes conspicuous by the falling of the hair and by the scabs or crusts with accompanying itching and discomfort. Other species of the same genus have been described which produce infected areas differing in detail but similar in their general characters. Favus.—Favus is caused by “Achorion schtnleinii, Remak, and affects man, cats, dogs, mice, rabbits, and fowls, and many wild animals. This is characterized by crusts, thickened at the edges and somewhat cup-shaped in center, composed of the mycelium of the mold cemented together into masses by glairy substance. Below, these crusts are in contact with the true skin. The fungus penetrates especially into the hair-follicles and hairs themselves, which later are shed. It attacks different species of animals with varying symptoms, but produces more serious lesions than those of Trichophyton. Favus is especially serious asitattacks man. Efforts to show that this fungus is merely a parasitic form of some species of higher fungi have failed. The diseased con- ditions have become so well defined and are reproduced so uniformly as to indicate a fixed habit in the organisms, whatever its source or relationship. ACTINOMYCOSIS* Actinomyces bovist This is a rather common disease of domestic animals, especially cattle. It prevails in Europe, North and South America, and is known by various names as lumpy jaw and wooden tongue. Cattle are most commonly affected, but humans, hogs, horses, sheep, and dogs are sus- ceptible. Actinomyces produces a local disease which never spreads widely or rapidly. Actinomycosis is to be considered as an infectious disease which spreads by inoculation. The disease produced by this microérganism usually runs a chronic course and is distinguished especially by enlargement of affected parts, * Prepared by M. H. Reynolds. | Actinomyces bovis has been classified by Frost (page 108) as a species of bacteria, but, because of many features, it is here inserted with the organisms strictly belonging to molds and yeasts.—Ed. 728 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS by hardening of the tongue, and by suppuration. The latter is one of the most constant and conspicuous characteristics. Head parts, in- cluding the facial bones, are commonly affected; lungs and various other internal organs and even the vertebre may be involved. The extent of injury done by this fungus depends on the location and size of the involved area. Usually the most conspicuous injury is impaired nutrition. There is probably but little risk to human health from actinomycosis in cattle as parts of the carcass mo$t commonly affected are not eaten Fic. 153.— Actinomyces bovis. The ray-fungus from cow. (Diagrammatic.) (After Williams.) and edible parts are usually cooked. It is generally considered that sound portions of carcasses which do not show generalized disease are fit for human food purposes. There are apparently several varieties of Actinomyces all of which are recognized for the present as Actinomyces bovis. The varieties of Actinomyces are to be regarded as members of a very complicated group of microdrganisms higher than bacteria and are generally spoken of as fungi. Actinomyces bovis is commonly known as the ray-fungus (Fig. 153). Its relation to the disease of actinomycosis is probably specific but it is frequently aided by pus producing bacteria. It is believed that the Actinomyces vegetate on various grasses, especially wild barley, and that infection occurs by inoculation with the awns and barbs of such grasses through the mucous membrane of the mouth or other portions of the alimentary tract. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 729 Infection by inoculation is the most common method of introducing the disease; but infection by inhalation evidently occurs in some cases. It seems probable that some special stage of development for the Act- nomyces is necessary either within the diseased animal body or upon some plants, in order that it may be able to infect animal bodies, for direct inoculation by pus has usually given negative results. Inocula- tion by bits of diseased tissue occasionally gives positive results. It is evidently not a producer of active toxins for the disease dis- turbances are apparently due to harmful growth in the tissues and to secondary infection. Suppuration is one of the conspicuous features as is also the develop- ment of much new granulation tissue which tends to degenerate at the center. Soft organs affected by this parasite show a tendency to multi- ple abscesses. _ ~~ — P Fic. 154.—Actinomycosis. Actinomyces bovis. Preparation from a pure culture. Xt1000. (After Williams.) Actinomyces bovis grows rapidly on a variety of laboratory media. On glycerin gaar the colonies develop into transparent drop-like bodies in four or five days at 73°. Old colonies become white or yellowish with a powdery surface. The cultural and other peculiarities vary much and according to the variety under observation. Some varieties appear distinctly aerobic and others anaerobic. As a rule it liquefies gelatin growing in spherical masses which settle to the bottom of the liquid. Fila- ments appear in artificial growth which are very long and slender, and about 6p 730 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS in diameter, and show true branching (Fig. 154). The young colony is a loose mass of filaments; older colonies become dense and felted. Rod-shaped and spherical forms appear in artificial cultures, and some filaments develop conidia. Cultures, _ especially those containing the round forms, are very resistant to heat, light, drying and disinfectants. Stains easily. Tissue section stained with carmine followed by Gram’s method gives good results, the thread showing dark and clubs red. Carmine followed by Weigert gives a beautiful stain. May be recognized as visible granules found floating in the pus in case of suppuration, or embedded in tissue. These granules vary in color; some are clear or yellow; others are quite dark. The colony as it appears in tissue section or pus smear consists of a rosette arrangement. , The central portion of the colony is a dense mass of mycelium and spherical bodies. From this felted central mass, there extend rays or club-like bodies. Club-shaped enlargements at the ends of filaments frequently appear and are regarded as a distinguishing characteristic of Actinomyces. This organism is usually destroyed at 75° for thirty minutes. Final diagnosis must rest upon actual demonstration under the microscope which is not difficult. The granular masses may be washed in normal salt solution; and examined unstained, or stained in diluted carbol fuchsin. Escape from the diseased body is usually in pus discharged from : actinomycotic abscesses. In case of open lung or intestinal lesions it may be discharged through the trachea or intestines. Very little is known concerning the disseminating agents except that the sharp awns of barley and some similar bodies from other wild grasses have been found carrying actinomycotic infection. There appears good reason for believing that such awns frequently serve to spread the disease. Actinomycotic pus scattered over fodder, mangers, and feed racks probably serves indirectly as a source of dissemination. Actinomycosis is not a disease of rapid or extensive dissemination. Control work is usually confined to isolation, to proper disposition of diseased animals and to suitable disinfection. It is recognized in sanitary legislation that very many actinomycotic carcasses are fit for food purposes and should not be condemned. ACTINOBACILLOSIS.—Actinobacillosis is probably to be distinguished from actinomycosis. It is very similar in subjects affected, in history and clinical evidence, but apparently different as to specific cause. The cause of actinobacillosis seems to be a very small bacterium found also in rosette-like masses resembling those of Actinomyces. Mycretoma (Mapura Foor)* This disease is endemic in India, especially in Madura, and is found in other warm countries. “Prepared by Edward Fidlar. ’ MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 731 It is a chronic inflammatory process found most commonly in the foot, occasionally in the hand but very rarely elsewhere. It is charac- terized by swelling and irregular deformities of the part with the occurrence of sinuses whence there is a purulent discharge containing granules suggesting those of actinomycosis. These granules may be whitish, yellowish, reddish, or black in color. The causative organism is generally regarded as a fungus. It is not unlikely that some cases of the disease may be confused with actinomycosis. Several different molds have been described, some of: which have been classed as Aspergilli, while others have been given new names. It is probable that, while the disease is a fairly well-marked clinical entity, the etiological agent varies in different localities. Successful inoculation of the monkey with:the white variety and of pigeons with the black variety has been recorded. Mycotic LympHancitis*t Saccharomyces farciminosus* The disease caused by this yeast-like fungus has been called Japa- nese farcy, epizootic lymphangitis, and mycotic lymphangitis. This disease was first recognized in the United Statesin 1907. It has already been found in Pennsylvania, Iowa, California, and North Dakota. Saccharomyces farciminosus produces a slow, chronic, contagious disease of horses and mules. Cattle appear susceptible but rarely show clinical symptoms of infection. This Saccharomyces involves especially the superficial lymphatic vessels and glands, but internal organs are occasionally affected. The disease is essentially local, constitutional disturbances being slight. The disease produced is fatal in about 10 to 15 per cent of cases affected but is much more serious than these figures would indicate. Other horses that do not die are rendered useless for service, the sale value being ruined in many cases. The lesions produced by this parasite resemble most closely the farcy form of glanders but may be easily distinguished by quite different * Prepared by M. H. Reynolds. t+ Work done by Paige, Frothingham and Paige, Meyer and others raises questions concern- ing specific etiology and proper classification, but it is deemed wise to continue this recognition and classification for the present. 732 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS ulcers. The pus is thick, creamy, and usually yellow, whereas the pus from the farcy buds is clear and viscid. Farcy cases respond to the mallein test; lymphangitis cases do not. It seems to have been well established that Saccharomyces farct- minosus is the direct cause of mycotic lymphangitis—at least of one form of it. The Saccharomyces grows in the animal tissues and by its presence and products acts as a direct exciting cause of the disease. Entrance is effected through inoculation wounds which may be very super- _ ficial and very trivial, most frequently perhaps on the legs, shoulders, and neck. The incubation period varies from a few weeks to several months. This Saccharomyces is distributed through lymph vessels, chiefly superficial ones, the nodules appearing first near the point of inoculation. The tissue changes produced are infection, inflammation, and suppuration of the lymph vessels and glands. At first the lymph vessels enlarge and harden; then nodules develop under the skin along the course of the vessels. These nodules suppurate and the small abscess cavity fills up with bright red granulation tissue. The entire limb may enlarge very greatly by reason of excessive connective- tissue formation, and the greatly thickened skin. Saccharomyces farciminosus is a yeast-like fungus, ovoid in shape and 3 to su long by 2.54 to 3.54 broad. This fungus grows slowly under artificial conditions on agar and bouillon after inoculation with pus from an abscess. It reproduces by budding and does not stain well by common laboratory stains. Claudius’ method of staining gives good results. Cases should be isolated and stables disinfected by the free use of very strong disinfectants as this Saccharomyces is not easily killed by ordinary disinfecting solutions. Another mycotic organism has more recently been reported* in the United States as causing a lymphangitis very similar clinically’ to the lymphangitis caused by Saccharomyces farciminosus. Cases supposed to have been plain cases of the Saccharomyces form showed on laboratory examination a Sporothrix acting as the direct cause. These workers* reproduced cases by inoculation and recovered an *Sporothrix and Epizootic Lymphangitis, Paige, Frothingham, and Paige. Journal of Medical Research, Vol. XXIII, No. 1. This has been previously reported by Shenck, Hek- toen, and others for the human. 1 ' MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS) 733 organism differing very materially from Saccharomyces farciminosus. The case history and lesions produced parallel very closely those pro- duced by the Saccharomyces. This Sporothrix seems to have great vitality, remaining virulent in dried pus at a temperature of —7° for three months or more. The same organism has been recovered from similar lesions of the human where it was apparently acting as the direct exciting cause. If this be confirmed, we have two very different organisms capable of producing a similar mycotic lymphangitis. DISEASES CAUSED BY BACTERIA* BoTrRYOMYCOSIS fT Botryomyces equi We have typically in this disease closed abscesses with very tough fibrous walls and slow development. These abscesses involve espe- cially subcutaneous and intermuscular connective tissue, although typical lesions have been found in various internal organs. This affection usually appears in horses, but botryomycosis has been found in cattle and swine. The identity and proper classification of a specific microérganism is still in dispute. Johné found M. ascoformans acting as an etiolog- ical factor. Kitt and others found micrococci which could not be dis- tinguished from M. pyogenes. Moore found a variety of pyogenic micrococci and streptococci apparently serving as causative agents and reports one case of an enlarged spermatic cord where he found a fungus resembling Actinomyces bovis. In his later work Moore appears to identify the botryococcus of Bollinger with Micrococcus pyogenes. Others identify Botryomyces equi with Staphylococcus pyogenes aureus, etc. Primary infection occurs by inoculation and not infrequently follows surgical operations, ¢.g., castration. The primary infection may then lead to involvement of internal organs by metastasis. The * Arranged alphabetically under each of the following families: Coccacee (Micrococcus, Streptococcus), Bacteriacee (Bacterium, Bacillus, Pseudomonas), Spirtllacee@ (Microspira). + Prepared by M. H. Reynolds. 734 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS local effect here is that of an irritant and both irritant and tissue response appear to resemble those that occur in actinomycosis. Botryomycosis is easily distinguished from actinomycosis on microscopic examination. Cases that resemble the farcy form of glan- ders are easily distinguished by mallein test, by laboratory animal in- oculation and by lack of adjacent lymph-gland involvement. GONORRH@A* Micrococcus gonorrhee Gonorrhcea is one of the most prevalent of the bacterial diseases and is found throughout the civilized world and is confined to the human race. The urogenital tract is the most frequent seat of infection but orchitis, severe conjunctivitis, arthritis and endocarditis are not uncom- mon and a septicemic condition may also occur. Ophthalmia neona- torum is due to this organism. The ordinary infections of the urogeni- tal tract have an incubation period of from two to eight days. The inflamed mucous membranes give rise to more or less pain and yield a thick yellow discharge. While the fatality due directly to Gonococcus infection is not high, the frequent tendency to chronicity renders it one of the most important diseases. Gonorrhcea has been known from the very earliest times. In 1879 the diplococcus was pointed out by Neisser as -the probable cause. Bumm in 1885 first cultivated it on coagulated human placental serum. The microdrganisms can be easily stained in the typical early dis- charges where it occurs in pairs and for the most part within cells (Fig. 155). For isolation, agar media should contain human blood or blood serum or ascitic fluid, though the swine-serum-nutrose medium of Wassermann is also good. The fluid must be sterile and must be added to melted nutrient agar at about 45°. The Gonococcus is about 0.64 to 0.84 in diameter. It is usually seen in pairs; where the adjacent sides of the cocci are flattened the long diameter of the pair reaches as much as 1.64; non-motile and forms neither spores nor capsules. It stains readily with the aniline dyes and is Gram-negative. The temperature range is 30° to 38.5° with an optimum of 37.5°. Aerobic conditions are preferred though a slight growth may * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 735 be obtained anaerobically. The most favorable reaction of artificial media is said to be about 0.6 per cent acid to phenolphthalein. On serum agar or similar suitable media, colonies appear in twenty-four hours as fine slightly elevated, translucent or opalescent spots frequently referred to as “‘dew-drop” colonies. They possess a faint bluish or grayish white color with a slightly marked concentric or radial striation with a scalloped margin and finely granular center. In serum broth there may occasionally be a uniform clouding though, as a rule, there is a finely granular sediment somewhat slimy with clear fluid above. Only in exceptional cases has growth been observed in gelatin because of the unfavorable temperature. On Fic. 155.—Gonococci and pus cells. XX 1000. (After Williams.) inspissated blood serum growth may sometimes be observed as discrete pale yellow- ish or brownish colonies. Dextrose is changed with the development of acid but no gas. Alkali is not formed in any medium by typical strains. No gas, indol or pigment are formed. The toxins are intracellular and quite thermostable. Resistance is very slight toward external influences. Cultures undergo rapid autolytic changes and die out at room temperature, often within forty-eight hours. A temperature of 41° to 45° will kill in a few hours. To light and drying they are also very sensitive, and are rapidly killed by the ordinary disinfectants. Animals inoculated subcutaneously or intraperitoneally show symp- toms of poisoning with suppuration and necrosis locally and may succumb. The virulence of the organism is variable. They may apparently lie dormant or at least they are very slightly active in chronic conditions in one individual but set up an acute gonorrhoea when transferred to a second person. 7 36 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS The organism gains entrance to the urethral mucosa or conjunctiva usually by direct contact and it is doubtful if the disease could be carried by any infected article later than twenty-four hours. The organism is found at the local lesion and has been obtained from the fluid of affected joints, and from the blood in the septicemic cases. A general immunity is seldom if ever developed in man following an attack of gonorrhoea. There has been demonstrated in the blood serum, however, a complement-fixing antibody which, while not absolutely constant, is present with sufficient frequency to be an aid in diagnosis. Injections of cultures into animals give rise to agglutinins, bacteri- cidal and complement-binding bodies. The diplococcus is eliminated only in the purulent discharge. Great importance attaches to the fact that persons may harbor the Gonococcus long after the acute condition has disappeared and when the coccus seems to be no longer harmful to its host. Such cases bring about untold misery and form one of the most difficult problems in both medical and social science. It has been stated that Gonococci have been found as late as twenty years after the primary infection. The most extended and successful prophylactic measures have been carried out in the armies and navies of various countries by the use of germicidal solutions whenever there has been any chance of exposure to infection. The use of germicidal solutions in the eyes of new- born infants is practically universal as a preventive measure against ophthalmia. EPIDEMIC CEREBRO-SPINAL MeENINGITIS* Micrococcus meningitidis Cerebro-spinal meningitis may be caused by different bacteria such as the pneumococcus, streptococcus, staphylococcus, influenza bacillus, tubercle bacillus, etc., but the greater proportion of cases of acute meningitis, those of the epidemic type, are due to the meningococcus or diplococcus intracellularis meningitidis. Epidemic meningitis has been described chiefly in Europe and Amer- ica and appears to have been first clearly defined in 1805. While sporadic cases occur, the disease usually exists in the epidemic form, * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 737 ° i beginning in the fall, continuing during the winter, and declining in the spring. Of late years it would seem to be on the increase. The incubation period is unknown. There is considerable variety in the character of the cases. Asa rule the invasion is sudden with headache and vomiting as prominent symptoms. The headache usually increases, with disturbances of vision, restlessness, and pains and rigidity in the muscles of the back and neck. The temperature is irregular and variable, the usual being about ror° to 102°. Herpes occurs frequently and a purpuric rash is common, especially in the severe cases, so that the term “spotted fever” has sometimes been given to the disease. The patient usually passes into a stuporous state, though delirium may occur before it. Death may occur in a few hours (fulminant type) or within a week, or occa- sionally may be postponed as late as six months. In all favorable cases the convalescence is slow. A fibrinous exudate which occurs chiefly at the base of the brain and the presence of pus cells in the cerebro-spinal fluid are prominent pathological features, but in the fulminant cases the gross pathological findings may be surprisingly insignificant. : The mortality of the disease before the advent of serum treatment was about 70 per cent of the number of cases, but this has been reduced to about 30 per cent according to Flexner’s figures. The demonstration of the organism in the cerebro-spinal fluid of the typical case may sometimes be an easy matter, but at other times may require a prolonged search. It appears as a Gram-negative coccus, single and in pairs, frequently within pus cells but occasionally extra- cellular. From the usual case it can be obtained in pure culture by sowing the sediment from the spinal fluid upon suitable media. The amount of material planted should be abundant and to supplement these first cultures it is well to incubate the fluid at 37° for twelve to eighteen hours and then make further inoculations. The media used are usually fresh blood agar, serum agar and occasionally Loeffler’s blood serum. As found in culture media the meningococcus will show swollen involution forms often in comparatively young cultures. There are no spores, flagella nor capsules. It can be stained readily by the aniline dyes and with methylene blue will sometimes show metachromatism. It is Gram-negative. 47 738 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS e The temperature relations are of some importance in identifying the coccus. It has a minimum temperature of about 25°, an opti- mum of 37° and a maximum of 42°. Its atmospheric requirements are those of an aerobe. Upon suitably enriched agar media the colonies are small, grayish and glistening with a smooth outline and granular center. In broth growth is slow and occurs at the surface. Only rarely is growth ob- tained on gelatin media chiefly because of the unfavorable temperature required. There is no change in litmus milk. Acid is formed from dextrose and maltose. The toxins of the meningococcus are probably intracellular. The resistance of the organism to unfavorable conditions is very slight, and it undergoes autolytic changes almost with the same rapidity as does the gonococcus. Meningitis due to this coccus does not occur naturally in animals, but it has been produced in monkeys artificially. Laboratory animals inoculated subcutaneously, intraperitoneally or intravenously with a sufficiently large dose will die without developing meningitis. Animals immunized by graded doses show specific agglutinins, opsonins and lysins. Horses so treated yield a serum which in some hands has given very favorable results. In recent epidemics in the British and Canadian armies, however, the mortality has ranged from about 40 to 50 per cent and the serum has been distinctly disappointing. As the germs leave the body in the discharges of the nose and mouth, the prevention and control of the disease would appear at first thought to be an easy matter, but the occurrence of carriers and ignorance of the factors which govern the virulence of the infective agent and the individual’s susceptibility make epidemic meningitis a very difficult problem from the standpoint of public health. INFECTIOUS MastitIs* Infectious mastitis or mammitis (inflammation of the udder) appears in isolated outbreaks and is serious for the individual owner and individual herd, but it never spreads widely. It may affect a large portion of the herd and cause heavy financial losses. Infectious masti- tis may have serious significance for children and others consuming * Prepared by M. H. Reynolds. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 739 milk; but there is little information on this point, based on careful work. This is to be considered as an infectious, enzodtic disease and prob- ably not specific. There is good reason to suppose that different out- breaks have been due to several different pyogenic or pus-producing organisms. We cannot consider any one species of bacteria as the specific cause. Various micrococci, streptococci, and staphylococci have been found acting as causal agents. Recent evidence indicates that udders of apparently healthy cows may contain a variety of bacteria and that the infections may remain more or less permanent. This is in part the explanation of recurrent cases of mastitis. In the animal body this infection is practically limited to the udder. Its discharge is either through the teat or rarely by external rupture of abscess. Transmission from cow to cow is indirect, and frequently on milkers’ hands. Entrance is usually effected by way of the milk ducts; thence into the milk cistern and to more remote parts of the gland. The infection may also come by way of the blood or lymph channels to the glands. A given case may thus be due to bacteria previously in the udder, the attack being determined by an ayea of lessened tissue resistance pro- duced by injury. In one class of cases, the gland structures are first involved; in other cases the connective tissue frame-work is first involved. In one type of this disease caused by streptococci these microdrganisms attack espe- cially the mucous membrane lining milk ducts and produce a catarrhal disease of that membrane. This is indicated by a cord-like swelling which extends along the milk canal through the teat to the milk cistern. This infection frequently leads to ‘“‘blind quarter;”’ z.e., to closure of the teat canal and loss of the quarter; or this infection may lead to the for- mation of one or more pea-like nodules along the teat canal and conse- quent obstruction. In many cases the lactose is decomposed by the invading organisms, leading to the formation of organic acids. These acids produce coagu- lation. The coagula soon obstruct the milk ducts and alveoli and the secreting cells degenerate. The invaded tissues may suppurate or even become gangrenous. 740 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS These infections are indicated by dullness, lack of appetite, fever, inflammation of the udder, and by small nodules or cord-like swelling within and lengthwise of the teat. It must be borne in mind that the infecting microdrganism is the thing to be controlled. Outbreaks of this disease frequently have origin in infected cows added to the herd. Some cows are unsuspected “car- riers.” New cows should be suspected until found free by careful examination. Affected cows should be isolated if possible, and always milked last. Their milk should be boiled and fed to hogs, and the milkers’ hand suitably disinfected. \ Matta FEvVER* Micrococcus melitensis This disease is endemic along the shores of the Mediterranean, in South Africa, India, China, the Philippines, and the West Indies. The period of incubation is usually about six to ten days. The ordinary variety shows an intermittent or undulatory fever which may be protracted to six months or more, accompanied by consti- pation and general debility with various complications such as neural- gias, arthritis, orchitis, etc. Relapses occur after periods of absence of symptoms. Malignant cases are described which may be fatal in a week or ten days. The mortality is 2 per cent and no characteristic pathological changes are found. The etoliogical factor is M. melitensis and was described by Bruce in 1887. The organism can be obtained from the blood and in many cases from the urine. The most recently reported favorable medium for blood cultures is peptone broth with the addition of bile. It is generally recognized as an oval coccus, although it is also described as a bacillus. Its maximum measurements have been found to be 0.8u by 0.53, its minimum diameters 0.554 by o.4u. It occurs singly, in pairs, in irregular groups and in short chains. (Recently the organism has been described as motile and possessing a single flagellum at the extremity of the long diameter of the oval coccus.) It stains by ordinary aniline dyes and is Gram-negative. It grows slowly at room temperature, better at body temperature and does not seem to be markedly sensitive , to acidity or alkalinity of reaction. It grows aerobically. On plain agar after * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 741 about forty-eight hours small whitish to yellowish colonies appear. Growth has been observed in broth in eighteen to twenty-four hours, on gelatin in eight or nine days, and the latter is not liquefied. It has been found to grow on acid potato and in acid or alkaline urine. ‘ Human beings and animals eliminate the organisms in the urine, and the milk of goats has been found to be a prolific source of infection. With proper regulations in regard to goats’ milk the disease has been greatly reduced. STaPHYLococcic INFECTIONS* Boils, Abscesses, Wounds, Osteomyelitis, Pyemia, Etc. Micrococcus pyogenes var. aureus, etc. Infections of this order are found throughout the world and because of the association of staphylococci and streptococci with the large majority of purulent inflammations, these organisms are called the pyogenic cocci. No specific disease is produced, but chiefly boils, circumscribed abscesses, infected wounds, osteomyelitis, pyemia,etc. The symptoms alone will not indicate whether staphylococci or streptococci are present, but a low grade of infection with more pus and less constitu- tional disturbance tends to indicate the former, and staphylococci tend to pyemia rather than to septicemia. Pasteur, Koch, Ogston and Rosenbach established the importance of these organisms. _ Staphylococci in pus stain readily with aniline dyes. Pure cultures can be obtained by plating or streaking on plain nutrient agar. While several ‘different forms are found in pathological conditions, the UM. pyogenes var. aureus is by far the most frequent, and it is de- scribed here as a type. M. pyogenes var. aureus is a spherical coccus about 0.7» to o.gy in diameter though forms 0.4u to 1.24 have been noted. On solid media the organism may be found solitary, in pairs, or in rows of three or four, but characteristically in irregular groups like bunches of grapes. In liquid media the single and paired arrangement is most frequent. No spores, no capsules and no flagella are found; the organism shows marked Brownian movement, like other cocci; Gram-positive. The temperature range of growth is from about 1o° to 43° with an optimum about 30°. Aerobe and * Prepared by Edward Fidlar. 742 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS facultative anaerobe. It grows readily on all routine media, preferring a reaction slightly alkaline to litmus. Growth on plain agar is rapid and abundant. After twenty-four hours there appear round grayish-white or yellowish colonies about 2 mm. in diameter, smooth and raised above the surface of the medium. Micro- scopically, the colonies are regular in outline and finely granular. The char- acteristic orange-yellow pigment may not appear until later or if already present in twenty-four hours, deepens with further growth. In broth, growth is also rapid and causes a diffuse clouding with a thin pellicle and a heavy sediment after several days. In gelatin, colonies are as on agar and sink into cup-like depressions as the medium is liquefied. Liquefaction is rapid with some strains and slower with others, and in old cultures is of a funnel or saccate type. It is due to a thermolabile ferment- like substance known as gelatinase. In milk, the staphylococcus grows readily and causes coagulation sometimes early but usually in three or four days’ time. On potato growth is usually abundant; it is not as moist nor’as smooth as on agar and is slower. Pigment is developed usually to the highest degree and sometimes cultures appearing white on agar develop pigment on potato. On imspissated blood serum growth is usually moist and abundant. Occasionally the growth sinks slightly into the medium suggesting partial liquefaction. In dextrose, lactose and saccharose media acid is produced, but no gas. Acid is a constant product. Formic, lactic, butyric and valerianic acids have been found and probably other fatty acids occur. Some authorities state that indol is formed but negative results are the rule. Nitrites are formed by the reduction of nitrates. A characteristic odor from cultures is due probably to the presence of fatty acids. The pigment appears in aerobic cultures and is absent in anaerobic cultures. It is insoluble in water but soluble in alcohol, chloroform, ether and benzol. The toxins are largely intracellular. A thermolabile, hemolytic substance may be found in the more virulent strains after about ten days’ growth in moderately alkaline broth and can be freed by filtration through porcelain filters. Another soluble toxic substance is found, causing the death of leucocytes—leucocidin. It is considerably less stable than the staphylo-hemolysin. The staphylococci are among the most resistant of the non-spore bearing bacteria. Sometimes 60° for a full hour or even longer is necessary to kill watery suspensions; 70° is usually necessary to kill in ten minutes. If organic material is present the resistance is, of course, much greater. Low tem- peratures have little effect and it has been stated that 30 per cent have survived thirty minutes’ exposure to liquid air. To direct sunlight and drying staphylococci also show considerable resistance and may be found in dried pus for several months. Resistance to germicides is also somewhat greater than that of other vegetative bacteria, and is increased especially in the presence of organic material. In watery suspensions staphylococci are killed by 1: 1000 mercuric chloride in ten to fifteen minutes, by 3 per cent carbolic acid in two to ten minutes and by 5 per cent formal- dehyde in the same time. Man seems to be considerably more susceptible to staphylococcic in- fections than animals. Of the latter rabbits and mice and guinea-pigs are susceptible in this order. ms MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 743 The virulence of the organism shows considerable variation and is usually increased by successive passages through animals of the same species while remaining unaltered for animals of other species. Subcutaneous inoculation usually results in abscess formation. Virulent cultures injected into the peritoneal cavity of animals may kill in forty-eight hours to a week or even longer with pyemic abscesses especially in the kidneys. Malignant or ulcerative endocarditis has been experimentally produced by intravenous injection when the heart valves have been injured, chemically or mechanically. Osteomyelitis has also been experimentally produced. : In man simple rubbing of virulent cultures into the skin is often sufficient to produce a furuncle. Upon entering the tissue the cocci are strongly chemotactic and pus inevitably results. With virulent cultures the leucocidal substance is more or less strongly active. The organism may be limited to the first abscess or by invasion of the blood stream multiple abscesses result. In these cases, which are usually fatal, the organism will be found throughout the body. Immunization can be secured by repeated injections of cocci dead or alive in graduated doses. The serums possess slight bactericidal and agglutinating properties, and a high degree of opsonic power. The latter property is probably the most important. The serums of immunized animals is protective only when used slightly before or along with the injection of the organisms and is con- sequently of little practical value. Active immunization, however, is being extensively practised particularly with the autogenous strains. Leucocytic extracts have also been successfully though not so widely used. The prophylaxis of staphylococcic infections is the same as for other pus-producing forms. Several other kinds of staphylococci have been found associated with pathological conditions, the most important of which are M. pyogenes var. albus, M. epidermidis albus (Welch), and M. pyogenes var. citreus. The first seems to be slightly pathogenic, and rarely produces severe infection. It is distinguished from thé aureus by lack of pigment. The second variety appears to be an attenuated form of the other. The third variety is distinguished from aureus and albus by the development of a lemon-yellow pigment. 744 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS STREPTOCOCCIC INFECTIONS* General Septicemia, Puerperal Septicemia, Pee Etc. Streptococcus pyogenes Streptococcic infections are endemic among all races and under,all social conditions. In the days before antisepsis and our knowledge of the transmission of infectious diseases, erysipelas and puerperal sep- ticeemia occurred in epidemics that were the scourges of surgical and lying-in hospitals. When the work of Pasteur and Lister became fully comprehended such epidemics ceased to exist. Natural streptococcic infections have been described in horses and cattle and among the laboratory animals, but as a rule such disease is much rarer in animals than in the human being. The period of incubation is probably about one to three days. The symptoms of septicemia begin with a rapid rise of temperature which may reach 105°F. or even higher. Chills accompany the fever and are often severe. The pulse is rapid, irregular and weak and the respiration labored. There may be vomiting and constipation or diarrhoea. Headache is more or less severe with sometimes delirium. In cases lasting for several days the skin appears slightly jaundiced. The urine is of the usual febrile type and, as a rule, shows the micro- érganism causing the disease. Death may occur in two or three days or within a week or in milder cases may be followed by recovery. After death from septicemia the body tends to putrefy rapidly. - The glandular organs all tend to be swollen and soft, especially the spleen, and parenchymatous degenerations are found to a greater or less extent. The lining membrane of the heart and vessels is blood stained, a rather characteristic feature of streptococcic septicemia. Bronchitis and broncho-pneumonia are usually found. Erysipelas is an inflammation of the skin, occasionally of mucous membranes, and the name is applied now only when the condition is brought about by streptococci. The inflamed area is very definitely outlined and may present blebs of a greater or less size. Oedema may be very marked where the skin covers loose tissue. Fever is present with its usual accompaniments. There may be vomiting, constipation or diarrhoea. There may be severe headaches or delirium, In fatal * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 745 cases, death may occur without any apparent complication, or it may follow meningitis, pericarditis, nephritis or some other sequel. In sim- ple uncomplicated fatal cases the liver, kidneys and spleen are swollen and soft and show degenerative changes in the gland cells. Pasteur, Koch, Rosenbach and Fehleisen divide the earlier honors in the gradual working out of the relationships of streptococci to disease. Blood culture in plain broth in the.case of septicemia or inoculation of plain nutrient agar from pus are practically always successful. Growth is never luxuriant on the ordinary media. Cultivation from cases of erysipelas is less easy because most of the organisms are found at the margin of the lesion and are difficult to reach. In exudates a stained smear will usually demonstrate the chain- forming coccus at once. The cocci vary in size from 0.4 to 1u. In shape the organisms may be rounded or oval or with one aspect flattened when occurring in pairs. The chains may be long or short and a grouping into pairs is frequent even within the chain. There areno true spores developed and the organism is non-motile. Capsules are not found on the majority of streptococci. Staining the organism is easily accomplished with the ordinary aniline dyes. It is Gram-positive. The temperature range in which streptococci are capable of growing is about from 15° to 45°, the optimum tem- perature is about 37°. Streptococci are, as a rule, aerobes and facultative anaerobes Strict anaerobic species are said to have been isolated from feces. The reaction of media should be slightly alkaline. Acid production is a striking feature of this organism and has a decided inhibitive effect upon its growth. Concerning the action on carbohydrates this organism typically forms acid from monosaccharides, lactose, saccharose, and salicin. Gas is never produced. Nitrates are reduced by some streptococci to nitrites. The production of hydrogen sulphide is characteristic of some forms which have been grouped as Strept. fecalis. No pigment is found other than the slight brownish tinge seen in some gelatin cultures. Typically actively hemolytic. This power may be lost on cultivation. The toxic products of the streptococci have been the subject of a great deal of investigation, but few definite facts have been discovered. When cultivated on plain nutrient agar the growth is visible in eighteen to twenty-four hours as small round translucent finely granular colonies, which possess an even or notched border, and a tendency to remain discrete except when thickly sown. The center is thickened and the margins thinner. In plain nutrient broth the majority of long-chained varieties produce at the bottom and along the sides of the tube a granular deposit, or small flocculi or large flakes, leaving the remainder of the broth clear. A few long-chained varieties cloud the broth uniformly. The short-chained streptococci, asa rule, produce a cloudiness in the medium which remains for a number of days even though a finely granular deposit accumulates at the bottom of the tube. On plates of plain nutrient gelatin the colony formation remains the same as that on agar. In stab cultures a finely 746 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS granular filiform growth appears which later may have a beaded appearance and sometimes a brownish color. The gelatin is not liquefied. Milk is a favorable medium for the growth of streptococci and a strong acidity and coagulation some- times takes place. Growth on potato is said not to take place, but in some cases an invisible growth seems to occur. Loeffler’s blood serum is also a favorable medium. Streptococci, asa rule, die out rapidly in cultures due to the accumulation of their own products. In pus, blood, sputum, etc., the organism may be found alive after several weeks or even months at room temperature. The thermal death- point is about 54° in ten minutes. Direct sunlight kills within a few hours, and they are readily killed by many disinfectants. Entrance of streptococci is afforded by any break in the surface of the body. A local suppuration may be the result or it may be followed by a general septiceemic condition. In erysipelas some local injury is also probably necessary as a starting-point. Following the local establishment of streptococci sufficient toxin is elaborated to produce greater or less systemic disturbance. If a septicemia supervenes the poisoning becomes extreme and the organ- isms are distributed throughout the body. Immunity following recovery from natural streptococcic infection is very slight if any, and never of a permanent sort. Septicemias once established are generally fatal, and erysipelas can recur frequently. Bactericidal substances, opsonins, agglutinins and precipitins have been demonstrated in immune serums, which, however, show little therapeutic success. Streptococci are eliminated in the discharge of local infections ames in sputum, etc., and are then probably more virulent. Infection by contact from such sources is particularly dangerous. In anginas and streptococcic infections of the respiratory tract, the epidemiology is practically the same as for diphtheria and pneumonia. Similarly erysipelas is to be treated as a contagious disease. _ In the prophylaxis of streptococcic diseases, greatest care must be shown where chances of infection by the virulent strains are possible. . Isolation of erysipelas is universally practised in hospitals. Similarly © cases of puerperal sepsis and any local disease should be kept from contact with other puerpere. -Streptococcic pus from all sources‘ ‘ is to be carefully destroyed. Streptococci seem to be always present on the exposed surfaces . of the body and are probably capable of giving trouble should any MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 747 local lowered resistance occur. The prevention of this may be accom- plished by strict antiseptic treatment of wounds. PNEUMONIA* Streptococcus pneumonia The occurrence of a diplococcus in the large majority of cases, especially of the lobar type of pneumonia, has caused this coccus to be regarded as practically specific and warrants the name of Micrococcus pneumonia, Diplococcus pneumonia, or Pneumococcus. As occasional causes of pneumonia should be mentioned Streptococcus pyogenes, Staphylococcus pyogenes var. aureus, B. coli, Bact. diphtherie, Bact. influenze, B. capsulatus mucosus (pneumobacillus), B. typhosus and Bact. tuberculosis. - Pneumonia is world-wide in its distribution and is estimated to form anywhere from 1 to 7 per cent of all cases studied in internal medicine. It appears to be more frequent in regions subjected to sudden changes of temperature. The incubation period is two or three days of rather indefinite prodromata. The onset of the disease is marked by a chill, pain in the side, and rise in temperature. The respirations become frequent. The fever, as a rule, runs between 102° and 105°F. for from five to ten days and then in favorable cases terminates by a sudden drop of temperature to normal within a few hours (crisis). The most striking pathological findings are a marked congestion and cedema of the lungs following which the lung becomes solid, airless and of a dark red color, the alveoli showing, microscopically, a fibrinous exudate with large numbers of red blood cells, some leucocytes and desquamated epithelium. Thereafter the lung becomes slightly softer and is of a gray color, while microscopically the red cells degener- ate and leucocytes are more frequent and more evident. The final stage, resolution, is marked by the liquefaction and absorption of the contents of the alveoli and the entrance of air. Death occurs from toxemia or complications such as carditis, men- ingitis, etc. Roughly about ro per cent of all deaths are due to pneu- * Prepared by Edward Fidlar. 748 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS monia and the fatalities form about 10 per cent of the total number of cases. The Streptococcus pneumonie was described, as found in the spu- tum, by C. Frankel in 1884. A Gram-stained preparation of the sputum is sufficient to detect the diplococci but cultures are necessary for positive identification. Some medium richer than the ordinary by the addition of blood or serum from man or animals is best, and may be inoculated from the blood and organs or from sputum and other contami- nated sources by streaking or plating. Injection of sputum into white mice or rabbits will often cause a fatal septicemia in these animals and the coccus may then be obtained in pure culture from the heart’s blood. It occurs as pairs of oval or lanceolate cocci, with their contiguous surfaces somewhat flattened and the distal] ends slightly pointed. From this type the organism may vary to spherical or short bacillary forms. It may occur also singly or in chains of varying length usually consisting of not more than about six or eight individuals. Well developed capsules which may surround the single organism or the pairs and chains may be found in exudates or in milk and serum media. There are no spores nor flagella. The cocci stain readily with the aniline dyes and are Gram-positive. The capsule can be demonstrated by several methods of which Welch’s and Hiss’ are the most common. The temperature range is from 25° to 41°. It is both aerobic and anaerobic, and grows most readily in a medium slightly alkaline to phenolphthalein. Besides serum or blood, glycerin, nutrose and dextrose are found to be favorable for its growth. On agar it grows in small, rather transparent, finely granular colonies, which are larger and more opaque when serum or ascitic fluid is present, Broth is faintly and uniformly clouded. Milk is a favorable medium for most strains and typically is acidified and coagulated. On potato, growth may occur but is invisible. Gelatin can rarely be used at a temperature high enough to allow growth. When occasionally growth is obtained the medium is not liquefied. On blood serum, growth appears as small clear colonies and on the whole is better than on agar. A number of special media are described of which one of the most valuable is the inulin-serum-water medium of Hiss. It typically ferments, with the production of acid, the majority of carbohydrates, even polysaccharides as inulin. On blood agar the typical organism produces a greenish zone in the medium about the growth, but not a clear zone of hemolysis as do most strains of streptococci. The dif- ‘ferentiation from other streptococci is sometimes a matter of difficulty, and the following characters are of importance—the lanceolate shape, capsule formation, fermentation of inulin, absence of hemolytic powers, agglutination in antipneu- mococcic sera, susceptibility to lysis by the action of bile salts. Acid is an im- portant and characteristic product and, if allowed to accumulate, rapidly kills the organism. The toxic products appear to be closely united with the cell bodies and are only.released when these are broken up. The resistance to heat is not great and its thermal death-point is 52°. Light is germicidal if the cocci are not pro- tected in thick masses of sputum. Drying is resisted rather well in sputum or the G MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 749 blood of infected animals. To germicides the Pneumococcus is very sensitive and is killed in a few minutes by the common disinfectants in their usual strength. The pathogenic properties of the Pweumococcus for animals is some- what variable. Natural infection is not common. To artificial infec- tion mice and rabbits have been found most susceptible, while guinea pigs, dogs, rats and cats are more resistant, and birds are practically immune probably because of their high body temperature. Mice and rabbits succumb to subcutaneous or intraperitoneal injections of viru- lent cocci from cultures or in sputum with the development of a sep- ticemic condition, and in the latter case a peritonitis. By special. methods lobar pneumonia has been produced in rabbits as has also endocarditis. Variations in virulence of the Pueumococcus are very marked. The virulence can be increased by passage through susceptible animals until an extremely small dose will killa mouse. Cultures obtained from man may vary considerably in their virulence for animals. The organism gains entrance through the respiratory mucosa and asa matter of fact appears to be a common inhabitant of these regions. However the organism may reach the lung (the lobar distribution sug- gests sowing by the blood stream), it is certainly frequent to find posi- tive blood cultures during the disease—a fact which. accounts for the development of such complications as meningitis, endocarditis, etc. The toxemia probably arises from lysis of the organisms and it has been shown that the autolysis of cultures in salt solution gives rise to a soluble toxic portion and an insoluble non-toxic portion. Immunity to Pneumococcus infections can be shown to exist after an attack but only for a short time. Pneumococct may be considered as inhabiting the mucous mem- branes of the respiratory tract in the majority of people and acquire virulence only under some special circumstances lowering the general vitality. In pneumonia and some kinds of bronchitis as above men- tioned it should be remembered that sputum and mouth spray may contain large numbers of virulent organisms. Specific therapeutic agents such as antipneumococcic serums, vac- cines of dead cultures and autolysates, as well asleucocytic extracts, have been tried and all with some promising results. The earlier failures with serum therapy have been found to be due in part to the occurrence of different strains. By arranging these strains into three groups with 750 MICROBIOLOGY. OF DISEASES OF MAN AND DOMESTIC ANIMALS 4 corresponding antiserums more success has been obtained. Not one of these methods, however, has been sufficiently widely applied with suc- cess enough to warrant general adoption. The prophylaxis of Pxeumococcus infections lies in general hygienic measures, in the destruction of sputa and avoidance of possible infection by mouth spray, etc. ANTHRAX* Bacterium anthracis* Also called splenic fever or charbon; and in man, wool-sorter’s dis- ease or malignant pustule. The disease has been known for centuries. It is thought that it was one of the plagues of Egypt, mentioned as a murrain on beasts, and boils and blains on’man and beast. The first accurate characterization of the disease was made by Chabert about 1800. Pollender in 1849 and Rayer and Davaine in 1850 reported that they had seen “filiform bodies” in the blood of animals which had died of anthrax, and in 1860 Davaine announced he had succeeded in transmitting the disease to healthy animals by inoculating them with blood from an anthrax in- fected animal, and asserted that these filiform bodies or bacteria were the cause of the disease. This result was attacked, and for ten years there was a fierce controversy over this idea, which was finally stilled by the convincing experiments of Robert Koch in 1876. Koch culti- vated the bacterium of anthrax from the blood, showed that the inocu- lation of these cultures in susceptible animals produced anthrax, worked out the life history of the organism, and enunciated the cardinal require- ments—which constitute the proof of the pathogenic nature of an organ- ism, what later bacteriologists have named the rules or postulates of Koch. GEOGRAPHICAL DISTRIBUTION.—The disease is very widespread, occurring all over the world in tropic, semitropic, and temperate cli- mates. Wherever stock are found in large numbers anthrax is usually present. The disease ravages the herds and flocks in Russia, Siberia, India, Argentina and parts of Hungary, France and Germany. Local epidemics occur constantly in England, Canada and the United States. In the delta of certain rivers the organism probably grows in the soil * Prepared by F. C. Harrison. 2 allen eer in ee MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 751 as in the deltas of the Mississippi and Bramaputra, and the disease is also common along the banks of many rivers (Vistula, Rhine, Seine, etc.). The anthrax organism is a large, non-motile rod, from 5u to tou long and tp to 1.54 broad. In cultures it frequently forms long threads or filaments (Fig. 156). The free ends are slightly rounded, but those in contact are quite square, and slightly larger in diameter than the middle of the cell. Involution forms are obtained by culture on potato or at temperatures of 40° to 42°. It forms oval spores without distortion of the mother cell (Fig. 157). Free oxygen is necessary for the development of these bodies, and a temperature between 18° and 41°. Spore ger- mination is polar. By culture at 42° an asporogenous variety is formed. It stains Fic. 156.—Bact. anthracis. Showing Fic. 157.—Bact. anthracis. Spore pro- the thread formation of colony. (After duction. (After Migula.) Kolle‘and Wassermann from Stitt.) readily with the aniline dyes and also by Gram’s method. Under certain conditions a capsule may be seen. The organism is aerobic, in the body it grows as a faculta- tive anaerobe. Its optimum temperature is 37°, minimum 12°, maximum 45°. It forms characteristic wavy and filamentous colonies on gelatin and agar, it liquefies _ gelatin, produces an arborescent growth in gelatin stab cultures, coagulates and peptonizes milk with an alkaline reaction. Thermal death-point of the spores in liquids is four minutes at 1oo°, in hot air 140° for three hours. Mercuric chloride, 1 :1000, destroys the spores in a few minutes, and 4 per cent carbolic acid with hydrochloric acid 2 per cent in one hour. Zodlogically, anthrax is the most widespread of infectious diseases; white mice, guinea-pigs, rabbits, sheep, cattle, horses and man are susceptible. Old rats are insusceptible. Von Behring, Metchnikoff 752 MICROBIOLOGY OF DISEASES OF MAN AND- DOMESTIC ANIMALS and others have shown that the serum of white rats contains a lysin capable of dissolving the bacterium in vitro. Pigs are occasionally infected; the carnivora generally are refractory, the bear and cat being less resistant. Most birds are insusceptible, but some small birds, like the sparrow, are more susceptible. Cold-blooded animals . . . are refractory. Infection occurs: Through the food, giving rise to intestinal anthrax. Cattle and sheep are usually infected in this manner by spores, the bac- terium being destroyed by the gastric juice. In man infection through food rarely occurs. Through the air. Infection by inhalation through the lungs occurs in man through the medium of dust contaminated by anthrax spores, hence the name “‘wool-sorter’s disease. Through wounds. This method usually occurs in man and also in sheep. Cutaneous infection comes through a scratch or wound, and gives rise to a carbuncle—hence the name “malignant pustule.” It occurs most frequently among employees of tanneries, wool-sorters, veterinary surgeons, and those whose occupation brings them into touch with infected animals, their hides or products. The incubation period is a short one, even in the naturally occurring disease; inoculated laboratory animals die in twenty-four to forty. eight hours. The bacteria appear in the blood about fifteen hours after inoculation, and at death the blood simply swarms with the or- ganism. The veins are turgid, and the blood is often very dark, and coagulates slowly. The bacteria abound in the capillaries (Fig. 158). The spleen is enlarged and contains enormous numbers of the organisms. In the kidney the glomeruli and tubules are gorged with the bacteria, which pass into the urine. The bacteria can pass into the milk of females in lactation. The bacteria are also numerous in the liver, lungs and mesentery, but few are found in the muscles. Post-mortem examination of subcutaneously inoculated laboratory animals shows subcutaneous cedema and enlarged spleen. The organism is eliminated from the body in urine, faeces, mucous discharges, etc. Pastures become infected from burying anthrax carcasses which have been opened or have been skinned, thus favoring the formation of spores. If buried too near the surface, the rise of’ the ground water, or the castings of earth worms, bring spores to the surface and on to the herbage, where they may be ingested by graz- MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 753 ing animals. Tanneries using anthrax-infected hides may be the cause of distributing the organism by means of effluent water which has been used for steeping hides. Many such cases have been traced in Dela- ware, Wisconsin and in Ontario. Hay from an infected pasture may be transported to a distant farm, and cause an outbreak of the disease. In Brazil, vultures feeding on anthrax carcasses disseminate the spores by means of their excrement, and thus spread the disease. Blood- sucking flies may also be instrumental in transferring the bacterium from one animal to another. Fic. 158.—Anthrax. The organisms of anthrax in the capillaries of the liver of a mouse. (After Williams.) Season is a contributing factor. In years in which the spring floods have been very high, followed by a hot dry season, anthrax is most prevalent. There are a few preliminary symptoms; there is usually sudden loss of appetite, trembling and convulsive movements. Often blood is seen in urine or feeces or discharged from the nose. The mucous membranes are often bluish in color, and boils or pustules may occur on various parts of the body. Death in cattle occurs in two to five days and in sheep in twenty-four to thirty-six hours. The mortality is high and intestinal cases are fatal in 80 to go per cent of the animals attacked. 48 754 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS The usual post-mortem appearances are enlargement of the spleen, ‘blood thick and tarry, bloody extravasations in the muscles and organs, and bloody fluids escaping from mouth, nostrils or anus. In anthrax-infected districts vaccination should be used. The vaccines are prepared by cultivating the bacterium at a high tempera- ture—42° to 43°—thus forming an asporogenous race, according to methods devised by Pasteur in 1881. Two vaccines are often used, the first of very low virulence, the second more virulent. Between 1882 and 1907, 8,000,000 sheep and 1,300,000 Cattle have been vaccinated in France against anthrax, with excellent results. Vaccina- tion by toxin has been advocated by Toussiant, Hawkin, Marmier and others, but this method has not had the success of that described above. For treatment of the disease in man, Sclavo’s serum has been of considerable benefit. This serum is obtained from the sheep or ass. The animals first receive the two vaccines of Pasteur, then more viru- lent cultures in gradually increasing doses. A serum is then obtained which in a dose of 2 c.c. or less protects a rabbit against a lethal dose — of the anthrax organism. Animals dead of anthrax should never be opened or skinned. If doubt exists as to the nature of the disease, an ear may be cut off and sent to a laboratory for examination. Anthrax-infected carcasses may be either burned or buried at'a depth of 1.8 m. (6 feet), and covered with quick-lime, and as an extra precaution the burial ground may be fenced off. The prime necessity is to prevent the formation of spores, as it has been shown experimentally that they remain in this condition for eighteen years and produce the disease when inoculated. Soiled litter, forage and the excretions of animals dead of the disease should be collected and burned. The stalls, stables, implements and anything that has been in con- tact with the diseased animals should be disinfected by burning, boiling . or the use of some disinfectant like 5 per cent carbolic acid. BACILLARY WHITE DIARRHGA OF YOUNG CHICKS* Bacterium pullorum The epidemic type of diarrhoea which is characterized in part by a whitish diarrhoeal discharge, and which is now known as “bacillary * Prepared by L. F. Rettger. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 755 white diarrhoea,” is caused by a bacterium which belongs to the colon- typhoid group of organisms. It may be cultivated easily on the ordi- nary laboratory media, but its growth on slant agar containing Witte’s peptone is delicate and bears a striking resemblance to that of Strepto- coccus pyogenes. This finely beaded growth is an important aid in the identification of the bacterium. The specific organism, Bact. pullorum, is present in the liver, lungs, kidneys, spleen, heart and unabsorbed yolk of affected chicks, being most easily obtained from the liver and yolk, when the latter is present. Some of the most common post-mortem appearances of the organs are those of the liver and intestine, the former showing pale and congested areas, while the intestine is colorless and to a large extent void of contents. The disease seldom manifests itself in chicks after they have attained the age of four or five weeks. The greatest mortality usually occurs within the first two weeks. The chicks become listless, and are inclined to huddle together for warmth. There is loss of appetite and emacia- tion. The wings droop, the back seems to shorten and the abdomen protrudes out of proportion, causing the chicks to look stilty. The characteristic whitish discharge from the bowel may be absent from individual chicks, but is usually noticeable in groups of any appreciable size. Bacillary white diarrhoea may be transmitted to young chicks under five days old through infected food and drinking water, as has been demonstrated repeatedly. Furthermore, chicks are often infected with Bact. pullorum before they are hatched. This is due to the fact that the yolk of infected hens carries the specific organism in it from the time of its formation in the ovary. Hence, the mother hen is the source of infection, having retained within it the bacterium in question from the time she was an infected chick, or having acquired it later in life through contact with diseased fowls. In laying hens the infection is localized in the ovary which becomes decidedly abnormal in appearance. The partly developed ova are discolored, misshapen and of all degrees of consistency. Ovarian infection may be determined by the macroscopic agglutina- tion test which has proven itself very valuable and practicable in the organized campaign against bacillary white diarrhoea that has been conducted in the State of Connecticut for the past two years. This 756 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS method of diagnosis has been found to be much more valuable than the bacteriological examination of eggs. Eradication of infected laying stock is the solution of the white ‘diarrhcea problem. Flocks which are at all doubtful, or which have given a history of infection, should be tested, and the reacting fowls eliminated. Better still, no eggs should be used for hatching which have come from flocks that have shown an appreciable degree of infec- tion, although reacting individuals have been removed. CHICKEN CHOLERA* Bacterium cholere gallinarum The bacterium causing this disease was first noticed by Perroncito and Toussaint; later, in 1880, it was described by Pasteur, and was the first organism in which the French savant succeeded in attenuating the virulence and the first disease for which a vaccine made from atten- uated organisms was prepared. Koch in 1878 described an organism of similar pathogenicity as the bacterium of rabbit septicemia and in 1886 the term hemorrhagic septicaemia was given by Hueppe to a num- ber of infectious diseases of the lower animals in which hemorrhagic spots were found in the tissues and internal organs. In 1900 Ligniéres discussed these bacteria, and named them as a genus, Pasteurellose, the specific name given depending on the animal for which it was most pathogenic. Thus he distinguished avian, porcine, ovine, bovine, equine and canine Pasteurelloses. The specific characters of this group are small ovoid bacteria, often showing bi- polar staining when treated with the aniline dyes, non-motile, no spores, Gram- negative, polymorphic, not liquefying gelatin, no visible growth on naturally acid potato, milk unchanged, no indol production, generally aerobic but also a facultative anaerobe, virulence changeable, but usually very pronounced. The bacterium of fowl cholera, Bact. cholere gallinarum, or avian Pasteurellose, is from 0.5 to 1.254 long and 0.25 to 0.40u broad. It develops best at 37°, and very slowly at 20°. It loses its virulence in cultures very quickly, and it succumbs readily to desiccation. The disease is of frequent occurrence in Europe, but not often seen in North America but some outbreaks have been reported in the United States and Canada. Unfortunately it has been confused by poultry- * Prepared by F. C. Harrison. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 757 men with any disease characterized by excessive diarrhoea. The symp- toms first noticed are the yellow color of droppings soiling the cloacal feathers, then diarrhoea sets in, the character of the discharge varying, being at times a fluid greenish mass, or a brown-red mucus, or a viscous transparent and frothy fluid. The bird becomes uneasy, drinks copi- ously and with a rise in temperature to 42° to 44° the bird becomes drowsy and death follows. The period between the first noticeable symptoms and death varies from one to three days. Chronic cases sometimes occur and in these the bacterium is found with difficulty. The birds become infected by way of the digestive tract, from eating and picking up material infected by the discharges of diseased birds. Post-mortem indications are blackened combs, congestion of the blood-vessels in the organs and intestines, and punctiform or large hemorrhages of the duodenum, intestines and heart. The bacteria are numerous in the blood, the pulp of all organs, and in the intestinal con- tents. It is a true septicemia. If the disease breaks out in epidemic form the best and quickest method of getting rid of it is to kill off all the fowls, disinfect the houses, and dig or plough up the poultry runs, and leave them two weeks before re-stocking. CHRONIC BACTERIAL ENTERITIS* The disease produced by this bacterium has been demonstrated in Germany, Belgium, Switzerland, Holland, Denmark, and perhaps other European countries. It is known by various names, as Johné’s disease, chronic bacterial dysentery, and chronic bacterial enteritis. This bacterium produces a chronic infectious disease of cattle in- volving especially the intestinal mucous membrane. Other animals do not seem susceptible. The disease produced is usually fatal. Usually the most conspicuous general symptom is unthrift in spite of good appetite and good food. This microdrganism is a rod-shaped bacterium from 2p to 3u long and about o.sy broad and is strongly acid-fast. The production of active toxins is to be presumed since the amount of disturbance is fre- quently out of all proportion to the lesions found on examination post-mortem. * prepared by M. H. Reynolds. 758 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS The bacteria are present in the feces, intestinal mucosa, and sub- mucosa, most frequently in the small intestines. The large intestines -may be involved later. This microdrganism produces chronic, inflammatory changes of the intestinal mucous membrane, the whole intestinal wall becoming greatly thickened. This bacterium resembles closely avian tubercle bacteria, but may be distinguished by the fact that the avian tubercle bacterium is rather easily grown on artificial media. This organism does not have the same pathogenic peculiarities as the avian tubercle bacterium. It seems well demonstrated that many cases of chronic bacterial enteritis do probably react to avian tuberculin; but this does not prove identity. So far as known the bacterium is eliminated in the. manure of affected cattle and disseminated in this way. Wider dissemination is made by diseased animals moving from place to place. The most important considerations in controlling this disease are careful disposition of contaminated manure and isolation of suspected animals. The manure should be used only where it can not serve to spread disease to other cattle. Sick animals should be carefully isolated and premises thoroughly disinfected. ’ \ ConTacious ABORTION OF Domestic ANIMALS* Bacterium abortus The premature discharge of the products of conception from the uterus is a not infrequent occurrence among domestic animals, and’ doubtless various factors may from time to time operate in its causation. Injury, excessive fermentable food, or poisonous food may at times: produce this result. For a long time, however, practical husbandmen have recognized an ‘epizoétic form or a contagious abortion, a definite transmissible disease, of which the loss of the foetus is the most promi- nent characteristic. This disease appears to be generally distributed in all agricultural communities.. Cows, especially, are affected, but a somewhat similar if not identical disease also occurs in other domestic animals. In 1897 Bernhard Bang discovered in the uterine exudate of a cow, slaughtered during an attack of this disease before the abortion had * Prepared by W. J. MacNeal. adhe Se ci MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 759 occurred, a small bacterium which he was able to grow in pure culture, and, by inoculating pure cultures of this organism, he produced the disease in cows, sheep, goats and rabbits. The microbe is a short non-motile rod, staining with moderate ease, and decolorized by Gram’s method. It does not form spores but the vegetative forms are fairly resistant to drying and may, perhaps, live for some weeks under ordinary conditions in pastures and stables. Its artificial culture requires special technic because of its peculiar oxygen requirement. The bacterium usually fails to grow in the presence of the atmospheric air or under anaerobic conditions. It requires for its de- velopment a partial pressure of oxygen somewhat less than that of the atmosphere. When inoculated into deep serum-gelatin-agar tubes and incubated in the air, the colonies develop only in a particular zone about five millimeters beneath the surface of the medium. When cul- tures are placed in the proper atmosphere, development on the surface may be obtained. Prolonged cultivation on artificial media obscures this peculiar property of the microbe so that old culture strains grow well under ordinary aerobic conditions. — In the diseased animal, the specific bacteria are found in the pla- centa and amniotic fluid, frequently also inside the foetal intestine, sometimes in the tissues of the foetal organs, and in the wall of the maternal uterus. The placenta appears to be the particular organ favorable to the development of the germ, and when this has been discharged from the body the abortion bacilli no longer flourish, al- though the infection may continue as a chronic uterine inflammation for a long time. The general health is only slightly disturbed. At the next pregnancy the disease is practically certain to reappear, and pos- sibly again also at the succeeding one. After two or three abortions the animals appear to have acquired an immunity to the infection, and may sometimes breed normally thereafter, although some animals are per- manently sterile aftey a few attacks of the disease. The organisms escape from the diseased animal in the products of conception at the time of the abortion, and in the chronic uterine dis- charge which may continue for a long time afterward. The disease may be conveyed to other animals by contact with this material and by eating grass or other feed soiled with it. Doubtless the male is an im- portant factor, possibly the most important factor, in transmitting the disease, although no serious inflammation is produced in him. 760 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS The control of the disease depends upon the isolation of the infected animals, cremation of infected foetus, placenta and discharges, and thorough disinfection of the premises. Heifers and healthy cows should not be allowed to mingle with cows which have aborted, nor should they be served by a bull which has covered infected animals at any time. Local antiseptic treatment of the cow which has aborted diminishes the danger of the persisting discharge. Contagious abortion also occurs in other domestic animals, espe- cially in horses, sheep, goats and swine. Inoculation experiments have shown that the Bact. abortus of Bang can infect some of these animals. Its importance as a factor in the epizodtics of abortion occurring natu-. rally among them is stilluncertain. In horses at any rate another organ- ism appears to be more frequently involved. DIPHTHERIA* Bacterium diphtheria The disease is epidemic in all large communities ‘especially in Europe and America... It is, however, almost absent from tropical regions. Epidemics and pandemics occur in cycles. Essentially diphtheria is a disease peculiar to man. Avian diphtheria, however, is known, al- though seemingly due to another cause, and on rare occasions natural infection has been found in the horse. The period of incubation is said to be two to five days. In man the disease usually begins with lassitude and fever followed in a few hours by “sore throat.” The inflamed area on the pharyngeal wall, tonsils, larynx or wherever it may be becomes in typical cases the seat of degenerative changes in the epithelium and underlying tissues with abundant fibrinous exudation resulting in the formation of a com- paratively tough membrane or pseudo-membrane, which is a striking and characteristic feature of the disease. This local lesion is almost always found on mucous membranes though occasional instances of infection of wounds have been noted. The bacterium of diphtheria was described in 1883 by Klebs in sections of typical membranes. The organisms were isolated and dif- ferentiated in 1884 by Loeffler, who was able to fulfill Koch’s postulates for pathogenic microbes. Accidental infection of the human being has * Prepared by Edward Fidlar. : ' MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 761 happened in the laboratory and confirmed the findings of animal inocu- lation. The success of antitoxin treatment is further evidence of causal relationship. The organism is detected in the following manner: A sterile swab is rubbed gently over the inflamed area or against any visible membrane, care being taken to touch other parts as little as possible. Theswab is then immediately inserted into a tube of specially prepared medium—Loeffler’s inspissated blood serum—over the surface of which it is rubbed back and forth. The swab is returned to its own tube or left against the serum and the culture and swab sent to the laboratory. After twelve to fee Fic. 159.—Bacterium of diphtheria. 1000. (After Williams.) eighteen hours of incubation, at 37°, smears are made from the cultures and stained with Loefflers methylene blue. The diagnosis is made on the morphological char- acters of the bacterium. Occurring in pure cultures the form of the bacterium is subject to remarkable changes according to the medium and length of cultiva- tion. Its size as it appears in exudates and from serum media varies from tu to 7u in length and 0.254 to 2u in width. The rods are straight or slightly curved, usually not uniformly cylindrical but with swellings at the ends, or in the middle, or irregularly disposed (Fig. 159). Both ends may be rounded or both pointed, or one rounded and the other pointed. Branching forms are not infrequently found. The bacteria may appear in pairs end to end; more frequently and typically they are inclined to one another at a greater or less angle and may assume a parallel arrange- ment or the form of a short zigzag chain. The arrangement is most clearly under- stood and remembered by considering the peculiar “snapping” type of fission characteristic of the group. There are no flagella and no spores. The cell mem- . 762 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS brane is possessed of great elasticity as evidenced by the post-fission movements. The bacteria stain readily with all the aniline dyes and retain the primary stain fairly well by Gram’s method. With Loeffler’s methylene’ blue they stain in a character- istically irregular manner, and show metachromatic “granular” forms, “barred’’ and “solid-stained” forms (Fig. 160). On a basis of morphology and staining properties, Wesbrook, Wilson and McDaniel have devised a classification which is very convenient for descriptive purposes. The minimum temperature of growth 18° to 19°, optimum 35° to 37°, maximum 40° to 41°. The bacterium grows most readily in the presence of oxygen. Under certain conditions it will grow anaero- Fic. 160.—Wesbrook’s types of Bact. diphtherie. u, c, d, granular types; a’, c’, d’, barred types; a2, c?, d®, solid types. 1500. (From McFarland.) @ bically. The optimum reaction of blood serum media is about +0.8. The amount of acid which the bacterium can endure varies with the kind of acid. Gelatin is not liquefied, neither are the proteins of blood serum nor of milk. Caseinogen is not changed to casein. Some carbohydrates are broken up with the production of acid. All authorities find that the bacterium forms acid from dextrose. It is generally agreed that acid is produced from lactose, galactose and maltose. Action on dextrin, lactose, saccharose and glycerinis variable. The majority of workers find mannit is unchanged. An acid reaction in plain broth by fermentation of muscle sugar may be followed by the production of alkali. Gas is not produced under any circumstances. No indol is formed. Most strains cause hemolysis of red blood cells. A true diffusible toxin is formed for the artificial production of which in broth cultures peptone, absence of sugar, an alkaline reaction and free access of oxygen are favorable factors. Growth on plain nutrient agar is not so abundant as on Loeffler’s blood serum. Colonies of two types may be found: (a) most common is small grayish-white, rounded, slightly raised, almost translucent with MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 763 more or less granular surface and dark center, the margins varying in irregularity, and often with a thin extension spreading out from the edge; (0) less common, larger, more luxuriant, white, rounded, raised, granular to nearly smooth and some- what moist. Plain broth must be slightly alkaline to litmus. About one-half of cultures grow readily and half very feebly. The characteristic growth is a finely granular deposit at bottom and along sides of the tube leaving the broth clear; a few cultures produce a diffuse cloudiness with more or less well-developed pellicle. Growth on gelatin is scanty chiefly owing to temperature at which this medium must be kept. The gelatin is not liquefied. In milk growth takes place at com- paratively low temperature (20°) without coagulation but with acid production. On potato growth occurs especially if slightly alkaline; in the majority of cases invisible; it may appear as a thin dry glaze or with a whitish or slightly yellowish tinge. On Loeffler’s blood serum growth is rapid. In eighteen to twenty-four hours colonies are rounded, grayish-white with a slightly yellowish tinge moderately translucent except toward the center, smooth, moist and shiny. The margins are only slightly irregular. With age the colonies become dull and opaque, the surface becoming marked by concentric lines and sometimes also exhibiting radial striation. Thermal death-points are 58° to 60° for ten minutes, 70° for five minutes, 100° for one minute. On the other hand —190° for seven days and —252° for ten hours have failed to kill in some instances. Diffuse light hinders growth. Direct sunlight kills within two hours to-a few days according to the medium in which the organisms are suspended. The organism gains entrance into the body through the mouth and nose. The bacteria usually remain localized; they can almost always be demonstrated when a definite membrane is present and often when there is none. They are practically always found in the lung of fatal cases because of direct infection. Entrance of the bacteria into the blood stream with resulting infection of the internal organs has occurred in fatal cases. The protective apparatus concerned in diphtheria is probably dif- ferent at the beginning from that involved late in the disease. Experi- mentally, agglutinins, bacteriolysins and opsonins have been demon- strated in exudates and serum. While these properties may be important in warding off an infection they appear to be of little influ- ence once the bacteria are established, and thereafter on the amount of antitoxin will rest the outcome of the disease. The toxin is strongly antigenic, the cell bodies feebly so. Aggressins have not been demonstrated. The organisms escape by the secretions of the mouth and nose. Direct infection by coughing, sneezing and speaking probably takes 764 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS place frequently not only from the sick and convalescents but also from healthy carriers. Control of the disease is sought by quarantine of all sick persons and the placing of restrictions if not actual quarantine on those exposed and showing the bacteria in the nose and throat. DYSENTERY* t Bacterium dysenteriae Two chief types of dysentery are known, one due to a protozoan —Ameba Entameba tetragena; the other due to a bacterium—Bact. dysenterie. ‘ Only the latter is here dealt with. Acute dysentery in an epidemic form is found chiefly in Asia, some- times in Europe and in the Philippines. In this country occasional small epidemics and certain types of summer diarrhceas of infants have been shown to be due to Bact. dysenteri@. The disease occurs naturally only in man. Dysentery is an intestinal disorder usually acute and, in its epidemic form, very severe, marked by a flux in which there is the frequent pass- age of blood and mucus with severe tenesmus and pain in the abdomen. The fever accompanying this may reach 104° and in the severe cases delirium and death may result. In Japanese epidemics the fatality has reached 25 per.cent or more. The pathological findings are most marked in the intestine whee the mucosa is swollen and hyperemic, with more or less hemorrhage and extensive necrosis. Shiga in 1898 discovered a bacterium in the stools of persons suffer- ing from the disease and the organism was agglutinated by the blood serum of the patients. He found the same organism repeatedly in a considerable number of cases. The results of many other investigations have demonstrated the presence of several forms conforming in general to the type described by Shiga but showing some difference in fermentation properties; these are sometimes known as para- or pseudo-dysentery bacteria. The constant presence of the organism in the epidemic type and the fact of agglutination leave little doubt as to the etiological relation. A * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 765 criminal fed with a pure culture of Shiga’s organism developed the typical disease. The organism can occasionally be isolated in almost pure culture from bits of mucus in the stool. Ordinarily, however, special methods are required for isolation. Bact. dysenterie@ (Shiga) is rather short with rounded ends and closely resembles the typhoid bacillus in gross morphological features. It does not possess flagella. It stains readily with the aniline dyes and is Gram-negative. It grows best at body temperature, is aerobic and facultatively anaerobic. It prefers a slightly alkaline medium. On agar, broth, and gelatin growth resembles that of the typhoid bacillus. In litmus. milk an alkaline reaction usually follows a slight primary acidity without any further apparent change. On potato growthitisat first invisible but may appear later of a brownish color. Acid is formed from dextrose, levulose, and galactose. (Other types described differ from this in the fermentation of mannit and sometimes of maltose.) Gasisnever formed. Indolisnot formed. (Other types usually form indol.) The toxinsare probably chiefly endotoxins, though soluble poisons have also been demonstrated by some workers. The bacterium remains alive for months when preserved under the proper conditions. The thermal death point is 60° and re- sistance to low temperature is considerable. It is sensitive to the usual strength of ordinary disinfectants. , Dysentery does not occur in animals under natural conditions. By artificial methods, however, it is claimed the disease has been repro- duced in dogs. Cultures, living or dead, are often extremely toxic to small animals, especially the rabbit, and produce, after intravenous injection, violent intestinal symptoms, due evidently to the excretion of an irritating poison. Nervous symptoms are -also more or less marked and paralysis sometimes occurs before death. Immunity pro- duced artificially in animals is accompanied by the production of lysins and agglutinins and lately antitoxins have been described in accord with the demonstration of diffusible toxins. The agglutination in man is of diagnostic value. The epidemiology of dysentery is the same as for typhoid fever. In a few instances the bacilli have been demonstrated in the feces of healthy persons, and convalescents may remain carriers for several months. Some success has been recorded from the administration of animal immune serums and has been attributed to both lytic and antitoxic action. Active immunization as a means of prophylaxis does not seem to be of much value. 766 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS Fow. DIPHTHERIA* This disease, popularly known as Roup, and in its later stages canker, is characterized by a grayish-yellow fibrinous exudate, called a false membrane. which forms upon the mucous membrane of the eyes,. nasal passage, mouth, pharynx and larynx. Roup, or fowl diphtheria, may be caused by a number of different organisms, among them the well-known Ps. pyocyanea (green or blue pus organism), B. cacosmus and other species which give rise to a com- plex suppurative process. The pus formed is semi-solid, cheese-like and yellowish-white in color without any tendency to become soft and liquid or to perforate the surrounding skin. The organisms have a tendency to penetrate the deeper layers of the mucous membrane or sub-mucous tissues, and hence swabs or cultures taken from the false membranes may not contain the causal microérganisms which are retained in the depths of the tissues. Sections of membranes. from affected fowls show large numbers of pus cells, some regular masses, débris of epithelia) cells and bacteria, and thus they differ from the diphtheritic membianes of man. The organisms inentioned above have been isolated from affected fowls, not only in America but also by Hauser in Europe. Several investigators have described other bacteria producing false membranes, and there are a few who think that coccidia are associated with the disease. Both Ps. pyocyanea and B. cacusmus are able to produce false mem- branes when inoculated.into healthy birds, typical croupous and diph- theritic membranes in the mouth and eyes; tumors in the subcutaneous tissues, the contents of which are firm, cheesy and yellowish-white; purulent conjunctivitis, blindness, purulent ophthalmia, and cheese-like exudations in the bronchial tubes. These indications are identical with the symptoms of “roup.” . The disease is of variable virulence, and is apt to become chronic, especially in unhygienic surroundings, and in draughty, badly ventilated damp houses. A common cold is a predisposing factor, and favors the invasion of the organisms mentioned. Treatment of severe cases is useless, and demands too much time. Diseased birds should be isolated and the buildings thoroughly disin- * Prepared by F. C. Harrison. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 767 fected. Slight cases may be cured by a 2 per cent solution of potassium permanganate, in which the bird’s head is plunged for a few seconds. This treatment should be given twice a day and continued until all symptoms have disappeared. The most effective preventive is to keep fowls in good sanitary conditions—in dry, clean and well-ventilated houses, free from draughts.. Besides the organisms mentioned, Loeffler has described the B. diphtherie columbarium, and Loir and Duclaux the B. diphtherie gallin- arum as causing fowl diphtheria, but the diseases produced by these organisms are very dissimilar from the well-known “Roup” of North America. The Klebs-Loeffler bacterium of human diphtheria has no pathogenic effect on fowls. GLANDERS* Bacterium mallet Glanders is a very common and serious disease, most common among equines. It iscommunicable to the human being by inoculation and by the same process may affect sheep, goats, and laboratory animals. Cattle are not susceptible. Bact. mallei and the disease it produces are widely scattered over the civilized world wherever horses are numerous. This infection produces a disease which may be acute or chronic according to the virulence of the microérganisms and resistance of the animal. Mules and donkeys are less resistant than horses and usually have the disease in more acute form. The characteristic features of the disease produced are inflammatory changes of the lymph glands and lymph vessels, ulceration of mucous membranes, the tubercle, the farcy bud, the lymph cord, and the peculiar, clear, viscid discharge. ‘There is considerable fever in acute cases, much less marked or absent in chronic cases. In a very common type of the disease there frequently occurs a destructive inflammation of the nasal mucous membrane which results in ulcers and consequent nasal discharge. Glanders in man is rare considering the frequent opportunity for infection. There are usually inflammatory swellings with involvement * Prepared by M. H. Reynolds. 768 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS of local lymph glands, and constitutional disturbances soon follow the local symptoms. Human glanders is to be always regarded as very serious with a probability of fatal termination. Ulcers may develop in the nose or mouth with more or less discharge. Pustules appear involving the skin, and abscesses involve deeper structures in various portions of the body. The distribution of Bact. mallet in the animal body is shown by the most common appearance of its disease in the skin, subcutaneous tissue, mucous membranes, lymphatic system, lungs, liver, spleen and kidneys. The etiological factor is a small bacillus with rounded ends known as Bact. mallet, discovered by Loeffler and Schiitz and several others in 1882, and well demonstrated to be the specific cause of glanders. Entrance is usually effected by way of a mucous membrane, fre- quently the intestinal; sometimes by inoculation. The period of incubation seems variable and uncertain under natural infection, but in artificial inoculation with virulent cultures, is very brief. Bact. mallet probably produces toxins in artificial media and also in body tissues; the well-known preparation called mallein may be considered in this class awaiting more definite knowledge. This toxin (mallein) produces a distinct reaction by inoculation into glandered animals, but is practically non-toxic for healthy equines.. So far as known Bact. mallei attacks the animal tissues as do many other microérganisms, the harm resulting chiefly from bacterial toxins which give the local tissue reactions leading to the lesions characteristic of glanders. In its action on tissues Bact. mallet resembles Bact. tuberculosis; but shows a more rapid development of lesions and more active in- flammation. Lesions are of two types—a well-defined nodule followed by ulcera- tion and areas of diffuse infiltration. The nodule as it appears in glanders consists largely of lymphoid cells. Nodules die at the center, suppurate, and discharge. This occurs especially in the external form of glanders, which affects more commonly the legs and head. Occasionally defined enlargements appear in the involved lung areas. Pulmonary lymph glands are frequently enlarged, and hardened. The superficial skin lesions are in the form of nodules previously mentioned, which usually suppurate MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 769 and ultimately heal. In the deeper subcutaneous tissues there is a tendency to abscess formation. Small nodules or tubercles commonly appear in the lungs of affected horses. These vary in size from millet seed to as large as garden peas. Various degrees of broncho-pnuemonia appear and more or less pleurisy. Bact. mallei shows no flagella and is non-motile. Itisasmall bacteriumo.25u to o.4u thick by 1.54 to 3u long with rounded ends (Fig. 161). Spores have not been demonstrated. It is generally single. Coccus forms sometimes appear and even short threads when grown on certain media; e.g., potato. It decolorizes by Gram’s method and is not easily stained by the aniline dyes. This bacterium grows fairly well between 25° and 42° on potato, glycerin agar, or blood serum. The guinea pig gives a reliable diagnosis by inoculation, showing a diagnostic reaction within four or five days. Diagnosis may also be confirmed by the agglutination test in dilution of about 1:1000 or more and by the complement fixation test. Satis- factorily stained in tissue section by Kuehn’s carbol-methylene blue. Its growth is limited at an upper range of about 42° and the culture is killed at 55° in about five minutes. Bact. mallei is difficult to isolate by culture methods being a slow grower and easily lost beside faster growing organisms. It can be better isolated by guinea- pig inoculation. In growth it is both aerobic and anaerobic. Fic. 161.—Bacterium mallet. From pure culture on glycerin agar. X 1000. (From Migula.) The virus escapes from the body in various ways. Elimination is most common in morbid discharges from the nose, pharynx, trachea, and in pus from farcy buds and abscesses. ; Bact. mallei may be spread directly from the diseased animal to the susceptible animal, or the dissemination may be by way of intermedi- ate objects; e.g., troughs, feed boxes, water pails, etc., and perhaps also by inhalation. 49 770 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS In man, infection occurs usually by inoculation. Cases produced in this way, occasionally appear among laboratory workers. Bact. mallei is to be regarded as purely parasitic and limited in its natural activities to the animal body. Bact. mallei is easily destroyed by a variety of unfavorable condi- tions. It is destroyed by drying in fifteen to twenty days and is easily killed by heat or antiseptics. All plain cases of glanders in domestic animals should be promptly destroyed. Exposed horses should be tested with mallein. Those that react should be destroyed or quarantined, and contaminated premises properly disinfected. INFLUENZA* Bacterium influenze The natural disease appears to be limited to man. The incubation period is very uncertain—probably very variable. While the clinical manifestations of infection with Bact. influenze are variable the ordinary form begins with sudden severe headache accom- panied by great prostration, pains and aches in the back and bones, and a rapid rise in temperature. The fever lasts from three to five days and leaves the patient eebtertely: weak, and depressed in both - mind and body. Pfeiffer in 1892 described a bacterium which occurred in large num- bers in the purulent bronchial secretion expectorated by influenza patients. The causal relationship has been quite definitely established. An examination of the sputum furnishes good presumptive evidence, but cultivation is necessary for positive diagnosis. The purulent material is streaked out in a drop of sterile blood upon an agar plate. Agglutination and complement fixation tests are valuable means for identification. In pure cultures the bacterium is 0.24 to 0.3u wide by o.5u long with occasional threads up to 2 inlength. The ends of the rod are rounded. The arrangement is usually single, occasionally in pairs end to end and rarely in chains. The bacterium is non-motile and does not show capsules or spores. It does not stain readily with ordinary aniline dyes. It is Gram-negative. Polar staining is shown sometimes * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 771 The temperature range of the influenza bacterium is about 26° to 41°. It is aerobic. It grows on artificial media only in the presence of blood pigment. On plain agar, glycerin agar, or blood serum with a thin layer of rabbit or human blood the colonies in twenty-four hours are very small, round and transparent, and remain discrete unless very thickly sown. The center of older colonies may acquire a yellow-brown color. In blood broth growth occurs quite readily if the medium is used in thin layers. It shows very much less resistance than the majority of non-spore-bearing bacteria. It is destroyed at 60° within about one minute. It is especially sensitive to drying. The bacterium gains entrance through the mouth and nose and finds suitable soil in the mucous membrane of the respiratory passage. The toxemia is due to absorption from this site rather than to the presence .of the bacteria in the blood. The bodies of the bacteria are distinctly pyogenic. Secondary infections are prone to follow upon influenza so that abscess, gangrene of the lung, and empyema are not infrequent. Influenza bacteria have been found in pneumonia, otitis media, and meningitis. Immunity after natural infection is transitory. The organisms are eliminated through the gates of entrance. Infec- tion is for the most part direct, and follows close contact with a patient or carrier. The bacteria are said to persist for many years in the bronchial secretions of convalescents and healthy individuals. Therapeutically there is no specificmeasure for the control of this disease. Remembering that the infective agent is ejected during the coughing and speaking of the patient and is present in great numbers in the sputum, personal hygienic measures in both patient and contact should prove very effective. Wuoopinc CoucH* Bacterium pertusis Whooping cough accounted for the death of 4,856 children in the United States in 1907. The causative agent, according to Bordet and Gengou, is an influenza-like bacillus. It is a non-motile coccoid bacillus, stained faintly by aniline dyes and Gram-negative. It is distinguished from the influenza bacillus by * Prepared by Edward Fidlar. 772, MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS agglutination and complement deviation tests and by the fact that it can be gradually adapted to ordinary media. The production of pertussis in young animals has been claimed. The organism has an endotoxin which produces local necrosis after subcutaneous injection. Further evidence on the etiology of whooping cough is afforded by the observations of Mallory and others who have found large num- bers of small microérganisms corresponding morphologically with Bact. pertussis occurring between the cilia on the epithelial cells lining the respiratory tract in fatal cases of the disease. HamorruHacic SEPTicamtial Bacterium bovisepticum Hemorrhagic septicemia belongs to a class of similar diseases grouped under the general head of Pasteurelloses. This disease has been reported from many portions of North America, from some sections of South America and many European countries. It is known under a variety of names, as cornstalk disease, buffalo disease, pneumo-enteritis, etc. Bact. bovisepticum produces a serious disease and affects a wide variety of domestic and wild animals. The domestic animals most commonly affected are cattle, sheep, and goats; the disease being much more common among cattle than among other classes of stock. The period of incubation in the artificial disease appears to be short, six to forty-eight hours. The onset of disease is usually sudden, and the case acute. Hemorrhagic septicemia does not spread from herd to herd but appears in isolated outbreaks usually at wide distances apart. It is a common experience to find a serious outbreak in one herd without any appearance of the disease in another herd in an adjoining pasture, with only a barbed wire fence between. Appar- ently the virus exists locally and, under as yet unknown conditions of increased virulence or lowered resistance, is able to start a local outbreak. Hemorrhages found at autopsy constitute the most specific and char- acteristic clinical evidence of this disease. Its mortality is very high, running from 50 to go per cent. $ Prepared by M. H. Reynolds. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 773 Hemorrhagic septicemia of cattle, chicken cholera, and a number of other diseases belonging to this group are very similar in clinical featurés and the bacteria which cause these diseases are very similar in cultural and microscopic features. Yet all evidence points to the fact that Bact. bovisepticum acts as a specific causal agent for hemorrhagic septicemia of cattle. Body infection probably occurs by both inoculation and ingestion. This disease does not appear to spread by simple association or ordinary contact and there is no general atmospheric distribution of Bact. bovi- septicum. Acute and rapidly fatal cases where the autopsy shows only trifling lesions would indicate the formation of active toxins. The character- istic hemorrhages indicate the production of substances actively toxic for the endothelial cells of capillaries. The fact that these hemorrhages vary in different cases from extensive subcutaneous areas to those that are scarcely visible would seem to indicate that this toxin is produced in greatly varying quantities or of greatly varying toxicity. — The lesions produced by this bacterium indicate a general distribu- tion through the body. The characteristic features as previously mentioned are the hemor- rhages which are either subcutaneous, submucous or subserous. Lymph glands are frequently infiltrated with extensive hemorrhages. Cases have been reported as showing high fever. Those studied by the writer have, as a rule, showed slight disturbance of temperature until near death. When voluntary muscles are involved the hemor- rhages invade connective tissues rather than muscle tissue proper. Bact. bovisepticum resembles so closely the bacterium of chicken cholera, the bac- -terium of rabbit septicemia, Bact. swisepticus and other members of this group (Pas- teurelloses) that laboratory differentiation from other members of the group is exceedingly difficult. It is a very small bacterium with rounded ends, closely resembling a diplococcus. It is from 1p to 1.5u long and from 0.3 to o.6u thick. Involution forms sometimes appear. It shows bipolar stain, decolorizes by Gram’s method, produces no spores, has no flagella, and is non-motile. The disease resembles anthrax in some general characteristics but is easily distinguished by microscopic examination of the blood and failure to find the large anthrax bacterium and by the fact that the blood from the general circulation is apparently normal in hemorrhagic septicemia. This disease also resembles symptomatic anthrax (black- Mt 774 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS leg) but is easily distinguished in that external swellings are slight if present at all and do not show gas, both of these features being charac- teristic of blackleg. The bacillus of symptomatic anthrax may be recognized by microscopic examination as so different from Bacterium bovi septicum that there could be no mistaking one for the other. \ Little is known concerning elimination of this bacterium from the diseased body and concerning methods of dissemination. Hence we are very much in the dark when attempting to deal with the disease produced by it. Isolation and disinfection are to be recommended on general prin- ciples. LEprosy* Bacterium lepre Leprosy is a disease almost as old as history itself but modern leprosy cannot be definitely identified with the leprosy of the Old Testament, and to day is found chiefly in oriental countries and in Norway, Iceland and Russia. The disease is present in some of the provinces of Canada - and in the States of Louisiana, California and Minnesota, and practic- ally limited to Scandinavians in the latter states. The natural incuba- tion period is difficult to ascertain but is probably a matter of months or years. Clinically there are two main types of the disease, the tubercular or nodular and the anesthetic types. In the first form, nodules develop in the face or other parts of the body usually preceded by an erythe- matous patch. The mucous membranes become affected more or less extensively and the hair and eyebrows fall out. In the anesthetic type after various disturbances of sensation which may sometimes be. followed by macule there develop areas of anesthesia. Bulle, ulcers and necrosis may occur with resulting deformities or again this type may exist for years without leading to such results. The bacteria of leprosy were first described by Hansen in 1879 and almost at the same time Neisser published similar descriptions. Culti- vation of Bact. lepre has been successful in the hands of Clegg, Duval and others. The microérganisms can be shown in tissue by the use of the Ziehl- Neelsen or Gabbet methods. * Prepared by Edward Fidlar. es MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS) 775 In tissue the bacterium closely resembles the bacterium of tuberculosis, but usually appears somewhat longer (su to 7) and thicker (about 0.5) straighter and less beaded. Flagella have not been demonstrated. The bacterium can be stained with the ordinary aniline dyes. It is Gram-positive. The staining reactions on the whole are like those of Bact. tuberculosis but Bact. lepre stains more readily and also decolorizes more readily; 30 per cent nitric acid followed by 95 per cent alcohol will totally decolorize them while Bact. tuberculosis resists. The optimum temperature for growth ranges from 32° to 35° when grown in symbiosis with ameebe. The reaction of the media upon which successful isolation takes place is 1 to 1.5 per cent alkaline to phenolphthalein. In recently isolated cultures growth is extremely slow and appears on the surface of the special media in four to six weeks as moist grayish-white colonies elevated centrally, with an irregular wavy margin arid attaining a diameter of 2mm. Oldercultures on glycerin agar are moist and abund- ant, and develop an orange-yellow pigment. In glycerin broth a thin membrane is formed at the surface after several weeks, while a small amount of sediment collects at the bottom of the tube leaving the medium clear. The resistance to heat is much greater than that of ordinary vegetative bacteria, so that cultures may be freed from contamination by the latter by simply heating to 60° for one hour. The resistance to drying is probably considerable. ~ Human leprosy appears to be confined naturally to man and only lately has the disease been transmitted artificially to animals. In the Japanese dancing mouse, and less frequently in the white mouse and the monkey small nodules may be found on the peritoneum about four to eight weeks after intraperitoneal inoculation. The animals do not show any symptoms of illness and must be killed in order to find the lesion. More recently Duval has produced an apparently typical leprosy in monkeys by repeated injections of cultures from artificial media. It is generally considered that the usual path of entrance of the bac- terium is the naso-pharyngeal mucous membrane. The organisms seem to be distributed slowly over the body and according to their location produce the different types of the disease. They are found in the nodules of the nodular type and in the nerve trunks of the anesthetic type. Agglutinins have been demonstrated in the blood of lepers. Com- plement deviation with various antigens has been investigated and indicates an antilipoid immune body which not infrequently gives a positive Wassermann reaction. Lepers react frequently to tuberculin inoculations and this is not considered to be always due to associated tuberculosis. The chief source of elimination of leprosy bacteria is the nasal 7 76 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS mucosa. The bacteria have been demonstrated in this region in about 40 per cent of the macular types, 80 per cent of the nodular and mixed types. While a great deal of popular fear exists against this disease it is decidedly less infectious than pulmonary tuberculosis. Lepers have unquestionably been subjected to a great deal of wholly unnecessary persecution. ‘ Prophylactically, isolation has certainly demonstrated its value and the reported increase of leprosy in certain parts of Europe has been attributed to a decrease of this custom of segregation. PLacuE* Bacterium pestis Epidemics have been recognized since the second century. About . half the population of the Roman Empire died in the sixth century. An epidemic of the fourteenth century destroyed half the inhabitants of Europe. In India during 1901 to 1904 about 2,000,000 died of the disease. In China, in Egypt, South Africa, and in sea ports of the Western hemisphere, plague has been found. Among animals the disease has been found chiefly in rats and squirrels. Dogs may occasionally become infected. Four types are described, the ambulant, bubonic, septicemic, and pneumonic. The bubonic type forms three-quarters of the cases. Physical and mental depression accompanied by a high fever, often with a remission about the third day, occurs. Collapse may then follow with death. Glandular swellings (buboes) appear in the groin and axilla and these may suppurate. Hzmorrhages beneath skin and mucous membranes are common. The third type is a very rapid form, causing death before the development of buboes. The fourth type is also a short and extremely fatal form, marked by the occurrence of broncho- pneumonia due to the plague bacteria. The bacterium of bubonic plague was described by Yersin and Kitasato independently in 1893. They found it in glands and through- out the body in fatal cases. The organism is readily grown from the buboes, the blood, and the sputum in the pneumonic type, by simple inoculation of ordinary media of a slightly alkaline reac- * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 777 tion. The bacteria are 1.54 to 1.7u long by 0.55u to 0.74 wide with rounded ends occurring singly or in pairs and short chains in exudates and sometimes in long chains in broth. Involution forms, large swollen spheres, clubs, etc., are char- acteristic in artificial media. There are no spores. It is non-motile. Some observers have demonstrated a gelatinous capsule. Occasionally very distinct branching occurs (Hill). It stains easily with aniline dyes, particularly at the poles which may show round or oval granules. It is Gram-negative. Its minimum temperature for growth is about 12°, the optimum 30°, the maximum 40°. It is Fic. 162,—Bact. pestis. (After Versin from Williams.) aerobic. Agar after twenty-four hours shows small granular grayish colonies with a thickened center and indented margin. Broth shows a granular deposit and sometimes a pellicle with dependent outgrowths, the medium remaining otherwise clear. Gelatin growths are as on agar, and the medium is not liquefied. Litmus milk may show slight acid formation and no coagulation. Potato shows nothing characteristic. The toxins appear to be largely endotoxins, though soluble poi- sons have been found in old cultures. No indol is formed. Resistance toward heat is not great, boiling kills in a few minutes. Light kills in a few hours. They do not resist drying well, but in a moist condition remain viable for over a year. The usual strengths of ordinary disinfectants kill in about ten minutes. Rats, mice, guinea-pigs, rabbits, and monkeys are particularly susceptible to inoculation and even insects die from infection. The bacterium enters the body through the (usually abraded) skin or respiratory tract. After involvement of the nearest lymphatic glands the bacteria are distributed through the blood. iq Single attacksimmunize. Theantibodies developed are agglutinins, 778 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS probably bacteriolysins, and possibly antitoxins. The agglutination reaction is of value in diagnosis. The organisms are eliminated in the exudates from suppurating buboes, in the sputum in the pneumonic type, and are present through- out the body after death. The dead bodies of human beings and of rats are sources of infection for other rats. There seems good evidence of these animals becoming chronic carriers though showing no symp- toms of disease, and may thus be important factors in maintaining and spreading plague. The disease is largely communicated by means of fleas which have ' become infected by living on other human beings or even upon rats. Prophylaxis consists of isolation of pneumonic cases, thorough disinfection involving the killing of fleas, and chiefly the destruction of rats, squirrels, and other animals which may serve as carriers. Haff- kine’s vaccination method has also been shown to be a valuable prophylactic measure. The serum of immunized animals has been tried as a therapeutic © agent and gives encouraging results when administered in the early _ stages. SWINE ERYSIPELAS* Bacterium rhusiopathie suis Swine erysipelas is an infectious disease of hogs characterized by red or violet discoloration of the skin and mucous membranes. Swine erysipelas does not exist in the United States but is very prevalent in continental Europe. It is caused by a very small, slender, non-motile, non-spore-bearing bacterium (Bact. rhusiopathie suis) which stains by Gram’s method, and grows feebly on the ordinary culture media. Development is. best under anaerobic conditions. In gelatin stab cultures, after three or four days, a white growth can be seen along the needle puncture. Radiating from this are a number of delicate tufts which give the growth the appearance of a fine test-tube brush. White and gray mice, white rats, and pigeons succumb to the inocula- tion of minute amounts of the culture. The bacteria tend to collect within the bodies of the leucocytes. This microdrganism is closely related to and possibly identical with the bacterium of mouse septicemia . * Prepared by M. Dorset. : ae $e gy ob 4 MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 779 (Bact. murisepticum). Preventive inoculation with attenuated cul- tures has long been practised successfully in Europe. TUBERCULOSIS* Bacterium tuberculosis Consumption, phthisis, scrofula, pearl disease, etc., are synonyms of the term tuberculosis. This bacterium in its several varieties produces a very universal disease; practically all common animals and man are subject to it. Cattle and swine among the domestic animals are especially susceptible to this infection and wild animals in captivity easily become affected. The normal progress of tuberculosis is slow. Its characteristic . feature is the tubercle or nodule of various sizes. Tuberculosis is probably the most common and serious of all diseases for either animal or man. In 1906, 138,000 persons died from tuberculosis in the United States, or at the rate of 164 per 100,000 population. Based upon these facts,t it is estimated that about 5,000,000 of those now living in the United States may die of the disease. It is claimed that the disease alone costs the United States from $400,000,000 to $1,000,000,000 each year (Fisher). “ If the loss from wage earnings, the cost of the patient in suffering, medical treatment, medicines, nursing, board, and care, also the suffer- ing and sacrifice entailed by near relatives, friends, and communities are considered, the loss to the country mentioned above does not appear so enormous. It is estimated by the United States Bureau of Animal Industry that 2 per cent of hogs in the United States are tubercular, and that losses of stock in the United States, due to tuberculosis, amount to $23,000,000 annually. Of 400,000 cattle tested in the United States prior to 1908 9.25 per cent were tubercular. The highest prevalence of tuberculosis in cattle is among pure bred herds and in city dairy stables; 7.e., among the cattle kept most closely confined. It is most common in old cattle and rare in calves under six months old. Tuberculous infection is quite generally scattered among cattle of civilized nations. * Prepared by M. H. Reynolds. fF 1911. 780 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS Tuberculosis appears in man usually in the form of lupus (tubercu- losis of the skin), or scrofula (tuberculosis of the cervical glands), or phthisis (tuberculosis of the lungs). It also frequently appears in the mesenteric glands and other glands of the body, and may appear in any of the tissues. It is quite possible, judging from autopsies, that many persons have tuberculosis without realizing its existence in the body, and without its being detected in any way. It is questionable, how- _ . ever, whether under such circumstances the disease is transmitted or disseminated. As a rule affected cattle show no definite outward signs of the dis-. ease. Badly diseased animals frequently appear poor and thrifty. Many cases are mild and latent. A few tubercular animals cough; some show harsh hair and skin and other expressions of ill health. While these symptoms do not necessarily indicate tuberculosis, they are very suggestive. Bact. tuberculosis may invade almost any tissue or organ of man or the animal body and produce a variety of lesions. Man usually gives some evidence of the disease either objectively or subjectively, and in many instances the disease assumes a definite form which is easily rec- ognized by medical men, unlike its presence in animals. The symp- toms are more evident in swine than in cattle. Affected hogs are often unthrifty and show glandular enlargemets and degenerations of the enlarged glands. Avian tubercle bacteria are becoming disseminated among poultry, and to a serious extent in some sections of the country.. Among the more prominent symptoms of avian tuberculosis are emaciation with marked anemia and weakness. Examination of the carcass shows dis- ease most frequently in the liver, but intestines, spleen, lungs, and even the skin may be invaded. The avian tubercle bacterium varies in certain respects from the bovine variety; it usually is shorter, measuring from 1p to 4u in length with a general average of 2.74 and grows best at higher temperatures. Danish authorities report* serious outbreaks among swine due to avian tubercle bacteria. It has long been firmly established that Bact. tuberculosis is the spe- cific cause of this disease. But while this bacterium is to be regarded as the specific cause it must be understood that this organism is fre- quently associated with pus-producing bacteria which are responsible * Dunne, Trans. Jour. Bd Agr. (London), 22 (1915), No. 1. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 781 for certain phases of the disease as commonly seen. It should be under-. stood also that persons and animals become more susceptible and have greater opportunities for infection under close confinement and lack of exercise. There has been great difference of opinion concerning the unity of the tubercle bacterium, and the probability of inter-trans- mission between man and the lower animals. A large number of bac- teriologists now hold that the several types of tubercle bacteria are but environmental variations of the same species. In any case, man clearly appears susceptible to both human and bovine types at least. The entrance of the germ may occur in four ways, namely, by way of the digestive tract; it may occur by way of the respiratory organs; it may occur by inoculation; and infection may possibly occur before birth. Some authorities hold that the most common infection is by -way of the digestive tract and in early life. Others hold that inhalation ‘tuberculosis is most common. This bacterium produces a slow toxemia, and it is this toxemia to- gether with physical embarrassment of the vital organs by extensive lesions which together harm the affected body. Various toxins are produced, as indicated by the fact that killed cultures by subcutaneous - injection may destroy local tissues and produce abscess, debility, and emaciation. Production of toxins is indicated by the further fact that certain antitoxicimmunity may be produced by minute doses of killed culture gradually increased. -Tuberculin is a common and well known product or mixture of pro- ducts produced by this bacterium. One of its constituent products is a fever producer. Another product has been reported which reduces tem- perature, and still another which produces convulsions, in sufficient dose. Tuberculosis may be very general. Almost any tissue or organ in the body may be invaded; but as a rule, not many organs are badly affected in the same case. Distribution occurs by way of both the blood and lymph streams, especially the latter. It seems probable that tuber- cle bacteria may be distributed in the body by wandering phagocytes. The Bact. tuberculosis has a characteristic tendency to produce tuber- cles or nodules which may be large or small and which have.a tendency to central necrosis and degeneration. Mucous membranes, under this infection, tend to develop superficial ulcers. The lesions produced by this microérganism may vary from the tiniest tubercles to extensive areas of large organs. Lymph glands 782 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS frequently enlarge and undergo cheesy or calcareous degeneration. Tubercular masses of various sizes may appear upon the lining mem- branes of the chest and abdominal cavities and upon various internal organs. Cheesy abscesses may appear in the depths of soft organs. In cows the udder is occasionally enlarged and shows hard masses with little or none of the heat usually occurring in connection with in- flammatory changes. Bones and joints are often involved especially in the human; these structures increase in size, produce pain, and suppurate. Bacterium tuberculosis is a slender rod-shaped organism with rounded ends and under certain conditions shows granular forms. It varies between 24 and su in length, and 0.3u to 0.54 in width. This bacterium is usually straight, but may be bent; it appears either singly or in groups or branched, non-motile, and is probably not a spore producer (Fig. 163). Glycerin agar, blood serum, egg slant, and bouillon may all serve as satisfactory nutrient media. It is aerobic and its tem- perature limits for growth appear to be 29°-42°C. With the exception of young and rapidly growing forms it is strongly acid-fast. Tubercle bacteria may be eS Fic. 163.—Bact. tuberculosis. Branching Fic. 164.—Bacteriwm tuberculosis. forms froma culture. (After Migula.) Sputum preparation uncolored. (After Migula.) demonstrated in cover-glass smears from diseased tissues and fluids and in tissue sections (Fig. 164). In human tuberculosis the bacteria are frequently deter- mined in the sputum, in bovine tuberculosis the bacteria may be occasionally dem- onstrated in the nasal discharges andin the manure. Positive diagnosis may usually be made by guinea-pig inoculation. For microscopic examination a cover-glass smear is fixed in the usual way, then stained with hot carbol-fuchsin three to five minutes or in cold stain fifteen to twenty minutes. It is then decolorized, e.g., in ro per cent nitric acid, and counterstained with methylene blue for about one minute, after which it is rinsed and ready for examination. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 783 It is conceded that tubercle bacteria do not multiply in nature out- side the animal body and, therefore, dissemination must depend wholly upon the dissemination of infected people or animals and materials infected by diseased men and animals. Tubercle bacteria escape from open ulcers or from tubercular lesions which connect with digestive or respiratory organs. ‘They may reach the surface in other ways; é.g., by the discharge of abscesses. In controlling tuberculosis among humans at the present time, several methods are in vogue. In some localities, an effort is made to segregate tuberculous patients during the day for the purpose of treating them as well as teaching them how to care for themselves. This method aims to instruct how to prevent dissemination and transmission of the disease, to prepare suitable nourishment, and to secure the advantages of open-air influences. This instruction not only extends to the patients but others with whom the patients may mingle. Sanitaria are also constructed to receive patients suffering from the disease, and care for them under suitable medical supervision by proper treatment, nourish- ment, and open-air life. Again, the policy is being inaugurated to instruct tuberculous patients, where it is impossible to reach them by other means, to care for themselves in their own homes. By these general hygienic measures, much good has been accomplished, not only for the patients but, also, in a diminution of the number of new cases developing. The animal disease is carried to distant points, most commonly by breeding stock. Locally the disease spreads either by the movement of affected cattle, or frequently Fic. 165.—Bact. tubercu- losis. Glycerin agar culture. (After Curtis from Stitt.)” 784 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS by infected milk. Hogs receive their infection from the milk of tubercular cattle or from the manure or carcasses of such cattle in feeding yards. Unventilated stables are favorable for the spread of this disease because with insufficient ventilation the bacteria are not carried out, but become constantly more numerous. The tubercle bacterium is quite resistant to drying, but is rather sensitive to sun- light. It is usually destroyed by moist heat in six hours at 55°; in twenty minutes at 60°; and generally in five to twenty minutes at 95°, depending upon the protection it may have. Conditions of sensible sanitation are of the utmost importance. These include exercise, sunlight, and ventilation, particularly sunlight. In order that effective control work may be done among animals, tuberculin must be used freely and conscientiously. : The method of dealing with diseased herds depends upon breeding and value. Common cattle are usually dealt with most economically and efficiently by slaughter with a view to using such carcasses as may pass inspection. Valuable cattle, especially pure bred animals, may be used for breeding purposes, gradually building up a sound herd and gradually displacing the diseased animals. This latter plan is usually © unprofitable and unwise except for valuable cattle. Foot Rot oF SHEEP* Bacillus necrophorus This is an infectious disease of sheep characterized by an ulcerative inflammation of the tissues just above the horny part of the cleft of the hoof. It is seen in Europe, England, Australia, and the United States. Sheep are made lame and if the disease is not checked by appropriate treatment, the hoof becomes greatly distorted, the sheep being finally unable to walk. Mohler and Washburnf state that foot rot is caused by B. necrophorus, this organism being associated with pus-producing micrococci. B. necrophorus, which is a strict anaerobe, tends to grow out into long filaments; it is stained by the ordinary aniline dyes, but not by Gram’s method. Rabbits and white mice are susceptible to inocu- lations of this bacillus, but guinea-pigs appear to be immune. The disease is treated by causing infected sheep to walk through a disinfecting solution, such as a 3 per cent solution of carbolic acid. * Prepared by M. Dorset. ~ { Bull. 63. Bur. An. Industry, U. S. Dept. Agr., 1904. i MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 785 MALIGNANT (EDEMA* Bacillus edematis maligni The disease occurs as the result of infection of wounds with dust or soil. The wounds must involve the tissues deeply as in compound fractures and deep cuts. Any animal may be infected, although the dog and cat are said to be rather more resistant than others. The incubation period is short, from one to two days as a.rule. The usual case begins with sudden spreading hemorrhagic sub- stances, oedema and high fever. Practically no gas is formed. The fluid shows bacilli both with and without spores. Where soil contami- nation exists, mixed infections with gangrene are common. Pasteur in 1877 and Koch and Gaffky in 1881 found and studied the organism and by passing from animal to animal established the causal relationship. Glucose agar or glucose gelatin is inoculated with the suspected fluid, plates poured and placed under anaerobic conditions. The organism is 0.84 to 1 wide. Filaments may occur. The rods without spores are uniform in width with slightly squared ends. They are usually single, though pairs end to end are frequent and chains are also found. Oval spores are formed somewhat variable in their position, with a diameter usually larger than that of the vegetative rod, bringing about a spindle shape. Peritrichic flagella have been demonstrated, about twenty in number. It stains readily with aniline dyes, usually Gram-negative though some- what variable and indefinite in this regard. Growth takes place at both 20° and 37°. It isa strict anaerobe. Like anaerobes in general it prefers the presence of a fermentable carbohydrate such as glucose. On agar the colonies are small, whitish, and irregular in outline. Gelatin and blood serum are digested, caseinogen is changed to casein which is then digested. In both protein and carbohydrate media a gas is produced which has a very disagreeable odor. The spores are very resistant. This resistance accounts for its continuous presence in earth and dust and as a constant inhabitant of the intestine of animals, especially of herbivora. MILKSICKNESST Bacillus lactt morbi Milksickness, a disease so called because of it being most commonly transmitted to man through milk of infected cows, is caused by the * Prepared by Edward Fidlar. t Prepared by Norman Mac L. Harris. 50 . ‘ 786 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS growth of and consequent absorption of toxic principles from the infective agent lodged primarily in the small intestine, and secondarily in the liver and other organs of the body. The disease appears to be peculiarly an American one, and has been reported at intervals for many years from several of the Middle Western States and Kentucky, Tennessee, Virginia and North Carolina, and more recently from New Mexico. The bacterium, Bacillus lactimorb1, regarded by Jordan and Harris as the causative agent, appears to ‘flourish chiefly in the moist, shaded pasture lands of creek bottoms, or, as in New Mexico, on pasture lands that are subject to irrigation or flooding. Bacillus lactimorbi is an aerobic, spore-bearing, non-liquefying, non-gas-forming microérganism, which, when stained with Loeffler’s methylene blue, shows one or more metachromatic granules within its body. In young cultures it is Gram- positive, slowly alkalinizes milk, which at times becomes clarified through the great accumulation of hydroxylions. Agar plate colonies resemble those of streptococcus, and there frequently is developed a delicate film growth over the plate. It, or members of its group, are seemingly widely distributed in nature, as Luckhardt has found bacteria practically identical with Bacillus lactimorbi on alfalfa obtained | from Wisconsin, Illinois and Indiana, as well as on plants of several species from New Mexico. Certain of these strains possessed pathogenic properties, others showed none; therefore, it is believable that the bacterium may, under peculiar environmental conditions, develop varying degrees of virulence and pathogenicity. It may be properly regarded as a facultative pathogen. ; Pathogenicity is demonstrable for young rabbits, guinea-pigs, dogs, cats, lambs, and calves, although it must be borne in mind that its virulence at any time is of low degree. Infection, either through natu- ral or artificial channels, occurs only after considerable quantities of infective material are swallowed. In man, the disease is progressively marked by lassitude, loss of appetite, nausea, gastric pains, vomiting, obstinate constipation, a fall in body temperature, odor of acetone /on the breath, the presence of acetone, diacetic-and B-oxybutyric acids, and the absence of dextrose in the urine. Large doses of sodium bicarbonate are relied on to effect a cure. In the herbivora, muscular weakness, tremors, constipation, odor of acetone on the breath are prominent features; in horses, profuse sweating may be a prominent symptom. Degenerative and fatty changes of the parenchymatous organs is the especial pathological feature met with. Eradication of the disease in localities where it occurs may be ‘ MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 9787 effected by, either the cultivation of the infected pasturage, or by securely fencing it in. BIBLIOGRAPHY Jordan, E. O. and Harris, N. MacL., Jour. Infect. Diseases, 1909, vi, 401. Luckhardt, A. B., ibid., p. 492. Walsh, W. E., Illinois Med. Jour., 1909, xv, 422. Moseley, E. L., Med. Rec., 1909, lxxv, 839; 1910, lxxvii, 620. Clay, A. J., Illinois Med. Jour., 1914, xxvi, 103. Harris, N. MacL., Reference Handbook of the Medical Sciences, 1915, vi, 522. SympToMATIC ANTHRAX OR BLACKLEG* Bacillus anthracis symptomatict Blackleg, black quarter, symptomatic anthrax, quarter ill, are syno- nyms employed to designate this disease. Symptomatic anthrax is a very old disease and until recent years has been confused with true anthrax.. This disease is widely distributed, affecting practically all countries and climates. It is enzootic, never spreading widely or rapidly, and is often found in certain infected valleys and in relatively small areas. Young cattle, generally under two years of age, are most commonly affected, but sheep and goats are susceptible to this infection. This disease is infectious by inoculation, perhaps also by in- gestion, and usually acute. Subcutaneous and muscular tissues are especially affected. Its most prominent and characteristic feature is swelling, affecting most frequently, the front or hind quarters, and never extending below the knee or hock. As a rule, the bacillus of symptomatic anthrax produces a very acute disease with severe constitutional disturbances. The bacillus of symptomatic anthrax has been clearly demonstrated to be the specific cause of blackleg, infection occurring by inoculation. The period of incubation in the natural disease is uncertain. Under artificial inoculation this period varies from two to three days and is occasionally as short as one day. This bacillus produces in culture a very active toxin. This toxin is quite resistant to heat. That the bacillus of symptomatic anthrax stimulates the production of antibodies and that the injury is done by * Prepared by M. H. Reynolds. 788 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS toxins, is shown by the fact that immunity against virulent culture may be produced by treatment with presumably sterile filtrates of virulent cultures. | The bacillus of symptomatic anthrax is rarely found in the general blood before death; but is abundant in the affected muscle and over- lying subcutaneous tissue. It also occurs in great numbers in the bile and intestinal contents. Mucous membranes become congested and then very dark. There is a high fever. Local swellings occur which are at first sensitive and later insensitive and gaseous. There is usually developed a very marked swelling of a front or hind quarter or of the neck, with rapid formation of gas. The serous membranes, particularly the pleura and peritoneum, develop severe inflammation with hemorrhages and infiltrations and corresponding exudation in the cavities. General decomposition is rapid and the swelling may show a slight acetone odor. The local subcutaneous tissues are infiltrated, hemorrhagic or gaseous. The local lymph glands are swollen and hemorrhagic or cedematous. Muscle fibers show various degenerative changes. The abundant gases are mostly hydrogen and carbon dioxide. B. anthracis symptomatici is about 3u to 6u long by 0.5 to 0.84 thick. This is a spore-bearing bacillus of drum-stick shape or spindle shape and is anaerobic. It grows best at about 37°. It stains either by the simple aniline dyes or by Gram’s method. In artificial cultures, it sometimes shows long forms. This organism is motile for a short time, but soon loses this power, probably on account of the oxygen to which it is exposed. It shows well-defined flagella and develops spores. The specific organism may be demonstrated by the microscope in the blood without staining if done soon after death. ‘The bacillus of symptomatic anthrax is easily demonstrated in cover-glass smears from the affected tissues, and is very different from the bacteria of anthrax and hemorrhagic septicemia, the only diseases © liable to be mistaken for blackleg excepting malignant cedema Anthrax gives a surface growth and is aerobic. Symptomatic anthrax gives no surface growth and is anaerobic. This organism: may also be demonstrated by animal inoculation. The guinea-pig serves well for this purpose; it is very susceptible to inoculation and gives a characteristic blackleg reaction in both symptoms and lesions. From the lesions thus produced the characteristic bacilli are easily demonstrated by the microscope. ‘ MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 789 Elimination of this virus from the body occurs chiefly in the various discharges, and especially in the manure and also in general decomposi- tion of the carcass. Dissemination of this disease is chiefly if not ex- clusively by diseased carcasses and parts of carcasses and by the dis- charges. Contaminated soil plays a very important part in the prevalence of this disease. It appears probable that the specific bacillus may even multiply in the soil. . Carcasses should be burned if possible; otherwise very deeply buried and covered with lime. Contaminated grounds, or stable floors must be thoroughly disinfected, for the infection is very persistent and difficult to eradicate except by most vigorous effort since the spores are very resistant to heat and drying. Preventive inoculation after the method of Arloing and Kitt is very satisfactory. Their vaccine consists of specially treated muscular tissues from the diseased part. TETANUS* Bacillus tetani This disease is found throughout the world but more frequently in warmer than in colder climates. Certain localities are particularly affected. Man and domestic animals are susceptible. The incubation period varies from a few hours in experimental in- oculation of small animals, to several days or weeks in cases of natural infection in man. The disease follows a wound of a punctured type with contamination by earth, especially in injuries of hands and feet. It is characterized by tonic spasms of the voluntary musculature usually beginning in some one group of muscles and finally becoming general. The parts first affected are, in cases artificially produced, those at the site of inoculation, but in natural infections in man it is more common for the disease to manifest itself by stiffening of the muscles of the neck and face, producing what is ordinarily termed “‘lock- jaw.” In less severe infections in man local pain and stiffness are the first indications. The spasms occur in paroxysms which are spon- taneous or excited by effort. They are more or less prolonged and ex- hausting and are accompanied by greater or less pain. Death results * Prepared by Edward Fidlar. : ‘ 790 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS \ from general loss of strength or involvement of the respiratory muscles. The shorter the incubation period the higher the mortality. Few recover when the incubation period is less than ten days, about half the cases recover when the period is more than fifteen days. The nerves may show injury as indicated by swelling and redness and microscopically nerve cells have been observed in a state of granu- lar degeneration; there is a more or less distinct general congestion of the organs. While lockjaw has been known clinically for centuries, it was not until 1884 that the infectious character was demonstrated when Carlo and Rattone and Nicolaier were successful in animal inoculations. Kitasato obtained pure cultures of the bacillus in 1889. The organisms may be detected occasionally by examination of stained préparations of the pus from the wound. Pure cultures may be obtained by inoculating an alkaline dextrose broth with pus or tissue, incubating under anaerobic conditions for about forty-eight hours until sporulation, then exposing half an hour to a temperature of 80° to kill all vegetative forms and subsequently making subcultures. If other spore-bearing bacteria are present considerable difficulty may be en- .countered. Subcutaneous inoculations of mice or guinea-pigs is a good method for demonstrating the presence of the organism, but pure cultures should be combined with some aerobe (say B. coli) to secure results. The B. tetani is about 2y to su in length by 0.3 to 0.84 in width with rounded ends. The vegetative rods are uniformly cylindrical but the terminal spores give 4 “drum stick” appearance (Fig. 166). The arrangement is usually single, but threads may occur especially in old cultures. The organism forms round terminal spores which have a diameter of tu to 1.54. The young bacilli are motile and Possess 50 to-70 peritrichic flagella. Motility is lost with sporulation. The bacillus is ‘ stained by the aniline dyes and is Gram-positive. The spores are readily dem- onstrated by the special stains. The range of temperature for growth is from about 14° to 45° with an optimum about 37°. The organism is usually considered an obligate anaerobe though experimentally aerobic strains have been developed but with loss of pathogenic and toxogenic properties. Pure cultures do not develop in an atmosphere of carbon dioxide. Media for the cultivation of the bacillus should be slightly alkaline and should contain for best growth about 2 per cent of glucose or 1.5 per cent sodium formate. The addition of pieces of fresh raw sterile tissue is valuable. On agar at 37° colonies appear in forty-eight hours which show microscopically, a mass of tangled threads resembling colonies of B. subtilis or Bact. anthracis. In broth a cloudiness is produced in twenty-four, to MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS - 791 thirty-six hours with the development of gas and a very disagreeable odor. In gelatin the colonies develop more slowly than on agar and show liquefaction. In old stab cultures a pine tree growth occurs. Gas is usually produced. In milk growth occurs without coagulation. Acid is produced in some carbohydrate media. Gas is produced during the action upon protein and consists chiefly of carbon dioxide but also of hydrogen sulphide and certain volatile organic compounds commonly found in putrefactions. The tetanus bacillus forms two soluble toxins, tetano- lysin, and tetano-spasmin. The former is less stable and dissolves red blood- corpuscles. The latter produces the characteristic spasms of the muscles. This poison may be obtained after one to two weeks growth in slightly alkaline salt- peptone-bouillon under anaerobic conditions at 37.5° and separated by filtration Fic. 166.—Tetanus bacilli showing Eee (After Kolle and Wassermann from till. through porcelain filters. When taken by the mouth the toxin is ineffective, given intravenously it produces a generalized tetanus, while after subcutaneous injection the disease begins with local spasms. The central nervous system is reached by ascent of the toxin along the motor nerves nearest the point of inoculation. A dose of toxin injected directly into the nerve trunk of an animal may produce a fatal result when it is innocuous intravenously. The spores often withstand 80° for one hour and live steam for about ten minutes. Direct sunlight destroys them in time. They survive drying for several years and resist the ordinary disinfectants for a considerable length of time, 1: 1000 mercuric chloride for three hours, 5 per cent carbolic acid for about ten hours. Practically al] mammalia are susceptible to tetanus though rats are but slightly so. Very minute doses of toxin suffice to kill mice and guinea-pigs. Birds show but little susceptibility and the hen is said it 792 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS to be three hundred thousand times more resistant to tetanus toxin than the horse. Reptiles and amphibians are practically immune to very large doses when kept at low temperature. Natural infections probably do not occur without the presence of other microérganisms. The bacillus and its associated material gains entrance through some break in the tissues. The organism is prac- tically confined to the site of inoculation, but it is sometimes found in the blood and internal organs after death. Against toxin-freed cultures phagocytosis is probably the process which overcomes infection. The toxin is highly antigenic and animals can be immunized against it in a manner similar to that for diphtheria toxin. While direct infection of one person from another has occurred, cases of human tetanus are very rarely responsible for others. Horses are chiefly responsible for its distribution, the tetanus bacillus being common in manure, which accounts for the occurrence of tetanus in soil-contaminated injuries. Cattle probably are also carriers of the bacillus. Tetanus antitoxin as a cure has been a keen disappointment, especially if symptoms have fully developed after a short incubation period. Very large dosage, however, may have the desired effect. In prophylaxis the antitoxic serum is widely and successfully used in all suspicious cases, and in Fourth of July injuries in particular. TYPHOID FEVER* Bacillus typhosus Typhoid fever is one of the most widespread of bacterial diseases and is found endemic in practically all the countries of the world. Epidemics frequently occur because of the infection of some local public utility related to food or drink, particularly water or milk. _ Typhoid fever occurs naturally only in man. Intraperitoneal inocu- lation of susceptible animals may result in death with acute peritonitis, but lesions are in no way specific and can be produced by the colon bacillus. * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 793 The period of incubation varies ordinarily from five to twenty-one days, with an average of fourteen days. The first week of the disease in man begins with a train of rather indefinite symptoms such as headache, loss of appetite, digestive dis- turbances, lassitude, and sleeplessness. Nose bleed is a peculiar and rather constant feature. The temperature and pulse gradually rise until by the end of five to seven days the former has become high, 103°F. to ro4°F. and constant. The temperature continues thus through the second week during which a gradual stupor and occasional _ delirium, diarrhoea, and enlargement of the spleen occur. The pulse is often dicrotic and there is a rash consisting of isolated flattened rose- colored macules or spots which may be few or numerous and occur in successive crops. During the third week in mild cases these symptoms gradually subside. In severer forms no abatement is shown and com- ' plications are liable to occur. The fourth week shows beginning - convalescence in the typical case. The characteristic pathological findings are swelling and ulceration of the lymphoid structures of the lower part of the small intestine best seen in the Peyer’s patches of the ileum just above the ileo-cecal valve. The mesenteric glands and spleen are hyperemic. Parenchymatous degenerations more or less severe may be found in other organs. The characteristic histological feature is the crowding of the lymph spaces by proliferated endothelial cells. Perforation and hemorrhage of the bowel, peritonitis, myocarditis, thrombosis, etc., render typhoid fever a dangerous disease. The fatality varies considerably; at one time estimated at 25 per cent, it has been brought down to ro to 15 per cent by modern methods of treatment and has been given in Minnesota as low as 4 per cent. -Eberth found the organism in 1880 by the examination of the mesenteric glands and spleen of fatal cases. Gaffky cultivated it in 1884. The causal relationship has been a matter of gradual acceptance through evidence furnished by the study of such immunity processes as agglutination, bacteriolysis, and complement deviation, and finally by the high percentage of positive blood cultures. Conclusive evidence is afforded by the development of typhoid fever following the ingestion of pure cultures with suicidal intent. The agglutination reaction of Gruber and Widal is universally employed in diagnostic laboratories. The blood serum of typhoid 794 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS patients, after a certain period of the disease, will cause a characteristic clumping of the bacilli when mixed with pure cultures. The fresh. serum from a clot may be used, or, more conveniently, dried blood. from which a watery extract can be made. In positive cases thé reac- tion is present in at least the one-fiftieth dilution and usually in the one- hundredth or higher dilutions. The culture employed should be eighteen to twenty-four hours old; it should be freely agglutinable and show no artificial clumping, characters not possessed by all strains, especially those recently isolated. Cultures killed by a small per- centage of carbolic acid have been recommended for constancy in place of the living organisms. When the microscopic method is used the » reaction should be distinct in about one hour. An immune body capable of binding complement in the presence of typhoid antigen is said to occur in typhoid sera before the agglutina- tive property appears. The detection of the typhoid bacillus in the circulating blood has recently been very widely successful and furnishes the best support for the diagnosis of the disease. While blood culture may be hardly practical in public health laboratories, it has become a routine measure in the modern hospital. Blood is taken aseptically from a vein and about 1 to 5 c.c. is introduced into culture media, of which fluid media containing ox-bile and agar plating media containing glucose have been most strongly recommended. The fluid media are used in 100 to 500 c.c. amounts, which serves to dilute the antibacterial properties of the blood while the bile acts as an anticoagulant and possibly also as an antibactericidal measure. Plating lessens the diffusion of the anti- bacterial properties and thus favors growth. : The urine and faces have sometimes to be examined for the presence of B. typhosus. It then becomes necessary to differentiate the colonies of this bacillus from those of the colon group. For this purpose many ‘special media have been devised, some depending on the motility of the . typhoid bacillus to form a different shaped colony in suitable soft media, others based on the fact that some substances such as fuchsin, crystal violet, malachite green, etc., inhibit the growth of associated organisms while permitting the typhoid bacillus to develop more or less luxuriantly. As found in pure cultures, the bacillus is about ry to 3.5m in length and o. 5h to o.8u in width (Fig. 167). Filaments are sometimes found several times the length MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 795 of the single organism. It is quite regular in shape, straight with rounded ends. The bacilli usually occur singly; occasionally two may be attached end to end for a short time. There are ten to fourteen comparatively stout flagella about two or three times the length of the organism peritrichic in their arrangement. There are no capsules and no spores. They stain with all aniline dyes, and not infre- quently exhibit more deeply staining areas at the poles. They are Gram-negative. Biological and biochemical characters. The minimum temperature is about 10° the optimum 37°, maximum 40 to 41°. It is aerobic and facultatively anaerobic, The slight preference for oxygen is probably of little account when such sugars as glucose are present. The bacillus is not very sensitive to the reaction of media and will grow in the presence of either slightly alkaline or acid reaction. Alkaline gern ie bo fy : ~_* A eit he Yay ze | = ae Fic. 167.—Bacillus of typhoid fever. XX 1000. (After Williams.) substances are produced from peptone. Acid is formed from dextrose, levulose, galactose, mannit, maltose, and dextrin. Lactose and saccharose remain un- changed. Gas is never formed. It is the rule that the Bacillus typhosus does not form indol; certain strains, however, forma trace. The toxins of the bacillus have been very widely studied and several different opinions are held with regard to their nature. Most evidence supports the idea that the poisons are only set free by the destruction of the bacterial bodies. This may be brought about experi- mentally by various means such as the use of lytic or bactericidal sera, by the disintegration occurring in old cultures, by extraction under great pressures, by triturating after freezing in liquid air and by emulsifying cultures, sterilizing by heat, then extracting with salt solution. These endotoxins, however obtained out- side of the host, have been found to produce by injection into animals only lytic 796 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS. and bactericidal sera and not an antitoxin. More recently; however, some ob- servers claim to have shown in comparatively young cultures the presence of a sub- stance which upon injection into animals yields an antitoxin and thus comports itself after the manner of a true diffusible or soluble toxin. Agar streak cultures show an abundant filiform whitish or bluish-gray translucent growth with no special char- acteristics. Broth is‘uniformly and moderately clouded and only occasionally a delicate pellicle may develop. Gelatin colonies are bluish white in color, trans- parent and with somewhat notched margins. Stab cultures show more growth at the surface, while in the depth the growth is filiform and less abundant. The medium is not liquefied. Milk is not coagulated. In litmus milk there may be a trace of acid formed at first, followed by a return to neutral or very slightly alkaline reaction. Potato was at one time considered a very valuable differential medium. The growth of the bacillus upon it is quite abundant, glistening, but invisible, when the potato is acid. A more alkaline reaction allows a rather heavy yellowish growth indistinguishable from B. coli. Special media are used in the cultivation of the typhoid bacillus, chiefly for differential purposes. The cultural features on these do not show sufficiently striking characters to make it worth while to review the many that have been devised. Specific agglutinating and bacteriolytic sera as well as the complement binding reaction are valuable aids in identifying the bacillus. Resistance to heat and light is not different from that of the average non- spore-bearing species. Its thermal death-point is about 56° for ten minutes, 60° for one minute. Exceptionally resistant forms have been found alive in ice after three months. Sometimes the bacilli will remain viable for a month after drying. At other times they die out rapidly. They have been found to be viable for ten days in distilled water, while pure sodium chloride dissolved exerted an unfavorable influence. In faces the length of life is from a few hours to several days, or even as high as five months in winter. Their life in privies and cesspools is ordinarily brief but has been found to extend for thirty days. Of the non-spore-formers the bacillus appears to be rather more resistant than the average but succumbs within five minutes to 1 : 5000 mercuric chloride or 5 per cent phenol. _ The organism enters the body through the mouth by means of infected fingers, food, milk, and water, etc. On reaching the intestine the organism probably propagates to some extent before penetrating the intestinal mucosa. It enters into the blood stream and is disseminated throughout the body. According to: the endotoxin theory it must slowly be dissolved by the lytic sub- stances which have been gradually accumulating in response to the primary intoxication. The organisms have been cultivated from the rose spots and have been found in vomit without the presence of blood, and in sputum. Typhoid meningitis and osteitis occur occasionally. At autopsy the spleen and gall-bladder yield the highest number of positive cultures. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 107 It is of interest to note too that while the highest percentage (89-90 per cent) of positive blood cultures occurs in the first week and that the percentage diminishes from then on, the number of positive findings in the feces, on the other hand, runs in the opposite direction. Generally speaking, one attack confers immunity. Upon what antibodies immunity and recovery depend is a matter of controversy. ‘The elimination of the bacilli from the body will largely depend upon the stage of the disease, since the blood, especially early in the illness, practically always contains the specific organism; epistaxis is not an ‘unimportant feature asa possible means of disseminating the germs. The bacilli can also escape in the faces, urine, sputum, and vomit. _ In the control of this disease the best place to begin is at the bedside. . Disinfection of all excreta 4nd of everything which comes into con- tact with the patient should be rigorously carried out and in the case of the faeces and urine should ideally be continued until examination can | be made showing absence of the organism. It has been estimated that, as high as 5 per cent of convalescents continue to excrete living typhoid bacilli for varying periods from months to years after the disease; the longest time noted has been forty-six years. The recognition of typhoid carriers will depend absolutely on the finding of the specific germ in the feces or urine as the case may be. Where there are large numbers of suspects, the opsonic index is claimed to be an aid in exclusion of the improbable ones, as well as the agglutinin reaction. In a general way, prompt recognition of the source of infection such as milk, polluted water, bacilli-carriers, etc., together with instruction of the individual and the public are often effective in limiting and end- ing an epidemic. , While a great many sera have been used therapeutically with some success, prophylaxis promises more where it can be widely employed as in armies and navies. The artificial immunity is brought about by in- jection of dead cultures. A difference of about 25 per cent has been noted between the percentage of cases in vaccinated and unvaccinated persons in civil life. In the United States Army, however, where antityphoid inoculation is now compulsory, most remarkably favorable results have been shown during the year 1913. Amongst 90,646 men, both American and native troops, only three cases of typhoid fever occurred and two of these were 798 MICROBIOLOGY OF DISEASES OF. MAN AND DOMESTIC ANIMALS infected before enlistment; there were no deaths. When comparison is made with the best results obtained in the army from sanitary measures alone: without the vaccination, Major Russell estimates that in 1913 there’ was “only one one-hundred-and- -siaty-seventh of the loss of time from duty because of typhoid fever.” ASIATIC CHOLERA* Microspira comma The disease is endemic in parts of India whence epidemics have spread throughout the world. America has been visited by several epidemics and at the sea ports more frequently, chiefly New Orleans. The disease occurs naturally only in man. The incubation period is from part of a day to ten days, usually about three days. In its most characteristic form the disease begins with few or no prodromata. It is marked by fever, sudden onset of purging and vomiting followed by cramps and severe depression. Evacuations finally become almost a colorless liquid, “‘rice-water stools.” The cramps may occur in the whole muscular system most frequently in the legs and are often extremely painful. A stage of complete collapse finally occurs. There are, however, many variations from these typical cases. The mortality is usually given at from 45 to 50 per. cent. After death there are found extensive acute degenerative changes in the kidneys; the gastro-intestinal tract shows marked changes in the.... lining membrane which may be necrotic, sodden and in some places stripped away. The cholera vibrios may sometimes be seen in enormous numbers in smears from typical stools. For a positive diagnosis, however, the organism must be cultivated. The usual method is to inoculate a 1 per cent peptone solution from the stool, in- cubating at 37° for from four to eight hours and sowing plates from the very surface of the liquid, either of gelatin or alkaline agar or both. The vibrios are 3u to su long by about o. Au wide, and are curved slightly like a comma or sometimes in a half circle. These comma forms are best seen in broth cultures. The ends are usually rounded. In young cultures the organisms are usually arranged singly, occasionally two may be found end to end in the form of an “S.” There is no capsule, and no spore formation. There is a single terminal flagellum, and the * Prepared by Edward Fidlar. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 799 organism is exceedingly motile. Does not stain as readily with the ordinary aniline dyes as many other bacteria. Fuchsin gives the best result. It is Gram-negative. The optimum temperature for growth is 37° with a minimum of 8° and a maximum of 42°. Plain agar—moist, shining, grayish yellow, and rather thin and transparent as compared with the colon type of colony. A rapid growth takes place in broth, causing a uniform clouding with a more or less well-developed pellicle. In gelatin plates colonies are visible in twenty-four hours and are round, even, and yellowish white, later they become irregular and their surface presents fine refractile granules; within forty-eight hours the colonies are found to be sinking into a small round pit due to liquefaction of the medium (Fig. 169). Concentric rings may appear as ‘\\ liquefaction progresses from day to day. In old cultures the liquefaction assumes a Fic. 168.—Microspira comma. X 1000. (After Williams.) funnel or turnip shape with an air bubble at the surface due to evaporation. Growth in milk occurs without any visible change in the medium. At 37°, on potato, an abundant moist brownish growth. Blood serum is liquefied rapidly. The vibrios prefer the presence of oxygen, yet it is probable that organisms grow under practically . anaerobic conditions in the intestine. The reaction of all media must be very dis- tinctly alkaline and even very small amounts of acid are inhibitive. Neither gas nor acid is formed. The production of indol and the formation of nitrites from nitrates occurs regularly. The addition of sulphuric acid is sufficient to give the nitroso-indol reaction, which from its association with this bacterium has been called the cholera red reaction. No pigment is produced. Majority of freshly isolated cultures have hemolytic powers. It is generally considered that there is only an endotoxin, but it is strongly asserted by some that a soluble toxin is formed. Thermal death-points are 60° for ten minutes, 95° to 100° for one minute. Vibrios are quite sensitive to low temperature and at most have been found viable in ice only after a few days. The vibrios are quite susceptible to the ordinary disinfectants. 800 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS The cholera organism gains entrance through the mouth. Having succeeded in passing the acid secretions of the stomach the vibrios probably develop with great rapidity in the small intestine. The peculiar conditions favorable to the development of the organism in the intestine are unknown. A previous gastro-intestinal disturbance is probably necessary even though slight. The organisms have rarely been demonstrated in blood cultures. The gall-bladder gives the highest percentage of positive cultures. Fic. 169.—Microspira comma. Colonies on gelatin plates. a, Twenty-four hours old; 6, thirty hours old; c, forty-eight hours old. (After Fraenkel and Pfeiffer from Williams, Highly lytic and agglutinating sera can be developed experimentally, but little or no antitoxic power can be demonstrated. Protective inoculation has shown considerably more encouraging results than serum therapy. The cholera vibrios are eliminated in the discharges. Water and uncooked food becoming contaminated with cholera excreta are the chief means by which the epidemic is spread, so that its epidemiology is similar to that of typhoid fever. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 801 DISEASES OF UNKNOWN CAUSE* SCARLET FEVER, MEASLES, GERMAN MEASLES, DUKE’s DISEASE, SMALLPOx, CHICKENPOX, Mumpstf These diseases constitute a group the actual biological causes of which are unknown, yet which show analogies to diseases the causes of. which are known, so close as to make tenable the hypothesis that they are due to similar causes. Mumps is in a class by itself, its characteristics, well known to the laity, marking it off from the others sharply. Like the others it is infectious; it is derived only from a preceding case; it has a more or less definite incubation period (i.e., an interval between the date of infection and the first development of symptoms, during which ordinary health is enjoyed), and a prodromal stage (i.e., a period in which fever, headache, and other more or less marked opnstl tudinal symptoms exist without any marked characteristic symptom). Then appears the swelling of the parotid salivary glands just in front of the ears with some pain. The symptoms usually amend after a few days and the patient goes on to full recovery. There is no rash nor any great disturbance of the intestinal tract or internal organs as a rule, although metastases, affecting the mamme, ovaries or testicles de- velop at times; and secondary complications sometimes are found. - Smallpox and chickenpox together form a group quite often confused clinically, especially in the early stages and especially when smallpox is prevalent in mild form. They have incubation periods, approxi- mating about twelve days, in smallpox varying little from this period, in chickenpox varying widely from it. Smallpox has rather severe prodromes, backache, headache, fever, and sore throat, the rash appear- ing on the third or fourth day. Chickenpox usually has light or no prodromes, the rash appearing on the same day or within twenty- four hours, as a rule. In both diseases the face, chest, back, arms, hands, legs, and feet are likely to show eruption, but chickenpox tends to show the greatest number of spots “under cover,” i.¢., on the parts ‘usually covered by clothing, while smallpox tends to show the majority upon the face, neck, arms, wrists, hands, legs and feet rather than on the body. The skin lesions themselves differ very markedly, the typical lesions of chickenpox being superficial, thin walled, high, rounded, * Arranged alphabetically except group of diseases placed first. f Prepared by H. W. Hill. 51 802 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS and filled with clear liquid, those of smallpox being deep seated, tense, opaque, with a tough covering of epithelium. There are many other points of distinction, and any one familiar with the two diseases can hardly fall into error when dealing with typical cases at whatever stage they are encountered. To the layman’s eye, however, the two are ‘often indistinguishable. Scarlet fever, measles, German measles, and Duke’s disease are often likewise confused by the laity and even by physicians who have not had opportunities for extensive study. ' German measles is clinically related to true measles somewhat as chickenpox is to smallpox, i.¢., they are wholly distinct diseases yet show characteristics easily confused on superficial consideration. Duke’s disease is perhaps not a distinct entity; much has been said on this point and a satisfactory decision will probably never be reached until the causative agents have been found. It may be described briefly for clinical purposes as a variety of German measles having a scarlatiniform instead of a measly rash. Scarlet fever has an average incubation period of about five days, or perhaps sometimes less. ‘The prodromes are those usual to all these infections—headache, fever, and sore throat, but the latter is especially severe. Within twenty-four hours the rash appears usually: on the chest first, a bright scarlet superficial punctate flush, extending rapidly over the body. In measles the incubation period is longer, averaging nine or ten days, almost without any variation. The prodromes, headache, fever, and sore throat, are accompanied by very marked coryza and photo- phobia, catarrh and “cold on the chest.” . The rash appears about the fourth day, appearing on the face ead back but rapidly extending. It is darker, bluer, and deeper than the scarlet fever rash and unlike the latter is palpable. Koplik’s spots appear on the buccal membrane early in the disease. In German measles the prodromes are so indefinite that it is difficult to determine their length; very commonly the rash is the first thing no-, ticed. It appears on the face, chest, back, and arms as a light sub- cuticular mottling (measles type) or a more uniform pink flush (scarla- tiniform or Duke’s type); with this rash the eyes show some injection and slight photophobia develops. The attack passes off quickly, without complications. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 803 CANINE DISTEMPER* This diseasé (Maladie des jeunes chiens; Fr.) is so widespread that ‘the great majority of adult dogs may be regarded as having suffered from an attack and recovered. It is practically confined to very young animals and, so far as known, no species except dogs are susceptible. The disease is attended by more or less extensive coryza with a dis- charge from the eyes. There is an eruption on the skin and frequently nervous disorders of various kinds. The animal becomes emaciated and may die from bronchial pneumonia. No organism has been fully accepted as the cause of this disease. Carré has reported that he has succeeded in passing the infectious agent in nasal discharges through earthen filters, the filtrate reproducing distemper in characteristic form, Ferry has announced the discovery of an organism as the causal agent. Some attempts have been made to produce a protective serum. CatTTLE PLAGUE* This disease (rinderpest), which is probably the severest and most contagious of all cattle diseases, is characterized by high fever and lesions of the intestinal tract. It does not exist in the United States but is found in Europe, S. Africa and Asia. Extensive outbreaks have occurred in the Philippine Islands. The cause of cattle plague has never been isolated and the indications are that it is caused by an invisible microdrganism. Cattle plague was the first disease in which the process of “hyperimmunization’”’ was practised. Immune cattle receive massive injections of blood from diseased cattle. After this: treatment the blood serum of the immune is used to protect non- immunes. Enormous quantities of this serum are prepared and applied yearly by the British government in India. CHICKEN PEst* This disease (Hiihner Pest; Ger.: Peste aviaire; Fr.) of fowls, which is to be distinguished from chicken cholera, is not known in the United States, but has caused extensive losses of fowls in Europe, particularly in Italy. Affected chickens cease eating, the feathers become ruffled and the comb darker in color. The lesions found at autopsy are not * Prepared by M. Dorset. 804 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS constant, but a pericarditis is usually seen. There may be, also, con- gestion of the lungs, liver, and kidneys. The intestinal lesions are not as marked as is the case in chicken cholera. Chicken pest has been shown to be due to an invisible microérgan- ism which is present in the heart blood and in practically all of the organs of the body. Most fowls are susceptible; guinea-pigs and mice are refractory to the disease. The virus passes through Berkefeld and Chamberland F cylinders; it is quite resistant to drying but is destroyed by an exposure of half an hour to a temperature of 60°. ‘Several authorities have passed the filtered virus through four or more hens successively, thus demonstrating positively that the filtered virus is capable of multiplication. ContTacious BovINE PLEURO-PNEUMONIA* This disease affects cattle only; it is highly infectious and produces an inflammation of the lungs and pleural membranes. Thirty years ago bovine pleuro-pneumonia was quite prevalent in the United States but has since been eradicated through the efforts of the Federal Bureau of Animal Industry in codperation with State authorities. It still exists in European countries. The microdrganism of bovine pleuro-pneumonia is generally classed among the invisible viruses, though unlike the other organisms of this class it has been cultivated artificially and is just visible at a magni- fication of 2,000 diameters. The artificial cultivation of this virus was accomplished by Roux and Nocard through the use of the very in- genious “‘collodion sac method.” A smal amount of virus from a diseased cow was placed within a small thin-walled sac of collodion; after being hermetically sealed the sac was placed in the peritoneal cavity of a rabbit where it remained for several weeks. At the end of this time the unbroken sac was removed and the previously clear fluid within was found to be slightly opalescent. Microscopic examination revealed numberless minute motile bodies so small, however, that their exact form could not be determined. Later the organism was suc- cessfully cultivated outside of the animal body in a specially prepared bouillon. These cultures produced the disease when inoculated into susceptible cattle. When the virus is diluted it will pass through the * Prepared by M. Dorset. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 805 Berkefeld and Chamberland F cylinders, but not through the Chamber- land B cylinder. Cowrox, Horsrpox, AND SHEEPPOX* Variola refers to a condition or disease in man and animals, charac-. terized by fever and the appearance of skin eruptions which succes- sively assume the form of papules, vesicles and pustules. ‘The disease is frequently found in the human species (smallpox), cattle (variola vaccinia, cowpox), horses (variola equine, horsepox) and sheep (variola ovina, sheeppox). It is possible that some other species may be susceptible. On account of the fact that vaccination of man with virus from cases of cowpox affords remarkable protection against smallpox, it appears reasonable to believe that cowpox virus or smallpox vaccine is a modified form of smallpox virus. This fact, together with the occa- sional positive results of various experiments in which other species of animals have at times evidenced susceptibility to cowpox virus, strongly suggests the possible etiological relationship of the diseases in different species to each other and to smallpox in man. However, conclusive proof supporting this suggested relationship does not exist. The specific causative factor of smallpox or of cowpox is not known. Cowpox is a very common disease, perhaps having been prevalent in England and Europe for centuries. Its presence has frequently been observed in various countries since 1796 when Jenner contributed to the world his important discovery relative to smallpox vaccination. Many attempts have been made to isolate the causative factor of cowpox. Early investigators frequently secured mixed and pure cultures of various organisms, including different species of micrococci, streptococci and bacilli from vaccine lymph. None of these organisms were peculiar to the virus, and at present there exists no definite evidence that the infectious agent of vaccine lymph is of bacterial nature. Pfeiffer, Guanieri, Plimmer, Councilman, Mac- Grath, Brinckerhoff and others, after observing the presence of apparent cellular elements, or relatively large flattened bodies in vaccine lymph, have suggested the possible protozoan nature of the causative agent. Attempts have been made, with more or less success, to cultivate these * oenaned by W. E. King. 806 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS bodies in collodion capsules in the peritoneal cavities of experimental animals. According to some investigators the virus has been passed through a Chamberland filter. The failure to discover the causative factor, according to the present methods, may be due to the inability of microbiologists to cultivate or stain the specific agent. Cowpox is characterized by eruptions which usually occur on the skin of the teats and udder. The material contained in these pustules is transferred to other animals by the hands of the milker and through other possible means of dissemination. The chief channel of infection appears to be through an abrasion in the skin. The period of incuba- tion of cowpox is about two days. The virus possesses relatively weak resistance to heat, light and chemicals. The control of the disease depends chiefly upon precautions relative to the transmission of the virus on the hands of the milker from infected to healthy cows. Horsepox may be diagnosed by the appearance of the characteristic pustules usually upon the skin, nasal mucosa and buccal membrane. Sheeppox is characterized by the presence of the typical skin eruptions, following a rise of temperature. DENGUE* This disease (break-bone fever) of man occurs in all parts of the world. It is characterized by a sudden attack, intense prostration and severe pains in the muscles and joints. The fever during the attack ‘shows a characteristic curve. There is a sudden rise of and main- tained temperature for several days. Then a remission and a second rise of temperature which is less than the first. » Our knowledge of the cause of this disease rests chiefly upon researches of Ashburn and Craig (The Journ. of Infectious Diseases, Vol. X, p. 440, 1907). These authors conclude that dengue is not con- tagious in the ordinary sense but that it is transmitted through the bite of the mosquito (Culex fatigans). No visible organism could be, demonstrated in either fresh or stained specimens of blood from patients affected with dengue although such blood was capable of pro- ducing a typical attack of dehgue when inoculated intravenously into healthy men. The authors likewise show that blood from a case of dengue retained its infectiveness after passage through a filter made of diatomaceous earth. The organism of dengue fever is therefore probably of ultramicroscopic size. * Prepared by M. Dorset. ‘ MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 807 Foot-AND-MoUTH DISEASE* Foot-and-mouth disease is primarily a disease of cattle, though the other domestic animals and man may be attacked. The disease is very contagious and is characterized by the eruption of vesicles in the mouths, on the udders and on the skin surrounding the hoofs of cattle. It is very prevalent in European countries. There have been two outbreaks in the United States both of which were promptly eradi- cated by vigorous repressive measures instituted by the Federal authorities. The cause of this disease is an invisible microérganism which exists in the lymph from the vesicles which form in the mouths and on the feet of cattle. This virus has never been cultivated artificially. It passes through the Berkefeld cylinder but not through the finer-pored Kitasato filters; it is quickly destroyed by formaldehyde, carbolic acid and similar disinfectants. The disease is readily transmitted from one animal to another by contact and the contagion may persist for some time in the manure, or straw from infected stables. The milk of infected cows has been said to produce the disease in children. Animals which recover from an attack remain immune for a short time only; it is therefore not surprising that no satisfactory means of artificial immunization has been devised. Hoc CHOLERA* The first recorded outbreak of hog cholera in the United States occurred in Ohio in the year 1833 and it now exists in practically all sections of this country. Hog cholera is most prevalent in the late summer and fall, although outbreaks are reported at all seasons of the year. All races of hogs are susceptible and the average mortality is about 80 per cent. In the United States alone the losses from hog cholera are estimated to average at least $15,000,000 annually. Hogs only are attacked. This disease is supposed to have been introduced into the United States through the importation of hogs from Europe, where it is known under the names “swine fever”’ (Br.), ““schweinepest” (Ger.), and ‘“‘peste du porc” (Fr.). * Prepared by M. Dorset. 808 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS The essential features of hog cholera may be briefly summarized as follows: Extreme contagiousness. Symptoms of severe illness accom- panied by fever, loss of appetite, weakness and diarrhoea. Hzmor- rhagic lesions in the various organs and lymphatic glands and round button-like ulcers in the large intestine. Immunity in hogs which recover. Fic. 170.—Hemorrhagic points on kidneys of hog-cholera hog. (Original.) The etiology of hog cholera has long been the subject of scientific controversy, but it is now generally acknowledged that the cause of this disease is an invisible microérganism which exists in the blood, the internal organs, and the urine of infected hogs. The fact that this disease is caused by an invisible microdrganism was demonstrated as follows:* The blood serum of hogs infected with hog cholera acquired in the * Bulletin 72, Bureau of Animal Industry, U. S. Dept. Agriculture, 1905. “MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 809 natural way is very infectious for non-immune hogs, the disease being . readily transmitted by\the subcutaneous injection of small amounts. The disease produced by this subcutaneous injéction is identical in all respects with the disease as it occurs in nature. If, now, this infectious -serum is diluted with normal salt solution or with ordinary bouillon (1 to 10) and passed through either a Berkefeld or Chamberland filter, the filtrate, though free from all visible microdrganisms, still re- tains the power to produce hog cholera by subcutaneous injection. The disease which is produced in this manner by the filtered hog cholera serum is identical in all respects with the disease produced by the unfiltered serum and also with the disease as it occurs in nature. The hogs which receiye the filtered serum present the symptoms and lesions of hog cholera. The disease set up in this manner is very contagious and hogs which recover from the inoculation of filtered serum are thereafter immune against hog cholera. By repeated inoculation and filtration this virus may serve to infect successively a large number of hogs. The invisible virus of hog cholera, in view of its ability to pass through the Chamberland B filter, must be regarded as one of the smallest of the invisible microdrganisms. It has never been cultivated artificially, hence, aside from its disease-producing qualities, we have little knowledge concerning it. We do know, however, that the virus is quite resistant to such common disinfectants as carbolic acid and bichloride of mercury and that it is quickly destroyed by a 3 per cent solution of liquor cresolis compositus (U. S. P.) as well as by a 5 per cent solution of antiformin. When preserved.in sealed glass bulbs in a cool dark place, the virus retains its activity for six months or longer. Rabbits, guinea-pigs, and other small animals are entirely insusceptible to inoculations of the filtered virus in amounts which would prove fatal to hogs. / The virus of hog cholera is known to be thrown off from the body . through the urine, and it is probably also eliminated through the feces. Therefore any agency which would serve to carry a particle of dirt from infected hog yards might be the means of disseminating the virus. As many sick hogs find their way to the public stock yards through ship- ment by rail, all stock cars and stock yards are to be regarded as permanently infected. It appears to be impracticable to prevent the spread of the disease by methods of quarantine and disinfection, \ : 810 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS_ - owing to the impossibility of enforcing such measures thoroughly. It has recently been found that a protective serum against hog cholera may be produced by “hyperimmunization.” The process consists in giving immune hogs large doses.of blood taken from hogs sick of hog cholera. As a result of this blood treatment their serum acquires the power to protect non-immunes. Injections of serum from hyper- immunized animals confers a passive immunity, while the simultaneous injection of serum with a small amount of virus produces an active immunity. Bacittus CHOLER Surs (B. suipestifer)—No description of the etiology of hog cholera would be complete without a reference to this bacterium which was long regarded as the cause of hog cholera. It is found after death in the blood and organs of the majority of hogs affected with hog cholera and in this réle of secondary invader itno doubt tends to increase the mortality from the disease. B. cholere suis is a small, very actively motile, non-spore-bearing bacillus with rounded ends, which stain readily with the ordinary aniline dyes. It does not stain by Gram’s method. This organism is easily cultivated — on the ordinary media; gelatin is not liquefied; milk is not coagulated but acquires an acid reaction at first; this changes after a week or more to an alkaline reaction. Gas is produced in bouillon containing dextrose, but lactose and saccharose are not affected. Rabbits and guinea pigs succumb within four to ten days to small doses of this organism. Hogs are much more refractory. It is only after the ad- ministration of large doses that they show any symptoms of illness following subcutaneous injections. By feeding pure cultures of B. ' cholere suis or by injecting these intravenously a considerable number of hogs will succumb and at autopsy present lesions which correspond quite closely to those seen in naturally acquired cases of hog cholera. There.are, however, certain important differences between the disease produced by B. cholere suis and the natural disease hog cholera. For example, hogs infected with B. cholere suis do not transmit the disease - -- to other hogs by contact. The blood of hogs infected with B. cholere suis does not produce disease when injected subcutaneously into other hogs, and, in addition, hogs which recover from illness produced by injections or feedings of pure cultures of B. cholere suis have no immunity against the natural disease hog cholera. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 811 Horst SICKNESS* This disease affects the equine species only and appears to be con- ined to South Africa. It is most prevalent in summer and appears to de transmitted by the bite of an insect, as it is not contagious but may 2€ communicated to susceptible horses through blood inoculations. This disease manifests itself by producing severe inflammatory changes n the lungs and in the tissues of the head and neck and is attended oy a high mortality. No visible organism has been found which will oroduce horse sickness and as McFadyean and Nocard have shown that the virus is capable of passing through the finest bacteria-proof filters, this disease is probably caused by an invisible microdrganism. Blood containing the microdrganisms of horse sickness may be kept in sealed bulbs in the dark at room temperature for more than two years without losing its infectiveness. The virus is quite resistant to drying and may survive heating for ten minutes at a temperature of 75°. INFANTILE PARALYSIS* As indicated by its name, this disease (epidemic poliomyelitis) is usually seen in children. It has long been known to exist in both Europe and America, occurring generally in sporadic form. During the last decade, however, its prevalence has greatly increased and a number of well-defined epidemics have been reported. Though the character of this malady long ago led to the belief that it was caused by 4 microdrganism, this fact was not definitely proven until the year 1909 when Landsteiner and Popper in Germany, and Straus and Huntoon and Flexner and Lewis in the United States, succeeded in transmitting the infection to monkeys. So far as is now known, none of the lower animals except monkeys are susceptible. , The symptoms and effects of infantile paralysis-are extremely variable. Paralysis is by no means constant, many cases being very mild and thus possibly escaping detection. In the severer forms of the disease paralyses of various types and degrees are seen. Whenrecovery takes place the paralysis may appear to improve only to be followed by atrophy of certain groups of muscles, resulting in deformity and per- manent lameness. These effects are caused by the destruction of certain nerve centers in the spinal cord. * Prepared by M. Dorset. 812 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS As stated above, the microbial origin of infantile paralysis was first demonstrated by the inoculation of monkeys, Flexner and Lewis having successfully carried the infection through a long series of monkeys by successive intracranial injections of an emulsion of the spinal cord taken from infected animals. The microérganism passes through the Chamberland and Berkefeld filters with little or no loss in disease- producing power. ‘Flexner and Noguchi, employing the technic previously used for cultivating pathogenic spirochetes, have succeeded in obtaining from infected tissues cultures of a minute round organism which they believe to be the cause of infantile paralysis.” The virus withstands freezing or drying for long periods of time but is quickly destroyed by heating at a temperature of 50°. It is likewise quickly: killed by the ordinary disinfectants. Monkeys may be infected by the subcutaneous, intraperitoneal, intravenous, or intracranial injection of material from an infected spinal cord, but attempts at infection through feeding have been unsuccessftl. The virus appears to be eliminated from the body through the nasal mucous membranes. It appears probable that one attack of the disease protects from a second attack. No cases of a second attack have been reported. Furthermore, monkeys which have recovered from the infection appear to be entirely immune as shown by Flexner. Active immunity in monkeys has been established by repeated infections of gradually increased amounts of the virus. The blood of human beings and of monkeys that have recovered from an attack of the disease is capable of neutralizing a certain amount of the virus. This protective quality of the blood serum may be increased by repeated inoculations of virus, and infection in monkeys can be prevented by injecting simultaneously the virus into the brain and the serum into the sub-arachnoid space. The serum treatment of this disease is, however, not developed to such a state that it can be regarded as of practical use. Lovupinc ILL—TREMBLING IN SHEEP* This disease is known only in Scotland and is essentially a disease of sheep. It is characterized by a variety of nervous phenomena, such as trembling, irritability, and convulsive movements, which, are followed later by partial or complete paralysis. The chief lesions are found in * Prepared by M. Dorset. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 813 the meningeal membranes. This disease is supposed to be transmitted by ticks, not the wingless fly which is generally called a “sheep tick” in the United States, but true ticks, belonging to the genus Ixodes. The specific microdrganism has not been discovered. / PELLAGRA* Pellagra is a disease of man characterized by the annually recurring manifestation, each spring or summer, of erythema on the backs of the hands and forearms and sometimes on the face and neck, feet’ and ankles, coupled with digestive disorder and more or less well-marked mental disturbances. During the winter the signs of the disease usually disappear. At present there are two main groups of theories concerning the causation of pellagra, each of which includes a multitude of hypotheses. According to one group of theories pellagra is a food poisoning due to eating maize (Indian corn); according to the other, pellagra is a specific infectious disease not necessarily associated with the ingestion of corn. None of the theories concerning causation is supported by conclusive evidence. Theevidence against the corn theory marshalled by Sambont and others{ has greatly weakened the almost general belief in this theory which formerly obtained. Some prominent zeists§ have recently shown a tendency to ascribe pellagra not essentially to the use of maize but to a supposed deficiency or lack of a necessary something in the diet. This change of opinion has been caused in part by the failure of the maize theory when put to the test of actual observation and in part by an eager application to pellagra of the facts learned in the study of another disease, namely, beriberi. To the writer it seems very improbable that this new phase of the dietary theory will survive as long as has the maize theory proper, although it has recently received enthusiastic support from the U. S. Public Health Service. || * Prepared by W. J. MacNeal. re {7 Sambon, Brit. Med. Journ., Nov. 11, 1905; Journ. Trop. Med. and Hyg., 1910, Vol. XIII 271-282; 287=300; 305-315; 319-321. } Siler and Garrison, Amer. Journ. Med. Sciences, July, 1913, Vol. CXLVI, p. 42; ibid., Aug., 1913, Vol. CXLVI, p. 238; Siler, Garrison and MacNeal, Journ. A.M.A., 1914, Vol. LXII, ne cant: Trans. Soc. Trop. Med. and Hyg., 1913, VI, p.p. 143-148; Weiss, Riv. Pellagrologica Italiana, 1913, XIII, 90; Driscoll, Southern Med. Journ., 1913, VI, pp. 400-401. ik Goldberger, Joseph, and collaborators, Public Health Rep., June 26, 1914, Vol. XXIX, p. 1683; #bid., Sep. 11, 1914, Vol. XXIX, p. 2354; thid., Oct. 22, 1915, Vol. XXX, p. 3117; idid., Nov. 12, 1915, Vol. XXX, p. 3336. 814 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS According to the second theory, pellagra is a specific infectious dis- ease, in which poor nutrition is one of the important predisposing factors. The epidemiological study* of pellagra, as it has developed and spread in certain parts of the southern United States, has brought to light evi- dence of its infectious nature which, to the writer, seems very convinc- ing. The same investigation has also strongly suggested that the infection is intestinal and transmitted in much the same way as is typhoid fever. A specific microbic cause of pellagra has not been identified. / Rasrest Lyssa or Rabies, the madness of dogs, was recognized as a definite disease of animals and man by the peoples of ancient times. The disease is generally distributed throughout the civilized world except in those places where special measures to stamp it out have been enforced. It does not arise spontaneously but is an infectious disease transmitted from animal to animal. Rabies is primarily a disease of wolves and dogs, and the bite of a mad dog is the most frequent cause of the disease in other animals and in man. It is not uncommon in horses and cattle, and all mammals appear to be susceptible to it. In animals inoculated by injection of the most virulent virus (fixed virus) directly into the brain, the symptoms of rabies appear in four to six days and death usually occurs on the seventh day. Accidental inoculation by the bite of a rabid animal (street virus) rarely causes the symptoms to appear before three weeks, and the onset may be delayed for six months or a year. Not all persons or animals bitten by rabid animals take the disease; probably not more than one in four or five. This variability depends upon several factors, the most important ones being the virulence and the amount of disease virus, and the part of the body into which it is introduced. Bites upon the face or hands, because of the rich nerve supply of these regions and the lack of protec- tion by clothing, are likely to result in rabies sooner than bites elsewhere. After the disease has developed, death is inevitable. In all animals the symptoms are those of a nervous disorder. At first there is excita- i) * Siler, Garrison and MacNeal, Arch. Int. Med., 1914. Vol. XIV, pp. 292-373; ibid., 1914, Vol. XIV, pp. 453-474; Journ. A.M.A., 1914, Vol. LXIII, pp. 1090-1093. + Prepared by W. J. MacNeal. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 81 5 ° ‘ tion, and this is followed by paralysis and death, the relative length of the two stages varying in different animals. In the dog the disease runs its course in six to eight days. It begins with altered behavior of the animal; itching of the infecting scar, changed appetite, and slight fever. The dog swallows grass, stones, and pieces of wood. As the stage of excitement becomes more fully developed, the animal may run away and may travel fifty miles or more, snapping and biting from time to time, as the fits seize him, everything in his path. Finally the excitement is succeeded by paralysis, beginning in the lower jaw, which hangs down. Then the hind legs fail, and soon the dog, no longer able to drag himself along, lies completely paralyzed, greatly emaciated, and soon dies. In the rabbit the stage of excitement is hardly notice- able, but the animal passes quickly into the paralytic stage, dying after two or three days. This type of paralytic rabies sometimes occurs in dogs, but is more commonly observed in herbivorous animals. In man there is a first psychical change, irritation in the scar of the infecting wound and rise of temperature. The first diagnostic symp- tom is usually a sudden spasm of the pharynx upon an attempt to swallow water. This convulsive seizure is repeated upon every at- tempt to drink, and soon even the sight of water or the thought of it brings on the attack. The cramps extend to other muscles of the body, and the patient may die in a convulsive seizure, or may pass into the succeeding paralytic stage and die peacefully. The dread of water which is often so prominent a symptom in man has given the name of hydrophobia to the disease. Consciousness and general intelligence are not particularly affected. The duration of active symptoms of the disease is from three to six days. Rabies can be transmitted with certainty by injecting a small amount of emulsified spinal cord of the rabid animal into the brain of a rabbit or guinea-pig. Inoculation under the skin is not quite so certain; and inoculation into the blood stream, or by feeding, generally fails to transmit the disease. When first removed from a rabid dog, the virus (street virus) kills rabbits in from two to four weeks, but after repeated transfer from rabbit to rabbit in series, the period of incuba- tion is shortened until death occurs quite regularly in six or seven days after inoculation. Beyond this there is no further increase in virulence for rabbits, and this six- or seven-day virus is called the “fixed virus.” "The localization of virus in the body of the rabid animal has been * 816 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS. worked out by experimental inoculations. The central nervous system is always virulent, as are also the salivary glands and the saliva. The peripheral nerves frequently contain the virus, less commonly other glands and secretions such as the tears, urine and milk. The virus has never been found in the liver or spleen, or in the blood. Under ordinary conditions, the chief source of danger is the saliva of the rabid animal, especially when this is introduced into a wound. Rabies-may be recognized in a dog in one of the three ways: observa- tion of the course of the disease; autopsy; inoculation of test-animals and observation of the course of the disease in them. If the suspected dog is chained or caged, the question of rabies may be settled in afew days, for, if mad, the raging stage will be succeeded by the character- istic paralysis and death. If the dog has already been killed, a careful, autopsy may show the absence of normal food from the digestive tract and the presence there of abnormal ingested material, highly suggestive of rabies: Microscopic examination of the central nervous system is, in the hands of an expert, a reliable method of diagnosis, which in this case depends upon the finding of the characteristic Negri bodies in the specimen. For confirmation of the diagnosis, a portion of the brain or spinal cord, removed without contamination, should be injected into the brain of test animals, and the effects observed. This last test carried out by experienced observers is justly regarded as the most trustworthy of all. Tue Necri Bopres.—The peculiar bodies found by Negri in the central nervous system of rabid animals seem to occur invariably |. , and exclusively in this disease, and it is probable that they represent stages in the development of the infectious agent. These bodies are especially numerous and most easily demonstrated in the Ammon’s horn of the brain in cases of the natural disease in dogs (street rabies). Excellent results may be obtained by the method of Lentz.* Transverse sections, 2 to 3 mm. in Ehietetibs) of the Ammon’s horn of the sus- pected brain are hardened in acetone at 37° for one hour, then transferred to melted paraffin (melting point 55°) in the paraffin oven at 58° for one and one-half hours and embedded. Sections, 2 to 3u in thickness, are then cut with the micro- tome, floated upon lukewarm water and mounted upon perfectly clean flamed glass slides. The excess of water is carefully removed with filter paper and the slides are then completely dried on a warm plate at 45° or in the incubator at * Lentz, Otto, Ein Beitrag zur Faerbung der Negrischen Koerperchen, Centralbl. f. Bakt etc., I Abt., Bd. XLIV, pp. 374-378. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 817 37°. The sections adhere perfectly as a rule and are dry enough to proceed with after ten to fifteen minutes. The slides are next transferred to xylol to remove the paraffin and thence to absolute alcohol. The staining procedure is as follows: z. One minute in alcoholic eosin. Eosin extra B-Hoechst, 0.5. Alcohol, 60 per cent, 100.0. 2. Wash in water. 3. One minute in Loeffler’s methylene blue. Saturated alcoholic solution of methylene blue, B-Patent Hoechst, 30.0. Potassium hydroxide solution, o.or per cent 100.0. . Wash in water. 5. One minute in Gram’s solution. { Iodine, 1.0. ‘Potassium iodide, 2.0. Distilled water, 300.0. . Wash in water. , . Methylic alcohol until the preparation becomes entirely red. . Wash in water. . Loeffler’s methylene blue again for thirty seconds. to. Wash in water. 11. Dry carefully by pressing with filter paper upon the preparation. 12. Differentiate in alkaline alcohol until only a weak eosin color remains in the preparation. : Absolute alcohol, 30.0 c.c. Sodium hydroxide, 1 per cent solution in absolute alcohol, 5 drops. 13. Differentiate in acid alcohol until the collections of ganglion cells in the gray matter are still faintly blue while the rest of the section is free from blue (macroscopic). Absolute alcohol, 30.0 c.c. Acetic acid, 50 per cent, 1 drop. 14. Wash quickly in absolute alcohol. 15. Xylol. 16. Balsam and cover-glass. - Oo COND DN Steps 5 to 9, inclusive, may be omitted to save time at some sacrifice in the final result. The Negri body is stained pink with blue granules in its interior. The nerve cells are stained pale blue. Although sections are most satisfactory for diagnostic purposes and especially to show the relation of the Negri bodies to the ganglion cells, it is usually possible to recognize the Negri bodies in smears, after a little experinece. For this purpose a portion of the gray matter of the Ammon’s horn is crushed by gentle pressure between two perfectly clean flamed slides and spread upon them by carefully slip- ping the slides apart. The moist smears are at once fixed in methyl alcohol for one minute, then washed in absolute ethyl alcohol, whereupon they are ready to be stained by the procedure outlined above. 52 818 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS The Negri bodies (Fig. 171) appear as round or somewhat tri- angular structures, for the most part inside the ganglion cells. Their size varies considerably, from 1m to 274 in diameter, the majority measuring about su. In the interior of the Negri body, smaller structures of variable size and number can be seen. These granules eee Fic. 171.—Section through the cornu ammonis of brain of a rabid dog; stained by the method of Lentz. Five Negri bodies of different sizes are shown, enclosed within the ganglion cells. The smallest contains only three minute granules. (After Lentz, Centralbl. f. Bakt, 1907, Abt. I, Vol. XLIV, p. 378.) may be differentially stained as in the Lentz method. Some careful students of rabies regard the Negri bodies as protozoa and consider them to be the infectious agent. Proof of this belief is still lacking inasmuch as it has not yet been conclusively shown that they are actually living organisms. A wound infected by a rabid animal should be thoroughly cauter- ized, under anesthesia if desired, at the earliest possible moment, and x MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 810 this cauterization should not be omitted even if twenty-four hours have elapsed. Cauterization cannot be relied upon to prevent the develop- ment of rabies, but it does serve to prolong the'incubation period. The. Pasteur treatment should then be instituted as soon as possible, and it has proved to be practically an absolute preventive, provided the incubation period of the disease is sufficiently prolonged for the treat- ment to become effective, and this is usually the case. The treatment consists in the daily subcutaneous injections of altered fixed virus for a period of about three weeks, and is most effectively given at Pasteur Institutes devoted especially to this work. Valuable animals as well as man may be successfully treated in this way. The general prevention of ‘rabies depends almost solely upon the efficient control of all dogs ina community. General muzzling, strictly enforced, is a certain preventive of rabies, and in countries where this is done rabies is practically unknown. Swamp FEVER* This is a comparatively new disease of horses so far as definite infor- mation is concerned, but is in reality an old disease that has been described under a variety of names for many years. It is known by various names as infectious anemia, malarial fever, horse typhoid, “plains” paralysis, and pernicious anemia, and has been recognized in many portions of the Western United States and Canada. This disease is usually of chronic type, but acute cases have been reported. There is usually a long illness extending from a month to a year or more, and marked by periods of fever and debility, alternating with periods of apparent recovery. The phase of apparent illness is characterized by mild fever, general weakness, and staggering gait, and the disease terminates fatally, as a rule. The peculiar features of the disease are the alternating periods of illness and recovery, unthriftiness in spite of unusually good appetite, pallor of mucous membranes, dropsical swellings of the belly and limbs. It has been satisfactorily proved that swamp fever is caused by filterable virus present in blood, urine, and feces. Under artificial inoculation with blood, the period of incubation varies from ten to forty days. The natural method of infection is * Prepared by M. H. Reynolds. 820: MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS unknown, but there are reasons for believing that infection does not easily occur by way of the digestive organs nor through the respiratory organs. The disease is apparently not communicated by simply stabling diseased animals with healthy animals. Distribution in the body is very general, as shown by the wide dis- tribution of characteristic lesions, and as shown by the fact that the blood is infectious. The virus which causes swamp fever reduces greatly the number of red blood corpuscles and also produces local hemorrhages which are most frequently small and sharply defined. The reduction of red blood cells produces marked pallor, and there gradually develops noticeable emaciation. * Post-mortem lesions in many cases are slight. The hemorrhages. may involve subcutaneous and intermuscular tissues, spleen, and lymph glands and are rather common on the lungs and heart. Any of the abdominal organs may show the characteristic hemorrhages. The bone marrow has been reported in some cases as distinctly changed in color, the yellow marrow of long bones becoming dark red. In some cases the liver shows degeneration and necrosis or tissue death. TypHus FEVER* 1 Typhus fever (ship fever, jail fever) has been known to exist for centuries but until very recently we have been without precise knowl- edge concerning its cause. Typhus is found in all parts of the world; it affects man only and is characterized by a high fever and an erup- tion on the skin. The course of the disease is limited and lasts for only about twelve days. In the years 1909 and 1910 Nicolle, working in Tunis, and Anderson and Goldberger, and Ricketts and Wilder working in Mexico, showed that typhus is communicated from man to man by means of the body louse (Pediculus vestimenti), and that the disease is not contagious in the ordinary sense of the word. Nicolle states that after biting a typhus fever patient the louse cannot convey the infection until the fourth day thereafter and that it loses this power after the seventh day. This indicates a similarity between the micro- érganisms causing yellow fever, malaria and typhus. The disease may be communicated to monkeys by subcutaneous inoculations of blood * Prepared by M. Dorset. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 821 from a typhus fever patient. The virus may be transferred from one monkey to another indefinitely. In monkeys recovery from severe attack produces a firm immunity. No microdrganism has been dis- covered which can be regarded as the cause of the disease. Attempts to pass the virus through filters have been unsuccessful with the possible exception of certain experiments by Nicolle. The virus is destroyed by heating from 50 to 55°. YELLOW FEVER* Yellow fever is an acute infectious, non-contagious disease of man which is seen in tropical and sub-tropical countries, particularly the West Indies, South America, and the west coast of Africa. The most notable symptoms of the disease are fever, jaundice, and hemorrhages from the mucous membranes, this latter resulting in severe cases in what is known as “Black Vomit,” which consists chiefly of extravasated blood which has been changed to a brown or black color by the action of the gastric juice. Prior to the brilliant researches of Walter Reed and his associates on the United States Army Commission in the year 1900, it was generally believed that yellow fever was contagious, and that the disease was transmitted directly from infected to non-infected in- dividuals, and furthermore that the clothing, bedding, and all materials which came into contact with the infected subject were capable of transmitting the disease. Reed and his associates, during the American occupation of Cuba, secured a number of volunteer subjects to serve the Commission in its studies. This Commission demonstrated posi- tively that yellow fever could not be transmitted to man in any other way than by the bite of a particular mosquito, Aédes (Stegomyia) calopus (Meigen). These mosquitoes were allowed to bite patients suffering from yellow fever at different stages of the disease. Sub- sequently these same mosquitoes were allowed to bite healthy men at different periods of time following their application to the infected individual. It was proved that the mosquito, in order to be capable of conveying the disease, must bite an infected individual during the first three days of the fever and at least twelve days must elapse thereafter before the mosquito is capable of transmitting the disease to a sus- ceptible individual. * Prepared by M. Dorset. 822 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS DISEASES CAUSED BY PROTOZOA* Raizopopa (von Seibold) The amcebe are the most important of the parasites belonging to the rhizopods. Various species of amcebz are parasitic in the intestines of cattle, horses, mice, frogs and fish as well as human beings and most of them, like Entameba coli of man, are harmless. One species, Entameba histolytica (tetragena), produces a very severe dis- ease of man. LEntameba meleagridis is the cause of a fatal disease of turkeys, (page 824). Entameba gingivalis (buccalis) is a parasite which is frequently found in a diseased condition of the gums characterized by peridental abscesses but. is also frequent in apparently healthy mouths. The parasitic species lack the con- tractile vacuole which is a feature of the free living species that are commonly , encountered so that it is not difficult to distinguish the two types. Amc@BIc DYSENTERY Entameba histolytica—Schaudinn, 1903 Syn.: Entameba tetragena—Viereck, 1907 Distribution —Ameebic dysentery occurs most frequently in tropical, or sub-tropical, countries, but cases of it occasionally occur in Great Britain, in Central Europe, and in the United States. Intestinal amebe; Entameba coli Lésch (Fig. 172) and Entameba histolytica (tetra- _gena) are both parasites in the human intestine. They measure from 15m to zou in diameter and, when examined in freshly passed feces, may be seen in active motion. Their cytoplasm contains a nucleus, vacuoles, and food particles. Both may multiply by binary and by multiple division; the appearance of certain of the encysted forms in both species indicates a process of autogamy. Both of the parasitic amcebe of the human intestine produce a characteristic number of daughter amcebe in the course of their multiplication. E. coli com- monly divides into eight small amcebe so that these organisms may present any number of nuclei below this number and occasionally they contain several more. The encysted forms of this. species also divide into approximately eight small amosbe. In E. histolytica the number produced as the result of division is more regular being almost invariably four whether in the division of the motile trophozoite or the encysted forms. The character of the division thus furnishes the most certain criterion in differentiating the two species. Multiplying forms are not always readily found, however, and it is necessary to take other characteristics into consideration. The non;pathogenic species (E. coli) is more sluggish in its movements, is generally larger, dull grayish in appearance, and. with no sharp differentiation into ectoplasm and endoplasm. E. histolytica is active, of a greenish hue and the ectoplasm is well defined and very clear in portions extruded as pseudopods. The nucleus in the harmless species, commonly centrally situated, is larger and shows a larger amount of chromatin. The nucleus of the * Diseases arranged generically. * Prepared by J. L. Todd and revised by E. E. Tyzzer. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 823 dysentery ameebz is smaller, poorer in chromatin and commonly peripherally sit- uatedin thecytoplasm. This species also devours red corpuscles in large numbers but the absence of these in intestinal amcebe is not sufficient basis for considering them to belong to the harmless species. The cysts are passed in the feces; and it is through the ingestion of food or drink, contaminated by encysted amebe, that in- fection is accomplished. If unencysted amcebz are swallowed, they are digested by the acid juices of the stomach, whereas encysted amcebz pass through the stomach unaltered and become active in the alkaline contents of theintestine. The dysentery ameeba is pathogenic to certain lower animals, and kittens have been found to be most favorable for the experimental production of the disease. Monkeys are also sus- ceptible to a certain extent. A Fic. 172.—Entameba coli Lésch 1875. A-C, various forms of motile amcebe; D, the 8-nuclear stage; E-G, cysts with nuclear fragments; H, bursting cyst; 7. young motile ameebe. (Afler Casagrandi and Barbagallo, from Doflein. ) , Entameba histolytica may be present in an intestine for months without marked symptoms resulting. It may, however, enter the glands of Lieberkihn and pass through it into the submucosal layer of the intestine. Bacteria accompany these amcebze and they, with the latter, cause an ulcer which spreads through a local destruction of the submucosa and undermines the mucosal layer of the intestine. In severe cases, when the ulcers have spread widely, large areas of the mucosa may be sloughed off. The amcebe lie at the edge of the ulcer and cause it to enlarge by working their way into sound tissue; once an ulcer is started, it is not impossible that Entameba coli as well as the dysentery amcebe may be found init. The latter live upon the red cells or fragments of intestinal cells. In chronic cases, the wall of the intestine becomes greatly thickened. 824 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS Ulcers caused by amcebe are almost always situated in the large intestine; consequently, the symptoms of amcebic dysentery are those of inflammation of that part of the body. There is usually abdominal pain, accompanied by the passage of frequent, blood-stained stools with mucus. The infection may, however, be accompanied by no marked symptoms and there may be no diarrhoea. There are usually developed more general symptoms, such as fever and loss of flesh. The onset is frequently very gradual and insidious and the disease runs a chronic course. If amcebic dysentery causes death, it is usually does so by per- foration of the bowel with resulting peritonitis, by hemorrhage from the erosion of a blood vessel, or as the result of abscess of the liver. Liver abscesses occur not infrequently in amcebic dysentery. Ameebic dysentery is cured with difficulty although emetine, a product isolated from ipecac, has recently been found of great value. Since the encysted ameebe are killed by heat, it can be avoided by eating and drinking only foods and liquids that have been cooked. ENTERO-HEPATITIS OF TURKEYS Emtameba meleagridis—Smith, 1895 Entero-hepatitis, or black-head, of turkeys is caused by Entameba meleagridis. The disease is widespread throughout North America. It is a very fatal affection and on many farms it makes the raising of turkeys a difficult problem. The disease is characterized by thick- ening and ulceration of the ceca, and by extensive necrosis of the liver. Entameba meleagridis is a small amoeba measuring about 8p to towin diameter.. Turkeys probably become infected with this parasite by swallowing its encysted forms; young turkeys may possibly become infected from encysted amcebe, which adhere to the shells from which they were hatched. ° There has been no efficient treatment devised for the disease, since it is usually not noted until far advanced, but it can be avoided through keeping healthy stock on land which has never been infected by drop- pings from infected birds, and by carefully wiping eggs intended for hatching with formalin. Fraceiiata (Cohn emend. Biitschli) The herpetomonads and the trypanosomes are the most important of the para- sitic flagellates. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 825 LEISHMANIA (Ross, 1903) The three parasites belonging to this genus which require mention are included by some authorities in the genus Herpetomonas but the differences with respect to habit of life justify the recognition of a distinct genus. Herpetomonads live in the alimentary tract of various insects, for example, of the common blow fly and are extracellular parasites. Their bodies in general are rigid. Leishmania is, on the other hand, an intracellular parasite and in the flagellated phase of its develop- ment its body is plastic and bends during locomotion. Three species are recognized in association with three distinct types of disease. Kata AZAR Leishmania donovani—Laveran and Mesnil, 1903 This disease occurs in certain parts of Asia being first noted in Assam, Northern India. It is caused by L. donovani (Fig. 173). The parasite is rarely found in the blood; when it is seen there, it almost never occurs free but is found in variable numbers Fic. 173.—Leishmania donovani. Free organisms and several within cells. (After Donovan, from Doflein.) within phagocytic cells. It is, usually, easily found by an examination of the juice obtained from the spleen or lymph nodes by puncture with the needle of a syringe. The liver is enlarged and it, also, contains parasites. As the organisms are seen in preparations of spleen juice, they are small ovals measuring about 2 in length and 1.54 in width. They consist of cytoplasm, in which lie two chromatic bodies, one of them large and rounded, the other small and rod-like. This form of the parasite may multiply in the body of the host, by binary or multiple division. If spleen pulp, or blood, containing such organisms be placed on a suitable culture 1 826 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS medium, they will develop in three or four days, into herpetomonad forms. The large nucleus becomes the trophonucleus of the flagellate form, while the smaller, rod-like, mass becomes the kinetonucleus, from which arises the flagellum. The method by which the infection is acquired i is unknown; it is probably by the bite of an insect, perhaps a bedbug. Kala azar is a chronic disease ‘characterized by emaciation, by an irregular fever and by considerable enlargement of the spleen. There is great loss in strength and energy. Although there may be periods of apparent amelioration, the disease . usually progresses steadily, in spite of treatment, to a fatal termination. INFANTILE Kata AZAR " Leishmania infantum—Nicolle, 1908 Most authorities recognize the generalized leishmaniasis which occurs in various countries bordering on the Mediterranean as a distinct disease. It is confined almost wholly to young children in whom it usually runs a fatal course. This disease has been transmitted to cep? PON lower animals and the infection occurs naturally in dogs, especially ~~ those of infested districts. Recent investigations indicate that the dog furnishes a source of infection for human beings and that transmission is affected through the agency of a species of flea. Locatizep LEISHMANIASIS (DELHI Bott) Leishmania tropica—Wright, 1903 The localized forms of leishmaniasis occur in widely distributed localities throughout the tropics, and numerous local names have been applied, Aleppo Boil, Oriental sore, Bagdad Boil, Biskra Button, etc. In South America likewise a number of local names have been applied, Espundia, Uta, Bubas, Braziliana and Forest Yaws. In certain forms of the disease the mucous membranes are invaded with loss of tissue of the nose and palate causing great deformity. The parasites are found at the spreading edge of the lesions. As they occur in the ulcer they are oval parasites, almost identical with those which are found in the spleen of persons suffering from kala azar. If infected material be placed on a culture medium, flagellated forms will be developed. In many cases the organisms are difficult to find. Delhi boil is a painless ulcer, covered by a dry scab, which usually occurs about the face, or other uncovered portions of the body. If the MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 827 ‘ sore be left untreated, it cures itself after some months., In countries where it occurs, Delhi boil is particularly liable to form at the site of a cut or abrasion. It is possible that, in some cases, the infection may be carried to a wound by house flies. The condition may be treated by free excision although it runs a self-limited course. In places where it is endemic, care should be taken to avoid the possibility of infection by carefully protecting all wounds, no matter how small. ra TRYPANOSOMA (Gruby, 1843) Trypanosomes are parasitic in insects, fish, reptiles, birds, and mammals in: all parts of the world. Many of them seem to be harmless parasites; others cause very serious diseases. Sleeping sickness since it affects human beings is regarded as the most im- portant of the diseases due to trypanosomes. ’ S SLEEPING SICKNESS 1 Trypanosoma gambiense—Dutton, 1902 Sleeping sickness is a disease of man caused by Trypanosoma gambiense; it is usually transmitted by the bites of Glossina palpalis, a tsetse fly. Sleeping sickness occurs only in those parts of Africa where this species of fly is found. Fic. 174.—Trypanosoma granulosum. n, Tropho-nucleus; m, undulating membrane: ¢, kinetonucleus; f, flagellum. X 2000 diam. (After Laveran and Mesnil from Doflein.). 1 Trypanosoma gambiense (Fig. 174) is somewhat fusiform in shape and measures about 174to 254 from the posterior extremity to the tip ofits flagellum. A large 828 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS. tropho-nucleus is situated near the center of the trypanosome; a smaller, kineto- nucleus lies near its posterior end. From this smaller nucleus a filament arises, which runs the whole length of the parasite and extends from its anterior end as a free flagellum. Where the filament runs along the body, the periplast extends over it to form the undulating membrane. The trypanosome moves by means of the undulating membrane and flagellum and also through the contraction of the myonemes which lie in the ectoplasm. In the blood, Trypanosoma gambiense multiplies by binary division. It is not impossible that it may multiply in other ways, as do other trypanosomes; for example, a trypanosome of frogs loses its locomotory apparatus and forms a sphere, then the sphere divides into many small, spheres, each of which becomes a trypanosome. Sometimes Trypanosoma gambiense loses its locomotory apparatus and forms a sphere; these forms are found in the organs of infected animals. They are probably more resistant, resting forms and a single trypanosome may be formed from some of them. 4 Trypanosomiasis is easily transmitted to susceptible animals byinoculation. Itis possible that the disease may be transmitted occasionally, in this way, by the mere Fic. 173.—Trypanosoma gambiense. (After Minchin, from Doflein.) mechanical exchange of infected material, through an insect’s bite, from an infected to a healthy individual. But, as a rule, the disease can only be transmitted by the bites of Glossina palpalis in which the organism has developed for some time (Fig. 175); this fly is not usually infective until three weeks after it has fed on an infected person, and it retains its infecting power for some months. An incubation period of at least ten days intervenes between the bite and the appearance of symptoms, but this period may be much longer, for trypanosomiasis may manifest itself in apparently healthy negroes several years after they have left localities in which the disease could have been acquired. The disease sometimes causes death within three or four months; but it may last for one or more years. It is a chronic, wasting affection, characterized by loss of strength and energy, and by an irregular fever. A change in the MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 829 Fic. 176.—Glossina palpalis. (After Doflein.) 830 MICROBIOLOGY .OF DISEASES OF MAN AND DOMESTIC ANIMALS mentality, red blotches on the skin, and enlargement of the lymphatic glands, are all early signs of the disease. In the later stages, headache, mania, uncontrollable sleep, and other nervous symptoms may be present. Death rarely results from trypanosomiasis alone; the patients usually succumb to one of the secondary infections, to which their reduced condition makes them especially liable. No toxin has been isolated in trypanosomiasis, but from the nature of the infection no great amount would be necessary to produce symptoms. ‘The para-. sites not only live in the blood and other fluids of the body but are found in the tissues of various organs. They are. distributed throughout the tissue of the brain and their presence.is associated with infiltration of the tissue about the blood-vessels with large numbers of lymphocytes. : The recognition of trypanosomiasis depends upon the demonstra- tion of the parasites. They may be found in fresh or stained prepara- tions of the blood, in the juice obtained by aspirating an enlarged lymphatic gland, or in the cerebrospinal fluid. The examination of the blood is the simplest method of searching for trypanosomes; the examination of gland juice is the most efficient one. The improvement in the methods of treating trypanosomiasis during the past ten years (1901-1911) affords an excellent example of the value of laboratory work. Before 1901.arsenic, given in some inorganic form, was the only drug known to have any effect on trypanosomiasis. Inor- ganic arsenic drives the parasites from the blood and improves the patient’s condition. Unfortunately, the trypanosomes usually reap- pear and, then, they have become resistant to arsenic so that the patient succumbs in spite of repeated doses of the drug. Many organic com- pounds of arsenic were experimented with in the hope of finding an efficient trypanocide and several valuable drugs have been found: “ Atoxyl”’ which is the sodium salt of para-amido-phenyl-arsenic acid, acetylated atoxyl, and arsenophenylglycin, are all organic compounds of arsenic. They are much more effective than is arsenic itself. Similar °° organic compounds of antimony and tartar emetic are almost as effective, while certain aniline dyes have a distinct trypanocidal value. It has been found that trypanosomes may become resistant to any one of these drugs, and that drugs may destroy some stages of the trypano- some while they are unable to destroy others. In order to give the parasites no opportunity of acquiring resistance to any drug, and in MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 831 order to destroy them at all stages of their development, the following general rules are now observed in the treatment of trypanosomiasis. The drugs employed should be alternated, and they should be given as early in the disease as possible, and in as large doses as possible.' It is probable that these principles will be found to be of value in the treatment of other diseases caused by protozoa. The prevention of the disease depends upon the avoidance of the water’s edge, where Glossina palpalis exists, and of the proximity of persons infected by trypanosomiasis. The most usually successful way of recognizing infected persons is by the discovery of trypanosomes in the fluid aspirated from their enlarged lymphatic glands. Homan TRYPANOSOMIASIS OF SoUTH AMERICA Trypanosoma cruzi—Chagas, 1909 This disease is caused by Trpyanosoma cruzi (Endotrypanum cruzi) and is transmitted by the bites of areduviidinsect, Conorrhinus megistus. It has only been found in Brazil. \ Trypanosoma cruzi may be either free in the blood plasma or lie within a red cell. It multiplies, in the organs, by losing its locomotory apparatus and forming a sphere which divides into eight portions: a new trypanosome develops from each portion. : The disease is a chronic one, characterized by irregular temperature, by wasting, cedema, and enlargement of the spleen and lymphatic glands. It occurs chiefly in young children and is often fatal. It may be prevented by avoiding the insect which transmits it—the habits of the Conorrhinus resemble those of a bed bug. TRYPANOSOMIASES OF ANIMALS Several diseases, of great economic importance, which affect domestic animals, are caused by trypanosomes. The following are the most important. Tsetse-fly disease, or nagana, of Southern Africa, is caused by Trypanosoma brucei (Plimmer and Bradford) and it is trans- mitted by the Tsetse fly, Glossina morsitans; it affects all domestic animals. In South America, Mal de caderas, a disease of horses, is caused by 832 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS Trypanosoma equinum (Voges); it is probably transmitted by a biting fly, Stomoxys. All through Asia, Surra, caused by Trypanosoma evansi (Steel), isa severe disease of cattle and equines; it is probably transmitted by a biting fly. Trypanosoma dimorphon (Laveran and Mesnil) and many other trypanosomes, more or less closely allied to it, cause diseases, of horses, cattle, and other domestic animals in many parts of Africa; they are probably all transmitted by the bites of flies. Fic. 177.—Colonization in Trypanosoma lewisi (Kent). (From Doflein.) One of the commonest trypanosomes is Trypanosoma lewisi (Kent). It is usually a harmless parasite and it is found in rats in all parts of the world. It is transmitted by the flea and possibly by the lice which infest these animals. It is not transmissible to other mammals. Dourine or maladie de coit, is a serious disease of equines; it is caused by Trypanosoma equiperdum (Doflein). This disease was MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 833 brought to North America by an imported Percheron stallion. It is now endemic in some of the western states and in part of southern Alberta, in Canada. It is transmitted by coitus and, perhaps, rarely by the bites of fleas. A very large trypanosome, Trypanosoma theileri (Bruce), occurs in cattle in southern Europe and in Africa and a large trypanosome, Trypanosoma americanum, resembling this one, has been found in cattle in the United States. These trypanosomes seem to do no harm to their hosts. Although there are slight differences, the symptoms are much the same in all the trypanosomiases of animals, and they much resemble those which occur in the diseases produced in men by trypanosomes. Occasionally, as in nagana, an animal trypanosomiasis may run an acute course, and kill the host in two or three weeks, but usually, they are diseases of long duration, characterized by irregular fever, edemas and progressive loss of strength, weight, and energy. Localized areas of cedema beneath the skin and about the genitals are especially seen in dourine; Trypanosoma equiperdum is most easily found by examining serum obtained by puncturing these oedemas. Sporozoa (Leuckart, 1879) This class contains many very important pathogenic parasites. ; Coccrp1a (Leuckart) Coccidia of various species are parasitic in the epithelial cells lining the intestines of mice, horses, cattle, pigs, goats, and other animals. In Europe, Eimeria stiede (Coccidium cuniculi) sometimes causes an enteritis of cattle; in East Africa, a coccidium causes a serious disease of cattle. Other coccidia kill many young pigeons, grouse, and chickens. Coccidia have been reported as infecting the intestinal tract of man. Coccmpiosis OF RABBITS Eimeria stiede—Lindemann, 1865 Syn.: Coccidium cuniculi The coccidium causing this disease is the best known of the coccidia infecting mammals. 53 834 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS This coccidium is parasitic within the epithelial cells of the intestine and within the epithelium lining the bile ducts. Adult, asexual forms measure from 20n to 50u in diameter and they produce from 30 to 200 merozoites. ‘The merozoites infect other epithelial cells where they may again multiply asexually or they may develop ‘into male and female forms destined to multiply sexually. One of the micro- gametes, produced by a microgametocyte, fertilizes a macrogamete and an odcyst is developed. Within the odcyst a number of sporoblasts form, which contain two spores each. The oocysts are passed with the feces and if they are ingested bya suitable host the spores are set free. When the cyst reaches the intestine, the sporozoits are liberated, and a new infection is commenced. Since the cells parasitized by the coccidia are destroyed, it is evi- dent that a severe infection may do a great deal of harm and interfere with the functions of both intestine and liver. The disease may be limited by making it impossible for uninfected animals to come into contact with the droppings of infected stock. AvIAN CoccIDIOosIS Coccidium infection is of frequent occurrence among birds and es- pecially those of domestic varieties without causing serious symptoms. It is known, however, to cause severe epidemics in certain species, and when present in milder form should be regarded as antagonistic to health. Entameba meleagridis the organism of “Black head” in turkeys from its peculiar relationship to the tissues has been erroneously regarded as a form of coccidium. HamosporipiA (Danilewsky emend. Schaudinn) The most important parasites of this order are those, belonging to the Genus Plasmodium, which cause malaria in man. Organisms similar to these are parasitic in the red blood cells of higher apes and monkeys. Proteosoma and Hemoproteus are two genera parasitic in the red blood cells of birds. It was the study of these avian parasites which led to the discovery of the way in which malaria is transmitted by the bite of the mosquito. PLasmopium (Marchiafava and Celli) At least three species of this genus are.parasitic in man: Plasmodium vivax (Grassi and Feletti), the cause of tertian malaria, Plasmodium mal- ari@ (Laveran), causing quartan malaria, and Plasmodium falciparum (Welsh), which causes aestivo-autumnal malarial fever. Loe MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 835 MALARIA Malaria is a disease caused by an amceboid parasite of the red blood corpuscles and is transmitted by the bite of anopheline mosquitoes in which the.parasite has completed the sexual cycle of its development. It exists in all parts of the tropical and subtropical world (Fig. 178). A young malarial parasite or sporozoit, derived from the mosquito enters a red cell and supports itself by living upon the cell’s substance. The parasite grows, proceeds to multiply asexually and divides into a number of merozoites which are set free a9 GY @ SED, Scnizocony (Asexual Generation) as in MAN. © SPOROGONY (Sexual Generation) in the Mosquito = : Oocypst. Ooeyat with Sporoblasts. Fic. 178.—Diagram illustrating the human and mosquito cycles of exist the malaria parasite. (After Martin’s General Pathology.) peers by the rupture of the red cell. ~ Those of the merozoites which escape ingestion by the white cells of the blood enter red cells where they may again multiply asexually, or they may develop into sexualforms. When blood, containing malarial parasites, a gested by a suitable mosquito, all the parasites, except the adult sexual ones, are di- gested and die. Soon after they are ingested, the macrogametocyte extrudes polar 3 36 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS bodies and become a macrogamete and the microgametocyte produces several micro- gametes, one of which enters and fertilizes the macrogamete. Through the fusion of macrogamete and microgametea copula is formed, which since it is motile is called an odkinet. This makes its way until it comes to lie just beneath the outer surface of the mosquito’s stomach. There it develops, as an odcyst, until it reaches several times its original size. It divides into a number of areas, or sporoblasts, each of which subdivides to form many very small, hair-like sporozoites. When the odcyst bursts, some of the sporozoites pass forward to find their way into the salivary glands of the mosquito, and, when it bites, they are extruded, with the saliva, into the body of the person from whom blood is being sucked. The entry of a sporozoite into a red cell recommences the cycle of development which has just been described. If the adult sexual parasites are not taken up by a mosquito they die off in the blood, but some of the female forms may live for years and then divide parthenogenetically, without a precedent fertilization, to produce several young parasites. It has been suggested but never demonstrated that the sporozoites may enter eggs lying in the ovaries of infected mosquitoes and that mosquitoes, hatched from such eggs, may, inherit the infection from their parent and that they, also, are able to transmit malaria. In fresh preparations of blood, a malarial parasite is seen as a body of varying size, which is more refractile and of a lighter color, than the red cell which contains it. In its growing phase it has distinct amoeboid movement and the pigment granules lying in it are in active motion. In preparations, stained by a modification of Romanowsky’s method, every malarial parasite is seen to possess a definite purple nucleus surrounded by blue-staining cytoplasm. Young parasites measure less than a fifth of the diameter of a red cell in width; adult parasites may completely fill the cell which contains them. Malarial pigment is the waste product which results from the digestion of the hemoglobin of the red cells by a malarial parasite, and consequently, since they have digested more hemoglobin, the older parasites contain more pigment than do the younger ones. A mature asexual finally segments into a number of merozoites; Plasmodium vivax forms about eighteen, Plasmodium malarie about eight merozoites. The adult sexual forms of Plasmodium falciparum are shaped like a crescent. The three malarial parasites of man may be distinguished from one another by these peculiarities as well as by other, lesser differences in themselves and in the red cells which they parasitize. When a mature, asexual, malarial parasite bursts, it sets free young _ parasites and a toxin. Practically all of the parasites, present in a person suffering from typical acute malaria, mature and burst at the same time and the considerable amount of toxin, set free in this way, produces a paroxysm characterized by. chills and fever. The parasites of Plasmodium vivax mature in forty-eight hours. Conse- quently, a person infected by it has a chill when schizogony occurs, on every third day, and the disease caused by it is called a tertian fever. Plasmodium malarie matures in seventy-two hours, causes DESCRIPTION OF PLATE I. (Reproduced from Greene’s Medical Diagnosis) Plasmodia of three varieties. Stained by Wright’s stain. In this plate the chromatin of the parasites is shown in red while the pigment granules appear as black dots. THE QUARTAN PARASITE (P. malaria) 1-g. Asexual multiplication. ro. Adult gametocyte. 11. Normal red cell. 12. Flagellating microgametocyte. THE TERTIAN PARASITE (P. vivax) 13-21. Asexual multiplication. 22. Flagellating microgametocyte. THE ASTIVO-AUTUMNAL PARASITE (P. falciparum) 23-31. Asexual multiplication; 25 and 26 are doubly infected cells. 32-35. Gametocytes. 36, 37. Flagellating microgametocyte. PLATE L THE QUARTAN PARASITE THE AESTIVO AUTUMNAL, PARASITE 25 24 25 MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 837 an attack of ague on every fourth day and the disease produced is called quartan fever. Patients infected by Plasmodium falci- parum often have a quotidian fever with a daily rise in the temp- erature, although a three day period may be recognized in some cases. The disease is called estivo-autumnal fever. ‘There are three stages in the paroxysm: during the chill, the patient feels cold; in the hot stage he feels warm—his temperature is above normal during both stages; in the sweating stage the temperature falls to normal and the patient’s discomfort becomes much less. The regularly recurring chills and fever constitute the only symp- toms characteristic of malaria and a regular rise in temperature on the third or fourth days of an illness is strongly suggestive of a malarial infection. The type of disease and the symptoms, produced by a malarial infection, may vary almost indefinitely according to the pre- cise way in which the host is harmed by the infection. Consequently, an enumeration of the clinical manifestations of malaria is of less importance to a student than is an understanding of the way in which the malarial parasites harm their host. The malarial parasites de- stroy the red cells and thus cause an anemia with the symp- toms which result from it. Secondly, they produce toxins which may cause both acute and chronic intoxications. The acute intoxications are seen in the elevation of body temperature and in unconsciousness in some pernicious forms of malaria; malarial neuritis is an example of chronic intoxication. Lastly, malarial para- sites may do harm by blocking the capillaries and causing the death of the cells which are cut off from the circulation, the symptoms which result depending upon the functions of the cells which are destroyed. If the disease be long continued, with a high temperature, the de- generative changes which usually result from chronic disease and constant fever are produced in the patient. The definite diagnosis of malarial fever depends upon the demon- stration, in a patient, of the malarial parasite, or of the pigment pro- duced by it. Quinine has a specific action on the malarial parasite and is the most valuable drug available for the treatment of the disease. It must be given in full doses and treatment continued until all parasites dis- appear from the blood. Malaria, since it is of the type of disease which is produced by 838 MICROBIOLOGY OF DISEASES OB MAN AND DOMESTIC ANIMALS an insect-transmitted parasite, may be prevented by measures directed either against the parasite or against the transmitting agent. Malaria is caused by a Plasmodium and transmitted by the bites of mosquitoes belonging to the Anopheline. The disease may be com- bated by destroying the parasite, in infected persons with quinine, and by isolating such persons under mosquito nets so that mosquitoes may never have an opportunity of ingesting the parasites which they harbor in their blood. Malaria may also be prevented by destroying the mosquitoes which transmit it. The most efficient way of getting rid Fic. 179.—Longitudinal section of Anopheles. A, head; B, thorax; C, abdomen 1, esophagus; 2, salivary glands; 3, dorsal reservoir; 4, ventral reservoir; 5, cana entering stomach; 6, stomach; 7, malpighian tubes; 8, hind-gut; 9, rectum; 10, wings 11, legs. (After Grassi, from Lang and Doflein.) of mosquitoes is to make it impossible for them to breed. The eggs of a mosquito are laid in water, and water is absolutely necessary for the larval and pupal stages, which must be passed through before the adult mosquito is produced. Fish destroy developing mosquitoes and large sheets of water are also too rough for them—so mosquitoes must have, for breeding, rather small collections of fresh water free from fish. Mosquitoes will soon disappear from a locality if all such col- lections of water, within a quarter of a mile of it, are filled up, drained, or covered with a film of coal oil so as to make it impossible for the mosquitoes to breed in them. ‘Those who live in a malarious district should protect themselves from mosquito bites by the careful use of mosquito-netting. By the simple observance of these evident indi- cations, malaria has already been banished from several localities in which it was formerly endemic. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 839 Basesta (Starcovici, 1893) This order is often called PrropLasmA. It includes several species of parasites, which cause diseases of considerable economic importance in horses, cattle, sheep, and dogs. One of the best-known species is Babesia bigemina, which causes Red-Water or Texas Fever of cattle. The parasites which are associated with the numerous babesiases are distinguished from one another by the host in which they are found, by slight differences in their morphology and by their inoculability into various animals. Rep WATER : Babesia bigemina—Smith and Kilborne, 1893 Red ‘water is one of the names given to a disease of cattle which is characterized by hemoglobinuria; in the United States it is often called Texas cattle fever. It is caused by Babesia bovis (bigemina) (Fig. 180). The parasite is transmitted by the bites of a tick, in North America, by Rhipicephalus annulatus. Red water occurs not only in the southern portion of the United States but almost everywhere in the tropics and in many of the warmer parts of the temperate zones. The parasite is a pear-shaped organism which usually lies within a red cell. It measures from 2u to 4u in length and about ry in breadth. In fresh preparations they appear as refractile bodies possessed of slight amoeboid movement; in stained preparations they are seen to consist of a blue-staining cytoplasm which contains a mass of chromatin at its broader end. Multiplication is accomplished by simple division into two or more parts; it is possible that schizogony and sporogony may also occur. ‘The parasites are often very scarce in the peripheral circulation but are much more numerous in the organs and particularly in the spleen. The disease can be transmitted, experimentally, from bovine to bovine by the inocula- tion of blood which contains parasites; normally, it is transferred from animal to animal by the bites of a tick. The species of tick which carries red water is not the same in all parts of the world. Ten days intervene between the bite of the infecting tick and the first sign of the infection. The temperature rises, it may be, to 106°, or more, and it remains high for a week. The animal is evidently very ill, it has no appetite, and it rapidly loses strength and weight. Many red cells are destroyed and anemia may be marked. The urine is albuminous and it is red because of the hemoglobin which it contains. Death may occur in very acute cases as early as the second day. Animals which recover from a severe attack are usually immune to the 840 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS disease. The immunity is not an absolute one, however, for blood taken from such recovered animals is often infective; the parasite probably exists in them in a latent form through the establishment of a tolerance on the part of the host. There is no specific treatment for babesiosis. Some of the aniline drugs, used in the treatment of trypanosomiasis, such as trypan-blue, are of some value. Many districts are kept free from red water by not allowing cattle coming from infected districts to enter them. Where it exists, the dis- ease is controlled by destroying the ticks on cattle with poisonous A B C. D E ‘g & Si 8 & 8 | ® F G H J K Fic. 180.—Babesia bigemina. Various stages of development in red blood cells. A, young parasite; B, a twin-form; C-E, a multiple division; F-K, large pear-shaped forms. (After Doflein.) washes and by occasionally plowing, or burning over, the pastures in order to destroy ticks which have dropped to the ground. In the United States, cattle on some farms are kept free from ticks, and conse- quently from red water, by a manceuvre which takes advantage of the way in which the tick transmits the disease. The adult tick remains upon her host until she is ready to deposit her eggs; she then drops off, lays her eggs and dies. The young ticks, hatched from these eggs, attach themselves to new hosts and it is through their bites that the disease is transmitted. Therefore, since the disease is transmitted by MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 841 the progeny of ticks which have fed upon infected mammals, susceptible cattle may be protected from the disease by preventing young ticks from reaching them. This may be done by not allowing them to feed over fields where ticks may have been dropped until sufficient time, about ten months, has elapsed for all the ticks and their progeny to have died of starvation. There are a number of parasites which although closely related to the Babesias are usually placed in other genera. Most of these have been shown to be transmitted by ticks of various species. East Coast FEVER ne: hemaocytozoin parvum—Meyer (Theiler, 1903) Syn.: Theilerio parvum The parasite causing this disease which is characterized by severe anemia is found in the red blood corpuscles of infected cattle. The intracorpuscular forms vary in form, some being slender and rod- shaped, the others being more rounded or pear-shaped. They may be arranged to form a cross, but this is not due to segmentation but to fortuitous grouping in heavily infected cells. They are regarded as gametocytes, for although such blood is infectious for ticks it will not produce infection when injected into normal cattle. The multipli- cative or asexual phase of the organism is restricted to certain organs, especially the lymph nodes, spleen and bone marrow. The tissue from these organs when injected into normal cattle produces infection. Theileria (Babesia) mutans is a parasite of cattle closely resembling T. parvum since it furnishes many rod-shaped forms in the red blood corpuscles. The cross-forms which occur in this species result from segmentation and since the blood is infectious when injected into normal animals it is evident that the asexual phase is present in the blood. This parasite produces no serious symptoms and does not appear to affect the health of the animal. OrovA FEVER Bartonella bacilliformis—Strong, Tyzzer and Sellards, 1915 A human disease characterized by rapidly developing and severe anemia associated with an irregular fever occurs in certain mountain valleys in Peru. The red blood corpuscles harbor slender rod-shaped 842 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS and small rounded organisms in numbers varying with the severity of the disease. The rods are frequently arranged in chains of two, three or even four, and present deeply stained granules at one or both extremities. Examined in fresh preparations they are found to move slowly without marked change of shape through the interior of the red cell. Cross-forms are rare and probably represent fortuitous arrangement rather than segmentation of the organisms. The endo- thelium of the blood-vessels of the lymph nodes, spleen and liver contain organisms in various stages of development, small rod-shaped forms similar to those of the red cells eventually being formed. The distention of the endothelial cells is often sufficient to occlude many, of the blood-vessels and in the lymph follicles of the large intestine this has apparently led to the necrosis of the surrounding tissue and ulceration. The organism of this disease is smaller than that of East Coast fever, the rod forms being very slender and nuclear material is not so readily differentiated. Its resemblance in other respects together with the similarity of its distribution in the tissues indicates a relationship to this group of organisms. The disease has not been transmitted to lower animals. Carrion, a Peruvian student, who inoculated himself with the blood of a patient suffering from Verruga peruviana died from a disease which may have been Oroya fever, although the evidence on this point is inconclusive. Oroya fever and Verruga peruviana not infrequently occur simul- taneously in the same individual, just as the latter disease is frequently complicated by malaria, and this together with the result of Carrion’s experiment led many Peruvian physicians to the erroneous belief that Oroya fever and Verruga were different stages or manifestations of a single infection. Verruga is, however, readily transmitted to lower animals. The mode of transmission of Oroya fever has not been conclusively determined. ANAPLASMOSIS In a pernicious anemia of cattle and at times in babesiasis the red blood corpuscles contain minute deeply stained, rounded bodies. These are frequently in pairs and are commonly situated near the margin of the cell so that they have been given the name Anaplasma marginale (Theiler), by those who are convinced of their parasitic nature. They have also been found in other domestic animals and similar structures MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 843 are found normally in all individuals of certain species, as for example, the mouse. Since the bodies of this general appearance which occur in normal animals are evidently to be regarded as nuclear material certain investigators are inclined to doubt the parasitic nature of Anaplasma.’ SarcosporipiA (Balbiani) Different species of this order are frequent parasites of all the domestic animals, of mice and, occasionally, of man. Mice are killed by them and it is possible that they may produce ill effects in men and domestic animals but no definite disease is associated with their presence. Though they may occur in any part of the body, they are most numerous in certain muscles, such as those of the larynx and cesophagus, which are near the alimentary canal. For this reason it seems possible that they may enter the bodies of their hosts with food, but our knowledge of their life history is incomplete. Myxosporm1 (Biitschli) There are many species in this group which are parasitic in fishes and certain arthropods but not in higher animals. The classification is based largely on the character of the spores produced. The latter are provided with a resistant membrane or shell and with polar capsules, each of which contains a coiled filament which when extruded serves to anchor the spore to the surface. The spores are produced continuously within the protoplasm of the mother organism which may be situated in any part of the body of the host. Severe infection with myxosporidia may cause boil-like lesions and the death of large numbers of fishes. Microspormi1A (Balbiani) Protozoa belonging to this order do not produce disease in man. They are the cause of a disease of bees, and they are of particular interest because one of them, Nosema bombycis, causes Pébrine, a serious disease affecting silk-worms (page 656). Inrusorra (Leddermiiller, 1763) Most of the parasitic infusoria occur in the alimentary tracts of their hosts. Harmless infusoria are found in the stomachs of many 844 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS herbivorous animals and also in the large intestine of the frog. Balantidium coli is a common and apparently innocuous parasite of the cecum and large intestine of the pig, but it may cause a severe inflammation of the large intestine in man which not infrequently proves fatal. One or two other infusoria occasionally produce similar symptoms in man. Other species of infusoria are parasitic on fish. Some of these are harmless, but some by finding their way into the _ gills or beneath the scales, cause serious diseases. BALANTIDIUM ENTERITIS Balantidium coli—Malmsten, 1857 Balantidium coli is the most important of the infusoria parasitic in man and may cause a form of dysentery. This organism measures about 150u in length and sou in breadth. It is covered with cilia; its cytoplasm is differentiated to form oral and anal areas and it contains digestive and contractile vacuoles. It multiplies by simple transverse division, either with or without a precedent conjugation. It may encyst, and this is the form in which the parasite is transmitted from one host to another. High enemata of mild antiseptics have been used in the treatment of this infection. PARASITES OF UNCERTAIN POSITION In Panama, there is a disease of man, somewhat resembling one form of tuberculosis, which is caused by a protozoén called Histoplasma capsulatum. The only known stage of this parasite greatly resembles the non-motile form of Leishmania donovani; but it contains only one, not two masses of chromatin. This organism is certainly a protozoén; although the genus to which it belongs has not been determined. Cutamypozoa (Prowazek, 1907) This name is given to certain bodies because their presence excites the cell containing them to produce a substance which surrounds them like a cloak. The exact nature of these bodies is disputed; it is even doubtful whether they are para- sites, or whether they are merely the expression of some morbid change, produced in the cells, by an unseen virus which causes the disease. They have been found in trachoma, a disease of the eyelids of man, in hydrophobia, in Molluscum contagiosum, a skin disease, in smallpox, in vaccinia, and in scarlet fever. They are mentioned with the protozoa because, if they are parasites, they are probably more nearly allied to the protozoa than to the bacteria, They are extremely small bodies, some measuring only 0.254 in diameter. They are spherical and occur within MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 845 the cells. In preparations stained by Romanowsky’s method they are colored like chromatin. ULTRAMICROSCOPIC VIRUSES (See page 116) SprrocH#ta (Ehrenberg, 1833) Many spirochetes are, apparently, harmless parasites in shell fish, in the ali- mentary canals of various animals and in the blood of fish, birds, and many mammals; other spirochetes produce disease in men and in lower animals. Several spirochetes are parasitic in man. Spirocheta dentium and Sphiro- cheta buccalis are harmless organisms which are found in tartar, about the teeth. Spirocheta vincenti occurs in great numbers in a certain form of sore throat. Other spirochetes have been found in foul ulcers, and others have been found in association with bronchitis and enteritis. All these are comparatively unimportant parasites. Spirocheta duttoni, Spirocheta obermeieri and Spirocheta pallidula are more important ones. AFRICAN TICK FEVER Spirocheta duttoni—Novy and Knapp, 1906 African tick fever is a disease caused by Spirocheta duttoni and transmitted by the bites of a tick Ornithodoros moubata (Fig. 181). EMenbinat = Fic. 181.—Ornithodoros moubata. (Murray from Doflein.) This disease exists in Central and Eastern Africa, wherever Ornith- odoros moubata occurs. A disease, which is probably caused by a spirochete, is transmitted by another tick, Argas persicus,in Persia. 846 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS In Central America a spirochete, which causes a disease almost identical with tick fever, is carried by Ornithodoros chinche. - Spirocheta duttoni is a slender organism measuring from 14y to 16u in length; its thread-like body lies in a number of waves, which vary greatly in number, according to the way in which the preparation is made; consequently, the number of waves is not a constant character which can be relied upon for the identification of this species of spirochete. This spirochete is composed of an outer ectoplasmic sheath, and of an interior composed largely of chromatin; the sheath extends at either end into flagellum-like prolongations. Multiplication is accomplished by transverse binary division. Sometimes, perhaps most often toward the end of an attack of fever the spirochetes coil up tightly, within a cyst-like matrix. Such encysted forms may lie within cells, z.¢., liver cells, and spleen cells; they are seen most frequently in the liver and spleen, and they are always present in the alimentary canal of ticks which have ingested spirochate-infected blood. The chro- matin of both free and encysted spirochetes may be frag- mented, more or less regularly. In the tick, cysts, containing Fic. 182.—Spiroc- 4 spirochete with fragmented chromatin, burst and set free heta duttoni. (After P a es a : Doflein.) the granules of chromatin. Some investigators believe that each granule develops into a spirochete, others that this represents a degeneration and destruction of the parasite. It is not impossi- ble that some such method of multiplication occurs in man. The form and the exact way in which the spirochete is transmitted by the tick is not known. It is probable that a tick, once infected, never loses its power to trans- mit the disease; the infection may be transmitted, from mother to daughter, through at least three generations of ticks. The ticks hide during the day and feed at night. The wound produced by theis bite is insignificant. , An incubation period of about five days intervenes between the tick bite and the appearance of eee The fever is characteristic; it rises rapidly to, perhaps, 105° and it remains high for from three to five days. | It then falls suddenly and there is no fever for from five days to two weeks. Then the temperature rises again and there may be from three to six such recurrences of fever before the illness ends. The definite periodicity of the relapses probably depends upon some more or less regular developmental change in the spirochetes since the latter are always most numerous in the blood during the height of the fever. The disease is not often fatal and “606” is a specific treatment for it. It can be easily prevented by avoiding tick bites. MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 847 RELAPSING OR RECURRENT FEVER Spirocheta recurrentis—Lebert, 1874 This is still a common disease in some parts of Europe. Its symp- toms are almost identical with those of tick fever; and the spirochete causing it, formerly called Spirocheta obermeieri, can only be dis- tinguished from Spirocheta duttoni by the fact that an animal which has recovered from an infection by one of these parasites is immune to inoculation with it, but is susceptible to an inoculation with the other spirochete. The means by which relapsing fever is transmitted is not known; it is probably carried by the bites of lice, or bedbugs. TreponeMA (Schaudinn, 1905) Two species of this genus are very important parasites. SYPHILIS Treponema pallidum—Schaudinn, 1905 This disease, in all its diverse forms, is caused by Treponema pallidum. The treponema is an exceedingly slender, thread-like organism, with a waved body which measures from 6y to 14» in length (Fig. 183). It greatly resembles the spirochetes, but differs from them in having each end drawn out to resemble a very slender flagellum. Very little is known of the life history of the treponema except that it multiplies by transverse division. It is transmitted by the contact of a lesion, containing the parasites, with the broken skin, or with a mucous mem- brane of an uninfected person. The symptoms of syphilis are attributable to continuous mild injury, and destruction of the tissues of the infected persons by the treponema and by its toxic effects. It is a parasite of the connective tissue. Mercury and potassium iodide were formerly almost exclusively employed in treating syphilis. The search for an efficient drug for the treatment of trypanosomiasis has led to the discovery of other drugs which are of value in the treatment of syphilis, such as atoxyl (the sodium salt of para-amido-phenyl-arsenic acid) and its acetylated derivative, and of dichlorhydrate-diamido-arseno-benzol. The last- named drug has proved of great importance in the treatment of syphilis. 848 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS YAWS OR FRAMBOGSIA Treponema pertenue—Castellani, 1905 This is a disease of the tropics which was formerly confused with syphilis. It is characterized by the presence, on any part of the body, of more or less numerous warty fungoid lesions which tend to ulcerate. In this disease a primary lesion appears after a period of incubation and this is followed after another interval by a general eruption. It is not BA Fic. 183.—Treponema pallidum (in centre) and the Spirocheta refringens. (Greene’s Med. Diagnosis) a venereal disease as is the case with syphilis. It is caused by a slender spirochete, Treponema pertenue, which is morphologically identical with the organism of syphilis. Animals which have been immunized to one of these diseases are found to be still susceptible to the other. The organisms in yaws are present in enormous numbers in the hypertrophied and swollen epidermis of the lesions whereas in syphilis they are confined to the deeper tissues. The disease responds more favorably even than syphilis to treatment with salvarsan which may MICROBIAL DISEASES OF MAN AND DOMESTIC ANIMALS 849 be thus regarded as practically a specific. It responds positively to the Wassermann test and is more uniform with respect to this test than syphilis. OTHER SPIROCHZTAL DISEASES ULCERATION GRANULOMA OF THE PUDENDA is a tropical disease which has been claimed to be caused by a spirochete, although its etiology still remains uncertain. Spirochetes cause diseases of geese in southern Russia and of fowls in Brazil and in other tropical countries. The spirochete of fowls, Spiro- cheta gallinarum, P. Blanchard, is transmitted by a tick, Argas miniatus; the means by which the goose spirochete, Spirocheta anserina, Sacharoff, is carried is not known. 54 CHAPTER Iv* CONTROL OF INFECTIOUS DISEASES PRINCIPLES That the infectious diseases can be controlled depends upon the facts that they arise only in the presence of a specific living infective agent; that they pass from patient to prospective patient only because the infective agent passes from patient to prospective patient; and that therefore the prevention of effective passage will prevent. the spread of the disease. These preventive measures with their natural incidental developments constitute the practice of present public health relating to these diseases. In general the infective agent leaves the body of the patient by the mucus-lined orifices of the body, the nose and the mouth, the anus, the urethra, the mamma, and the genital organs. In general it must, if it is to infect successfully another person, reach one or more of the same mucus-lined orifices of that other person. Excluding the venereal diseases the ordinary infectious ‘diseases (tuberculosis, typhoid fever, diphtheria, scarlet fever, measles, whooping cough, smallpox, chicken- pox, pneumonic plague, leprosy) are received almost exclusively into the body through the mouth (or nose). While the passage is usually from mucous membrane to mucous membrane as above outlined, the infective agent may pass effectively from mucous membrane to cut or abrased skin (the uninjured skin is probably almost always resistant to these infections). Again, in those diseases where skin lesions are a: prominent feature (smallpox, plague, leprosy) the infective agent may pass from the skin lesions to a mucous membrane, or to a cut or abra- sion. But these are rare methods of transmission as compared with the mucous to mucous forms, except in syphilis and chancroid where they frequently occur. The routes of travel between the patient and the prospective pa- tient are many. At times, mucous membrane may be applied to * Prepared by H. W. Hill. 850 CONTROL OF INFECTIOUS DISEASES 851 mucous membrane as when a well person kisses a diphtheritic child; conveyance of particles through the air, sprayed from the mouth, may occur, as when a diphtheritic patient coughs into an attendant’s face; or mucous membranes may be applied to skin or vice versa, as in the kissing of a smallpox patient; but in general the discharges are con- veyed somewhat indirectly. The prime route from mucous membrane to mucous membrane is furnished by the hands. An attendant touches the patient’s lip or wipes out the mouth or otherwise performs toilet services and receives the discharges upon his fingers. The fingers go then to the attendant’s mouth directly, or touch something (the tines of a fork or the bowl of a spoon, etc.) which in turn goes into his mouth; or the attendant may touch the fork or spoon or food of others and thus they become infected. He may milk a cow and so get the discharges into the milk. With the infection in his own mouth he may kiss others and transfer it to them. It is impossible to outline the infinite combinations that may occur, but the principles are here made obvious. When the infective discharges handled are those of the bladder or bowel (as in typhoid fever, cholera, etc.) the same dangers of trans- mission are encountered and unfortunately too often realized. The wholesale discharge of sewage into water supplies is merely a gross example of the same principle of transfer of discharges from human bodies to the human mouth. Another factor in the transmission of disease (as distinguished from the transmission of the germ) is the condition of the infectee. The germ is analogous to a seed; the methods of transmission are somewhat analogous to the distribution of seeds in nature; the condition of the infectee is analogous to the character and nutritional condition of the soil which the seed reaches. If for any reason the germ will not develop in the soil where it is planted, or, still further, if it grows but fails to produce those poisons through which alone it acts, or finally if, growing and producing its poisons, the soil neutralizes the poisons, no disease results. Science, logic, and the law (each of which regards itself, and rightly so, as merely an apotheosis in its own line of “common sense’’) unite in the dictum that a disease exists only when the normal functions of the body are in some way interfered with to the detriment of the body. The mere in- fection of the body with a disease germ does not, in science, logic, or the law, constitute disease. Hence, the reception of a disease germ 852 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS into the body is but the first of three essentials, the other two being poison-production by the germ and poison-action on the _ tissues. Many persons are insusceptible to the poisons of one or more disease germs. In whatever way this insusceptibility originate, (natural, acquired by a previous attack, or acquired by artificial treatment) the existence of insusceptibilty tends to prevent the acquiring of the disease. PRACTICE Undoubtedly, the one wholly efficient method of preventing the spread of infectious diseases would consist in immunizing all the possible infectees against all the possible diseases. Unfortunately, we know of no practical immunizing methods except in the case of a very few diseases, notably smallpox and typhoid fever, paratyphoid and cholera. Our methods of control of any disease therefore begin with the attempt to destroy them at their origin in the body of the patient, but ‘ such methods are merely incidental to the destruction of the germs for the good of the patient himself, i.e., they belong rather to therapeusis than to public health. Unfortunately, also, scarcely any efficient method of destroying bacteria within the body of the patient without destroying the patient also is known and therapeusis along this line contents itself largely as yet in so controlling the patient’s condition as to permit and encourage to the highest the natural forces of the body to attack the germs. These natural forces, however, direct their chief energies and secure their chief results, not in destroying the germ but in neutralizing the poisons the germs throw off, and in practice, patients recover rather because they have neutralized the poisons than because they have killed or ejected the germs. For this reason a recovered patient often remains a breeding ground for the germs which caused the attack, but to whose poisons he is now resistant or immune. ‘ Practically, then, the germs must leave the patient’s body before they can be destroyed. It is at this stage that the most efficient control can be exercised, and that control consists in killing them before they become scattered. In practice the efficient disinfection of all the discharges of a patient will prevent the spread of any disease from him. But this is not as easy to do as at first might appear. Ridding the CONTROL OF INFECTIOUS DISEASES 853 body of its discharges in health is a process dependent on the individual, carried out by him consciously or unconsciously all his life by methods chiefly directed to conserve convenience rather than to prevent their spread. In health, the careless scattering of these discharges is not of great moment, but of course the habits of indifferent and careless discharge, acquired in health, persist after disease is con- tracted. The presence of an infective agent in the discharges renders the previously harmless scattering of the discharges the greatest menace that is known to the health of the associates. Hence one primary requisite in the personal warfare against the infectious diseases is to establish among all people such habits during health that even the normal discharges are not exchanged. This must be achieved by teaching the individual not to scatter his discharges and by teaching his associates not to receive them, if he does. Accepting conditions as they are, the care of the sick by watchful, well-trained nurses who will prevent the spread of the discharges must ‘ largely take the place of the earlier training of the patient. Usually this also is impossible. It would seem that at least 95 per cent of the total cases of infectious disease in this country are cared for at home by the home folks, z.e., untrained, worried, exhausted mothers chiefly, trying to learn in the actual face of the enemy, the technic and knowl- edge acquired quietly and systematically by the trained nurse. Hence, within the home, and at present, sanitary nursing to prevent spread of disease is a poor and often broken defence. The third method of control is the destruction of the germs in pas- sage from patient to prospective patient; and this must be largely con- fined to the actual discharges when accumulated in one place; the finer discharges thrown into the air can hardly be followed. Under this head may be classed the disinfection of feces and urine, the disinfection of bed clothing, eating utensils, etc., coming into con- tact with the patient, and especially the disinfection of the hands of attendants. The throats of attendants often contain the germ, espe- cially when diphtheria, scarlet fever, measles, etc., are concerned. Unfortunately, the disinfection of the throat is extremely difficult and the scientific nurse will take every precaution to avoid receiving the germ into the mouth, rather than try to dislodge or destroy it after its reception. The use of a respirator is useful. As outlined in the preceding section, the principles involved in con- 854 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS trolling infectious diseases are very simple, but in practice the individual cannot be trusted to avoid spreading his discharges, partly from ignor- ance, partly from carelessness, often from mere ingrained bad habits regarding the disposal of discharges, especially those of nose and mouth, indulged unconsciously by those who both know how and mean to be . careful. . This would matter little were the infected persons always so sick as to be confined to the house or to bed, especially if during such confine- ment their discharges were under strict surveillance by scientific trained nurses. But since many, perhaps half, of the infected persons are not sick enough (if sick at all) even to remain at home; since, also, even severe cases, under surveillance in bed during the height of the attack, have a prodromal stage and a convalescent stage during which they are going about although infective, it is not hard to see that the population of any community is likely to embrace at any time infective persons at - large—persons who may or may not be aware of their own condition. Theoretically and practically, then, the official control of infectious diseases must begin with the blanket assumption that the discharges of every individual must be confined to himself and especially prevented from reaching, through any public utility, the mouths of other citizens. Official control of the exchange of discharges by the individual within the family and in the absence of any specific proof that the discharges are infective, is impossible, although through various agencies the indi- vidual may be urged to that end. The moment, however, that the individual or the family engage in any occupation which permits them to inflict their discharges upon others, especially through food or milk, that moment should the individual or family come under official cognizance, their methods be inspected and their infectiveness esti- mated. The same arguments apply to aggregations of individuals | from different families. So long as private meetings are held, it is difficult to supervise or prevent exchange of discharges. But public and especially compulsory meetings, at school, at church, at theatre, etc., should receive official attention. Provision should be made con- cerning all such meetings that they be held only in suitable places, without overcrowding. The exclusion by the officers, attendants, or the general public of all known to be infected or suspected of- infection and of all who more openly disregard ordinary rules of decency in the CONTROL OF INFECTIOUS DISEASES 855 disposal of discharges (spitting, etc.), should be part of the duties of the health department. Finally, the strictest supervision of those concerned publicly and officially in the handling of public utilities on a large scale (water sup- plies, milk supplies, hotels, restaurants, food stores, etc.) should hold all strictly accountable for the contamination of such supplies with dis- charges whether these be normal or not. Hence official control of infectious disease divides itself naturally as follows: 1. The recognition and isolation of frank cases of the diseases in question, at home or, better, in a proper hospital. ‘2. The supervision of the attendants and immediate associates of such frank cases. (a) To detect among them that one from whom the frank case, already recognized, received his infection. (6) To detect at the earliest moment any other frank case about to develop from among those associates who may have been infected at the same time and from the same source as the frank case already found. (c) To prevent further spread from any already infected associates or those who may become infected by later association with the frank case during its existence as such. 3. The exclusion of the frank cases, their attendants and immediate associates, from participation in public life so long as danger continues and especially their exclusion from having to do with public utilities or public gatherings. Hence has arisen the crude drastic but efficient (when consistently and uniformly carried out in every case) system of isolation of the sick and quarantine of his associates. Unfortunately quarantine has become a mere letter-of-the-law pro- cedure, working great hardships on those who conscientiously sub- mit to it and yet failing to achieve its objects because of the great number of those who evade or escape it; moreover, because its provi- sions are unintelligently enforced. Of what avail is rigid quarantine of an infected family where milk continues to be sold from the same premises? Why quarantine the honest man who has an honest physician and whose case is reported, while his neighbor, having the same disease in his family, calls no physician, or a dishonest one, and therefore escapes official cognizance? The only remedy seems to be the recognition of the principle that harboring or having in possession a case of infectious disease, unknown 8 56 MICROBIOLOGY OF DISEASES OF MAN AND DOMESTIC ANIMALS to the proper officials, is a crime against society, and that the excuse that the person harboring such case did not know it to be such should be of no more weight than the plea of ignorance of the law which ‘is not allowed in other and often far less serious matters. The official isolation of infectious cases involves also official responsi- bilities regarding the release from isolation after the acute attack is over. Officially to declare a person dangerous to the community does no harm to the community if a mistake is made. An official declaration that a person is no longer dangerous and is therefore free to enter into the community life again may, if mistaken, result in a widespread outbreak. No more delicate task confronts the public health official than the making of this decision. In diphtheria, the examination of cultures from the throat and nose of the person in question and the repeated failure to find the bacterium of diphtheria is usually considered a safe criterion. In scarlet fever, complete and continued restoration of the throat and nose to normal conditions, together with absence of ear discharges, should be required, yet is not perfect; for it is not very unlikely that the scarlet fever infective agent, whatever it may be, can continue in a recovered scarlet fever throat as the diphtheria bacterium may remain in a recovered diphtheria throat. In other diseases the decision is based on similar lines—the disappearance of crusts in smallpox and chickenpox, of discharges in measles, on restoration to normal of whooping cough; but in all these diseases the analogy with diphtheria may hold to a greater or less extent. In tuberculosis, the patient is infective as long as Bact. tuberculosis can be found in the sputum; in typhoid fever the patient is likewise infective as long as the urine or faces show the typhoid bacillus. In these two diseases, however, quarantine or even isolation is not officially carried out nor release from restriction officially given to any great extent or with any marked uniformity. Full sanitary nursing precautions regarding a typhoid fever patient’s discharges should continue for an average of three months after recovery. i ' Methods of Handling the Common Infectious Diseases, London, Canada. January, 1916, Published by the Institute of Public Health, London, Canada ISOLATION PERIODS FOR THE PATIENT | Chickenpox, Fhooping 6 Mumps, and Smallpox Searlet Fever Measles Diphtheria Typhoid Fever The patient, whether at hospital, at Ch. Pox—After all scabs are ith nurse or at peat : About 6 weeks from firet appearance |About 3 weeks from the date first} Wh. Cough—About 6 weeks ter first whoop. roe, permitted to return ||After all scabs are gone, as deter-} of rash. provided recovery is com-| sick, provided recovery Aft negative b aaa weeks from first symptoms,|Mumps—4 weeks from to school, work, ete., in mined by M. O. H. plete, cs determined by M. O. H.| plete, as determined by M. O. H.| from the nose utiy throat. ded eee y is complete as de-|G. Measles—About 3m from first ap- Rimined by M. O. H. four diseases recovery is com- Cee Tate byM.0.H. | PLACARDING OF HOUSE IN WHICH THE CASE OCCURS If isolation of ‘ Chickenpox, Goma ets Mumps and patient is Smallpox Scarlet Fever Measles Diphtheria Typhoid Fever 1, At hospital. Is not placarded. 1a not placarded, Ts not placarded. Is not placarded. le not placarded. Ts not. placarded. ca e hous ej2. At home with nuree.|/[s placarded. Ia placarded. Is placarded. Is placarded. Ia not necessarily placarded, Is not necessarily placarded. re case . occurs, 3. At home withoue}[ts placarded. Is placarded. Is placarded. Is placarded. Is not necessarily placarded.. Is not necessarily placarded. » nurse, : rt QUARANTINE PERIODS FOR THE CONTACTS (THOSE EXPOSED) Classes of If isolation of Chick = i 3 contacts. patient is Smallpox Scarlet Fever Measles Diphtheria Typhoid Fever Boning eee eine After 3 i) tive cult Aten le 14 days if i tactal 3 (a) After 14 days, if non-immune| @), Alter 5 days, if non-immune con-|(a) After 14 days if non-immune con-| eI a a remaining (n the house. ee ey r ntined in ‘tou ie bas A. *1, At hospital. contacts are qui uarantingd inhouse.| tacts are quarantined tacts are auarantin use, |(b) On change of residence after 3 consec-| era Ate ace, rit no (a) As in corresponding case of Measles with Immunes (those| iy. At once, if no & ) At once, if not, 'b) At once, if n utive negative cultures from all con-/(c) At once on change of residence. of time as follows: who have had (c) At once, on change of residence.|(c) At once, on change of residence.|(c) At once, on ‘change of residence.| tacts in such residence. a pons alts 6 dave business in-|*2. At home-with nurse| ditto ditto ditto ditto ditto Moe Satter a diye ue volving tact with|*3. At home without! (a) at days after release of patient @), 5 days after release of patient|(a) 14 days after release of patient! 14 aft lease food. milk or! nurse. from leplation if Fon Unmnune con- froma isolaslon if jon tamu con-| ; from: isolation if pon lmmtinec con- Fr elation noxeamane paeiene {rom nm arej* quarantin use. re quarant in house. are quarantined in ieee ti rel aac i (On On feleane of patient ‘trom isola-| (o),0n On felease of patient from isola- ko) On release! of patient from isola-| ne. (by On release a patient from isolation,| ey At ones if ot serociatiae, wih Datient. er ion, if not. ‘ on, lon, (c) At once, on change of residence. |(c) At once, on change of residence. |(c) At once, on change of residence. (c) At co eace on change of residence. *1. At hospital. ‘As for Immunes, Class A. 1. At once. At. once. ‘Same as for immunes, Class A., except |At once. At once. 2 inetead of 3. ; tn. *2, At home with nuree]|As for Immunes, Class A. 2. At once. At once. ditto At once. . At once. are released, (a) At once, if not associated with/(a) At once, if not associated with patlent or non-immune contacts. atient or non-immune contacts, #3. At home without//As for immunes, Class A. 3. (b) At once on change of residence. |(b) At once on change of residence. ditto At once, if not associating with patient.|At once. nurse. : i bate Ch. Pox—after 16 days. AL #1. At hospital. After 14 days. After 5 days. After 14 days. Same as for immunes, Class A. After 14 days. Mpevsaeer 31 dave . fen iem im mui nes} iG. Measles—after 14 days. t a) the]*2, At home with nurse}/14 days after release of patient fi After 5 days. After 14 days. de tn oleh va y! v8. ditto After 14 days. ditto usiness ; volving con-|*3. At home without]/14 days after retease of patient from fay 6 days after release of patient! a) 14 days after release of patient! . release ‘i itto—the periods tac tow ith] nurse, isolation, from isolation, ‘ irom isolation. ee ditto eo ay efter of patient from) Dig of quarantine to date ee mil ‘food or (b) ), After 5 days on change of rest- (o) ), After 4 days on change of resi- (b) After 14 days, on change of residence. change of rae a pales leased, 8 iy ca wee ting dal: 3 once, if reporting daily to a| At once, i At once, if H ‘oe ‘e *1 At hospital. As fof non immues, Class A. 1, After 5 days. physician, ce nurse with knowledge/Same as for immunes, Clasa B. symptoms toa physician.” to a physician. any munes are r leased, © *2, At home with nurse||As for non-immunes, Class A. 2.- After 5 days. ditto ditto At once, if any At once, 6 symptoms to a physician. toa Shysician, a #3, At horhe without] After 5 days if not associating with|Ditto, if not associati nurse. JAs for non-immunes, Class A. 3. patieut of nonimmune contacta. | or other contra ote on sitio JAt once if not assoclating with patient. |At once, if not associating with patient or other Non-immune contacte. | REGULATIONS REGARDING VISITORS TO A QUARANTINED HOUSE If isolation of coall Chicke: ‘Whoopi Mi patient is Smallpox Scarlet Fever Measles Diphtheria ‘Typhoid Fever mpm eran Menakes Mumps and Fe ital. “|/Non-i ded. All child: id li Al nature of food poisons, 495 relations of nitrogen fixation, 339 Chemical preservation of foods, 479 after-storage changes, 480 of butter, 482 of fish, 480. of fruits, 482 influence on food, 479 of meats, 480 storage, 480 of vegetables, 482 Chemicals, influence upon organisms, 270, 393 Chemotaxis, 230, 231 Chemotropism, 230, 231 Chicken cholera, 756 pest, 803 pox, 801 Chitin, 148 Chlamydospore, 37 Chlamydothrix hyalina, 110 Chlamydozoa, 136 Chlorophyl, function of, 141 Chloroplastids, 20 Cholera, Asiatic, 798 INDEX Cholera, chicken, 756 hog, 807 Chondriosomes, 19 Chondrium, 42 Chromatic reduction, 35, 127 Chromatin, 16, 122 grains, 16, 122 Chromatium okenii, 91, 92, 103, 104 Chromidium, 18, 123 structure, 18, 19, 20, 42, 44, 46, 62, 63 Chromogens, 175 Chromophorous bacteria, 176 Chromoplastids, 20 Chromoporous bacteria, 176 Chromosomes, 28, 127 Cider, 530 composition of, 530 microérganisms of, 531 Cilia, 124 Citric fermentation, 164 Citromyces, 164 \ Citrus canker, 612 Cladosporium, 55 herbarum, 55 Cladothrix dichotoma, 112, 256 Claviceps purpurea, 494 Climatic influences on soil, 295 Clostridium americanum, 342 Coagglutinins, 714 Coagulating basins, 268 Cocci in milk, 384 Coccidiosis, 833, 834 Coccidium, 134 avium, 834 cuniculi, 135 hominis, 503 schubergii, 18, 126 Cocoanut rot, 620 Coenocytes, 40 2 Cold as preservative, 471 Coleosporium senecionis, 29 Colpidium colpoda, 307 Colpoda cucullus, 307 Colymbetes fuscus, 644 Commensals, 130 INDEX Complement, 706 deflection of, 708 deviation of, 707 fixation of, 709 Complementoid, 706 Complementophile, 706 Composition of cell, 148 cell contents, 148 wall, 148 moisture, 148 Composition (mechanical) of soil, 293 Compressed yeast, 551 Concentrated milk, 439 Condensed milk, 438, 463 7 sweetened, 438 unsweetened, 437 Conidiophore, 37 Conidium, 37 Conjugation, 128 Conjunctiva, 692 Contact infection, 668 Contagious abortion, 758 bovine pleuro-pneumonia, 804 Contents, xiii Control of infectious diseases, 850 practice of, 852 principles of, 850 Copper in soil, 362 Copra, dried, 455 Coprecipitin, 720 Copula, 128 Copulation, 128 Corn, canned, 466 sterilization, 466 Cow, body, 376 cleanliness, 377 individuality, 375 Cowpox, 805 Cream, cultures for ripening, 415 ice, 447 microérganisms in, 416 pasteurized, 415 ss raw, 415 ripening (spontaneous and con- trolled), 411 873 Crenothrix, 107, 108 polyspora, 107, 108 Cresol, 237 Crithidia, 132 Crown gall, 605 Cucumber, angular leaf-spot, 613 Cultures, commercial, 413 pure, 415 in beer, 528 in oleomargarine, 416 in pasteurized cream, 415 in raw cream, 415 in vinegar, 543 in wine, 518 Curd, acid, 351 manipulation of, 426 rennet, 425 Curdling of milk, 425 acid, 420 rennin, 420 Curing by chemical preservation, 479 Cutaneous orifices, 688 Cyanophycee, 17, 21, 22, 24, 30, 92, 95 nuclei of, 17 Cybister laterimarginalis, 644 Cycle of development, 97, 128 Cysts, 37 Cytase, 194, 525 Cytology, 15 bacteria, 87 elements of, 15 molds, 40 protozoa, 123 yeasts, 61 Cytolysins, 704, 709 Cytoplasm, 18, 42, 122 appearance, 18 properties, 18 Cytotoxins, 705, 709 DAIRY, microérganisms of, 265 Decomposition of butter, 417 of insoluble food, 189 of organic matter, 315 of urea, 146, 279 Delhi boil, 826 874 Dematium, 41, 44, 57 pullulans, 57 Dengue, 806 Denitrification, 333 environmental relationships, 335 experimental study, 333 Dermatomycosis, 726 Desiccation, 211, 450 Deviation of complement, 707 Dextrose fermentation, 142, 163 Diastase, 190, 194, 195 Digestive tract, 496 microérganisms, 496, 499, 500 - study of, 496 Dilution, influence on organisms in water, 264 Diphtheria, antitoxin, 576 fowl, 756 human, 766 manufacture of antitoxin, 575 toxin, 576 unit of antitoxin, 503 Direct division of cell, 34 Dirt in milk, 399 Disaccharide fermentation, 163 Diseases, animal carriers of, 667 contagious, 660 control of, 850 human carriers of, 667 infectious, 659, 724 non-inheritance, 685 of insects (below), 632 American foul brood, 646 bacterial disease of the gut epithelium, 652 European foul brood, 649 flacherii (silkworm), 635 graphitosis, 645 of the June beetle larve, 634 of locusts, 637 pebrine, 656 sacbrood, 653 septicemia, of caterpillar, 643 of cockchafer, 649 of larve of Lamellicornia, 651 wilt disease of gypsy moth, 654 INDEX Diseases of man and animals (below), 659, 724 abscesses, 741 actinobacillosis, 730 actinomycosis, 727 African tick fever, 845 amoebic dysentery, 822 anthrax, 750 Asiatic cholera, 798 bacillary white diarrhcea of chicks, 754 balantidium enteritis, 844 barbers’ itch, 726 boils, 741 botryomycosis, 733 canine distemper, 803 cattle plague, 803 cerebro-spinal meningitis, 736 chicken cholera, 756 pest, 803 pox, 801 cholera, Asiatic, 798 chicken, 756 hog, 807 chronic bacterial enteritis, 753 coccidiosis, 833, 834 contagious abortion, 758 contagious bovine pleuro-pneu- monia, 804 cowpox, 805 Delhi boil, 826 dengue, 806 dermatomycoses, 726 diphtheria, fowl, 756 human, 766 dourine, 832 Dukes disease, 801 dysentery, amoebic, 822 bacillary, 764 East coast fever, 841 entero-hepatitis of turkeys, 824 erysipelas, 744 favus, 727 foot- and-mouth disease, 807 foot-rot of sheep, 784 foul brood of bees, 846 INDEX Diseases of man and animals (below), fowl diphtheria, 756 German measles, 801 glanders, 767 gonorrhcea, 734 hemorrhagic septicemia, 772 herpes tonsurans, 726 hog cholera, 807 horsepox, 805 ; sickness, 811 human trypanosomiases, 831 infantile paralysis, 811 infectious mastitis, 738 influenza, 770 kala azar, 825, 826 leishmaniasis, 825, 826 leprosy, 774 louping ill, 812 lyssa, 814 malaria, 834 malignant oedema, 785 Malta fever, 746 measles, 801 milk sickness, 785 in milk, 385, 401 mumps, 801 mycetoma, 730 mycotic lymphangitis, 731 oroya fever, 841 osteomyelitis, 741 pebrine, 656 pellagra, 495, 813 plague, 776 pneumomycoses, 724 pneumonia, 747 puerperal septicemia, 744 pyemia, 741 pyorrhcea aveolaris, 498 rabies, 841 red water, 839 telapsing fever, 847 ting worm, 726 scarlet fever, 801 septicemia, general, 744 hemorrhagic, 777 puerperal, 744 875 Diseases of man and animals (below), sheep-pox, 805 sleeping sickness, 827 smallpox, 801 spirochetal diseases, 845 staphylococcic infections, 741 streptococcic infections, 744 swamp fever, 819 swine erysipelas, 778 symptomatic anthrax, 787 syphilis, 847 tetanus, 789 Texas fever, 839 thrush, 725 tick fever (African), 845 trichomycosis, 726 trypanosomiases of animals, 831 of humans. 831 tuberculosis, 779. typhoid fever, 792 typhus fever, 820 white diarrhea, amcebic, 834 bacillary, 754 whooping cough, 771 wounds, 741 yaws, 848 yellow fever, 821 of plants (below), 588 blight (stem) of alfalfa, 590 of beans (bacteriosis), 592 of lettuce, 593 of mulberry, 594 of oats (blade), 595 of pear, 597 ~ of peas (stem), 595 of peas (streak), 601 of tomato, 602 of walnut, 602 galls and tumors, 605 crown gall, 605 finger and toes, 608 olive knot, 608 tuberculosis of sugar beet, 611 leaf spots, 612 876 Diseases of plants (below), leaf spots, angular leaf spot of cu- cumbers, 613 citrus oe 612 larkspur, 614 plum and peach, 614 sugar beets, 615 tots, (black) of cabbage, 617 of calla lily (soft rot), 623 of carrots, other vegetables, 623 | of cauliflower, 622 of cocoanut (bud rot), 620 of hyacinth, musk melon, 624 of orchids (brown rot), 621 “of potatoes (basal stem rot), 619 of sugar beets, 626 Walker’s hyacinth disease, 618 wilts of cucurbits, 627 Irish potato, 629 sweet corn, 628 tobacco, 629 tomato, 629 predisposition to, 685 protection against (See protec- tion), 688 of vinegar, 549 of wine, 521 Disinfectants, 232 acids, 236 alcohols, 237 alkalies, 236 classification, 235 essential oils, 238 factors influencing, 234 gases, 238 hydrocarbons, 238 mode of action, 232 salts, 239 Disinfection, concurrent, 856 methods of, 857 terminal, 856 Dissemination of microérganisms, 667 Distribution of bacteria in soil, 308, 309 INDEX Distribution of pathogenic micro- organisms, 662 Dorset-Niles serum, 568 Dried foods, beef, 456. copra, 455 eggs, 456 extract of meat, 456 , fish, 456 gelatin, 456 jam, 455 jellies, 455 jerked meat, 456 macaroni, 455 milk, 456 molasses, 455 pemmican, 456 syrups, 455 somatose, 456 Drosophylla cellaris, 549 Drought in soil, 292 Drying, methods of, 452 Dukes disease, 801 Dust infection, 665 influence on milk, 374, 377 Dysentery, amcebic, 822 bacillary, 764 serum for, 581 Dyticus pisanus, 644 EAST coast fever, 841 Ectoplasm, 19, 122 Egg, dried, 456 refrigerated, 475 Eimeria stieda, 135 Electricity, influence of, 226 Emmenthaler cheese, 434 Empusa musce, 633 Emulsin, 191, 194, 197 Encystment, 129 Endomyces decipiens, 40 fibuliger, 40, 41 magnusii, 41, 42 Endoplasm, 19, 122 Endothelial tissues, action of micro- organisms on, 682 Endotoxin, 676 INDEX Energid, 15, 40 Energy of, aerobic processes, 155 anaerobic processes, 155 of food, 152 microdrganisms, 145 supply, 145 Entameeba, buccalis, 497, 498 coli, 502, 504, 822 . hystolytica, 18 meleagridis, 824 tetragena, 661 See amceba also. Enteritis, chronic bacillary, 757 Entero-hepatitis of turkeys, 824 Entomogenous fungi, 58, 634 Entomophthoracea, 58, 633 Enzymes, 178, 190 alcoholase, 194, 200 amidase, 194, 201 amylase, 194 casease, 427 cellulase, 194 ’ classification of, 194 coagulating, 194 cytase, 194, 525 diastase, 190, 194; 195, 525 emulsin, 191, 194, 196 endo- 194, 195, 196 enzymes of yeast, 525 erepsin, 194, 201 energy liberated by, 200 ereptase, 190, I9I, 194, 197 exo-, 194 hydrolizing, 195 inulase, 194 invertase, 190, 191, 194, 195, 200 katalase, 194, 202 lactacidase, 194, 200 lactase, 194, 195, 200 lipase, 190, 196, 200 maltase, 194, 195, 200 nuclease, 194 oxidase, 194, 198, 200 oxidase of vinegar, 194, 202 oxidizing, 202 pepsin, 190, 194, 198, 200, 525 877 Enzymes, peroxidase, 194, 202 properties of, 191 proteases, 194, 196 proteolytic, 194, 196, 201 upon albumin, egg, fibrin, gelatin, milk, serum, 199 pyocyanase, 244 taffinase, 194 reducing, 194, 202 reductases, 194, 202 rennet, I9I, 199 reversibility of, 205 steapsin, 194, 196 thrombase, 194, 199 trypsin, 194, 198, 200 tyrosinase, 194 urease, 194, 200, 201 zymases, 194, 200 zymatic, 194 Epidemics from milk, 401 Epiplasm, 27, 44, 67 Epithelial tissues, action of micro- Organisms on, 682 Epitoxoid, 704 Equations of fermentations, 158 Equatorial plates, 28 Erepsin, 201 Ereptase, 190, 194, 198 Erysipelas, 744 Erythrocytes, action of microérgan- isms on, 682 Eubacteria, 77 Euglena splendens, 31 viridis, 21 European foul brood, 649 Exhaustion theory of immunity, 721 Extracellular fermentation, 189 FACTORY refuse (canning), dis- posal of, 469 Facultative aerobes, 157 anaerobes, 157 parasites, 660 saprophytes, 46 Fat, fermentation, 168, 197, 278, 318 butter, 408 878 Fat, food, 151, 153 milk, 264, 408 ; sewage, 278 soil, 318 as food, 151, 153 Favus, 727 Feces, microérganisms of, 500 Feeding influencing milk, 374, 375 Fermentation, abnormal in milk, 396 acetic, 165, 512 alcoholic, 159, 164, 518 distilled, 536 of amygdalin, 197 beer, 527 bread, 552 of butter, 160, 166, 417 butter-milk, 446 butyric, 166, 515 of canned food, 469 carbon dioxide, 142 of cellulose, 167, 277, 303, 315 of cheese, 427 of cider, 530 citric, 164 curdled milk, acid, 395 sweet, 397, 425 of dextrose, 142, 163 of disaccharides, 163 energy of, 200 equations of, 158 extracellular, 189 of fats, 168, 197, 278, 318 of flax, 557 formaldehyde, 238, 486 ginger beer, 533 glycerin, 169 hippurric acid, 173 hydrogen, 150 hydromel, 531 indigo, 557 intracellular, 189 kepfir, 443 koji, 532 kumiss, 442 lactic, 153, 160, 164, 165, 512 of lactose, 164 INDEX Fermentation, leben, 444 of mannit, 514, mead, 531 mechanism of, 189 methane, 150 Mexican pulque, 532 of milk, 396 molds of, 47 moto, 532 organic matter, 315 perry, 530 pombe, 533 of protein, 276, 320 proteolytic, 169, 197, 276, 320 Tagi, 537 rice beer, 532 of saccharose, 163 sake, 532 sauer kraut, 554 of sewage, 276 of starch, 166, 195, 317 in starch manufacture, 555 of sugar, 153, 155, 163, 317, 555 in sugar manufacture, 555 tanning, 558 tobacco, 556 of urea, 173, 279 vinegar, 542 waxes, 318 wine, 506 yahourth, 445 Films in yeast, 76 Filterable organisms, 116 Filters, life in, 287 porous, 269 sand, 269 sewage, 285 ; Filtration of water, 267, 268, 269 Finger-and-toes, 608 Fish, canned, 465 dried, 456 poisoning, 491 preserved, 480. refrigerated, 473 sterilization, 465 Fixation of complement, 709 INDEX Flacherie (silk-worm), 635 Flagella, 102, 124 bacteria, 102 protozoa, 124 structure, 102, 124 Flagellata, 24, 25, 130, 131, 824 Flavor of butter, abnormal, 417 control of, 413 cultures for, 415 kinds of bacteria in cream, 410 number of bacteria in cream, 410 Flax fermentation, 557 Fluorescent pigment, 175 Food, alteration by heat, 459 amount required for bacteria, 153 appearance changes by heat, 459 assimilation, 297 biological changes of, 461 canning of, 467 changes in,' 460 changes in dried, 450 chemical change by heat, 460 desiccation of, 450 digestibility changes by heat, 460 drying of, 450 energy, 141, 152, 189 evaporation of, 450 for growth, 150 infection, 469 influence on organisms in water, 263 mechanical heat, 460 microbial changes in, 461 of microérganisms, 150 milk as, 264 mineral, 152 nitrogenous, 151 amino-acids, 151 ammonia, 151 nitrates, 151 nitrogen (free), 151 proteins, 151 urea, I51 non-nitrogenous, 151, 153 alcohols, 153 disintegration by 879 Food, non-nitrogenous, carbohy- drates, 151, 153 fats, 151, 153 hydrogen, 152, 153 methane, 152, 153 organic acids, 153 oxygen, 152 normal fauna of, 461 flora of, 461 nutritive value of chemically pre- served, 482 palatability changes by heat, 460 pasteurizaton of, 461 physical changes by heat, 459 plant, assimilation of, 297 poisoning, 488, 491 preserved by chemicals, 479 by drying, 450 by heat, 457 by refrigeration, 471 required, amount, 149 sterilization of, 464 vital disorganization of, 661 yeast as, 553 Foot-and-mouth-disease, 807 Foot rot of sheep, 784 Formaldehyde, 238, 486 Formation of soil, 354 Formic acid, 485 Foul brood of bees, 646 Fowl diphtheria, 756 Frambeesia, 848 Frozen milk, 218, 446 Fruits, canned, 466 Fungi imperfecti, 33 in soil, 302 Fusarium, 55 GALACTIMA succosa, 33 Galleria melonella, 633 Gametes, 34, 128 Gammarus zschokkei, 89 Gelatin, drying, 456 Gemma, 37 Gemmulation, 27, 125 Genito-urinary tract, 692 880 Geotaxis, 229 German measles, 801 Germination of ascospores, 70 Ginger beer, 72 Glanders, 767 Glaucoma piriformis, 19 Glossina palpalis, 829 Glycogen, 149 Gonorrheea, 734 serum for, 580 Gorgonzola cheese, 436 Gortyna ochracez, 633 Grapes, yeast on, 515 Graphitosis, 645 Gravity, influence of, 227, 228 Gregarine, 134 Growth, inhibition of, 232 stimulation of, 230 Guaranteed milk, 364 Gymnoascee, 59 HAIL, microérganisms in, 261 Haplosporidia, 136 Haptophile receptors, 700 Haptophore group, 700 Heat as preservative, 270, 457 Heat production, 187 Heliotaxis, 224 Heliotropism, 224 Hemogregarinz, 136 Hemolysins, 704 Hemoé6psonins, 713 Hemoproteus, 834 Hemorrhagic septicemia, 772 Hemosporidia, 135, 834 Hepatozoon, 136 perniciosum, 136 Herpes tonsurans, 726 Herpetomonas (Leishmania), 132, 825 donovani, 825, 844 furunculosa (tropica), 848 musce-domestice, 131 Heterogamy, 34 Heterotrophic organisms, 15 Higher bacteria, 105 Hippuric acid, 173 INDEX Histoplasma capsulatum, 136 History of microbiology, 1 of non-symbiotic nitrogen-fixa- tion, 339 of symbiotic nitrogen-fixation, 343 Hog cholera, 807 Holophytic organisms, 145 Holozoic organisms, 144 Hormodendron, 55 Horse pox, 805 sickness, 811 Human carriers, 667 Hydrogen, fermentation, 150 as food, 152, 153 oxidation, 294, 317 peroxide, 239 production, 315 sulphide, 358 Hydromel, 531 Hypha, 36 Hyposulphite oxidation, 146 ICE CREAM, 447 poisoning, 493 Immunity, acquired, 589, 687, 697 active, 589, 697 : agents of, 676 definition of, 684 exhaustion theory of, 721 familial, 688 general, 684 individual, 688 natural, 687 noxious retention theory of, 721 passive, 589, 697 phagocytic theory of, 722, plants, 589 racial, 687 ~* side chain theory of, 721 substances, 676 theories of, 721 Imperfect fungi, 39 Index, opsonic, 712 percentage, 712 phagocytic, 712 INDEX Indigo, 557 , Indirect division of cell, 28 Indol, 170 Infantile paralysis, 811 Infection, air-borne, 664 animal carriers of, 667 avenues of, 669, 674 carriers of, 667 cause of, 675 channels of, 659 contact, 663 defined, 659 droplet, 665 dust, 665 \ factors influencing, 624 food, 490, 666 human carriers, 667 manner of entering body, 664 milk-borne, 400, 666 number of organisms involved in, 674 resistance against, 675 routes of, 669 soil, 666 sources of, 664 variations of, 672 virulence of, 674 water-borne, 665 Infectious mastitis, 738 Inflammatory processes, 693 Influenza, 770 Infusoria, 136, 843 Inheritance of disease, 685 Inspected milk, 367 Intestines, 496 methods of study, 504 microorganisms of, 256, 499 protection by, 691 Intracellular fermentation, 189 Introduction, vii, ix Inulase, 194 Invertase, 190, I9I, 194, 195, 200 Invisible organisms, 116 determination of, 119 evidences of, 118 [odococcus, 497 é 56 881 Todococcus magnus, 497 parvus, 497 vaginatus, 497 Iodo-iodide solution, 45 Tron in soil, 361 Isogamy, 34, 128 fertilization, 128 Isoprecipitin, 718 JAM, 455 Jellies, 455 KALA azar, 825, 826 Karyokinesis, 28 bacteria, 87, 88 molds, 43 protozoa, 127 yeasts, 66 Karyosome, 17 Katabolism, 189 theory of, 204 Katalase, 202 Kepfir, 443 Kinetonucleus, 18, 132 Koji, 532 Kumiss, 442 LABOULBENIALES, 724 Lacnosterna, 634, 635 Lactacidase, 200 Lactase, 194, 196, 200 Lactic, bacteria, 378 fermentation, 164, 165 beer, 526 milk, 378 pickles, 554 wine, 512 Lakes, micro6érganisms in, 262 Lamblia, 133, 499 intestinalis, 133, 134 Lamellicornia, 651 Laverania malaria, 135 Leaf-spot of cucumbers, 613 of larkspur, 614 of plum and peach, 614 of sugar beets, 615 882 INDEX Leben, 444 Leishmania, donovani, 131, 825 tropica, 848 Leishmaniasis, 825, 826 Leprosy, 774 Leptothrix, 36 buccalis, 497 Lettuce blight, 593 Leucin, 170 Leucocytes, action on microérgan- isms, 612 Leucocytes in milk, 404 Leucocytotoxins, 710 Leucocytozo6n lovati, 27 ‘Leucoplastids, 20 | Light, heliotaxis, 224 heliotropism, 224. influence of, 222 upon organisms in water, 263 phototaxis, 224 phototropism, 224 production, 188 radium rays, 225 X-rays, 225 Lime in soil, 314, 354 Linin, 123 Lipase, 194, 197, 200 Locomotion, 24, 124 Louping ill, 812 Luetin, 586 Lungs, 691 Lysinogen, 705 Lysins, bacterio-, 704 cyto-, 704 hemo-, 704 structure of, 706 Lyssa, 814 MACROGAMETES, 128 Macronucleus, 18 Magnesium in soil, 354, 356 Malaria, 834 Malignant edema, 785 Mallein, 585 Malta fever, 740 Maltase, 194, 196, 200 Manganese in soil, 362 Mannitic, bacteria, 514 fermentation, 514 Market milk, 366 Mead, 531 Measles, common, 801 German, 801 Meat, canned, 465 jerked, 456 — poisoning, 491 preserved, 480 refrigeration, 473 sterilization, 465 Mechanical effects, influence of, 227 Media, beer as, 524 influence of physical structure, 181 must as culture, 506 soil as culture, 290 wine as culture, 506 Melolontha vulgaris, 643, 649 Membrane, 23 bacterial, 101 cell, 46, 100 undulating, 25, 124 Meningitis, cerebro-spinal, 736 epidemic, 736 Merozoits, 126 Mesomitosis, 34, 127 Metabiosis, 242 Metabolism, action of microdrgan- isms on, 189 mechanism of, 189 and microérganisms, I41 physical products of, 187 products of, 158 in protozoa, 143 theory of, 204 Metachromatic corpuscles, 20, 88 bacteria, 88, 99 molds, 43 protozoa, 146 yeasts, 61 ‘ Metamitosis, 30 Metaphase, 28° Methane, fermentation, 150 INDEX Methane as food, 152, 153 oxidation, 317 production, 315 Methods of studying bacteria in soil, 311 Methyl-guanidin, 171 Mexican pulque, 532 Microbiology, of air, 247 of alcohol, 506 distilled, 533 products, 506 of antisera, 575 of ‘beer, 524 of bread, 552 of butter, 408 of cheese, 420 of cider, 530 of compressed yeast, 557 of dairy (various) products, 438 of diseases of insects, 632 of food poisoning, 488 of foods preserved by chemicals, 479 by cold, 471 by drying, 450 by heat, 457 of ginger beer, 533 history of, I of human and animal diseases, 659, 724 of hydromel, 531 of immunity, 684 of indigo, 557 of mead, 531 of milk, 264 of perry, 530 of plant diseases, 588 of pombe, 533 of retting, 557 of rice beer, 532 of sake, 532 scope of, 11 sera, 575 of sewage, 274 of soil, 289 of starch, 555 883 Microbiology of sugar, 555 of susceptibility, 684 of tanning, 558 of tobacco, 556 of vaccines, 560! of vegetables (pickles, kraut, etc.), 554 of vinegar, 538 of water, 254 of wine, 506 Micrococcus aquatilis, 256 candicans, 256 coronatus, 256 epidermidus albus, 743 gonorrhoee, 573, 670, 671, 692, 734 : intracellularis var. meningitidis, 663, 670, 690, 736 melitensis, 740 nigrofaciens, 634 nivalis, 256 pneumonie, 573 progrediens, 118 pyogenes, 733 pyogenes var. albus, 573, 688, 690, 743 pyogenes var. aureus, 199, 227, 497, 573, 672, 682, 688, 690, 714, 733) 740, 747 pyogenes var. citreus, 573, 743 Microgametes, 128 Microglena punctifera, 21 Micronucleus, 18 ; Microdérganisms, acetic in vinegar, 538 in wine, 511 acid-forming in milk, 378 aerobic, in soil, 157, 294 in wine, 511 air, 247, 252, 257 alge in soil, 305 anaerobic, in sewage, 157, 286 in soil, 294 in wine, 512 autotrophic, 151 .B. coli in water, 257 B. coli-aerogenes in milk, 382 sater- INDEX Microérganisms, B. coli, enteritidis sporogenes in water, 257 typhosus in milk, 401 in water, 258 bacteria, in beer, 526 in bread, 552, 553 on grapes, 515 in productive soil, 309 in soil, 309 in unproductive soil, 309 Bact. bulgaricum in milk, 379, 383 : lactis acidi in milk, 367, 379, 380 lactici acidi in udder, 371 lactis aerogenes in water, 258 in body of sound individuals, 663 butyric in wine, 515 of cheese, 420 chromophorous bacteria, 176 chromoporous bacteria, 176 of cider, 530 classes in water, 255 cocci in milk, 384 complexity in sewage, 275 cosmopolitan saprophytes, 46 of cream, 410 decrease in water, 263 determination in air, 249 development in milk, 386 in digestive tract, 496 of diseases, 660 dissemination of pathogenic, 678 in dust of stable, 374, 377 effect on animal body, 680 elimination from body, 678 energy supply of, 145 entrance to air, 248 facultative anaerobes, 157 in feces, 500 in feed for cattle, 374, 375 food of, 148 food required for, 149 in foods, 450, 461, 471, 479, 490 general reactions on body, 680 on grapes, 507 Microérganisms in hail, 261 heterotrophic, 151 higher bacteria, 105 increase in water, 263 infusoria, 843 in intestines, 499 invisible, 116 kinds in air, 251, 252, 257 in beer, 524 in cream, 410 in milk, 265 in sewage, 274 in soil, 289, 302 in water, 255. in wine, 511 lactic in milk, 378 in wine, 512 in lakes, 262 local reactions in body, 680 mannitic in wine, 514 methods of study in soil, 311 Microspira comma in water, 258 in milk, 265 certified milk, 367 milk pail, 377 special milk, 367 in moisture, 207 molds of fermentation, 47 on grapes, 507 in soils, 302 morphological groups in soil, 310 in mouth, 496 nitric, 329 nitrous, 329 numbers in air, 249, 250 in cream, 410 in milk, 378 in soil, 308 nutrition of, 141 obligate anaerobes, 157 of oxidation, 153 parachrome bacteria, 176 parasitic, 47, 670 pathogenic, 282, 660 in butter, 419 in milk, 385, 401 INDEX 885 Microérganisms, period of incubation Milk, Bact. lactis acidiin, 367, 379, in body, 680 380, 395, 397 physiological group in soil, 310 bitter, 398 physiology of, 141 Boston certified, 368 propionic, 512 common, 366 proteolytic in milk, 385 butter-, 446 — prototrophic, 151 centrifugal separation of, 389 protozoa in soil, 305 certified, 367 pseudo-yeast, on grapes, 509 Boston, 368 putrefaction in sewage, 276 Brooklyn, 368 ' in rain, 260 : Chicago, 368 in rivers, 262 New Yrok City, 368 in sea water, 262 changes due to organisms, 265 in sewage, 274, 275 : chemicalsin checking bacteria, 393 sewage streptococci, 257 Chicago certified, 368 slime-forming in wine, 512 common, 366 in snow, 261 cocci in, 384 soil bacteria in water, 256 common, 365 sources in milk, 369 Boston, 366 in stomach, 499 Chicago, 366 subsidence in air, 249 Connecticut cities, 366 typical forms in sewage, 275 Ithaca, 366 in udder of cow, 369 Montclair, 366 in upland waters, 262 Rochester, 366 in water, 254, 262 "condensed, 438, 463 in wells, 261 concentrated, 439 in wine, 511 powdered, 441 yeasts of beer, 524 sweetened, 438 of bread, 552 unsweetened, 439 compressed, 551 contamination of, 375, 386 on grapes, 508 ‘ curdling by acid, 395 of vinegar, 538 by rennin, 425 of wine, 506 development of bacteria in, 386 Microspira aestuarii, 360 dirt in, 399 comma, 103, 199, 228, 258, 573, disease carrier, 400 660, 692, 705, 714, 718, 798 diseases, epidemics, 401 Microsporidia, 136, 843 non-epidemic, 402 Milk, acid bacteria and health of, 400 drinks, 441 acid curdling of, 378 butter-milk, 446 acid forming bacteria in, 378 kepfir, 443 aeration of, 386 kumiss, 442 air contamination, 374 leben, 444 . alcoholic, 399 yahourth, 445 analysis of, 403 dust, influence of, 374, 377 B. coli aerogenes in, 382 feeding, influence on germ con- Bact. bulgaricum in, 379, 383 tent, 374, 375 886 Milk, fermentation (abnormal) of, 396 as food, 264. frozen, 446 germicidal action, 394 guaranteed, 367 house, 374 inspected, 367 Ithaca, 366 leucocytes in, 404 market, 366 microbial content of, 265 microscopic method of analyses, 404 milker in relation to, 374 Montclair (N. J.), 366 neutral bacteria, 385, 401 New York certified, 368 odors, 264 ‘ pail, 377 pasteurization of, 391 pathogenic bacteria in, 401 periods of change, 394 plating method of analysis, 404 poisonous, 493 powdered, 441, 456 proteolytic bacteria in, 385 proteolytic changes in, 385 quality for cheese making, 421 refrigeration, 476 ripening of, 424 Rochester, 402 TOpy, 397 selected, 367 sickness, 785 slimy, 397 sources of organisms in, 369 special, 367 standards of, 404 straining of, 386 sweet, curdling of, 397 taints, 264 . temperature, influence on germ content, 390 tests for quality, 423 utensils for, 375 385, INDEX Milk, value of standards and analy- ses, 406 water supply in relation to, 375 Msp. comma in water, 258 Milk-borne diseases, 400, 666 Milker in relation to milk, 274 Milk-house, air of, 374 Milk-sickness, 785 Mineral food in soil, 152, 300 Mineralization of organic matter in - soil, 295 Mitochondria, 18 granular, 19 in molds, 42 in protozoa, 19 rod-shaped, 19 thread-shaped, 19 in yeasts, 62 Mitosis, 28 Moisture, 207 content of microérganisms, 148 influence of, 207 in soil, 209 Molasses, 455 fermentation of, 534 Molds, in disease, 724 of fermentation, 47 on grapes, 507 in soil, 302 structure, 40 Monas, 133 Monas termo, 26 Monilia, 57 candida, 57 Morphological groups of bacteria in soil, 310 Morphology, bacteria, 77 molds, 36 protozoa, 120 yeasts, 60 Motility, bacteria, 79 protozoa, 124 spirochetz, 124 Moto, 532 Mouth, 496 microérganisms, 496 INDEX Mouth, protection by, 690 Mucor, 22, 40, 42, 43, 48, 507, 558 circinelloides, 41, 49 javanicus, 49 mucedo, 48 oryz®, 72, 535, 537 plumbeus, 49 racemosus, 48 rouxii, 49, 535 Mumps, 801 Must of grapes, 507 Mutual influences, 241 Mycelium, 36 Mycetoma, 730 Mycetozoa, 121 Mycoderma, 74 aceti, 539 vini, 75 Mycorrhiza, 304 Mycotic lymphangitis, 731 Myonemes, 124 Myxobolus pfeifferi, 13 Myxosporidia, 136 NASAL cavity, 690 Neurin, 171 Neutral bacteria in milk, 385, 401 Nipa cinerea, 644 Nitrate disappearance, 332 as food, 151 reduction, 174, 279, 333 in sewage, 279° in soil, 333 transformation, 322 Nitrification, accumulation of ni- trates, 327, 332 of ammonia, 332 disappearance of nitrates, 332 environmental relations, 329, 332 . experimental study, 327 nitric bacteria, 329 nitrous bacteria, 329 Nitrite reduction, 174, 279, 333 in sewage, 279 in Soil,’ 333 Nitrogen, addition to soil, 303, 314 887 Nitrogen cycle, 185 Nitrogen fixation (atmospheric), 303, 339 aerobic species, 340 anaerobic species, 340 biological relations, 339 chemical relations, 339 cultures for inoculation, 352 development of organisms, 346 energy relations, 342 entrance of organisms, 346 environmental relations, 348 inoculations, methods of, 350, 352 mechanism, 348 physiological efficiency, 347 resistance of plants to, 347 specialization of organisms, 348 symbiotic, history of, 343 symbiotic (non), history of, 339 theories of, 338 variations of, 348 Nitromonas, 146, 329 Nitrosomonas, 146, 281, 329 Non-symbiotic nitrogen fixation, 339 aerobic species, 340 , anaerobic species, 340 energy relations, 342 history of, 339 Normal precipitins, 718 Nosema bombycis, 656 Notonecta glauca, 644 Noxious retention theory of immunity 721 Nuclease, 194 Nuclein, 16 Nucleolus, 16 Nucleoplasm, 15 Nucleus, 15, 122 bacteria, 87, 91 bi-, 131 diffuse, 17, 91 division, 28 fluid, 15 forms, 17 molds, 42 protozoa, 122 s 888 Nucleus reproduction, 28 reproductive, 18 structure, 15 value, 16 vegetative, 18 yeasts, 61 Number of bacteria, in air, 250 in butter, 410 in cream, 410 in milk, 378 in sewage, 274, 275 in soil, 308 in water, 255 Nutrition of microérganisms, 141 OBLIGATE aerobes, 157 anaerobes, 157 parasites, 660 saprophytes, 660 Odors in milk, 264 Oidium, 56 albicans, 496, 725 lactis, 56, 167, 216, 227, 242, 396, 429, 437 Oleomargarine, cultures for, 416 Olive knot, 608 Oomycetes, 38 Opsonic index, 712 Opsonins, 572, 711 hemo-, 713 index of, 712 Opsonogens, 711 Oroya fever, 841 Orchids, brown rot, 621 Organelle, 123 Organic acids, as food, 153 in soil, 319 Organic matter, in soil, 300 Origin of carbohydrates in soil, 315 fats in soils, 318 soil, 354 waxes in soil, 318 Ornithodoros moubata, 845 Oscillaria, 108 Osmotic pressure, 207 Osteomyelitis, 741 INDEX Outlines of plant, groups, 12 of protozoal groups, 13 Oxidase, 194, 202, 508 of vinegar, 194, 200 Oxidation, of acetic acid, 168 alcohol, 155, 167, 168 of ammonia, 146, 174 bacteria, 281 of carbon, 151, 313 of carbon monoxide, 317 of hydrogen, 317 of hydrogen sulphide, 358 of hyposulphite, 146 influence on organisms in water, 264 of methane, 317 of nitrites, 146, 174, 327, 329 of nitrogen, 313, 339, 343 rate in carbon, 313 in soil, 294 of sulphur, 172, 174, 281, 359 in water, 264 Oxygen, influence on different species, 152, 153, 157 Ozone, as preservative, 269 in water, 269 PACHYTYLUS migratoroides, 643 Parachrome bacteria, 176 Parasites, facultative, 129 obligate, 129 of uncertain position, 136 Parasitic microérganisms, 660 molds, 47 Parasitism, 129 Parenchymatous tissues, action of microorganisms on, 682 Parthenogenesis, 128 Pasteurization of beer, 461 of cream, 461 economic importance of, 461 of food, 461 of fruit juices, 461 of milk, 391 Pathogenic bacteria in air, 252 in butter, 419 INDEX .- Pathogenic bacteria in food, 488 in milk, 401 in sewage, 282 in soil, 666 in water, 665 Peas, blight, 595 canned, 466 sterilization, 466 Pebrine, 656 Pellagra, 495, 813 Pelomyxia palustris, 15, 16, 32, 33 Pemmican, 456 Penicillium, 41, 45, 47, 50, 164, 507, 558, 725 brevicaule, 52, 175 camemberti, 52, 201, 203, 437 expansum, 51, 52 glaucum, 42 roqueforti, 52, 436 Pepsin, 190, 194, 198, 200 Peptone, transformation, 322 Percentage index, 712 ‘Period of incubation, 680 Periplaneta americana, 635 orientalis, 92 Perithecium, 39 Peroxidase, 202 Perry, 530 Pestularia vesiculosa, 46 Phagocytic, index, 712 theory of immunity, 722 Phagocytosis, 711 -Phosphine, 172 Phosphorus, cycle of, 186 in soil, 357 Phototaxis, 224 Phototropism, 224, 231 Phycomycetes, 38, 40, 59 Physical influences of microérganisms, 207 Physiological groups, 310 of bacteria in soil, 308 variation, 161 Physiology of microorganisms, 141. Phytotoxins, 178 Pickles, 554 889. Pigment, 175 bacteriopurpurin, 177 carotin bodies, 177 chromogens, 176 chromophorous bacteria, 176 chromoporous bacteria, 176 fluorescent, 175 parachrome bacteria, 176 prodigiosin bodies, 177 solvents, 177 Plague, 776° é Plant diseases (See diseases of plants) 588 Plant food in soil, 297 groups, 12 Plasmodiophora brassice, 608, 610 Plasmodium, 136, 834 falciparum, 834 malaria, 834 vivax, 834 Plasmolysis, 208 colloidal substances, 210 salt solutions, 209 sugar solutions, 209 water, 208 Plastids, 20 Pleuro-pneumonia, bovine, 804 Pneumomycosis, 724 Pneumonia, 747. Poisoning, by cheese, 493 chemical nature of, 232, 495 classes of food, 491 due to saprophytic changes, 491 by fish, 491 by foods, 488 ice cream, 493 infections transmitted, 491 by meat; 491 by milk, 493 by sausage, 491 by shell fish, 493 by vegetables, 494 Poisons, 232 Pombe, 533 Potassium in soil, 360 Poultry refrigeration, 473 \ 890 Powdered milk, 441, 456 \ Precipitate, 718, 720 Precipitinogen, 718, 719 Precipitins, anti-, 719 auto-, 718, 719 co-, 720 forensic use, 720 iso-, 718 mechanism of, 718 normal, 718 specific inhibition, 719 Precipitoid, 720 Predisposition to disease, 685 Preservation by chemicals, 479 by cold, 471 by drying, 450 by heat, 457 Preservatives, alcohol, 486 benzoic acid, 486 boric acid, 484 effect on system, 483 formaldehyde, 238, 486 formic acid, 485 hydrogen peroxide, 239 inorganic, 484 in milk, 385 nitric acid, 481, 484 nitrous acid, 481, 484 organic, 485 ozone, 269 preserving by chemical action, 483 be by physical action, 483 salicylic acid, 485 sodium chloride, 483 sugar, 480 sulphurous acid, 484 vinegar, 486 wood smoke, 486 Pressure, influence of, 227 Prodigiosin bodies, 177 Promitosis, 32, 127 Prophase, 28 Propionic bacteria, 512 Prorodon teres, 26 Proteases, 194, 197 INDEX Protection against disease, 688 conjunctiva, 692 cutaneous orifices, 688 genito-urinary tract, 692 inflammatory processes, 693 intestines, 691 lungs, 691 mouth, 690 nasal cavity, 690 natural antibacterial substances, 695 natural antitoxins, 694 normal agglutinins, 696 hemolysins, 696 precipitins, 697 skin, 669 stomach, 691 Proteins, bacterial, 676 in fermentation, 276 as food, 151, 169 in sewage, 276 in soil, 320 toxic, 178, 676 Proteolytic bacteria, 169, 197, 276, 320 changes in milk, 385 in sewage, 276 in soil, 320 Proteosoma, 136, 834 Proteus group, 256 Protista, 43, 99, 120 Prototoxoids, 704 Prototrophic organisms, 151 Protozoa, classification, 130 functions, 143 locomotion, 124 flagella, 124 myonemes, 124 metabolism, 124, 146 reproduction, 125 anisogamous, 128 binary division, 125 chromosomes, 127 conjugation, 128 copula, 128 copulation, 128 INDEX Protozoa, reproduction, mental cycle, 128 encystment, 129 isogamous, 128 macrogametes, 128 merozoites, 126 microgametes, 128 mitosis, 127 parthenogenesis, 128 schizogony, 126 self-fertilization, 128 sporogony, 126 sporozoites, 126 zygote, 128 general, 120 groups, 13, 14 influence on organisms in water, 264 parasitism, 129 -commensals, 130 facultative parasites, 130, 660 obligatory parasites, 130, 660 saprozoic parasites, 130 pathogenic, 660 in soil, 305 structure, I21,,141 chromatin, 122 cytoplasm, 122 ectoplasm, 122 endoplasm, 122 linin, 123 - nucleus, 122 organelle, 123 technic, 137 in water, 264 Protozoal groups, 13, 14 Pseudomonas aptatum, 615 avene, 595 ' beticola, 611 campestris, 617 citri, 612 hyacinthi, 618 juglandis, 602 lachrymans, 613 medicaginis, 590, 591, 592 mori-, 594 develop- 891 Pseudomonas phaseoli, 592, 593 pisi, 595, 596 pruni, 614 pyocyanea, 103, 172, 176, 199, 244, 341, 503, 682, 696, 704, 756 radicicola, 151, 212, 242, 298, 341, 345, 346, 350 solanacearum, 629 stewartii, 618 syncyanea, 103. tumefaciens, 605, 607 viridilividum, 593 . Pseudopodia, 24, 124 Pseudo-yeasts, 73 on grapes, 509 Ptomains, 171, 495 Puerperal fever (septicaemia), 744 Pulsating vacuoles, 22, 144 Purification of water, by chemicals, 270 : electricity, 270 filtration, 266, 267, 268 heat, 270 ozone, 270 Putrescin, 171 Pyzmia, 741 Pyocyanase, 244 _Pyorrhoea alveolaris, 498 Pyrameis cardui, 633 Pyrinin, 16 RABIES, 814 serum for, 581 vaccine, 566 Radium rays, 225 Raffinase, 194 Ragi, 537 Rain, microérganisms in, 260 Ranatra linearis, 644 Rate of oxidation, 294, 313 carbon in soil, 194, 313 hydrogen, 294 nitrogen in soil, 294, 313 Reaction of soil, 298 Reductases, 202 892 Reduction of nitrates, 155, 174, 279, 333 nitrites, 174, 274, 333 sulphate, 174, 279, 360 Red water, 839 Refrigeration, 471 after-storage changes, 473 of butter, 476 chilling changes, 472 of eggs, 475 of fish, 473 of food-stuffs, 473 of fruits, 477 legal control of, 477 of meat, 473 of milk, 476 of poultry, 473 storage changes, 472 of vegetables, 477 Relapsing fever, 847 Rennet, 191, 194, 199 Reproduction, bacteria, 81 cell, 27 molds, 43 protozoa, 123 spirochetes, 105 trichobacteria, 106 ¥ yeasts, 61 Reserve products, 22, 43 Resistance of spores, 220 Retention theory of immunity, 721 Retting, 557 Rhinosporidium kinealyi, 136 Rhizoplast, 26 Rhizopoda, 130, 131, 822 Rhizopus, avanicus, 49 nigricans, 42, 48, 223 oryze, 49, 72, 193, 203 stolonifer (See mucor). Rice beer, 532 Ring worm, 726 Ripening of milk, 424 Rivers, microérganisms in, 262 Rivularia bullata, 17 nuclei, 17 threads, 17 INDEX Ropy milk, 397 Roquefort cheese, 436 Rot of cauliflower, 622 ” of hyacinth, 624 of lily, 623 of sugar beet, 626 of vegetables, 623 Rotation of elements, 184 Routes of infection, 669, 674 SACBROOD, 653 Saccharomyces, 59, 508, 530 albicans, 725 anomalus, 60, 61, 529 apiculatus, 73, 75, 510, 518, 529 cerevisiz, 16, 27, 59, 62, 63, 65, 71, 72, 155, 162, 227, 524 ellipsoideus, 59, 71, 72, 76, 509, 510, 511, 518, 519, 529 exiguus, 529 + farciminosus, 731 foetidus, 529 fragilis, 72 kefir, 443 ludwigii, 68, 69, 70 marxianus, 60 membranefaciens, 76 pasteurianus, 61, 73, 509, 527, 529 pyriformis, 72 (schizo) octosporus, 34, 68 (schizo) pombe, 75 vordemanni, 72, 537 zopfi, 555 Saccharomycetes, 59 Saccharose fermentation, 163 Sake, 532 Salicylic acid, as preservative, 485 Sand filter, 267, 285 life in, 287 Saprolegniacee, 58, 633 Saprophytes, cosmopolitan, 46 facultative, 660 obligate, 660 Saprozoic parasites, 130 Sarcina, 85, 529 INDEX 893 Sarcina auriantiaca, 251 lutea, 251, 256, 326, 327 ventriculi, 499 Sarcocystis tenella, 23 Sarcosporidia, 23, 121, 136 Sauerkraut, 554 Sausage poisoning, 491 Scarlet fever, 801 Schick test, 587 Schistocerca americana, 642 paranensis, 642 Schizogony, 126 Schizomycetes, 12 Schizosaccharomyces, 60, 67, 90 octosporous, 34, 61, 68 pombe, 73 Sclerotium, 37 Seasonal influence on soil, 309 Sea water, microédrganisms in, 264, 266, 267 Sedimentation, influence on germ content of water, 264, 266, 267 Selenopsis gemminata, 643 Self-fertilization, 128 Sensitized vaccine, 574 Septicamia, 744 of caterpillar, 643 of cockchafer, 649 hemorrhagic, 777 of larva of Lamellicornia, 651 puerperal, 744 Septic tank, 286 Septum, 36 Serum, antidiphtheritic, 576 antidysenteric, 581 antigonococcic, 580 antimeningococcic, 580 antimicrobial, 580 antirabic, 581 antistreptococcic, 580 Dorset-Niles, 568 preservation of, 581 Sewage, anaerobic organisms, 276 disinfection, 287 fermentation of, 276 Sewage, fermentation of, cellulose, 277 fats, 278 proteins, '276 urea, 279 filters, 285 longevity of pathogenic organism in, 282 microérganisms of, 274, 275 cultivation of, 284 destruction by biological proc- esses, 287 by chemical processes, 287 life in filters of, 283 oxidizing bacteria of, 281 pathogenic bacteria in, 282 prevalence of pathogenic, 282 reduction of nitrates, 279 nitrites, 279 sulphates, 279 septic tank for, 286 streptococci in water, 257, 274 tanks, 286 Sexual changes, 34 Sheath of higher bacteria, 1o1 spirochetes, 105 trichobacteria, 107 Sheeppox, 805 - Shell-fish poisoning, 493 Side-chain theory of immunity, 721 Skatol, 170 Skin, 669 Sleeping sickness, 827 Slimy milk, 397 wine, 512 Smallpox, 801 vaccine, 563 Snow, microdrganisms in, 261, Sodium chloride as preservative, 483 Soil, acidity, 298 causes of, 299 number of organisms, influ- enced by, .299 range of, 298 species, influenced by, 299 aeration, 293 804. INDEX Soil, aerobic activities, 294 Soil, drought, 292 alge in, 305 early, 297 aluminum in, 362 fermentations, cellulose, 303, 315 ammonia formation, 331 fats, 318 transformation, 336 fixation of nitrogen, 338 ammonification, 322 - organic matter, 289 climatic conditions influencing, proteins, 320 323 ‘ ; starches, 317 efficiency, 326 sugars, 317 of different species, 326 waxes and gums, 318 experimental study, 322 food (plant) assimilation of, 297 mechanism of, 323 (plant) production in, 297 molds, 303 formation of, 354 numbers involved, 325 general discussion, 289 species involved, 325 higher plants in, 308 anaerobic activities, 294 infection, 666 ‘bacteria, 308 inoculation, 352 aerobic species, 340 iron, 361 anaerobic species, 340 . late, 297 at different depths, 309 magnesium, 354, 356 distribution, 309 * manganese, 314, 362 groups (morphological and - medium, as culture, 290 physiological), 310 mineral food, 300 methods of study (quantita- mineralization of organic matter, tive and qualitative), 311 (295 nitric, 329 moisture relations, 290 nitrous, 329 rainfall, 291 number, 308 . range of, 291 productive soil, 308 molds in, 302 unproductive soil, 308 nitrates, transformation of, 336 in water, 256 nitrification, 327 bacterial substance available, 336 bacteria (nitric and nitrous), substance present, 335 329 ; - biological factors of soil-forma- environmental relations, 329 tion, 354 experimental study, 327 calcium, 314, 354 nitrates, accumulation, 332 carbohydrates (origin), 315 disappearance, 332 carbon dioxide in, 394 nitrogen, addition to, 314 carbon-nitrogen ratio, 321 nitrogenous compounds,’ trans- climatic influences, 295 formation of, 336 composition (mechanical), 293 nitrogen (atmospheric) fixation, copper, 362 aerobic species, 303, 340 cultures for soil, 352 anaerobic species, 340 denitrification, 333 ‘ biological relations, 339 environmental relations, 335 chemical relations,. 339 experimental study, 333 cultures for inoculation, 352 INDEX Soil, nitrogen (atmospheric) fixation, development of organisms, 346 energy relations, 342. entrance of organisms, 346 environmental relations, 348 inoculations, methods of, 350, 352 mechanism, 348 physiological efficiency, 347 resistance of plants to, 347 specialization of organisms, 348 symbiotic, history of, 343 symbiotic (non), history of, 339 theories of, 338 variations of, 348 organic acids, accumulation of,319 source of, 319 transformation of, 319 organic matter in, 300 origin of, 356 carbohydrates in, 315 fats, 318 waxes, 318 oxidation of carbon, 294, 313 carbon monoxide, 317 hydrogen, 294, 317 methane, 317 nitrogen, 294, 313. peptone, transformation of, 336 phosphorus, 314, 357 plants (higher in), 308 potassium, 360 production of hydrogen, 315 methane, 315 protein bodies in, 320 amount, 320 » quality, 320 protozoa in, 305 reaction of, 298 reduction of nitrates, 333 nitrites, 383 sulphates, 358 seasons, influence of, 309 sulphate reduction, 358 sulphur, 358 895 | Soil, temperature relations, 295 temperatures of, 295 transformation of nitrogen com- pounds, 322, 336 organic acids, 319 reactions of, 298, 311, 312 weathering process, 354 .Somatose, 456 Sources of organic acids.in soil, 319 carbon for microérganisms, 151, 152 hydrogen for microérganisms, 152, 153 infection, 664 microérganisms in milk, 369 minerals for microérganisms, 152 nitrogen for microérganisms, 151 _ oxygen for microérganisms, 157 Specific gravity of bacteria, 150 Spireme, 28 Spirilliaceze, 105 Spirillum, desulphuricans, 280, 360 rubrum, 103 rugula, 278 sputigenum, 497 undula, 87 volutans, 99, 101, 104 Spirocheta, 136, 845 anserina, 825 buccalis, 497 dentium, 497 duttoni, 845 gallinarum, 825 macrodentium, 497 media, 497 microdentium, 497 obermeieri, 845 pallida, 498 pallidula, 845 recurrentis, 847 vincenti, 845 o Spirochetal diseases, 845 Spirochetes, 105 form, 105 motility, 105 . reproduction, 105 896 INDEX Spirochetes, size, 105 Streptococcus, mesenteroides, 455, Spongomonas uvella, 26 555 Spores, 37 pneumoniz, 663, 670, 673, 691, resistance of, 220 692, 747 Sporogony, 27, 126 Sporoplasm, 69 Sporothrix, 732 Sporotrichum globuliferum, 634 Sporozoa, 130, 134, 833 Sporozoits, 126 Sporulation, of ascospores, 69 Stable, air of, 374 Staphylococcic infections, 85, 741 Starch, fermentation of, 555 _ for manufacture, 555 raw materials of, 555 Starters, for beer, 528 butter, 415 buttermilk, 446 cheese, 427 milk drinks, 441 vinegar, 543 wine, 518 Stauronautus maroccanus, 640, 642 Steapsin, 194, 196 Sterigma, 39 Sterigmatocystis, 53 ochracea, 55 Sterilization, of corn, 466 economic importance of, 464 of fish, 465 of fruits, 466 of meat, 465 of peas, 466 Stilton cheese, 436 Stomach, 691 microorganisms of, 499 protection by, 691 Straining of milk, 386 Streptococcic infections, serum for, 580 Streptococci in sewage, 257, 282 Streptococcus, 85 bombycis, 635 hemolyticus, 497 lacticus, 164, 166, 202, 245 pyogenes, 572, 662, 663, 670, 672, 673, 677, 682, 688, 690, 704, 744, 747 salivarius, 497 viridans, 497, 498 Subsidence in water, 264, 266, 267 Sugar, in fermentation, 153, 163, 196 in fermentation in soil, 317 manufacture, 555 Sugar-beet tuberculosis, 611 Sulphate reduction, in sewage, 279 in soil, 358 Sulpho-bacteria, 91, 97, 108, 358 Sulphur cycle, 186 oxidation, 172, 174, 281, 359 in soil, 314, 358 Sulphurous acid as preservative, 484 Surface washings in soil, 256 in water, 256 Susceptibility, 684 definition, 684 familial, 688 general, 684 hyper, 684 individual, 688 natural, 687 racial, 687 Suspensions for agglutination tests, 585 Swamp fever, 819 Swine erysipelas, 778 Swiss cheese, 434 Symbiosis, 241 Symbiotic nitrogen fixation, cultures ‘ in, 343 development of organisms, 346 entrance of organisms, 346 environmental relations, 348 history of, 343 inoculation, methods of, 350, 352 legume-earth for inoculation, 352 mechanism of, 348 INDEX . Symbiotic nitrogen fixation, physio- logical efficiency, 347 resistance of plants, 347 specialization, 348 variations, 348 Symptomatic anthrax, 787 vaccine, 565 Syphilis, 847 Syrups, 455 TAINTS of milk, 264 Tanks, anaerobic, 286 septic, 286 Tanning, 558 Tapej, 547 Telophase, 29 Temperature, cardinal points, signifi- cance of, 216 . endpoints of fermentation, 217 influence on germ content of milk, 390 of water, 263 maximum, 215 minimum, 214 optimum, 213 resistance of spores, 220 | of soil, 295 thermal death-point, 219 Tests for milk quality, 423 Tetanus, 789 antitoxin, 579 toxin, 579 Tetracoccus, 85 Texas fever, 839 Thamnidium elegans, 41, 50 Theories of nitrogen-fixation, 338 Thermal death-point, 219 Thermotaxis, 145 Thiobacillus denitrificans, 334 Thiothrix tenuis, 17 - Thrombase, 199 Thrush, 725 Tick fever, 845 Tobacco fermentation, 556 Torula, 73, 75, 209, 509, 511, 524 Toxic proteins, 178, 676 897 Toxins, 178, 575, 576 cyto-, 709 diphtheria, 576 endo-, 676 leucocyto-, 710 neutralization, 702 soluble, 676 tetanus, 579 unit of, 578, 579 Toxoid, true, 704 Toxon, 704 Toxophile receptors, 700 Toxophore group, 700 Transformation of ammonia, 336 nitrates, 336 nitrogen in soil, 336 organic acids in soil, 319 peptone, 336 Treponema pallidum, 661, 671, 672, 692, 708, 847 pertenue, 848 Trichobacteria, 106 Trichocyst, 26 Trichoderma kceningi, 327 Trichomonas, 499 eberthi, 133 intestinalis, 502, 503 Trichomycosis, 726 Trichophyton tonsurans, 725 Trimethylamin, 171 Trypanoplasma, 132 Trypanosoma, 132 americanum, 832 brucei, 661, 831 cruzi, 831 dimorphon, 831 equinum, 831 equiperdum, 832 evansi, 831 gambiense, 827 granulosum, 827 lewisi, 661, 832 rotatorium, 23 theileri, 832 tincee, 132 Trypanosomiases, animal, 831 898° Trypanosomiases, human, 831 Trypsin, 194, 198 Tryptophan, 170 Tuberculin, Koch’s old, 581, 582 Koch’s other (T. R., B. E., etc.), 584 Tuberculosis, 779 animals, 779 man, 779 sugar-beet, 611 vaccine, 584 Typhoid fever, 258, 259, 792 Typhus fever, 820 Tyrosin, 170 Tyrosinase, 194, 202 Tyrothrix, nivea, 108 UDDER of cow, 369 bacteria from quarters, 371 from entire udder, 370 Bact. lactis acidiin, 371 diseased, 372 exterior, 372 healthy, 369 interior, 369 wiping of, 376 Ultramicroscopic organisms, 116, 660 Ultramicroscopic viruses, 136 Undulating membrane, 25, 124 Upland surface waters, 262 Urea, 146, 153° fermentation, 173, 279 as food, 146 Urease, 200, 201 Uric acid, 173 / Urospora lagidis, 33 Utensils of milk, 375 VACCINE, anthrax, 570 Asiatic cholera, 573 bacterial, 571 black leg, 565 bubonic plague, 574 hog cholera, 568 kinds of, 562 manufacture of, 560 INDEX Vaccine, rabies, 566 sensitized, 574 smallpox, 563 tuberculosis, 571 Vacuoles, 22 pulsating, 22 Variation, physiological, 161 in structure of cells, 40, 59, 77, 121 Vegetables, 554 canned, 466 pickles, 554 preservation, 482 refrigeration, 477 sauerkraut, 554 Vegetable poisoning, 494 Vegetation, influencing on organisms in water, 264 Vinegar, acetic fermentation, 538 after-treatment of, 549 apparatus for manufacture of, 543 bacteria, 538 cultures for, 543 diseases of, 549 ' domestic method, 543 fermentation of, 542 German method, 543 manufacture of, 541 Orleans method, 543 Pasteur method, 543 as preservative, 486 raw materials of, 541 totating barrel method, 548 starters for, 543 yeasts, 538 Virulence of infection, 674 Virus, 561 attenuation, 562 Volutin, 149 WALKER’S hyacinth disease, 618 Wassermann’s test, 709 Water, analysis (qualitative and quan- titative), 265, 266 B. coli in, 257 enteritidis in, 257 INDEX : 899 Water, B. typhosus in, 258 Whooping cough, 771 bacteria of natural, 255 , Wilt, of curcubits, 627 Bact. lactis aerogenes in, 258 Irish potato, 629 -borne infections, 665 sweet corn, 628 classes of bacteria in, 255 tobacco, 629 ' coagulating basins, 268 tomato, 629 decrease of organisms in, 263 Wilt disease of Gypsy Moth, 654 dilution of, 264 Wine, 506 filters, 267, 269 i acetic bacteria in, 512 filtrations, 267, 268, 269 aerobic organisms in, 512 food influences, 263 anaerobic organisms in, 512 from hail, germ content, 261 butyric bacteria in, 515 increase of organism in, 263 composition of, 506 lake, germ content, 262 control after fermentation, 521 light, influence of, 263 of fermentation, 518 microérganisms in, 254, 255 on grapes, 515 Microspira comma in, 258 defined, 506 ' oxidation, influence on germ con- dry, 506 tent, 264 fermentation, 518 protozoa, influence on germ con- fortified, 506 tent, 264 lactic bacteria in, 512 purification, by chemicals, 270 mannitic bacteria in, 514 by heat, 270 as medium for cultivation, 506 by ozone, 269 microérganisms in, 511 from rain, germ content, 260 mycoderme in, 511 from rivers, germ content, 262 propionic bacteria in, 512 sea water, germ content, 262 slime-forming bacteria in, 512 sedimentation of, 264, 266, 267 white, 506 sewage, streptococci in, 257 yeasts, 506 from snow, germ content, 261 Wood smoke as preservative, 486 soil bacteria in, 256 Wounds, 741 supply for dairy, 375 Wisconsin curd test, 424 surface washings in, 256 temperature, influence in, 263 X-RAYS, 225 in upland surface, 262 vegetation, influence on germ YAHOURTH, 445 content, 264 Yaws, 848 from wells, germ content, 261,270 Yeast, in alcoholic fermentation, 535 Wax fermentation, 318 in beer, 71, 524 in soil, 318 in bread, 71, 552 Weight of bacteria, 150 cell of, 59, 61 Wells, construction of, 270, 272 classification of, 59 location of, 270, 271 compressed, 551 microérganisms in, 261 culture of, 74 White diarrhoea, amoebic, 834 cytology, 61 bacillary, 754 differentiation of, 64, 76 900 INDEX Yeast, division of, 64 Yeast, of wine, 71, 506 food value, 553 Yellow fever, 821 1 of ginger beer, 443 ' on grapes, 515 ZONOCERCUS elegans, 643 important, 71 Zygomycetes, 38 morphology of, 59, 61 Zygosaccharomyces, chevalieri, 68 of pombe, 533 Zygospore, 38 : pseudo-, 73 Zygote, 128 - of vinegar, 538 Zymases, 193, 200 MU i D ae ate Hl