f LT) □ BL/WHOI nj □ D m D □ -^— THE NEWER KNOWLEDGE OF BACTERIOLOGY AND IMMUNOLOGY THE UNIVERSITY OF CHICAGO PRESS CHICAGO, ILLINOIS THE BAKER & TAYLOR COMPANY NEW YORK. THE MACMILLAN COMPANY OF CANADA, LIMITED TORONTO THE CAMBRIDGE UNIVERSITY PRESS LONDON THE MARUZEN-KABUSHIKI-KAISHA TOKYO, OSAKA, KYOTO, FUKUOKA, SENDAI THE COMMERCIAL PRESS, LIMITED SHANGHAI THE NEWER KNOWLEDGE OF BACTERIOLOGY AND IMMUNOLOGY IL By EIGHTY-TWO CONTRIBUTORS Edited by EDWIN O. JORDAN and L S. FALK The University of Chicago THE UNIVERSITY OF CHICAGO PRESS CHICAGO • ILLINOIS COPYRIGHT 1928 BY THE UNIVERSITY OF CHICAGO ALL RIGHTS RESERVED. PUBLISHED APRIL I928 COMPOSED AND PRINTED BY THE UNIVERSITY OF CHICAGO PRESS, CHICAGO, ILLINOIS, U.S.A. PREFACE This book was prepared in an attempt to make the latest results of investigation in various lines of bacteriology and immunology available for students and active workers. It does not purport in any sense to be a textbook, nor does it pretend to be a comprehensive survey of the whole field. Our object has been primarily to obtain authoritative critical reviews of topics in which at the present time interest is par- ticularly keen or investigation most active. It is our hope that the book will serve to promote research both by furnishing landmarks of progress and by affording sugges- tions on significant unsolved problems. With these ends in view, great latitude has been given to the individual contrib- utors, each of whom assumes direct responsibility for the material presented. While the editors have endeavored to avoid serious duplication and overlapping, they have in a number of cases intentionally asked for and included papers with opposing views and interpretations in order to set clearly before the reader the divergences of cur- rent opinion. This independence of treatment has even extended to such a matter as the no- menclature of microbic types and species. It was early found in our preliminary cor- respondence that many of our contributors had very strong convictions regarding nomenclatorial practice, and that their convictions were widely apart. Since we did not ourselves feel that the time had arrived for insistence upon a uniform and rigid bacterial nomenclature, we chose to give full rein to individual preference. While we are aware that this course is open to criticism, we believe that our decision will at least serve to bring into yet stronger relief the almost hopeless confusion and di- vergence of opinion into which classification and names of bacteria have fallen. We considered it an important feature of our undertaking that the individual arti- cles should come to hand as nearly as possible at the same time in order to insure promptness and timeliness of publication. We are greatly indebted to our contributors for their generally hearty response to this request. Many of them have been able to comply with this condition only at serious personal inconvenience and even sacrifice. In a few instances illness has interfered with the preparation of an intended manu- script. Several of our European correspondents who originally promised articles have failed to send anything. We are under particular obligation to the Board of Trustees of the University of Chicago and to Mr. Gordon J. Laing, director of the University Press, for aiding the publication of this volume with a special fund. Among the many individuals who have given us signal assistance, we wish to acknowledge special indebtedness to Mr. Donald P. Bean and Miss Anabel Ireland, of the University Press, and to Miss Theodora Piatt, of the Department of Hygiene and Bacteriology, for their efficient and untiring interest in seeing the book through the press. The Editors February 15, 1928 ^^. . %►• ^ ^ / CONTENTS "^ ^ A » • • J^/' CHAPTER ^^Ci^ O K/*^' PAGE I. The Newer Knowledge of the Morphology of Bacteria~T^ — T . i Herbert C. Ward II. The Chemical Structure of Bacteria 14 Traugott Baumgdrtel III. Staining Reactions of Bacteria 19 Johfi W. Churchman IV. Morphological Changes during the Growth of Bacteria ... 38 Paul F. Clark V. Growth Curves of Bacteria 46 R. E. Buchanan VI. The Rise and Fall of Bacterial Populations 58 C.-E. A. Winslow VII. The Dissociative Aspects of Bacterial Behavior 84 Philip Hadley VIII. Bacterial Associations 102 W. L. Holman IX. Classification of Bacteria 120 Roger G. Perkins X. Atoms, Ions, Salts, and Surfaces 136 William D. Harkins XI. The Effect of the Surface Tension of the Menstruum upon Bac- teria and Toxins 179 W. P. Larson XII. Oxidation-Reduction Potentials of Dye Systems and Their Sig- nificance in Bacteriology 188 W. Mansfield Clark XIII. Anaerobiosis 198 Ivan C. Hall XIV. Bacterial Oxidations and Reductions .211 James Walter McLeod XV. Protein (Nitrogen) Metabolism of Bacteria 218 Leo F. Rettger XVI. The Utilization of Carbohydrates by Bacteria 227 Arthur Isaac Kendall XVII. Utilization of Aliphatic and Aromatic Compounds by Bacteria . 243 S. A. Koser XVIII. Gas Metabolism of Bacteria 250 M. H. Souk XIX. Enzymes of Bacteria 268 Selman A . Waksman vii 30 ^ viii CONTENTS CHAPTER PAGE XX. Synthetic Culture Media 279 H. W. Schocnlein XXI. Determinations of Thermal Death-Time 285 /. Russell Esty XXII. The Standardization of Disinfectants and Antiseptics . . . 301 George F. Reddish XXIII. Nature, Distribution, AND Functions OF Soil Micro-organisms . 310 Selman A . Wakstnan XXIV. Autotrophic Bacteria 322 Robert L. Star key XXV. The Root-Nodule Bacteria of Leguminous Plants . . . -332 • Edwin Broun Fred XXVI. Micro-organisms in Relation to Soil Fertility 341 Jacob G. Lipman XXVII. The Role of Bacteria in the Treatment of Sewage . . . -351 F. W. Mohlman XXVIII. Some Problems in Water Bacteriology 362 John F. Norton XXIX. The Action of Ultra- Violet Light on Bacteria AND Their Products 371 Johfi F. Norton XXX. Bacteria in Milk 378 Robert S. Breed XXXI. Bacteria in Dairy Products 395 L. A. Rogers XXXII. The Bacterial and Health Aspects of Pasteurization . . . 403 Milton J. Rosenau XXXIII. Mechanical and Engineering Aspects of Pasteurization . . . 419 George W. Putnam XXXIV. Contamination and Deterioration of Food 437 Charles Thorn XXXV. The Bacteria of Food Poisoning 443 Edwin 0. Jordan XXXVI. The Spirochetes 452 Hideyo Noguchi XXXVII. Current Problems on Yeasts 498 F. W. Tanner XXXVIII. The Aspergilli: A Typical Group of Molds 509 Charles Thorn XXXIX. FiLTEElABLE VIRUSES 5^7 Thomas M. Rivers XL. The Bacteriophage: Present Status of the Question of Its Nature AND Mode of Action 525 J. Bronfenbrenner XLI. Filterability of Micro-organisms 557 5". P. Kramer XLII. A Theory of Microbic Virulence 565 /. S. Falk CONTENTS CHAPTER XLIII. Elective Localization of Bacteria in the Animal Body . Edward C. Rosenow XLIV. Bacteria in Relation to Plant Diseases George K. K. Link XLV. Communicable Diseases of Laboratory Animals K. F. Meyer XL VI. Bacteria of the Intestinal Tract Leo F. Rettger XLVII. Bacteria of the Respiratory Tract D. J. Davis XLVIII. Intestinal Protozoa of Man and Their Host-Parasite Relations Robert Hegner XLlX. The Immunological Bases for Different Types of Infection by the Blood Protozoa William H. Taliaferro L. Antigens and Their Specificity H. Gideon Wells LI. The Chemistry of Antigens Sara E. Branham LII. Antigenic Properties of the Bacterial Cell and Antibody Re- actions Hans Zinsser and J. Howard Mueller LIII. Heterophils Antigens and Antibodies C. G. Bull LIV. The' Physical Chemistry of Toxin and Antitoxin Mary E. Maver LV. The Preparation and Purification of Toxins, Toxoids, and Anti- toxins Edwin J. BanzhaJ LVI. The Titration of Toxins and Antitoxins by the Flocculation Method Stanhope Bayne-J ones LVII. Sublethal Intoxications with Bacterial Products . J. P. Simonds LVIIL The Mechanism of Agglutination Johi H. Northrop LIX. The Functional Role of Agglutinins .... G. Howard Bailey LX. Bacterial Agglutinins and Their Applications /. G. FitzGerald and Donald T. Eraser LXI. Precipitins and Their Applications H. M. Powell LXII. The Complement Fescation Reaction with Bacterlal Antigens Augustus Wadsworth LXIII. The Complement Fixation Test for Syphilis .... Ruth Gilbert IX PAGE 576 S90 607 639 650 660 679 702 711 721 733 739 745 759 772 782 802 811 824 831 838 X CONTENTS CHAPTER PAGE LXIV. The Kahn Reaction 848 R. L. Kahn LXV. The Mechanism of Phagocytosis 861 W. 0. Fenn LXVI. Phagocytes and Phagocytosis in Immunity 870 W. B. Wherry LXVII. Local and Tissue Immunity 881 Frederick P. Gay LXVIII. The Human Blood Groups 892 K. Landsteiner LXIX. The Heredity of the Blood Groups 909 Reuben Ottenberg and David Beres LXX. Antibacterial Sera 921 F. M. Hunloon and R. H. Hutchison LXXI. The Use of Human Serum from Convalescent Cases in Prevention and Treatment of Disease 934 William H. Park LXXII. Control and Standardization of Biological Products . . . 947 G. W^ McCoy LXXIII. Anaphylaxis and Anaphylactoid Reactions 966 Howard T. Karsner LXXIV. The Technique of Experimentation in Anaphylaxis .... 989 W. H. Manwaring LXXV. Atopy 1004 Arthur F. Coca LXXVI. Tuberculin and the Tuberculin Reaction 1016 Esmond R. Long LXXVII. Origin of Antibodies 1035 Katharine M. Howell LXXVIII. The Isolation of Substances with Immune Properties . . . 1049 Arthur Locke and Edwin F. Hirsch LXXIX. Abderhalden's Dialysis Reaction and Theory of the So-called "Protective" Ferments 1056 /. Bronfenbrenner LXXX. Venoms and Antivenins 1066 Afranio do Amaral LXXXI. A Critique of the Ehrlich Theory, with an Outline of the Enzyme Theory of Antibody Formation 1078 W. H. Manwaring LXXXII. Non-Specific Protein Therapy 1086 William F. Petersen LXXXIII. Chemotherapy of Bacterial Diseases iioi John A. Kolmer Author Index 1135 Subject Index 1167 CHAPTER I THE NEWER KNOWLEDGE OF THE MORPHOLOGY OF BACTERIA HERBERT C. WARD School of Hygiene and Public Health, Johns Hopkins University For years bacteriology, dominated by the cellular doctrine of Virchow, has ac- cepted the thesis, more or less completely proved by Cohn and Koch, that each bacte- rial cell is derived from a previously existing cell of practically the same size and shape. Owing to the fact that a large number of highly diversified forms were included in the group of bacteria, considerable confusion existed before Koch devised his solid media and his plate-pouring methods. The proof that each type of cell, such as the spherical coccus or the elongated bacillus, came from a cell of the same type, a coccus or bacillus, was of enormous benefit in straightening out the confusion and in eventually reconcil- ing conflicting observations and opinions. While it was early recognized that the meth- ods by which the bacterial cells are derived from the pre-existing cells might differ in different species, two regular methods were definitely estabhshed: binary fission, where one cell divides transversely into two new cells which eventually attain the size and shape of the original; and spore formation, by which a single spore forms in a ba- cillus, this spore subsequently giving origin to a vegetative rod like the original rod be- fore sporulation begins. A more complicated cycle of development from conidia was established for certain species which were differentiated from the simple bacteria ex- hibiting transverse fission or spore formation as the "higher bacteria" and called "streptothrix" or "actinomyces." Exceptions to these regular methods of reproduction of the bacterial cells were frequently noted. With some species appearances were described which indicated that new cells might rise from old cells by a kind of branching of the cytoplasm, this phenomenon being described as "true branching" to distinguish it from the branching seen in certain plants related to the bacteria, where the cells divide transversely and eventually so crowd the sheath in which they are contained that this itself divides. Knoblike protrusions from the bacterial cells were noted occasionally, and the sug- gestion was made that these protrusions are in reality buds, capable of growth and de- velopment into adult forms. Large, irregular, distorted elements were described in old cultures, especially by Hueppe, who regarded them as true stages in the life-cycle of the bacteria and named them "arthrospores." Occasionally more than one spore was found in a single bacillus, and the idea naturally arose that spore formation may lead to an actual increase in the numbers of the cells. Such double spore formation was ad- mittedly very rare and generally doubted so that the thesis maintained by Kruse was usually accepted, to the effect that spores represent resistant stages of bacteria like the cysts of the protozoa which serve for the perpetuation of the species under adverse conditions, but not for multiplication. Finally, with the flexible spiral organisms, the spirochetes, differing from the ordinary bacteria in many of their characteristics, a pe- 2 THE MORPHOLOGY OF BACTERIA culiar type of transverse division was early noted and later worked out in great detail by Gross who described it as "multiplication by incurvation." It is of interest in this connection to note that some years ago Gotschlich, in his studies on plasmolysis and plasmoptysis, came to the conclusion that bacterial cells may break up into small bits of protoplasm, resembling in no way the original cell in size or shape but serving as starting-points from which new cells germinate. Gotschlich admitted that no positive evidence exists to show such a kind of reproduction, suggesting it merely as a possibil- ity which cannot be excluded. During the past few years a number of observations have been made which indi- cate that many of our earlier ideas in regard to the morphology of the bacterial cells must be subjected to rigid scrutiny, and the new conceptions recently advanced will, if proved, modify our entire point of view with respect to these microscropical organ- isms. PLEOMORPHISM It has long been recognized that bacterial cells exhibit what we regard as their characteristic morphology under certain favorable conditions which we find in the laboratory, usually in young cultures and on media peculiarly favorable to their growth. Our effort, indeed, is to subject organisms to constant conditions of light, temperature, and nutrition and so maintain constancy of form. Certain species are peculiarly susceptible to environmental influences and prone to exhibit departures from their standard morphology. Thus the cholera vibrio and Vibrio proteus assume in old cultures the most bizarre shapes, swollen spheres, crescents, half-moons, and distorted cells with knoblike protuberances. Similar changes are seen when these species are cultivated on acid media. Transfer of these distorted forms to media of the proper alkaline reaction yields normal vibrios as evidence of their potential viability. Large spherical forms are common in old cultures of the micrococci and are sometimes seen in the streptococci. The plague bacillus is especially likely to produce enlarged distorted forms on certain kinds of media (salt agar), and their production is regarded as practically diagnostic. In the animal body also departures of bacteria from their characteristic morphology have been frequently noted. One can find in streptococcus infections of the throat large cocci (megacocci) bearing little resemblance to ordinary streptococci, yet cultures from such throats may yield only streptococci. With the plague bacillus the appearance of enlarged, distorted forms is not uncommon in both natural and artificial infections. Organisms exhibiting an atypical morphology are usually called "degeneration" or "involution" forms. A number of investigations bearing upon their origin have ap- peared within fairly recent years. Thus Wilson' has found that when Bacillus coli, Bacillus typhosus, Bacillus enteritidis of Gartner, the Friedliinder bacillus, and the plague bacillus are cultivated on media containing urine, a great diversity of forms re- sults. The organisms are large and distorted and filamentous elements are common. Hata^ has shown that the addition of magnesium chloride to agar upon which dysen- tery and plague bacilU are grown leads to marked variation in the morphology of ' Wilson, W. J.: /. Path, b" Bad., ii, 394. 1906. 'Hata, S.: Centralbl.f. Bakteriol., 46, 289. 1908. HERBERT C. WARD 3 these organisms. Reed and Orr' attribute such morphological changes to the influence of the hydrogen-ion concentration in the media. Any acidity or alkalinity close to the limiting reaction for successful cultivation leads to the appearance of these aberrant forms. Changes in morphology cannot be attributed solely to the influence of the hy- drogen-ion concentration since Vay' had previously found that the addition of dyes like dahlia to the media leads to the production of long dye-stained threads by Bacillus typhosus and Bacillus paratyphosus. According to Henrici,^ who has studied the in- volution forms in Bacillus coli especially, variation in morphology seems to correlate with autolysis of dead cells. The rate of autolysis depends upon the degree of acidity or alkalinity of the medium. That bacteria may have different sizes at different stages has long been suspected. Evidence substantiating this view has been presented by Clark and Ruehl, and by Henrici. Clark and RuehP have noted that in general very young bacteria, in the first few hours of growth, are much larger than older organisms. This does not hold for all species, however, since these authors found that this enlargement does not occur with diphtheria and glanders bacilli. Henrici^ has found that morphological variations occur in the lag period of bacterial growth. The initiation of growth is marked by a transformation to embryonic or growing cells which are considerably larger than the cells produced during the phase of maximum development. The production of large, irregular involution forms regarded as characteristic of the plague bacillus has been found by Smillie^ in other representatives of the hemor- rhagic septicemia group. This work has not thus far been definitely confirmed. In- volution forms occur with such species as Bacillus suisepticus, but they are not as marked as are those in Bacillus pestis. Finally, a new point of view in regard to the branching of bacteria has been brought out by Gardner^ who believes that probably many pathogenic bacilli grow by three-point branching from Y-shaped forms. If Gardner's observations can be con- firmed they will give us a different conception of bacterial multiplication and may ex- plain many obscure points in morphology and physiology. FILTERABLE FORMS OF BACTERIA Under ordinary circumstances the passage of bacteria through the pores of filter material like the siliceous earth in Berkefeld candles and the unglazed porcelain in Chamberland bougies depends on the size of the pores and the size of the bacteria, al- though other factors of great importance may modify the results. The most important of these are the amount of pressure or suction applied to force the fluid through the filter mass, the composition of the fluid in which the bacteria are suspended, and its reaction. At the same time the type of the micro-organisms has an important influ- ence on their ability to pass through filter material, and it has long been recognized ' Reed, G., and Orr, J. H.: J. Bad., 8, 103. 1923. *Vay, F. : Cenlralbl.f. Bakteriol., 55, 193. 1910. 3 Henrici, A. T.: /. Infect. Dis., 39, 429. 1926. " Clark, P. F., and Ruehl, W. H.: /. BacL, 4, 615. 1919, s Henrici, A. T.: /. Infect. Dis., 38, 54. 1926. * Smillie, W. G.: ibid., 27, 378. 1920. ^ Gardner, A. D.: /. Path. 6* Bad., 28, 189. 1925. 4 THE MORPHOLOGY OF BACTERIA that the flexible spirochetes can pass filters more easily than other forms of bacteria. On numerous occasions bacteriologists have found that the effluent from the finest fil- ters may yield species like the original on cultures, although organisms cannot be found on microscopic examination. At the same time the effluent from suspensions of pathogenic organisms may produce disease in animals although organisms cannot be found on examination, or obtained in cultures. Unexpected results of this character were usually attributed to defects in the filter material permitting a small number of viable organisms to pass, or to a prolonged time in the process of filtration which per- mitted motile organisms to penetrate the filter mass, or viable bacteria to grow through. It was eventually recognized that this type of explanation did not always suffice to explain the results and that other possibilities must be considered. Theoretically, the bacterial cell may at times develop into morphological minia- tures of the original but with dimensions only fractions of the standard, or it may pro- duce a kind of spore or seed within its waUs, visible, filterable and viable, but morpho- logically unrecognized except as granules of characteristic size, shape, and staining re- action. Finally, the bacterial cell as we see it ordinarily may be but one stage in a complicated life-cycle, another stage being represented by filterable units, invisible by present methods, but viable in cultures, or viable only in the human or the animal body. While Schaudinn in his investigations of protozoa and spirochetes anticipated theoreticaUy much of the work of more recent years and expressed his belief that cer- tain species produce units which can pass filters, Gotschlich, as mentioned above, was the first to emphasize the possibility of minute units among the simpler bacteria. In his most recent publication Gotschlich' accepts filterable forms of bacteria as demon- strated and states that they may be differentiated from the filterable and inanimate products of bacteria by the fact that they are viable, in some instances capable of growth and culture, and in other instances capable of producing a characteristic dis- ease picture in experimental animals. With bacteria obviously too large to pass through filters Gotschlich again emphasizes his earlier opinion that a fractional part of the cell may escape and a normal adult cell regenerate from this portion. A vast amount of work has been carried out to substantiate views of this nature, among which may be mentioned the extensive studies of life-cycles of bacteria by Almquist, Enderlein, Fuhrmann, Lohnis, Mellon, and others. Of especial importance in this im- mediate connection is the work on the tubercle bacillus. Much^ in 1907 found small gram positive granules in the tubercle bacillus forming an integral part of the cell and which can be recognized outside it by their peculiar ap- pearance. With material containing these granules and no regular tubercle bacilli. Much believed that he had produced tuberculous lesions in experimental animals from which the characteristic acid fast organisms could be obtained. A little later Fontes^ pointed out that the Much granules are able to pass through Berkefeld candles. He inoculated guinea pigs with filtered granules and found no lesions except in the spleen. With spleens from the first series of guinea pigs he inoculated another series, the ma- ' Gotschlich, E.: Handb. d. path. Mikroorg., i, 33. 1927. » Much, H.: Beitr. 2. Klin. d. Tuherk., 8, 85. 1907. »Fontes, A.: Centralbl.f. Bakleriol., Abt. I, Ref., 51, 244. 1912. HERBERT C. WARD 5 jority of which showed tuberculous lesions from which the tubercle bacillus was culti- vated. On the basis of these expermients Fontes concluded that the Much granules represent a filter-passing stage of the tubercle bacillus. Spengler' noted, in tuberculous sputum, granules somewhat like the Much granules in morphology but acid fast in their staining reactions. In cultures from this material Spengler found the same acid fast granules together with small acid fast bacilli. After some time the cultures re- vealed acid fast bacilli of the normal size. These minute bacillary types Spengler named "SpHtter" and regarded as filterable stages in the life of the tubercle bacillus. Splitter forms were more frequently noted by Spengler in bovine lesions than in hu- man, and this has subsequently been confirmed. A number of observers, such as Hauduroy and Vandremer^ and Valtis,^ have em- ployed tuberculous discharges filtered free of adult tubercle bacilli and injected them into animals. These animals later developed tuberculous lesions from which tubercle bacilli were obtained. Valtis was unable to get cultures from his filtrates but found multiple glandular enlargements in his inoculated guinea pigs. Many acid fast tuber- cle bacilli were present in the visceral and pneumonic lesions. These animals reacted to tuberculin just as did the animals inoculated with the unfiltered tuberculous ma- terial. Valtis concluded that the filtrates from the tuberculous material contained the tubercle bacillus in some exceedingly small form capable of passing his filters. His conclusions have recently been confirmed by Arloingt who employed tuberculous fluids from a variety of lesions and cultures of the tubercle bacillus. With carefully controlled filters the filtrates were found to be sterile. In animals inoculated with the filtrates the lymphatic glands were enlarged in a characteristic manner, and micro- scopic examination revealed numerous tubercle bacilli. Finally, De Potter^ made use of materials containing the avian type of the tubercle bacillus and controlled his fil- trations with the minute organisms which cause chicken cholera. His filtrates were sterile while his experimental animals developed tuberculous lesions from which the avian tubercle bacillus was cultivated. Observations similar to those made on the tubercle bacillus have been reported for a number of other organisms — for the dysentery bacillus by Hauduroy; for the typhoid bacillus by Almquist, Fijgin, and Bergstrand; and for Bacillus coli by Izar d'Herelle, and Tomaselli. The conception of filterable stages in these organisms does not differ materially from that in regard to the tubercle bacillus, but the experimental results are by no means as clear. For the present we can only state that very strong evidence has been presented in favor of filterable stages in the tubercle bacillus, but that more extensive confirmation of the work is necessary before the question can be regarded as settled. SPECIAL ORGANS OF REPRODUCTION Intimately associated with the question of filterable stages is that of the presence of special, complete morphological structures in the simpler bacteria analogous to the ' Spengler, C: Ztschr.f. Hyg. u. Infektionskrankh., 49, 541. 1905. ' Hauduroy, P., and Vandremer, A.: Compt. rend. Soc. de biol., 89, 1276. 1923. J Valtis, J.: ibid., 90, 74. 1924; Ann. de I'lnst. Pasteur, 38, 453. 1924. * Arloing, F.: Bidl. Acad, de med., Paris, 96, 301. 1926. 5de Potter, F.: Compt. rend. Soc. de biol., 96, 138. 1927. 6 THE MORPHOLOGY OF BACTERIA conidia of the actinomyces which are definitely reproductive elements and serve to perpetuate the species. It is debatable whether it is advisable to employ such an anal- ogy since Drechsler has pointed out that the actinomyces form a group closely related to, and possibly forming a subdivision of, the hyphomycetes. Knoblike protrusions have been observed in many species of bacteria, and certain authors have come to re- gard them as conidia, thereby claiming a complicated cycle of development for the ordinary simple bacteria. Almquist, Enderlein, and Fuhrmann have emphasized this phase of development. Thus Almquist' has found, in cultures of the typhoid bacillus, the dysentery bacillus, and the cholera vibrio, spherical or globular elements and giant cells, often with distinct internal structure, from which normal forms are regen- erated. The transformation of these aberrant forms to the normal has been observed directly under the microscope by Almquist. Similar observations have been made in this country by Lohnis, Mellon, and Bergstrand, with diphtheria bacilli and with streptococci. Large spherical bodies had previously been observed by Swellengrebel in the larger spirilla but were regarded by him as no longer capable of multiplication. The ability of these knoblike protrusions and spherical bodies to regenerate normal individuals may be regarded as practically settled in view of Almquist's observations, but the in- terpretation of these bodies as true conidia can hardly be accepted at the present time. Almquist and Lohnis have further described a kind of sexual multiplication, consisting of conjugation or amalgamation of two forms. Interesting as such observa- tions may be, real proof for sexual multiphcation in bacteria is still lacking. It should be remembered, however, that if bacteria continue to multiply indefinitely without some kind of change which represents conjugation they form an exception to the laws governing other free-living plants and animals. INTERNAL STRUCTURE The characteristics of the internal structure of the simpler bacteria have been the subject of investigation since the earliest days of bacteriology, with particular refer- ence to the question of nuclear material. Opinions have ranged from the view that the entire cell represents the nucleus of higher cells to the view that no true nuclear material is present in them. Chemical analysis has thrown much light upon the kinds of chemical substances present in the bacteria, and the chemical demonstration of nuclear material has stimulated bacteriologists to approach the question again and make use of the more refined staining methods devised by the cytologists. As a re- sult, a kind of diffuse nucleation was established for the bacteria, nuclear material (chromatin) being distributed through the bacterial cytoplasm. Certain authors like Nakanishi' and Dobell,^ however, have always claimed the presence of true mor- phological nuclei. Thus Nakanishi has described minute spherical bodies in Staphylo- coccus aureus, in the middle of the cells, which he regards as nuclei. Dobell found true morphological nuclei in a spiral organism which he named Paras pii ilium vejdov- ski, but which was not cultivated. There is still some doubt whether Dobell's organ- ■ Almquist, E.: Ztschr.f. Ilyg. u. Infektionskrankh., 83, i. 1917. 2 Nakanishi, K.: Centralbl.f. Bakleriol., 30, 145. 1901. 3 Dobell, C. C: Arch.f. Proiist., 24, 907. 1912. HERBERT C. WARD 7 ism should be included among the bacteria. Douglas and Distaso/ however, working with a capsulated bacillus easily cultivated by them from cases of respiratory in- fections, were able to stain successfully certain cellular uaiits closely resembling true nuclei. Recently Gutstein,^ by staining methods, has made out a differentiation of the bacterial cytoplasm into ectoplasm and endoplasm. Tn the endoplasm he finds a macronucleus and a micronucleus. The ectoplasm of the gram positive bacteria Gut- stein^ later has shown to contain a basic ground substance demonstrable by malachite green and tannin, and an acid body demonstrable by Victoria blue. This acid sub- stance Gutstein regards as a lipoid. Somewhat similar observations have been made by Churchman-' in this country in his valuable studies on the gram reaction. He has found that Bacillus anthracis consists of two distinct parts, a gram positive cortex and a gram negative medulla. The term "cortex" as used by Churchman is equivalent to ectoplasm and the term "medulla" to endoplasm. The cortex may be removed by exposure to acriviolet or gentian violet and sometimes by hydrolysis in distilled water. The material lost is probably protein in character since the Berkefeld filtrate of a suspension of Bacillus anthracis exposed to gentian violet gives a positive nin- hydrin test. Bessubetz^ believes that the bodies inside bacteria and demonstrable by the use of the Giemsa stain are true morphological nuclei. Similar conclusions have been reached by Schumacher*' on the basis of chemical reactions. In view of the recent investigations on the internal structure of the bacteria we can only say that while true morphological nuclei cannot be regarded as definitely proved, material giving the staining reactions of chromatin may be found in a great many different bacteria. In stained specimens this chromatin is sometimes agglomer- ated in masses which resemble the nuclei of plant and animal cells. CAPSULES Various new methods for the demonstration of capsules have been devised during the past few years and our ability to fix and stain these structures has been consider- ably enhanced. In addition to these newer staining methods a number of investiga- tions have been made in regard to the chemical composition of the capsular substance. It should be noted that these investigations relate to the capsules of the true encapsu- lated bacteria like the pneumococcus, the Friedlander bacillus, and to certain intes- tinal organisms like Bacillus coli and not to saprophytic bacteria. Such saprophytes as Bacillus suhtilis and Bacillus mesentericus under certain circumstances are provided with beautiful capsules, and it is certain tiiat a great many different species may have a capsular material deposited about them under environmental stimuli. With the or- ganisms mentioned above capsules form an integral part of the bacterial cell. With such species as the Friedlander bacillus Toenniessen^ has shown that the capsule is a ' Douglas, S. R., and Distaso, A.: Ceniralbl.f. BakterioL, 63, i. 191 2. 'Gutstein, M.: ibid., 95, 357. 1925. 3 Gutstein, M.: ibid., p. i. 1925. * Churchman, J. W.: Proc. Soc. Exper. Biol. iT Med., 24, 737. 1927. sBessubetz, S. K.: Ceniralbl.f. BakterioL, 96, 177. 1925. ^Schumacher, J.: ibid., 97, 81. 1926. ' Toenniessen, E.: ibid., 65, 23. 1912; ibid., 85, 225. 1921. 8 THE MORPHOLOGY OF BACTERIA secretory product and made up of galaktan, a polysaccharide of galactose, and con- tains no protein. The investigations of Toenniessen were later extended by Kramar' who found that the capsule of the Friedlander bacillus consists of galaktan, that of Bacillus anthracis of a glycoprotein, and that of Bacillus radicicola of a dextran. The capsules of different bacteria thus differ among themselves radically in their chemical composition. Recently Avery, Heidelberger, and Goebel^ have approached the question of cap- sule formation in bacteria from still another angle. From the pneumococcus type II they have obtained the capsular substance and found it to be a nitrogen-free poly- saccharide. This substance was non-antigenic. From the bacterial cell they obtained a protein substance which was antigenic but had no type specificity. In order to get type-specific reactions in animals it was necessary to employ both the protein con- stituent of the cells and the capsular substance before separation of the cell mass into its constituents. A similar carbohydrate, a nitrogen-free polysaccharide, was obtained from a strain of the Friedlander bacillus. The carbohydrate obtained from the type II pneumococcus was the same as the substance found by Dochez and Avery^ in the blood and urine in pneumococcus infections in man. Julianelle,^ working with rabbits infected with a strain of the Friedlander bacillus, found specific substances in the urine and blood which were practically identical with those in the soluble portion of the or- ganisms. Finally, Smith^ has reported biochemical studies on certain encapsulated strains of Bacillus coli. She states that the capsule materials consist of 80 per cent of a special carbohydrate, a hexose, together with a small amount of glycuronic acid. In order to show that this carbohydrate really represents capsule substance, she employed strains of the encapsulated Bacillus coli deprived of their capsules through mutation cultivation (see Theobald Smith).'' On comparing the filtrates from the two organ- isms, she was able to show that the precipitin test was one hundred times less active specifically with the filtrate from the mutant than with the filtrate from the encapsu- lated strain. These observations on the kinds of substances which may be obtained from capsules are of fundamental importance and must, as they are further amplified, aid materially in the interpretation of the composition and especially the function of capsules.^ SPORES As already mentioned, the conception of Kruse that spores in the bacteria are resting bodies like the cysts of the protozoa has had wide acceptance. According to this conception, spores are produced under unfavorable conditions, especially as re- gards food supply, serve purely for the perpetuation of the species, and show no evi- dence of vital activity. A number of reports have recently been published which must change our interpretation of spore formation. Thus Ruehle^ has found that spores ' Kramar, E.: Centralbl. f. BaclerioL, 87, 401. 1922. ' Avery, O. T., Heidelberger, M., and Goebel, W. F.: /. Exper. Med., 42, 701, 709, 727. 1925. 3 Dochez, A. R., and Avery, O. T.: ibid., 26, 477. 191 7. -I Julianelle, L. A.: ibid., 46, 113. 1926. s Smith, D. E.: ibid., p. 155. 1927. 'See chaptet lii in this volume. ^ Smith, T.: ibid., p. 141. 1927- ' Ruehle, G. L. A.: J. Bad., 8, 487. 1923. HERBERT C. WARD 9 show evidence of enzymic activity even when no evidence of germination is present. Oxidases as well as gelatinase can be demonstrated in spore material. Magoon' be- lieves that spores are not dormant under ordinary conditions but are sluggishly ac- tive. Resistance to heat is not fixed but variable, being influenced by age, tempera- ture, heredity, etc. This resistance to heat may be increased by selective action (Ma- goon).^ In Bacillus mycoides certain organisms surviving after heating seem to have greater resistance to heat than the original spores. Daranyi^ has recently pointed out that the ability of certain species like Bacillus anthracis, Bacillus subtilis, and Bacillus anthracoides to form spores depends in general upon the same optimum conditions which lead to good vegetative development of the bacteria. Spore formation is brought about primarily by colloidal reactions; the most important of these is a diminution in the water content of the organisms, resulting therefore in a shrinking of the colloids. Under natural conditions spore formation begins when the organisms grow older. This aging is primarily a loss in water on the part of the colloids (hysteresis). The lack of food material for the bacteria has a favorable influence on spore formation only in that the organisms get poorer in water. With artificial dehydration, Daranyi was able to bring about spore formation in well-developed young bacilli. Koser and McClelland^ have added interesting facts in regard to the fate of spores in the animal body. The spores of Clostridium tetani, CI. putrificmn, CI. chauvei, and CI. oedematis-maligni are capable of withstanding the deleterious influence of the body tissues and may be transported from the site of inoculation to different organs, where they remain latent. Aerobic bacteria do not apparently suffer the same fate. Such observations are of considerable importance in an explanation of the occurrence of tetanus after wounds in cases where the wound itself is originally uninfected; it is pos- sible that the latter infection is due to the presence of latent spores in other parts of the body. Finally, mention must be made of the newer methods of determining the heat resistance of spores. Esty and Williarass have estimated resistance by heating a large number of tubes containing spores and plotting curves from the results. The rate of destruction corresponds in general to the rate of destruction of bacteria by dis- infectants worked out by Chick, by Madsen and Nymen, and by Eijkman. MULTIPLICATION IN THE SPIROCHETES The flexible spirochetes differ considerably in their morphology from the simpler bacteria but not sufficiently to separate them from the group in which the first repre- sentative was placed originally by Ehrenberg. In at least three particulars is their morphology interesting to us at the present time — their motility, method of division, and the possession of granules which are occasionally extruded from the cell and which may be reproductive bodies. In regard to motility it has now been satisfactorily proved that the spirochetes possess no organs of locomotion like flagella, as was orig- inally maintained. Flagella-like structures may occasionally be found, attached to the ' Magoon, C. A.: ibid., ii, 253. 1926. ^Magoon, C. A.: /. Infect. Dis., 38, 429. 1926. 3 Daranyi, J.: Centralbl.f. Bakteriol., Abt. II, 71, 353. 1927. * Koser, S. A., and McClelland, J. R.: /. Med. Research, 37, 259. 1917. 5 Esty, J. R., and Williams, C. C: /. Infect. Dis., 34, 516. 1924. A t I ^ ^ A R Y- o V 10 THE MORPHOLOGY OF BACTERIA outer rim of the organisms, but it has now been established that these are not flagella but bits of periplast torn off in fixation and staining. Under dark-field illumination no refraction of light can be detected like that seen with the ciliated bacteria. The crista present in some of the larger saprophytic spirochetes, the cristispiras, has been shown to lack independent motility. Motility in the spirochetes must therefore be attributed to streams of protoplasm passing through the bodies of the cells or to the presence of contractile elements within, like the myonemes of certain protozoa. No definite dem- onstration of myonemes has thus far been effected, although Noguchi has observed bodies which strongly suggest them. Tt is possible that streams of protoplasm do exist in these organisms and that their motility is due to changes of surface tension. A sat- isfactory explanation of motility is very much needed. It is now generally admitted that the simpler spirochetes (treponema, spironema, leptospira) multiply by transverse fission. It is true that appearances suggestive of longitudinal fission have often been noted, but these were explained as the separation of two organisms tightly coiled about each other. The chief evidence that spirochetes divide transversely and not longitudinally depends upon accurate measurements of the diameter of the cells, a method of study carried on especially by Shellack.' He as- sumed that the diameter of the newly formed cells must at some time be one-half of the diameter of the cells before division, if longitudinal division takes place. In careful measurements of certain spirochetes {Spironema duttoni, novyi, etc.) Shellack was never able to find organisms which were twice as thick as the thinnest forms. He was, however, always able to find organisms twice as long as the shortest forms. He there- fore concluded that the spirochetes always divide transversely and not longitudinally. Lange" has recently called attention to the fundamental misconception in our inter- pretation of measurements of all morphological types of bacteria. After equal binary fission of spherical organisms the diameter of the daughter-cells is not one-half that of the mother-cell, but equals the cube root of one-half the cube of the diameter of the mother-cell, or over three-quarters of it. With cylindrical organisms the daughter- cells are half as long as the mother-cells, after transverse division. After longitudinal division the diameter of the daughter-cells equals the square root of one-half the square of the diameter of the mother-cell, or a little less than three-quarters of it. Since the spirochetes are practically cylindrical organisms, the failure to find organisms whose diameter is one-half the diameter of the thickest cells cannot be regarded as ex- cluding longitudinal division. A large number of spirochetes, especially the pathogenic varieties, show minute spherical bodies in the interior of the cell, and these are occasionally extruded. These bodies have frequently been regarded as reproductive elements. This conception we owe primarily to Leishman who found numbers of granules in ticks infected with Spi- rochaeta duttoni but no spiral organisms. With these ticks he was able to convey spi- rochetal diseases experimentally. The matter was thoroughly investigated by Hindle^ and by Balfour.-* Hindle, studying ticks infected with Spirochaeta gallinarum, found ' Shellack, C: Arb. a. d. kais. Gesamle., 27, 364. 1908. "Lange, L. B.: J. Bad., 14, 275. 1927. J Hindle, E.: J. Parasitol., 4, 463. Cambridge, 1911. * Balfour, A.: /. Trap. Med., 10, 153. 1907; Brit. M.J., 2, 1130. 1907. HERBERT C. WARD n granules and both small and large spirochetes representing every gradation in size from granules to normal adult spirochetes. While he did not observe the actual growth of granules into spirochetes, he concluded from the varying numbers of granules and spirochetes that the change must occur in this direction. Balfour described a special type of spirochete concerned with fowl spirochetosis which he named S pirochaeta granulosa penetrans, and which he believed entered the red-blood corpuscles of the fowl and broke up into granules that were responsible for a peculiar recrudescence of the disease known as the ''after-phase." There can be Uttle doubt that granules are produced in a number of both pathogenic and saprophytic spirochetes, and are shed from the cells under certain circumstances. That granules are reproductive elements cannot be regarded as settled since Button and Todd have shown that adult spiro- chetes can sometimes be found on prolonged search in infective ticks along with the granules. Filtrates from spirochetal material showing an abundance of granules may also reveal a few spirochetes which because of their peculiar boring motility are able to pass through the filter substance. Recently Meirowsky" has observed swollen bodies and coiled forms in Treponema pallidum in secondary syphilis and has suggested that they are stages in the life-history of the organism. Szilvasi and Feher^ have found Meirowsky's forms in both primary and secondary lesions in syphilis and agree with him in regarding them as infective stages of the treponema. It may be noted in this connection that Aristowsky and Holzer-' have described peculiar coiled, twisted forms in Spirochaeta ohermeieri and believe that this organism also passes through a defi- nite life-cycle. THE RICKETTSIAE Following the discovery of pleomorphic bacteria-like bodies in Mexican typhus (tabardillo) by Ricketts a number of similar organisms were observed in various dis- eases and in their insect vectors. For this group the term "Rickettsia" was proposed by Da Rocha-Lima'' and the type species named Rickettsia prowazeki in honor of Ricketts who discovered the first representative and von Prowazek who devoted many years to its investigation. The significance of this group of micro-organisms will be considered in chapter xxxix of this volume, but one or two points should be mentioned here in regard to their morphology. Hertig and Wolbach^ and Cowdry^ in this country have noted their wide distribution in insects and arachnids. According to Arkwright, Atkin, and Bacot,^ the rickettsiae are characterized by their minute size, usually being less than 0.5 /x in diameter; their pleomorphism from round, coccus-like bodies and diplococci to minute bacilli and threadlike forms; their resistance to or- dinary aniline stains; their loss of gram stain and affinity for Giemsa; their absence of motility; their resistance to cultivation on ordinary media; and their occurrence in very large numbers in the gut of blood-sucking insects. In some cases they may be found ' Meirowsky, R.: Mimchen. med. Wchnschr., 60, 1870. 1913. 'Szilvasi, J., and Feher, D.: Cenlralbl. f. BakterioL, 95, 436. 1925. 3 Aristowsky, W., and Holtzer, R.: ibid., p. 175. 1925. 4 Da Rocha-Lima: Miinchen. med. Wchnschr., 67, 1381. 1916. 5 Hertig, M., and Wolbach, S. B.: J. Med. Research, 44, 329. 1924. ^Cowdry, E. V.: J. Exper. Med., 37, 431. 1923. 'Arkwright, J. A., Atkin, E. E., and Bacot, A.: /. ParasiloL, 13, 27. 1921. 12 THE MORPHOLOGY OF BACTERIA in other organs. Later Wolbach, Todd, and Palfrey^ emphasized the specificity of the rickettsiae for certain insect hosts. Hertig and Wolbach further ampHfied the defini- tion of the rickettsiae, suggesting that the term should be limited to proved pathogen- ic organisms which are pleomorphic, non-motile, gram negative, stain rather lightly with the aniline dyes, and have a tendency to an intracellular habitat. The rickettsiae have been cultivated in only a few instances. Noller^ has been able to grow Rickettsia melophagi on blood agar inactivated by heating to 57° C, and Sellards^ recently has cultivated a rickettsia-like micro-organism from tsutsugamushi disease. This organism was pathogenic and was named by him Rickettsia nipponica. In consequence it is difficult to come to any definite conclusions as to their nature and proper classification. It is quite clear that they are not mitachondria (Cowdry),^ and in many of their characteristics they resemble the bacteria very closely. Their pleo- morphism is not greater than that of many bacterial species. In size they cor- respond to many of the smaller bacteria. They have no definite internal structure al- though a morphological nucleus has been claimed by Epstein^ for Rickettsia prowazeki. They stain badly by the ordinary aniline dyes but no more so than certain bacterial species like the glanders bacillus and the cholera vibrio. When cultivated they grow on media similar to those used for the majority of bacteria. For the present the rickett- siae should probably be included among the bacteria. In this connection the obser- vations of Cowdry and of Wolbach and Schlesinger are of great importance. Cowdry^ has described a rickettsia (Rickettsia ruminantium) in Amblyomma hebraeum, the bont tick, which transmits the virus of the disease of sheep, goats, and cattle known as "heart water." These organisms lay in the endothelial cells of the renal glomeruli and in the superficial gray matter of the cerebral cortex. They were uniform coccus-shaped bodies, 0.2-0.5 ix in diameter, sometimes in diplo formation. They stained deep clear blue by Giemsa, and easily by Lofifler's methylene blue and other aniline dyes. They were gram negative. Such organisms would naturally be included in the group of rickettsiae as above outlined. Cowdry^ further described in a number of ticks (Ar- gasidae and Ixodidae) certain non-pathogenic, gram negative organisms characterized by their resemblance to bacteria morphologically, their large size and their intracel- lular habitat. Obviously such organisms would not meet the exacting requirements laid down by Hertig and Wolbach for the rickettsiae. Size and pathogenicity are somewhat doubtful characteristics for classification, and it is difiicult to exclude from any group organisms which possess in the main the character of the group but are larger and endowed with pathogenic action. For the present the non-pathogenic organisms described by Cowdry should probably be regarded as rickettsiae and included with them in a larger group of pathogens and non-pathogens. • Wolbach, S. B., Todd, J. L., and Palfrey, F. W.: The Etiology and Pathology of Typhus. Cam- bridge, 1922. ' NoUer, W.: Arch.f. Schijfs- n. Tropen-Hyg., 21, 53. 1917. ^Sellards, A. W.: Am. J. Trop. Med., 3, 529. 1923. "Cowdry, E. V.: he. cil. 'Epstein, H.: Centralbl.f. Bakteriol., 87, ^s^. 1922. * Cowdry, E. V.: /. Exper. Med., 42, 231, 253. 1925. T Ibid., 41, 817. 1925 HERBERT C. WARD 13 Finally, Wolbach and Schlesinger' have brought to cultivation the micro-organ- isms of Rocky Mountain spotted fever {Dermacentroxenus rickettsi) and typhus {Rickettsia prowazeki) in tissue-plasma cultures. These authors have shown that the parasite of Rocky Mountain fever shows a definite series of morphological changes in ticks, from coccus to bacillary forms. Such observations suggest a somewhat more complicated cycle of development than that accepted for the ordinary bacteria and bring further evidence in favor of the view that binary fission and spore formation are not the only methods by which the bacteria multiply. ' Wolbach, S. B., and Schlesinger, M. J.: /. Med. Research, 44, 231. 1923-24. CHAPTER II THE CHEMICAL STRUCTURE OF BACTERIA^ TRAUGOTT BAUMGARTEL Technischen Hochschule, Munich, Germany- Like all plant and animal organisms, bacteria require ten elements — carbon, hy- drogen, oxygen, nitrogen, phosphorus, sulphur, potassium, calcium, magnesium, and iron — as indispensable building stones for their body substance. While bacteria take the metals (potassium, calcium, magnesium, and iron) as well as the metalloids (phosphorus and sulphur) mostly in the form of simple mineral salts and satisfy their need for hydrogen and oxygen chiefly from water, they are able, in the assimilation of carbon and nitrogen, to utilize numerous and widely diverse sources of nutrition. All substances — from complex natural compounds like proteins and poly- saccharides down to their simple cleavage and decomposition products and the very elements like nitrogen and hydrogen — can be utilized by the bacteria in their metab- olism and modified in many ways. For example, in their carbon nutrition they util- ize in this way complex plant as well as animal substances, proteins and their cleavage and decomposition products, carbohydrates, fats, alcohols, acids, carbohydrates, car- bon dioxide, carbon monoxide, and methane. Thus, the bacteria, in spite of all ad- vantages and choice of many food substances, show an unparalleled ability for ad- justment to the sources of food offered them. Furthermore, the entire process of nu- trition in the bacteria is in large part dependent on the hydrogen and oxygen content of the culture medium as well as on the reaction and temperature. For these reasons, the chemical composition of different kinds of bacteria varies markedly; even in one and the same kind of bacteria there are considerable variations, so that generally valid statements about the chemical composition of the body sub- stance of bacteria can be made only with due consideration of the breadth of their bi- ological variations. THE WATER CONTENT OF BACTERIA In order to determine the water content of bacteria by chemical analysis, the usual procedure is carefully to scrape off the colonies grown on solid medium, centrifugate the liquid growth promptly, and weigh the material thus obtained in its moist, living condition ("fresh weight") ; afterward, dry it at ioo°-iio° C. and weigh it again ("dry weight"). The difference between the fresh weight and the dry weight — expressed per loo gm. of fresh mass of culture — is of course the water content. In this way it has been determined that the water content of most kinds of bacteria is rather high. On the average it amounts to between 75 and 85 per cent of the fresh weight. There are rather wide fluctuations according to the kind of bacteria and the growth of the organ- ism chosen (whether on solid or liquid medium), and also according to the age of the ' For a more extensive summary, cf . Baumgartel, T. : Grundriss der theoretischen Bakteriologie. Berlin, 1924. 14 TRAUGOTT BAUMGARTEL 15 culture. It is clear that a particular kind of bacteria shows a different water content according to whether one is dealing with a young, fully developed culture in liquid culture medium or an old dying culture on solid medium. Especially rich in water are the mucus-producing kinds of bacteria. Species which store lasting reserve material like volutin, glycogen, or fat, and spores contain proportionately little water. THE DRY SUBSTANCE OF BACTERIA Differences in the water content of the bacterial cells lead to differences also in their dry substance, in which the proportions can vary markedly between the com- bustible ("organic") material and the non-combustible ("mineral") material. In gen- eral, a particular kind of bacteria when cultivated on liquid medium contains more water than when grown on solid medium. On the other hand, according to whether an increased content of mineral salts or of carbohydrates is put into the liquid or solid medium, sometimes the mineral part of the dry substance of bacterial growths is greater than usual, sometimes the organic part is greater. In view of the high varia- bility of the bacteria, no general statements can be made beyond this. So far as re- liable researches have shown, 70-97 per cent of a bacterium's dry weight is made up of organic substance, and correspondingly 30-3 per cent is mineral substance. THE ORGANIC DRY SUBSTANCE Doubtless the organic dry substance of bacteria consists of numerous compounds, still in part wholly unknown, which on account of their common properties belong chiefly to the proteins, carbohydrates, and fats. Like the water content of bacteria, the chemical composition of the organic dry substance also varies and is different in any one species according to the cultural conditions under which the colonies chosen for analysis were grown. For example, it has been found that cultures of Bact. pro- digiosum grown on potato contain almost 50 per cent more organic dry substance than the cultures raised on yellow turnip, and that the cultures of the same species kept on potato at 33° C. have more dry substance than similar cultures preserved at 16° C. It is also demonstrable that four-to-six-day-old cultures on potato show a larger quan- tity of organic dry substance than cultures thirteen to sixteen days old. Because in building their organic body substances the bacteria can adapt them- selves, within definite but rather wide limits, to the nutrient medium on which they are growing, there can be no generalization as to the results of chemical analysis on the qualitative and quantitative composition of the organic dry substance. Ac- cording to the findings in many studies, the protein content of the organic dry sub- stance of most bacteria varies somewhere between 40 and 70 per cent, the carbohy- drate content between 10 and 30 per cent, and the fat or lipoid between i and 10 per cent. In general, therefore, proteins make up the greater part of the bacterial body; then follow carbohydrates and fats or lipoidal substances. As is evident from precipitation and agglutination reactions, different kinds of bacteria grown on the same nutrient medium and under conditions precisely similar in other respects can be differentiated with respect to their proteins, so that with the aid of the serological reactions they can be identified without further difficulty. Chemical analysis, on the other hand, offers at present no method of distinguishing the different i6 THE CHEMICAL STRUCTURE OF BACTERIA kinds of bacterial proteins, for the methods are not sensitive enough and do not give unconditionally reliable results; nor do they permit generalization. At least worthy of mention, however, is the hydraulic-press method which produces from moist, living bacterial cultures, without deep-seated changes in the cell substance, protein-rich fluids. For example, in order to obtain in this way the protein material of the cholera vibrio, colonies of this organism are grown on nutrient agar; the thick, growing layer of vibrios is carefully lifted off by means of a platinum spatula; the colonies thus ob- tained are finely pulverized with diatomaceous earth and quartz sand in a mortar and through the addition of salt solution worked into a pulpy mass. This is thrown into a strong filter cloth and laid under the hydraulic press. By a pressure which is gradually increased to 400 or 500 atmospheres a press fluid is obtained which comes through the thick filter at first as a light, clear, protein-rich liquid, later changing its color in the air to yellow and brownish— the so-called "vibrio plasma." As the chemical investiga- tion shows, the plasma is largely precipitable with acetic acid in the cold; it does not dissolve with excess of acetic acid, i.e., it acts like a nucleoprotein. In other respects the expressed fluid gives the usual protein reactions. With the help of a number of color reactions the protein chemical differentiation of many kinds of bacteria can be carried out. The best known in this connection is the usual color method of Gram, which depends on the fact that salts of pararosanilin (for example, gentian violet and methyl violet) unite firmly with iodine and that certain kinds of bacteria take the color compounds thus formed very firmly, i.e., when treated with alcohol later, they do not so readily give up the color substance they have taken as do certain other bacteria. In consequence, bacteria may be separated into those "colored by Gram's method" (gram positive) and "not colored by Gram's method" (gram negative). The differential behavior of bacteria in the color test of Gram is due to an unexplained difference in the physico-chemical structure of the protoplasm.^ In analogous ways certain protein or protein-like cell constituents can be detected in the bacteria. Among these may be included the reserve material (volutin) stored up in many bacteria in the form of colorless and strongly light-refracting spheres, vis- ible under the microscope, the presence of which, for example, is utilized practically in the usual bacteriological tests for the diagnosis of diphtheria. Volutin is dissolved by warm water (30°) within two or three days. In water at 80° C. volutin is dissolved in five minutes. Volutin fixed with heat or alcohol (or formol) is insoluble in boiling water. A 5 per cent or saturated aqueous solution of sodium carbonate dissolves volu- tin in five minutes, as does also caustic potash. A freshly prepared solution of Javelle water dissolves volutin in five minutes. A 5 per cent sulphuric or hydrochloric acid solution dissolves volutin in from five to ten minutes; a 25 per cent nitric acid solution dissolves it at once; and a i per cent acetic or osmic acid as well as a 5 per cent car- bolic acid solution dissolves it slowly. On the other hand, alcohol, ether, chloroform, and carbon tetrachloride do not dissolve volutin. Potassium iodide-iodine solution, Millon's reagent, vanillin hydrochloride, and zinc chloride-iodine give no reaction with volutin; and trypsin and pepsin are also without effect. Moreover, dyestuffs also give a characteristic behavior with volutin. Methylene blue and carbol-fuchsin in 10 ' See chap, iii (by Dr. Churchman) in this volume. TRAUGOTT BAUMGARTEL 17 per cent solution color volutin quite intensively, while eosin borax carmine and nigrosin do not stain it; safranin and Bismarck brown give a stronger color to volutin than to the cytoplasm. As with the protein materials, so with the carbohydrates of bacteria — little defi- nite investigation has as yet been done. Most of the work up to the present has con- cerned itself with the study of the carbohydrates in the composition of bacterial mem- branes and mucus. For example, the gelatin formed by Streptococcus mesenterioides has been analyzed. The purified masses of gelatin were extracted with 96 per cent al- cohol, then boiled for a rather long time with milk of lime and the thickened masses obtained in this way precipitated with carbonic acid. From the precipitated calcium carbonate the mucous solution was poured off, cleared with hydrochloric acid, and precipitated with alcohol. The substance thus obtained is called "dextran." It ap- pears that neutral lead acetate does not precipitate the concentrated dextran solution, while basic lead acetate produces a pastelike mass. Moreover, it happens that a heat- saturated solution of barium hydroxide precipitates from concentrated dextran solu- tion an oily substance, and Fehling's solution, without itself being reduced, precipi- tates a mucous substance. When boiled with dilute sulphuric acid, dextran goes over into dextrose slowly; when heated to 120° C, it goes over in a few hours. Here may also be mentioned the researches on the carbohydrate content of the pellicle which appears in cultures of Bad. xylinum. The leather-like colonies were first cleansed with water, then boiled with a 20 per cent caustic potash, washed with dilute hydrochloric acid or with water, and finally treated with bromine. In this way there were produced colorless, transparent, thin films which dissolved in ammoniacal copper oxide as well as in concentrated sulphuric acid and, by element analysis, showed the composition corresponding to the formula CcHioOj. When the film was dissolved in concentrated sulphuric acid and this solution — after preliminary dilution with water — boiled, neutralized with barium carbonate, and filtered, its reactions in regard to dextro-rotation and reduction power were like dextrose. The carbohydrates of the bacteria that are demonstrable by microchemistry are glycogen and iogen (granulose), which appear in the protoplasm of many cells as colorless, viscous-flowing masses. Glycogen and iogen differ microchemically in their behavior with iodine. If very dilute potassium iodide-iodine solution is added to bac- teria which contain glycogen and iogen, only the iogen wiU be colored at first (blue) ; with stronger iodine solution the glycogen will also take (a dark red-brown) color. If the carbohydrate of the bacteria is colored only red brown, then it consists solely of glycogen ; if, with a stronger iodine-KI solution it takes only a blue color, then only iogen is present. If carbohydrate-containing bacteria are boiled for five minutes in water, the carbohydrates are still demonstrable by means of iodine-KI solution. If pigmented carbohydrate-containing bacteria are boiled in water, they appear without color when examined on the warm stage of a microscope. On the other hand, they again show color when the iodine-KI solution is cold. If bacteria containing carbohy- drates are treated for three minutes with boiling concentrated sulphuric acid, the car- bohydrates are completely dissolved.' ' For a discussion of polysaccharides ("specific soluble substances") from bacteria, of. chap, lii (by Drs. Zinsser and Mueller) in this volume. 1 8 THE CHEMICAL STRUCTURE OF BACTERIA Fat in bacteria appears mostly in the form of strongly light-refracting drops which in the young cells are very finely distributed in great numbers in the protoplasm; with increasing age, however, the drops join to form larger drops which occasionally fill the whole cell interior. Analytical studies on the fat or lipoid content of bacteria have been frequently conducted. From these it appears that the amount and the composi- tion of the detectable fats, lipoids, and waxes vary for the several kinds of bacteria and are dependent upon the conditions of growth. Thorough investigations have been made most frequently with pure cultures of the tubercle bacillus, which has an un- usually high fat content. In a very detailed study of this fat, four-to-five-month-old glycerol-bouillon cultures of this organism were killed in the autoclave at iio° C, the colonies collected on filter paper and treated with hot water until all the ingredients of the nutrient medium were washed away. The material was then spread on porous earthenware plates and dried at 40° C. In order to measure the fat contents of the pul- verized bacterial masses, these were treated with various fat solvents in the Soxhlet extraction apparatus. For a more precise determination of the fat of the tubercle bacillus, the same bac- terial powder was extracted several times with chloroform, the different extract por- tions mixed, and, after distilling off the chloroform, dried at 100° C. In this way there was obtained a dark-brown, semi-solid mass with glassy wrinkles and with the odor typical for tubercle bacillus cultures, like good wax from linden or flower honey. The melting-point of this fatty substance was 46° C. Further chemical investigation showed that the fat of the tubercle bacillus is a completely homogeneous substance which dis- plays no resemblance to any other fat or wax. It seems to be rather a mixture com- posed of free fatty acids, neutral fats, fatty-acid esters, and higher alcohols (lecithin, cholesterin), and, in addition, a large quantity of extractives which are insoluble in water and which, when heated with alkalies, disintegrate in part to form products sol- uble in the water. The demonstration of bacterial fat is made by the usual microchemical methods. Glacial acetic acid and chloralhydrate dissolve fat; Javelle water does not dissolve it; osmic acid does not blacken it; iodine-potassium iodide solution colors it yellow brown. Caustic potash seems to saponify the fat. The behavior of the bacterial fat toward certain dyes is also notable. While the common stains (methylene blue, gentian violet, and fuchsin) do not color bacterial fat, staining succeeds with Sudan III, naphtol blue, and dimethylamidoazobenzol. The latter stains the fat yellow; Sudan III, red; and napthtol blue, a deep blue. THE MINERAL DRY SUBSTANCE Thorough investigations have been carried out on the quantitative analysis of the ingredients of the ash from many kinds of bacteria. It appears from these studies that the mineral dry substance of the bacterial body always forms only a small proportion of the cell substance, that it differs for the several kinds of bacteria, and that the amount and nature of the mineral substance in any particular species vary with the conditions of cultivation and the age of the culture.^ ' For a review, cf. Falk, I. S.: Abslr. Bad., 7, 44. 1923. CHAPTER III STAINING REACTIONS OF BACTERIA^ JOHN W. CHURCHMAN Cornell University Medical College, New York CHEMISTRY OF DYES Bacteriological stains belong almost entirely to the group known as "aniline dyes." Since a number of these, however, are not derived from aniline and bear no direct relation to it, and since all are derivatives of the hydrocarbon, benzene (CeHo), "coal-tar dyes" is a better term. Coal-tar dyes are monacid salts of color bases or alkali salts of color acids. "Ba- sic," "acidic," "neutral" — the descriptive terms usually applied to them — are not particularly fortunate terms since the dyes are not necessarily bases or acids, and even if called "basic" may have an H-ion concentration on the acid side of neutrality. Basic dyes are usually encountered as dye salts of a colorless acid such as hydrochloric, sulphuric, oxalic, or acetic acid; acid dyes as sodium, potassium, calcium, or am- monium salts of dye acids. The terms "acidic" and "basic" as applied to dyes really refer to the affinity of the chromogenic radicle for acidic or basic groups as the case may be. The term "chromogenic radicle" leads directly to a consideration of the cur- rently accepted theory as to the molecular structure of the dyes. The basis of this structure is the benzene ring of Kekule from which — as is well known — an almost infinite number of derivatives can be formed. When the deriva- tives contain certain groups of elements known as "chromophores," these groups impart the property of color. Benzene derivatives containing chromophore radicles are known as "chromogens." Chromogens, although colored, are not dyes since they may have little or no affinity for fibers or tissues ; the color they impart to fibers or tis- sues is a superficial coat easily removed by mechanical processes — the color does not "take." In order that a chromogen become a dye the chromogen derivative must contain, in addition to the chromophore, auxiliary groups which are known as "aux- ochromes." These have themselves little or no color and are not the cause of the color of the dye; but they impart to the compound the property of electrolytic dissociation, •furnish it with salt-forming properties, and thus convert it into a dye. The formation of the yellow dye, picric acid, from the yellow chromogen, tri- nitro-benzene, by the addition of the auxochrome, hydroxyl ( — OH), illustrates present-day conceptions of the chemical structure of coal-tar dyes. When three H- atoms in the benzene ring are replaced with the chromophore (— NOj), tri-nitro- benzene results: O.N. .. .NO, NO. ' The detailed history of staining in bacteriology may be found in Unna's articles, "Die Ent- wicklung der Bakterienfarbung," Centralbl.f. BakierioL, 3, 22-345. 1888. 19 20 STAINING REACTIONS OF BACTERIA This yellow substance, which is insoluble in water, is neither an acid nor a base. Since it cannot dissociate electrolytically, it is incapable of forming salts. It is not therefore a dye. If, however, one more H-atom in the benzene ring be replaced with the auxochrome hydroxyl (—OH), picric acid is formed: This yellow substance is an acid, capable of electrolytic dissociation and of form- ing salts with alkalies. It is a dye. Some auxochromes (e.g., the amine group, — NH) are basic; others (e.g., the hy- droxyl group, — OH) are acidic. The acidity or basicity of a dye — as these terms are used in the expressions "acidic" or "basic" dyes — is determined by the character of its auxochromes. According as the chromogen is united with acidic or basic groups, the dyes are known as "acidic" or "basic." If basic groups are united to an acid chro- mophore, the dye is more weakly basic than if the same basic groups were united with a basic chromophore. A dye retains its color only so long as its affinities for hydrogen are not completely satisfied. When they are satisfied, reduction occurs and colorless leukobodies are formed. The polychrome stains stand in a class somewhat by themselves. In principle, all depend upon a combination of eosin and methylene blue, these elements not only stain- ing as units, but acting together in combination. It is assumed that these compound dyes act on the protoplasm as follows: Certain parts of the cell have an affinity for the neutral stain and take it up as such. Others have an affinity for the basic dye and break up the neutral stain so as to obtain the basic portion of it or, if dissociation has taken place, take up the basic ion directly. Other parts of the cell, with an affinity for acid dyes, similarly combine with the acid portion of the stain. These three types of cell structures are known as "neutrophile," "basophile," and "oxyphile" elements, respectively.' Polychrome stains are used in bacteriology chiefly for the study of spirochetes, Vincent's spirilla, and protozoa; and for the demonstration of chromatin (Zettnow).* Two other types of substance besides dyes are used in staining, namely, mordants and decolorizers. Mordants are chemical substances which have the power of making dyes stain material which they would not stain otherwise. This method of staining, in which the presence of a third substance besides dye and material to be stained is re- quired, is called by Mann the "adjective or indirect" method in contradistinction to the "substantive or direct" method in which the chemical and physical natures of dye and material to be stained are so interrelated that the material acquires the color ' Conn, H. J.: Biological Slains. Geneva, N.Y., 1925. ^Zettnow: Zlschr.f. Hyg. u. Injektionskrankh., 30, i. 1889. JOHN W. CHURCHMAN 21 without the addition of a mordant. Pure mordants have a strong chemical affinity both for the substrate and the dye, and are used where an anchorage of dye in sub- strate is desired. Decolorizers are used to withdraw stains from certain tissues or organisms or parts of an organism and thus by a process of "regressive staining" to differentiate them. "Regressive staining" is contrasted with the ordinary method of "progressive staining" in which the process is stopped when only those substances with great af- finity for the dye are stained. STRUCTURE OF THE CELL To appreciate the significance of staining phenomena and to be able to discuss the mechanism involved it is not only necessary to understand the structure of the dyes but also to be familiar with present-day conceptions of the structure of the living cell. The cell is not to be thought of as a mere random mixture of cell constituents. These constituents have a permanent spatial distribution and physical state. This special structural constitution or organization is responsible for the special peculiarities of chemical behavior. Present evidence indicates that the basic protoplasmic structure has a closer resemblance to an emulsion type of structure than to that of any other simple physical system. The reactions which cells undergo proceed most actively — although perhaps not exclusively — at the boundaries of protoplasmic phases; in other words, the surfaces of membranes, fibrils, granules, and other solid cell structures have an accelerating or catalytic influence on these reactions. Many features of the chem- ical organization and behavior of living protoplasm appear to depend on the presence of thin films (apparently consisting chiefly of lipoid material) by which its structural elements are bounded and inclosed. The entire cell is inclosed by a thin, semiperme- able film — the plasma membrane; the internal protoplasm is probably partitioned by similar films. Apparently the intracellular partitions undergo increase of permeability or break down at death so that many chemical reactions which are absent or inappre- ciable during life proceed rapidly in dead cells. The type of structure characteristic of living protoplasm appears to be one by which free diffusion is prevented or restricted. Various theories have been advanced to explain cell permeability, most of which, though not all, presuppose a plasma membrane which exhibits differential properties permitting some substances to enter the cell easily (alcohol, ether) ; others with diffi- culty (most salts, sugars, etc.); still others not at all (most colloids). While not uni- versally accepted, the membrane hypothesis seems to be the one with which the known facts are best in accord. In the theory of Overton this membrane is supposed to be impregnated with lipoids, probably not ordinary fat but a mixture of lecithin and cholesterol. Others (Ramsden, Loeb, Crozier) emphasize the importance of protein in the membrane, the first calling the structure a "haptogen membrane" ; and Nathan- sohn postulated a membrane composed of a mosaic of both lipoids and proteins. The size of the molecule doubtless plays some part in penetrating power; and if molecular weight be very high, penetration is prevented. But it must not be thought that be- low a certain point there is any correlation between molecular weight and difficulty of penetration; indeed, the reverse is often the case; fatty acids apparently enter cells 22 STAINING REACTIONS OF BACTERIA with increasing ease as molecular weight increases, at least up to a certain poin*. Molecular weight alone has little to do with cell permeability except as a limiting factor.'^ MECHANISM OF STAINING The theory of the molecular structure of coal-tar dyes, which I have stated, while perhaps not certainly established in all details, accounts for all the known facts, and its adoption has been so fruitful that it is now generally accepted. About the actual mechanism of staining, on the other hand, there is by no means general agreement. Much of the discussion has been devoted to a debate as to whether the process is chemical or physical. This seems peculiarly futile since a rigorous definition of chem- ical and physical processes is difhcult if not impossible to formulate and since the two processes are ultimately one. Even the ardent adherents of one or the other theory are usually forced in the end to acknowledge the possible identity of the two. If a chemical process be defined as a reaction between two substances in which a new chemical substance is formed and a physical process as a reaction between two substances in which no new chemical substance is formed, it is clear that both proc- esses occur in staining. A dyestuff as a whole may enter into and be deposited upon a tissue or cell by a process which Michaelis describes as "insorption," in which case the coloring matter may be subsequently extracted by any chemically indifferent solvent. On the other hand, a dye may become chemically united to the cell protoplasm by the formation of a salt, and in such a case the color can be removed only by agents like free acids which are capable of decomposing salts. It is certain that Ehrlich's dia- grammatic conception of chemoceptors to account for selective staining can no longer be held. Many observations in recent years point to the fact that other properties of the molecule than its structural formula (upon which the Ehrlich theory laid so much stress) are concerned and are probably of greater importance. There is good reason to believe that chromophilic protoplasm, so far as it is di- rectly stainable (that is to say, without mordants), is amphophilic or amphoteric, i.e., it possesses basic (amido-) and acidic (carboxyl-) groups side by side in the molecule. It has therefore the structure of amido-carbonic acid. The chemical processes of staining go on best the more auxophoric groups there are in the dye and the greater the number and the adequacy of chromophilic groups in the material to be stained. The physical processes of staining consist of surface attraction, osmosis, diffusion, adsorption ; and the factors which promote these processes are proper size of the dye molecule and adequate pore-volume of the material to be stained. It cannot be said that a completely satisfactory description of the chemistry of bacterial staining can be given. This is perhaps not strange considering the enormous complexity of the chemical structure of dyes, the complicated structure of bacteria, the difficulties of chemical analysis of bacterial bodies which are constantly changing their composition during life, and the minuteness of the microchemical reactions on which we have to depend. One thing essential for the process of staining bacteria is water. Water-free, al- coholic solutions of dyes will not stain dehydrated bacteria, nor will water-free alcohol 'Lillie, R. S.: General Cytology, p. 167. Chicago: University of Chicago Press, 1924; Jacobs. M. H.: ibid., p. 99. 1924. JOHN W. CHURCHMAN 23 decolorize them. The demonstration that bacteria contain an abundance of nucleo- protein appears to explain their affinity for basic coal-tar dyes, and there are many grounds for the belief that— perhaps in the majority of cases — staining is due to a weak combination between nucleoprotein and basic dye, decoloration being due to dissociation of the dye-protein compound. Bacteria certainly behave in their staining reactions as if composed largely of protein. They were formerly thought to consist entirely of nucleus and cell wall without cytoplasm. Whether this be true or not, one other element in bacterial cells is of great — perhaps of prime — importance in many staining phenomena, namely, lipoids. Bacteria are known to contain fats and lipoids in varying amounts which, because of the marked effect on surface tension, would for purely physical reasons tend to become concentrated at the periphery of such a col- loidal system as the bacterial protoplasm. Whether or not there is a morphologically distinct limiting membrane, we can reasonably assume that the surface of the bac- terial cell is potentially lipoid. The presence of unsaturated fatty acids in the lipo- protein of the bacterial cell is thought by many to explain the mechanism of the gram stain. The amount of dye taken up by bacteria and the firmness with which it is held doubtless depends in large part on the H-ion concentration of bacterial protein. Since the pH of bacterial protein may change with age it might be expected that the stain- ing characteristics of bacteria would change, and it is of course well known that this does occur; the difference between the gram behavior of young and old cultures may be cited as an example. The chief physical factor of the staining process — emphasized by advocates of the physical hypothesis to explain the phenomena of dyeing, and which must in any event be taken into consideration — is the process of adsorption of the dye by the bacterial substance after it has passed through the wall membrane by osmosis. Adsorption is the property possessed by a solid body of attracting to itself by physical means from a surrounding solution certain compounds or ions present in that solution. In the case of the dyes it is assumed that, when once adsorbed, they remain in the stained tissue in solid solution. There are certain facts which point strongly to a physical explana- tion of staining. For example, there is no evidence of the formation of a new substance when tissue is stained; the colored tissue merely takes on one of the characteristics of the dye (color). It is usually possible to extract all or nearly all the dye by immersion in water or alcohol. Furthermore, tissue never removes all the stain from the dye solu- tion, no matter how dilute. These facts must be borne in mind, and physical processes must be thought of as playing an important role. But more and more evidence is ac- cumulating to indicate that strictly chemical processes are also concerned. THE GRAM STAIN Dyes are used in bacteriology for four purposes: (i) to make organisms visible, (2) to display their structure, (3) to reveal their chemical nature, and (4) to influence their growth. It is well known that most bacteria are stained easily and a few with difficulty, and that certain parts of the bacterial cell (the spores, capsules, and flagel- la) are not stained at all by ordinary methods. However, with the exception of the acid fast group, the vast majority of bacteria behave very much alike toward simple 24 STAINING REACTIONS OF BACTERIA stains, without mordants. A few instances of elective staining have been described — as, for example, the affinity of picric acid and neutral red for cholera vibrios and kresylechtiviolet for gonococci; but, on the whole, there is so little difference in the behavior of most bacteria to dyes that elective staining methods have not proved of striking value. To this statement there are of course two notable exceptions — the method of Gram and the method of Ziehl-Neelsen. The method of Gram illustrates well the fact that the progress which results from a new observation is often in a different direction from that in which the observer was at the time searching. Gram was attempting to develop a method for staining micro- organisms in tissue. The differential staining which he noticed proved to be of great value for the general identification and classification of bacteria. The ability to retain dye when stained by the method of Gram is not a property of living cells in general but is almost entirely confined to yeasts and bacteria. All tissue elements appear to be decolorizable save perhaps keratohyalin. Henrici found that sections of vegetable tissue contained no gram positive elements; and in animal tissue — while the nucleus retains the stain somewhat longer than the cytoplasm — all the elements may be ultimately decolorized. Molds stain irregularly, isolated granules in the mycelia retaining the stain, while large areas do not stain at all. Protozoa, spiro- chetes, and malarial parasites are gram negative. It is well known, of course, that the differentiation of bacteria into gram positive and gram negative is not hard and fast, that the gram characteristic of a given organism, like any of its other characteristics, may change with age or be disturbed by variations in environment and in other ways. But within its well-established limitations the method of Gram is an exact one. It is clear that the differential behavior of gram positive and gram negative bacteria must ultimately depend upon difference in chemical or physical characteristics (or both) of the bacteria themselves. A number of such differences have been proved to exist, and for certain others there is strong, if not conclusive, evidence. Of these diiferences, the principal ones are shown in Table I. It can be objected that the facts in nature are not quite so clear cut as a table of this sort suggests. Not all gram positive organisms are equally gram positive, and it has been shown that their gram behavior may in different groups of gram positive organisms rest on an entirely different anatomical basis. They therefore differ among themselves and cannot all be classified together. The same thing is doubtless true of the gram negative organisms. Such a statement, for example, as the one that gram positive organisms are as a rule much more susceptible to the bacteriostatic action of basic triphenyl-methane dyes than gram negative' is open to a criticism of this kind, and the criticism has in fact been made.^ Nevertheless, the statement as originally enunciated is correct: with about lo per cent of exceptions, gram positive aerobes, whether spore bearing or not, are extremely susceptible to the bacteriostatic effect of these dyes and, with about lo per cent exceptions, gram negative aerobes are resistant. The original statement of this parallelism was extended by Smith, Eisenberg, Simon, and Wood^ to include about sixty dyes, and the work of these and other investigators ' Churchman, J. W. : loc. cit. = Stearns, A. E., and E. W.: /. Bad., g, 493. 1924. 3 Simon, C. E., and Wood, M. A.: Am. J. M. Sc, 147, 247, 524. 1914. JOHN W. CHURCHMAN 25 TABLE I Gram.Positive Organisms Gram-Negative Organisms 1. Killed bacteria not digested by trypsin or pepsin (Kantorowiecz) * 2. Only a few are digested by gastric juice (Burgers) t 3. Resistant to alkalies; not dissolved by i per cent KOH (Kruse,t Smith§) 4. Lipoids resistant to fat solvents (Jobling and Petersen) 1 1 5. Degree of dispersion of nucleoproteins high (Hottinger)^ 6. Sensitive to weak electrolytes (dyes) Smith, § Burke**) 7. Optimum growth at relatively high pH (Burke)** 8. Resistant to non-electrolytes (Smith) § Q. Limited iso-electric staining range cen- tered about pH 2-3 (Stearns and Stearns) ft 10. For the most part (about 10 per cent exceptions) very susceptible to bacterio- static effect of triphenyl-methane dyes (Churchman) ft 11. May produce spores 12. In the living state, more readily per- meated by dyes (Benians)§§ 13. May be acid fast 14. More susceptible to quinine and hydro- cuprein derivatives (Traube);^|| 15. More susceptible to iodine (Traube)ll|| 16. More susceptible to mesohematin, mag- nesium, and manganese derivatives of mesoporphyrin (Kammerer)TfTf 17. More readily adsorbed because of surface lipins (Eisenberg)*** 18. Adsorb halogens less strongly (Breinl) fff 19. Unaffected by toluol provided no emulsoid be added (Benians)ttt * Munchen. med. Wchnschr., 56, 897. 1909. \ Zlschr.f. Hyg. u. Infeklionshrankh., 70, 119. 1911. X MUnchen. med. Wchnschr. 57, 685. 1910. ^Am. J. Hyg., 2, 607. 1922. II /. Ex per. Med., 20. 456. 1914. H Cenlralbl.f. Bakkriol., 76, 367. 1916. **/. Bad., 7, 159. 1922. tt Ibid., g, 463, 479, 491. 1925; 10, 13. 1925. ttJ. Exper. Med., 16, 221. 1912. §§ /. Palh. &* Bad., 17, 199. 191 2. nil Zlschr.f. Immunitdtsforsch. u. exper. Therap., 29, 2861. 'ill Arch. f. exper. Palh. u. Pharmakol., 88, 247. 1920. *** Centralbl. f. BaklerioL, 54, 145. 1912; ibid., 81, 72. 191 ttt Zlschr.f. Immunildtsforsch. u. exper. Therap., 29, 343. XXX Zlschr.f. Chemolherapie, 2, 28. 1913. 1. Killed bacteria digested by trypsin and pepsin 2. Majority digested by gastric juice 3. Dissolved by i per cent KOH 4. Lipoids less resistant to fat solvents 5. Degree of dispersion of nucleoproteins low 6. Resistant to weak electrolytes 7. Optimum growth at relatively low pH 8. Less resistant to non-electrolytes 9. Wider iso-electric staining range cen- tered about pH 5 ID. For the most part less susceptible to bacteriostatic effect of triphenyl-meth- ane dyes 11. Never (?) produce spores 12. Less permeable to dyes, in living state 13. Never acid fast 14. Less susceptible to quinine and hydro- cuprein derivatives 15. Less susceptible to iodine 16. Less susceptible to these substances 17. Less readily adsorbed 18. Adsorb halogens more readily 19. Killed by toluol 26 STAINING REACTIONS OF BACTERIA TABLE l— Continued Gram-Positive Organisms Gram-Negative Organisms 20. Less readily subject to auto- and sero- 20. More readily subject to auto- and lysis (Benians)§§§ serolysis 21. Less likely to form demonstrable anti- 21. More likely to form antibodies bodies in infected host (Benians)§§§ 22. Less susceptible to quinine (Graham- 22. More susceptible to quinine Smith)1f111f §§§y. Path, b" Bad., 23, 411. 1919-20. HHH/. H>'5-. 18, 1. 1919. suggests that perhaps the author's figures for the exceptions in each group are too high This clear-cut but not all-inclusive parallelism between gram behavior and triphenyl- methane behavior is striking, in spite of the fact that an exceptional gram positive organism (like the streptococcus) may be resistant and an exceptional gram negative one (like M. neisseri) may be susceptible; and in spite also of the fact that within each group there are quantitative differences in susceptibility to dyes. All the relations stated in the table are doubtless subject to similar qualifications which may be summarized as follows: 1. The division between gram positives and gram negatives is not absolute. Other factors enter in. For instance, the anaerobes form a class by themselves, and, although usually gram positive, may have many characteristics not shared by other gram pos- itives. They are really not included in the table. Again, organisms like the acid fast have peculiarities of structure which separate them chemically from others. 2. The gram positives differ among themselves, and gram positivity does not al- ways depend, in the various bacterial species, on the same mechanism. This point has been established by the observations of Deussen,' and more recently by the author,^ who has shown that two strongly gram positive organisms like B. anthracis and M. fretidenreichi differ markedly as to the stability — and probably as to the underlying cause — of their gram positivity. 3. A very interesting and important fact has been established by a number of ob- servers, that there is a group of organisms which — though gram negative — are inter- mediate between the two groups in most of their other characteristics. M. neisseri, for example, though gram negative is dye sensitive, falls w'ith the gram positives in opti- mum H-ion test, is quite resistant to lysis by KOH as compared with other gram neg- atives, and shows no cytolysis in peptic-digestion tests. There is some evidence (from peptic-digestion tests) that vibrios and spirilla also occupy in some respects a mid- position between the two groups. 4. The presence of spores also somewhat complicates the situation. The author^ has shown that certain dyes (acriflavine, acid fuchsin) exhibit a reverse selective bac- teriostatic action when tests are made between gram positive spore-bearers and the ordinary gram negative bacilli. 'Deussen, E.: Ztschr.f. Hyg. u. Infektionskrankh., 85, 235. 1918. 'Churchman, J. W.: Slain Technology, 2, No. i. 1927.. 3 Churchman, J. W.: J. Expcr. Med., 37, i. 1923; ibid., 38, i. 1923. JOHN W. CHURCHMAN 27 5. Not even all strains of a given species necessarily behave exactly alike toward dyes. The existence of "strains-within-a-species variants," as measured by the bac- teriostatic effect of gentian violet, has been described; and from a given pure culture of B. coli two strains have been isolated — one dye sensitive and the other not — which were identical in all cultural, morphological, and tinctorial respects ("strain-within-a- strain variant").' The facts are that the properties of the two groups stated in the foregoing table are probably to be regarded as "independent variables in the sense that any one of them may be possessed by a particular strain of bacteria independent of the other properties of that group." None the less, as Smith has stated, "this almost clear sep- aration of the families of bacteria on the basis of biochemical reactions cannot be without significance." It is inevitable that the dilemma as to the physical or chemical nature of staining which we have encountered in discussing the general mechanism of the staining proc- ess must also be faced in discussing the method of Gram. On the whole, the tendency is to emphasize the chemical rather than the physical factors of this reaction, and many observers (e.g., Deussen)^ regard the process as purely chemical. That physical structure is also involved seems none the less certain. Benians^ has shown that bac- terial disintegration induced by mechanical measures upsets the gram behavior; Churchman has shown that certain organisms (e.g., B. anthracis) are gram positive only in the cortex, which may be removed by chemical means, exposing a gram nega- tive medulla ; and that some sort of bacterial membrane plays a physical part in the process is taken for granted in most of the theories which attempt to account for the phenomenon. Benians has been the chief exponent of a purely physical explanation of the gram stain and has advanced perhaps the most convincing evidence for this view. From experiments in which bacteria were studied after disintegration by crushing, Benians^ drew the following conclusions: 1. The gram positive property is inherent in the physical structure of the bacte- rial cell, and — since mordants are not essential — is not conferred on it by the mordant. 2. Nothing in the nature of chemical fixation of the compound dye to bacterial substance occurs. 3. The effect of the mordant is to dissociate dye from its adsorption compound with the tissues, forming with it a large, compound molecular body which in alcoholic solution does not easily pass out of the gram positive bacteria. This conception of the role of the mordant supplanted Benians' earlier idea that it acted by preventing al- cohol from entering the cell. 4. Capacity for retaining the compound dye is chiefly dependent on the structure and integrity of the limiting membrane. 5. The essential character of the gram positive cell membrane is that it does not ' Churchman, J. W., and Michael, W. H.: ibid., 16, 822. 191 2; Churchman, J. W.: ibid., 33, 569. 1921. ^Deussen, E.: loc. cit. 3 Benians, T. H. C: J. Path. & BaQi., 17, 199. 1912. ''Benians, T. H. C: ibid., 23, 411. 1919-20. 28 STAINING REACTIONS OF BACTERIA readily permit the contained large, compound iodine molecule in alcoholic solution to pass through it. 6. Gram negative bacteria are of two types as regards cell membrane: (a) (Rep- resented by M. neisseri) allow attachment and probable permeation of the dye ; but from them iodine-dye precipitate is readily washed out by the decolorizer. These or- ganisms approximate the gram positives so far as they contain the dye, but the ab- sence of the necessary specific cell membrane does not permit the retention of the dye when alcohol is applied, (b) (Represented by B. coli) allow no penetration of the dye but only peripheral adsorption. 7. Not all bacteria which fail to retain the stain are similar in structure. The ap- parently permeable gram negatives (M. neisseri) probably have more in common with the gram positives than with the gram negatives. Burke corroborated Benians' conclusions by demonstrating a reverse gram re- action.' He found that an alcoholic solution of the iodine-dye complex stained the gram negatives but not the gram positives, so that gram positives are characterized by permitting the entrance of water-soluble dye but not the egress of the alcohol- soluble iodine-dye complex. The opposite is true of gram negatives. Brudny,^ on the other hand, considered the gram positives more permeable to io- dine so that in these organisms a deeper iodine-dye precipitation occurs which is less accessible to the decolorizers. Other observers have laid stress on the chemical con- stituents of the bacterial surface. Eisenberg found that ether extraction of staphy- lococcus reverses its gram behavior, concluding that the lipoid-protein compounds on the surface were the important factor; while Dreyer, Scott, and Walker were able to turn B. coli into gram positive organisms by treating them with lecithin. Stearns and Stearns^ called attention to the fact that bacteria exhibit the ampho- lytic character common to protein and tend to retain acid stains when in acid solu- tion and basic stains when in alkaline solution. They found that gram positive bac- teria can be rendered gram negative by increasing acidity, and that the reverse effect is produced by alkalies ; at the iso-electric point there is little staining, and the so-called "iso-electric range" is generally wider with gram negative than with gram positive bacteria. Mordanting, they suggest, is due to a mild oxidation which increases acidity and hence the affinity for basic dyes. They suggest further that gram positivity de- pends on the presence in the compound bacterial lipoproteins of unsaturated fatty acids which are partially oxidized by the mordant, intensifying the acid properties and increasing affinity for basic dyes. The presence of unsaturated fatty acids has been invoked by other observers to explain the phenomenon, but in a different way. Thus Jobling and Petersen'' suggested that gram positivity depended on a high fatty acid content and a high affinity for iodine; and Tamura found that the lipoid extract from bacteria contains the element responsible for retention of the dye. Hottinger,^ on the 'Burke, V.: loc. cit. = Brudny, V.: CcnlralU. f. Bakleriol., 21, 62. 1908. 3 Stearns, A. E., and E. W.: op. ciL, 9, 463, 479, 491. 1925; 10, 13. 1925. ■t Jobling, J. W., and Petersen, W. H.: loc. cit. sHottinger, R.: loc. oil. JOHN W. CHURCHMAN 29 other hand, thought gram positivity is solely dependent on the degree of dispersal of the nucleo-proteins: in gram negatives the stained nucleoprotein forms a colloid of high dispersion ; in gram positives it forms an emulsoid ; when gram positives become negative the dispersion is increased. Deussen' concludes his exhaustive study of the subject by stating that the gram reaction depends on the chemical nature of the cell contents which undergo — according to their chemical structure — a greater or lesser degree of hydrolytic splitting of the molecule. The reaction belongs in the complica- ted field of nuclein combinations. It is chemical and not physical. It has been for a long time believed on the basis of statements attributed to Unna^ that gram positivity depends on the formation of a peculiar iodine-dye-protein com- pound and that only para-rosanilin dyes could be used for this purpose. That the chemical process involved may not be quite so simple as this was shown by Eisenberg who reported that the reaction could be produced with deeply colored dyes of the acid class as well as with basic dyes and that the use of mordants is not necessary. None the less, para-rosanilin dyes are the best for the purpose, and much the most clear-cut results occur when mordants are used. Bacteria were for a long time treated as though their constitution were constant, and characteristics were assigned to them which — by implication at least — they were supposed to exhibit always and under all conditions. That they are, as a matter of fact, biological units showing all the changes of metabolism, that they carry on res- piratory processes, that they undergo dissociation, that they exhibit polymorphism — these things are now well known. It is clear, therefore, that many of their character- istics are constantly changing and that many of them may be made to change more or less at will by alterations in the environment. Reversibility of the gram reaction by acids (Deussen)^ and by coal-tar dyes (Churchman)^ has already been referred to; and it must again be emphasized that the gram reaction of a given organism is constant only when the conditions of examination are held constant. It must also be borne in mind that gram positive organisms differ among themselves as to the stability of their gram positivity. Neide,4 for example, used the term Gramdauer to indicate that since a number of factors influenced decolorization by alcohol (among them the con- centration and temperature of the alcohol used) gram positive organisms differed among themselves as to the length of time they could retain stain when exposed to decolorizers. Henrici^ showed that in yeast cells the cytoplasm is not homogeneous as regards gram positivity, certain granules appearing in the decolorizing cell which hold the dye longer than others. The author has recently made very clear the fact that not all gram positive organisms are equally stable in their gram positivity.* Certain of these organisms (e.g., M. freudenreichi) are extraordinarily stable in this respect; others (e.g., B. anthracis), though equally gram positive, are less stable. Exposure of ' Deussen, E. : loc. cit. 'Unna, P. G.: Monatschr.f. prakt. Dermatol., Supplement No. 6. 1887. 3 Churchman, J. W.: Stain Technology, 2, No. i. 1927. TDon, J. E.: J. Hyg., 9, 239. 1909. "Henrici, A. T.: Proc. Soc. Exper. Biol. &= Med., 19, 132. 1921; 20, 179, 293. 1922-23; 21, 215, 343, 345- 1923-24; 22, 197. 1924; Science, 61, 644. 1925; /. Infed. Dis., 37, 75. 1925. 42 MORPHOLOGICAL CHANGES DURING GROWTH During the early latent period, when growth is slow or completely lacking, no apparent morphological change is seen. Following this, during the logarithmic period when maximum reproduction occurs, the cells from the young cultures of many genera of bacteria attain their maximum size, two to six times larger than the cells from the twenty-four-hour parent-cultures. After four to eight hours the cultures pass gradu- ally into the stationary period, in which the number of organisms remains relatively constant. During this progression to the stationary period, the bacteria become gradually smaller in size until, by the time the cultures are eighteen to twenty-four- hours old, the classical textbook picture is presented. Cultures older than twenty- four and forty-eight hours present more and more of the so-called "involution forms," irregular staining, in many instances bizarre forms, and organisms averaging smaller than those found in the twenty-four-hour cultures predominate. Clark and RuehP studied by this method seventy strains in all, including cultures of the following species: COCCACEAE Strains Staphylococcus aureus 5 Staphylococcus albus 2 Streptococcus hemolyticus 6 Diplococcus pneumoniae 5 (including types I, II, and III) Neisseria gonorrheae Neisseria catarrhalis 2 Neisseria intracellularis Neisseria mucosis Spirillaceae Vibrio comma Vibrio metchnikovi Vibrio schuylkilliensis Bacteriaceae Proteus vulgaris Escherichia coli Escherichia communior Eberthella typhi 5 Eberthella dysenteriae Eberthella paradysenteriae Salmonella paratyphi Salmonella schottmiilleri Salmonella suipestifer Encapsulatus pneumoniae 2 Pseudomonas pyocyaneus Serratia marcescens Pasteurella avicida Hemophilus influenzae Hemophilus pertussis Bacillus anthracis Bacillus subtilis Bacillus megatherium ' Clark, P. F., and Ruehl, W. H.: loc. cit. PAUL F. CLARK 43 Bacillus vulgatus Bacillus mycoidcs Mycobacteriaceae Mycobacterium leprae Mycobacterium smegmatis Mycobacterium phlci Coryncbacterium diphthcriae 4 Corynebacterium hofmanii Coryncbacterium xerosis Corynebacterium hodgkinii PJeiferella mallei Henrici' studied in detail Vibrio comma, Escherichia coli, Bacillus megatherium. Bacillus cohaerens, and an unidentified member of Corynebacterium. The increase in size during the "youth" of the cultures occurs in all the organisms studied except the Corynebacteria and B. mallei. In these bacilli, more especially in the first named, exactly the opposite progression occurs; the individuals from the younger cultures (two to six hours) are the smallest, averaging in C. diphtheriae less than half the size of the rods from a twenty-four-hour culture. The minimum size is reached during the period of rapid reproduction ; during the phase of slow growth the size increases, reaching the maximum during the resting period. As the individual or- ganisms become smaller, the metachromatic granules disappear and the bacteria stain uniformly and deeply with Loffier's methylene blue. Not infrequently at this stage members of this genus form coccoidal chains. The peculiar pleomorphism, irregular staining, and metachromatic bars and granules, so characteristic of the diphtheria group, reappear as the cells increase in size again. The method employed in these studies hardly needs further description save to add that Henrici' developed an admirable adaptation of the negative staining method of Benians^ to distinguish between the living and dead bacteria in smears. This made it possible to correlate precisely the changes in morphology with the rapidity of cell division in the bacterial cultures. Members of the colon-typhoid group will serve as a basis for the more detailed con- sideration of the typical mode of progression. A large majority of the bacteria in a four-hour culture of B. typhosus are 4-6 micra long and 0.7-0.8 micron wide, so large that they resemble the vegetative cells of the common spore-formers rather than the usual picture of B. typhosus based on the examination of twenty-four-hour growth. The increase in size is greater in length than in breadth, so that the larger cells are relatively more slender. Chain formation occurs commonly at this stage even in those species which in older cultures are characterized by discrete organisms. The bacteria stain more intensely, and the outline is more sharply defined. The time when the maximum average size occurs varies somewhat in different strains of the same or re- lated species. This is dependent largely upon the duration of the period of "lag," which according to Chesney^ is an expression of injury to the bacterial cell. By plot- ' Henrici, A. T.: see various works cited previously. 'Benians, T. H. C: Brit. M.J., 2, 722. 1916. 3 Chesney, A. M.: /. Exper. Med., 24, 387. 1916. 44 MORPHOLOGICAL CHANGES DURING GROWTH ting the projected image of the bacterium divided by the length squared, Henrici' has obtained an index of the variation in form. The coefificient of variation in length of cells, as well as in the area-length index, is increased during the period of increased size. The various members of the Spirillaceae, because of their marked pleomorphism, offer especially interesting opportunities for the use of this method. The three species studied follow the usual series of changes in size. During rapid growth, the new cells are long and plump and relatively straight save that where several organisms have remained attached they show definite spirals. As reproduction becomes slower, the individuals become more slender, more curved, and distinctly granular in staining. During the period of decline unusual forms are observed — various bulging or budding organisms and coccoid bodies both large and small. Henrici points out that "these latter types are the forms which Lohnis and others have described as extraordinary reproductive cells but that the trend of the growth curve would indicate that this is not the case." The three members of the Mycobacteria studied need no especial comment save to point out that the "senile" forms are distinctly granular with many coccoid bodies present. Even the cocci pass through similar cyclical changes in morphology, although the proportional difference between the diameter of the young and the senescent forms is less than with most of the rods and spirilla. The four- to six-hour cultures contain many deeply staining cocci approximately twice the diameter of those usually seen. In studying B. megatherium, Henrici' has pointed out a number of factors which affect the rate of progression through the period of maximum reproduction and con- sequently the onset of the phase of decline. If the volume and constituents of the media are constant and if varying numbers of the bacilli are inoculated, the fewer the cells introduced the longer is the period of maximum reproduction and the greater is the maximum size of the organisms. With the same seedings, if the nutrient ingredients of the media are varied, then the richer the media the longer is the period of logarith- mic growth and the greater is the maximum size attained. When transplantations are made during the period of increasing size, the organisms in the subculture con- tinue to increase in size, progressing even beyond the maximum reached by the parent- culture. Transplantations made immediately after the parent-culture has returned to the original size show no evidence of lag; the subcultures increase rapidly in size again. After two or more hours in the stationary phase, however, subcultures show an appreciable lag and do not progress beyond the curve of the parent-culture. Al- though other organisms have not been studied with reference to these points, pre- sumably they will follow the same laws. It is interesting to note that spore formation begins toward the end of the ac- tive growth period, so that the factors which lengthen the phase of positive accelera- tion in growth delay spore formation. It would appear, then, that even apparently simple bacteria growing under stand- ardized conditions admirably fitted to suppress variation and to increase uniformity pass through a series of cyclical changes which indicate a progression from youth ' Henrici, A. T.: see various works cited previously. PAUL F. CLARK 45 through maturity to old age. These three phases show marked differences in meta- boHc and reproductive rate, and correlated changes in size and structure. Sherman and Albus' have shown that other physiological correlations exist, in that young bac- teria are destroyed by brief exposures to cold and 2 per cent sodium chloride while cells from older cultures are not injured. Differences in agglutinability have been known for many years. There is no evidence that conjugation occurs, but there is ample evidence that, as Child^ has shown in higher forms, youth and rejuvenescence may occur apart from sexual processes. Observations such as the foregoing suggest an interesting correlation between the growth of bacterial cells and that of other living organisms. Protoplasmic changes, similar in kind to those cellular changes we associate with youth, maturity, and senil- ity in metazoan forms, occur even in these supposedly "immortal" single cells. Def- inite changes in gross morphology and in finer structure are apparent. A logical as- sumption that bacteria, in common with their more highly organized relatives, regain their youth and vigor of reproduction by the injury and stimulus of conjugation is not borne out by these observations. It must be borne in mind, however, that these observations were all made under conditions admirably suited for growth. Just as numbers of species of fungi have for years been classified with the Fungi imperfecti but later, by more complete studies under varying conditions of growth, have been shown to have definite sexual reproduction and, consequently, have been removed from this family, so it would seem to be highly probable that more detailed study will remove bacteria from their unique position and link them more closely with the rest of the biological world. 'Sherman, J. M., and Albus, W. R.: J. Bad., 8, 127. 1923; 9, 303. 1924. => Child, C. M.: Senescence and Rejuvenescence. 1915. CHAPTER V GROWTH CURVES OF BACTERIA R. E. BUCHANAN Iowa State College The following discussion of growth rates of bacteria and their graphical representa- tion in growth curves will be concerned solely with rates of increase. CHARACTERISTICS OF GROWTH CURVES The general characteristics of growth curves may be developed through considera- tion of the changes in numbers of bacteria which follow inoculation into a medium suitable for growth. For the purpose of preliminary discussion it is advan- tageous to assume that the inoculum consists of bacterial spores, for such material will permit of the maximum opportunity for differentiation of stages or phases of growth. Examination of such a culture at suitable intervals will show that an appreciable time elapses before any increase in numbers occurs, i.e., some time is required before any of the spores germinate and vegetative cells develop and divide. This may be termed the "initial stationary phase." It is scarcely to be anticipated that all the spores will germinate at the same instant. However, after cell division has been initiated it will pro- ceed with a considerable degree of regularity. Finally, all the viable spores will have germinated, and the culture will have completed its second or "lag phase." For a time thereafter the numbers of cells will increase more and more rapidly, with the rate of growth per cell remaining nearly uniform. This is the third or "logarithmic phase," during which there is a geometrical increase in cells with time. Conditions eventually become less favorable, and the rate of growth decreases. This is the phase of "negative growth acceleration." Finally the bacteria cease to multiply, and the "maximum stationary phase" is instituted. It is thus possible under favorable conditions to differentiate some five different growth phases. 46 a 10 jz J4 Time in Hours 20 22 2^ Fig. I. — Growth curve with various phases a-h. Initial stationary phase h-c. Lag phase c-d. Logarithmic phase d-e. Phase of negative acceleration e-/. Maximum stationary phase R. E. BUCHANAN 47 These facts and relationships may be shown graphically in several ways. The standard growth curve such as that noted above may be graphed (Fig. i) by plotting numbers of bacteria against time. In- spection of the graph shows the ex- istence of some four readily differen- tiable phases; the distinction between the lag phase and the logarithmic phase is not easily made by examination of this type of curve. A second method of representing increase in numbers is to plot the total increase in numbers of bacteria in each equal interval of time against time, thus developing a "rate curve." Such a curve corresponding to the growth curve of Figure i is given in Figure 2. A third type of graph may also be used to illustrate growth rates: one in which the successive rates of increase per cell (or, conversely, the generation Fig. 2. — Rate curve of Fig. i. Time in Hours curve corresponding to growth time) may be plotted against time. Curves such as those in Figure 3 may be thus secured. In this graph the identification of the five growth phases is more readily accomplished than in Figures i and 2. A still clearer differentiation of the various growth phases is to be secured by a fourth type of graph in which the logarithms of the numbers of bacteria are plotted against time, as in Figure 4. During the initial stationary phase (a-6) a straight line with o slope is developed, during the lag phase (&-c) a curved line, during the logarithmic phase a straight line {c-d) with posi- tive slope; during the phase of nega- tive acceleration a curved line {d~e), and finally during the maximum stationary phase (e-/) a straight line of o slope. A study of the typical growth curve (Fig. i) shows it to be more or less S-shaped. Its exact form in each case will depend upon the type of organism, its immediately antecedent history, and the various environmental influences. 1 V ' \ e y 1 c d c A _d a .y ^-^e / 13 20 JZ2 2^ '? c e JO 12 14 I Time in Houns Fig. 3 A . Graph of rate of growth per cell B. Graph of generation times a-h Initial stationary phase h-c Lag phase c-i Logarithmic phase d~e Phase of negative acceleration c~j Maximum stationary phase GROWTH CURVES OF BACTERIA Two principal types of explanation or interpretation have been used for the form assumed by bacterial growth curves. The first of these (a succession of growth phases) is the one developed above. In its essentials it was apparently first outlined by Lane-Claypon/ and later expanded by the writer.^ A somewhat different method of attack was suggested by McKendrick and Pai,^ and emphasized among others by Robertson^ and Lotka\ These authors conclude that the sigmoid shape of the growth curve is evidence of the resemblance of growth to the phenomenon of autocatalysis (or, in the terminology suggested by Ostwald, "autocatakinesis"). While these two interpretations are not essentially antagonistic, they represent somewhat different points of view, and require separate treatment. GROWTH CURVES CONSIDERED AS A SUC- CESSION OF GROWTH PHASES It was noted above that in some cases at least as many as five different growth phases may be observed in a culture of bacteria. They will be con- sidered in order. flnne in Hours Fig. 4. — Growth curve graphed as logarithms of numbers of bacteria against time. a-h Initial stationary phase h-c Lag phase c-i Logarithmic phase d-e Phase of negative acceleration e-j Maximum stationary phase INITIAL STATIONARY PHASE During this phase there is no in-, crease in numbers. In the illustration above its existence was ascribed to the time required for spores to germinate. Experience shows, however, that this phase is sometimes evident in a bac- terial transfer although the organism is one which does not sporulate. It is apparent that bacterial cells from old cultures (in the maximum stationary and later phases) possess some of the same inertia and slowness to develop under favorable environ- ment usually regarded as characteristic of spores. They may be regarded as in some respects the physiological, though not the morphological, equivalents of endo- spores. In some cases this phase may be prolonged for days or weeks. Such prolonga- tion has been noted particularly by Esty and Meyer^ in heated cultures of the bacil- lus of botulism. This phase is not found when the inoculum contains any considerable proportion of actively multiplying bacteria. ' Lane-Claypon, Janet E.: J . Hyg., 9, 239. 1909. " Buchanan, R. E.: /. Infect. Dis., 23, 109. 1918. 3 McKendrick, A. G., and Pai, M. Kesave: Proc. Roy. Soc, Edinburgh, 31, 649. 1911. ■t Robertson, T. B.: /. Physiol., 56, 404. 1922. sLotka, Alfred J.: Elements of Physical Biology. 1925. ' Esty, J. R., and Meyer, K. F.: J. Infect. Dis., 31, 650. 1922. R. E. BUCHANAN 49 LAG PHASE During this phase the average rate of growth per cell is increasing to the maxi- mum characteristic of the succeeding (logarithmic) phase. Some authors do not dif- ferentiate between this phase and the preceding, terming the two together the "lag phase." It is evident that during this period the number of bacteria present is a function of the time; and several attempts, both empirical and theoretical, have been made to formulate the mathematical relationships. In the analysis of certain data Ledingham and Penfold' found that a graph of the logarithms of the logarithms of the numbers of bacteria and the logarithms of the time is a straight line. This leads to the formu- lation: h=Beki' (i) in which J = Number of bacteria after time i 5 = Initial number of bacteria k and 5 = Constants which require evaluation for each particular set of experiments e = Base of natural logarithms These authors found the value of s to vary from 1.56 to 2.7. LOGARITHMIC PHASE During this phase the generation time is a constant, as is also the rate of growth per cell. This phase is the one most susceptible to simple mathematical analysis, and is of major importance in the study of the effect of environment upon bacteria. Methods for estimating the generation time and number of generations during this phase were apparently first developed by Buchner, Longard, and Riedlin.^ If it be assumed that the cells are multiplying regularly by binary fission, and jB = Initial number of bacteria 6= Number of bacteria after time t «= Number of generations in time / g=Length of one generation, i.e., time required for the bacteria to double in numbers then, b=B2» (2) and, since « = - , ^ • ^ b=Bis (3) ^^log^-log5 (^) log 2 / log 2 log ^— log B (5) ' Ledingham, J. C. G., and Penfold, W. J.: /. Hyg., 14, 242. 1914. * Buchner, H., Longard, K., and Riedlin, G.: Centralbl.f. BaklerioL, 2, i. 1887. 5° GROWTH CURVES OF BACTERIA If the numbers of cells present at any two times during the period of logarithmic growth are determined, the values of n and g may be readily derived. Nomograms for such determination have been developed by the writer/ If the numbers of bacteria are plotted against time a curve will be developed whose equation is (3) (see Fig. 5). Equation (5) may also be written in the form, log&=— ^+Iog5 (6) This indicates that if the logarithms of the numbers of bacteria are plotted against time a straight line will be developed, with slope log and with Time Fig. 5. — A. Growth curve during logarithmic growth phase. B. Plot of logarithms of numbers of bacteria against time. the intercept on the y-axis at log B (Fig. 5). This is a convenient criterion for determining whether or not a cul- ture is in the logarithmic phase. For some purposes it is advisable to determine the rate of growth per cell. If the cells are increasing regu- larly in geometrical progression, the rate of increase in the number of cells -,- ) is constantly proportional to the at J number of cells, i.e., db dt = kb (7) in which k is the proportionality constant termed the "velocity coefficient" of the rate of growth. Since db k=dt b the velocity coefficient is the rate of growth per cell. Integration of (7) gives the rela- tionship In Z) = ^/+ Constant of integration When i = o, the constant of integration is equal to l)i B, and In b = kl+ln B 1, h ^ (8) (9) ' Buchanan, R. E.: Iowa Stale College J . Sc, i, 63. 1926. R. E. BUCHANAN 51 Equation (8) is in the form of an equation of a straight line. It follows, therefore, that if the logarithms (to base e) of the numbers of bacteria are plotted against time, a straight line will be developed with slope k (rate of growth per cell) and intercept on the y-a,xis at In B. It follows that b = Bck^. (10) This is another form of equation (3), the equation of the logarithmic growth curve. It is sometimes convenient to evaluate k (rate of growth per cell) in terms of g (generation time) . Since the generation time is inversely proportional to the rate of growth per cell, • g=J- (II) The value of the proportionality constant C may be determined from equations (5) and (9). g=^ (12) and C=ln 2 = 2.307 log,o2 = 0.692 PHASE OF NEGATIVE GROWTH ACCELERATION This phase succeeds the logarithmic phase when conditions become progressively more unfavorable to growth, due either to decrease in concentration of nutrients or to the accumulation of toxic products. Mathematical analysis of this phase has been attempted, but the adequacy is questionable. MAXIMUM STATIONARY PHASE This is reached when the cells cease to increase in numbers. A count at this time gives the maximum crop yield. The rate of growth is o. SPECIAL MODIFICATIONS OF GROWTH CURVES In some cases growth curves are found to be more complex than the type indicated above. They may, for example, exhibit more than one logarithmic phase. One may find cultures of organisms which both produce CO. and are stimulated by it. A small seeding of such an organism (in the logarithmic growth phase) would for a time show a constant rate of growth per cell, later the CO2 would increase to a point where its stimulating action would be manifest, and the value of k would increase with increase in concentration of CO.. Eventually saturation with CO2 would occur and the rate of growth per cell would again become constant. Similarly, more than one of certain other growth phases may be manifested. GROWTH CURVES INTERPRETED AS AUTOCATAKINETIC PHENOMENA McKendrick and Pai (loc. cit) and later Robertson {he. cit.) and Lotka {loc. cit.) have suggested that the entire growth curve shows marked resemblance to a curve of autocatalysis. The relationship may be derived as follows: It has previously been shown that under constant environmental conditions the rate of increase of bacteria is constantly proportional to the number of bacteria pres- 52 GROWTH CURVES OF BACTERIA ent. It may be assumed that the rate of increase is also proportional to the concen- tration of the available nutrients, or to that of some single nutrient which acts as a limiting factor. If concentrations of cells and of nutrients are the only two factors governing the increase, the rate will be jointly proportional to the two. A convenient method of estimating the amount of available nutrient is to determine the total max- imum number of bacteria which may be produced in the culture. The difference be- tween the number of bacteria present at any instant and the maximum number of bacteria which may be developed is proportional to the available remaining nutrients. If /3 = maximum bacterial count, then Upon integration, When t = o,b = B, and j^=Kb{^-h) (13) ln-^^=K t+C (14) Equation (14) shows a straight-line relationship between In ~ — r and time, the straight line having a slope, K. This relationship may be used to determine whether in any case the growth curve resembles that of autocatalysis, or whether (according to Ostwald) the growth curve is autocatakinetic. The equation of an autocatakinetic growth curve may be derived from equation (14): A convenient evaluation of C may be made by taking /i as the time which has elapsed to the instant when b = -, i.e., until the number of bacteria has reached one-half the 2 maximum. C=-Ki^ and ^w«ir5=^' (^-^') i^r^+FKins- <"' A curve of this type is illustrated in Figure 6. It will be found to be symmetrical, and asymptotic to the lines 6 = and 6 = /(3. The point of inflection occurs at - after time ti. The y-axis is cut at b = B. R. E. BUCHANAN 53 Data of bacterial growth as studied by McKendrick and I'ai and by Lotka have been found to conform measurably well to such autocatakinetic curves. Other cases may be cited, however, in which the agreement is not good. This is particularly true when there is manifested a prolonged initial stationary phase or lag phase. Then, too, the rate of growth per cell may not be directly proportional to the concentration of the available nutrients. Other factors may also alter the form of the growth curve, and in consequence it may be unsymmetrical. The equations and relations of such curves are more complex, and are not subject to ready analysis. Even if the data se- cured are found to iit symmetrical curves, comparisons between curves secured under varying environmental conditions are /? not as readily made as between cor- responding (particularly the logarith- mic) growth phases. EFFECT OF AGE OF PARENT-CULTURE UPON FORM OF GROWTH CURVES The work of Lane-Claypon {loc. cit.) and others has shown quite clearly that the phase of growth of the culture from which a transfer is made influ- ences markedly the form of the growth curve in the daughter-culture. In most cases the following results will be secured: (i) Transfers from the initial stationary phase will show a continua- tion of this phase, followed by lag phase, etc., in normal sequence. (2) Transfers from the lag phase will usually show a continuation of this phase, followed by the logarithmic phase, etc., in normal sequence. (3) Transfers from the logarithmic phase usually show a continuation of the logarithmic phase. In some cases allelocatalysis (see below) may cause the culture to show an initial lag phase. (4) Transfers from the phase of negative growth acceleration will usually show a lag phase. (5) Transfers from the maximum stationary phase may show an initial stationary phase or a lag phase. EFFECT OF SIZE OF INOCULUM UPON THE FORM OF THE GROWTH CURVE It has been shown by Robertson^ that growth in a subculture of certain organisms, particularly protozoa, is stimulated by the presence of other cells of the same type. Single-cell transplants to hanging drops exhibit a much slower initial rate of growth per cell than do seedings of a larger number. This phenomenon of mutual or self- stimulation he terms "allelocatalysis." Wliile in general results with bacteria do not show this effect under the usual conditions of culture, there is evidence that with some forms, as the pneumococcus, single-cell isolations are very difficult. The work of Valley and Rettger^ and others seems to indicate that many organisms grow very ' Robertson, T. B.: Biocliem. J ., 15, 595. 1915. ^ Valley, George, and Rettger, Leo F.: J . Bad., 11, 78. 1926. Time Fig. 6. — An autocatakinetic growth curve 54 GROWTH CURVES OF BACTERIA slowly or not at all until a certain minimum concentration of carbon dioxide is pres- ent. This would lead to the development of a definite lag phase which would be much longer with small than with large seedings. Slator has shown that when very large inocula of yeast are used there may be no logarithmic phase. EFFECT OF CONCENTRATION OF CONSTITUENTS OF SUBSTRATE UPON GROWTH CURVES The various constituents of the culture medium may act either as accelerators or inhibitors of growth. The effect upon rates of increase is in general a function of the concentration. The exact relationship may be most satisfactorily evaluated usually by comparison of the rates of increase per cell {k) in different concentrations during the logarithmic growth phase. In chemical reactions generally it is found that the velocity coefficient of the rate of the reaction varies directly as some constant power of the concentration of the reactant. It is frequently advisable, as a first approxima- tion, to test the hypothesis that a similar relationship holds between rate of increase and concentration of a nutrient or inhibiting agent. This would give the relationship Integration yields in which f^=KC^b (i8) In b^KCH+ln B (19) /ir = Constant C= Concentration of chemical «= Constant It is apparent that the velocity coefficient (rate of increase per cell, k) is evaluated as k=KC^ (20) A determination of the validity of this relationship with varying concentrations may be made by plotting the logarithms of the velocity coefficients {k) against the log- arithms of the concentrations. Since log ^ = w log C+log i? (21) a straight line should be developed with slope n. If increase in concentration increases the growth rate, n will be positive; if it inhibits, n will be negative. EFFECT OF TEMPERATURE UPON THE FORM OF GROWTH CURVES The effect of temperature changes upon rates of increase is usually best evaluated by comparisons of the velocity coefficients {k) during logarithmic growth. It is cus- tomary to designate the ratio between the velocity constants at the higher and at the lower temperature as the temperature quotient (Q). The temperature interval for which determinations are usually made is 10° C. For this interxal the quotient is commonly designated as ()i„. It is frequently desirable to determine whether temperature effects upon growth R. E. BUCHANAN 55 rates resemble the effects upon rates of chemical reactions. It is commonly found that chemical reactions are accelerated by rise in temperature, and in ranges of o°-ioo° C. frequently doubled or trebled in rate by each increase of io°. This tendency to double or treble the rate has come to be termed the "R.G.T." {Reaktionsgeschwindigkeit Temperatur) rule. Studies upon rates of bacterial growth in certain ranges have shown values of Qw frequently equal to two or three. It has been shown that in chemical reactions the value of Qw tends to decrease with rise in temperature. This relationship has been developed by van't Hoff and Arrhenius into the generalization : The rate of change in the logarithm of the velocity coefficient of a chemical reaction with temperature is inversely proportional to the square of the absolute temperature, i.e., dink _ A . , It was also shown that the constant^ may be substituted by-^ , in which ju is a con- K stant characteristic of the reaction (thermal increment) and R the gas constant (numerically equal to 2). Integration of equation (22) yields lnk = -j,+C (23) Conformity of a reaction to the relationship of equation (23) may be determined by plotting the values of In k against the reciprocal of the absolute temperature; agree- ment is manifest by the development of a straight line, with slope - . It is of interest to determine whether growth rates (and growth curves) of micro- organisms are similarly related to temperature. The work of Crozier ef al.^ indicates that results of value may be secured by studies of the values of thermal increments (m). It is contended that growth rates are controlled by rates of chemical reactions, and changes in the latter due to temperature changes should produce corresponding changes in the former. Since the growth is probably the resultant, in many cases at least, of a catenary series of reactions, the rate of growth would be controlled by the slowest rate. Changes in temperature may therefore modify the rate of growth in the same manner as they modify the rate of the slowest reaction. From equation (23) the following relations are evident: k. H-ffi) Q,o=eT'T. (25) in which ^2 and kj represent the velocity coefficients at the higher and lower tempera- tures (absolute) T2 and Ti, respectively. ' Crozier, W. J., el al.: J. General Physiol., 7, i8g. 1924. 56 GROWTH CURVES OF BACTERIA .OOBee .0036I .OO3S<0 .O03S/ .00346 .003-1-I .00336 .0033I 0032S Reclproca/ of Absolute lemperafure. Fig. 7 i ; ; ; )^-"''^ -, -^^ r-^ i < ; ; / \ ■ ( 1 1 J \ 1 Reciprocal of /Absolute Temperature Tig. 8 R. E. BUCHANAN 57 The size and constancy of the thermal increment ^ may be determined either by substitution of values in equation (24) or by plotting the logarithm (base e) of k against the reciprocal of the absolute temperature. Such a graph is given in Figure 7 for the effect of temperature upon the growth of a bacterial culture between 0° and 30° C. It will be noted that the points apparently determine in this case two intersecting straight lines. Crozier and others interpret a finding of this type as indicating a change at a certain temperature from one basic reaction in the catenary series to an- other as governing the growth rates. Graphs of this type are made most conveniently by using a semilog paper in which the abscissae are indicated as temperatures centi- grade but are spaced in proportion to the value of the corresponding reciprocal of the absolute temperature. The data of Figure 7 are plotted on this type of co-ordinate paper in Figure 8. It is evident that if values of the thermal increment are known, and the relation- ships outlined above hold, it is possible to predict the form which growth curves will assume at different temperatures. CHAPTER VI THE RISE AND FALL OF BACTERIAL POPULATIONS C.-E. A. WINSLOW Yale School of Medicine I. THE LIFE-CURVE OF A BACTERIAL POPULATION In a study of the distribution of bacteria in their various natural habitats the bac- teriologist is inevitably impressed with a sense of a relatively stable adjustment be- tween a specific environment and the numbers and kinds of bacteria which will gener- ally be found therein. The botanist knows that on a certain kind of soil in a certain climate such-and-such trees and shrubs will be present, about so many to the acre. So, in our microscopical realm, we find that uncultivated sandy soils may yield 100,000 bacteria per gram while garden soils show 1,500,000. A given river in a dry summer will contain from 1,000-2,000 bacteria per cubic centimeter while a lake will contain only 50-150, and the deep waters of the ocean or those of a driven well will show only 5-10. From day to day, and from year to year, both the numbers and kinds of mi- crobes in a given habitat will remain extraordinarily stable — provided that the en- vironmental conditions themselves remain approximately constant. If, on the other hand, the conditions of the habitat change, or if a section of the bacterial population be transferred to a new environment, the balance is upset. A new and active struggle for existence is initiated, such as has occurred among the higher forms of life when a glacial epoch has changed the climate of a continent. With our short-lived forms of life, capable of completing a whole cycle of evolution in twenty-four hours, we can trace the course of such a struggle in a fashion which should be the envy of the ecologist; and as we do so, we find a remarkable degree of constancy underlying even the phenomena of change which characterize such a period of adaptation. The curve which marks the rise and fall of a bacterial population in a new environ- ment is illustrated graphically and schematically in Figure i ; and it may be claimed that this curve is a widely representative one for all conditions, with the limitation that according as the environment is more or less favorable the subdivisions of the curve may be relatively increased or decreased or even suppressed entirely. 2. THE PHASE OF ADJUSTMENT The first phase in the cycle of a bacterial population (if the environment be not too severe) is what may be called the "phase of adjustment." In a medium which is highly favorable this phase will be indicated, as in the solid line AB of Figure i, by a relatively slow increase, the period of lag or dormancy, as described by Rahn (1906), Barber (1908), Lane-Claypon (1909), Coplans (1909), Penfold (1914), Chesney (1916), and Sherman and Albus (1924). The first careful studies of the rate of bacterial multiplication and the first de- S8 C.-E. A. WINSLOW 59 scription of the lag phase in a favorable medium were due to Miiller (1895) who intro- duced the now-familiar formula for generation time: log 6— logo where G= minutes per generation, r = elapsed time in minutes, a = initial number of bacteria, and 6 = final number of bacteria. He showed that G increased with the age of the primary culture used for inocu- lating the medium in which generation time was measured. Thus typhoid bacilli inoc- ulated from a 2.5-3-hour mother-culture gave a generation time of 40 minutes while Fig. I. — Ideal curve of a bacterial population cycle. Ordinates = numbers; abscissae = elapsed time. A-B. Phase of adjustment D-E. Phase of decrease B-C. Phase of increase E-F. Phase of readjustment C-D. Phase of crisis those from a 6j-hour culture completed a generation in 80-85 minutes, and those from a 14-16-hour culture in over 160 minutes. He attributed the slower generation in media inoculated from older cultures to what is now called a "lag effect." Hehewerth (1901) confirmed these results. It was Rahn (1906), however, who first studied the preliminary lag period intensively, using B. fliwrescens in broth. He inoculated from (I) 20-hour broth, from (II) 20-hour agar, and from (III) 4- month broth, and obtained such results as are shown in Table I. If the medium be less favorable, the phase of adjustment will be marked by a de- crease followed by an.increase. This latter phenomenon was noted at least as early as 1894 (Fuller, 1895) in studies made at the Lawrence Experiment Station of the Massa- chusetts State Board of Health which showed that a bottled sample of sewage orig- inally containing 1,190,000 bacteria per cubic centimeter fell off to 1,085,000 after 2.5 hours, then rose steadily to a maximum of 23,100,000 after 25.5 hours, and then fell 6o THE RISE AND FALL OF BACTERIAL POPULATIONS steadily to 2,341,000 after 8 days. Whipple (1901) noted the same effect — an initial fall, followed by a rise, as a universal phenomenon in portions of natural waters stored in sample bottles under various conditions. This type of reaction, indicated by the dotted line AB in Figure i, does not appear to be substantially different from the lag phase in a richer culture medium and, indeed, it seems probable that whether the ob- served net effect be an increase or a decrease there is going on during this period a multiplication of some cells and a death of others, the relative rate of these two proc- esses determining the end-result. The fact that the bacterial count of milk shows an initial decrease was pointed out by Fokker (1890), and a long controversy has been carried on in regard to its cause (excellently summarized by Heinemann, 1919). Many investigators claim that freshly drawn cow's milk possesses special germicidal proper- ties due to the presence of agglutinins and other bactericidal substances, the presence of living phagocytes, or the restraining action of lecithin. Heating the milk destroys this "bactericidal power," but it must always be remembered that heating also changes the nutritive qualities of a medium and may therefore make it more favorable. In general, the phenomenon seems to be a special case of the lag period in a medium TABLE I Generation Time in Minutes Hours I n III 0-^ ^-6 1,083 113 60 1,309 77 63 997 212 6—12 12-24 62 more or less unfavorable to certain types of bacteria which have gained access to the milk. Ledingham and Tenfold (1914) and Slator (1917) have attempted to formulate mathematical expressions for the multiplication rate even in the highly variable lag period of the population cycle; but if the conclusion be justified that the phase of ad- justment may, according to circumstances, be characterized by a decrease followed by an increase or by a gradually accelerating increase, the futility of any attempt at mathematical analysis will be apparent. The inflection and the slope of the curve of the bacterial population will vary with three general factors: the type of bacteria involved, the medium into which they are introduced, and the temperature. The first two factors may in a sense be reduced to one — the suitability of the particular medium for the particular bacteria in question. If the medium be entirely inadequate, the bacteria simply die off and the phase of ad- justment merges into the phase of decrease. On the other hand, as I shall point out, if the medium be ideal and the bacteria in the right condition, the phase of adjustment may be reduced to proportions which are not measurable, and the logarithmic in- crease will begin at once. There may be an infinite number of gradations between these two extremes, giving a longer or a shorter phase of adjustment. Coplans (1909), for example, found that in transferring from peptone water to peptone water the lag period lasted about i hour while in transfer from one dulcitol medium to another it C.-E. A. WINSLOW 6i was longer. In fresh unsterilized milk the lag lasted 6 hours, but was apparently abol- ished by previous heating of the milk. Cohen and Clark (191 9) note that the onset of the period of maximal increase for Bad. coli was 2-4 hours in peptone phosphate broth atpH values between 6.1 and 8.1 but was increased to 3-5 hours at pH values below 5.5, and to 10-12 hours at pH 8.9 (37° C). A high temperature, of course, decreases the length of the phase of adjustment. According to Whipple's data for water bacteria stored in sample bottles, the period of lag averaged about 8 hours at 20^-24° C. and 17 hours at 12° C. Lane-Claypon (1909) reports a lag period in culture media varying from i hour at 42° to 6 hours at 20° C. An extreme case of lag may probably be found in the fact that both spores and vegetative cells occasionally show an exceedingly slow development in entirely favor- able culture media. Thus Burke, Sprague, and Barnes inoculated broth and agar tubes with approximately one cell of Bad. coli per tube. While 85 per cent of the 473 tubes which gave growth did so within 2 days and 97 per cent within 6 days, there were 10 tubes which developed only on the fourteenth day, 4 only on the fifteenth, and 4 only on the sixteenth. Spores of B. subtilis remained dormant under similar conditions for 39 days and spores of B. megatherium for 90 days. The fundamental causes of the lag phenomenon have been exhaustively discussed, particularly by Rahn (1906), Tenfold (1914), Chesney (1916), and Buchanan (1918). The phenomenon must be considered in the light of the observation of Miiller (1895) and Hehewerth 1901) that the rate of increase of bacteria in a given medium bears a generally inverse relation to the age of the mother-culture from which this medium was inoculated. Hehewerth found that the generation time for Bad, coli in broth, when transferred from a young broth culture, was 21-27 minutes while when transferred from an older broth culture it was 43 minutes. It is of cardinal sig- nificance to note that lag disappears entirely if transfer is made to an identical medi- um while the mother-culture is in its phase of logarithmic increase (Tenfold, Barber, Chesney). Furthermore, Tenfold shows that if the growth in a culture be stopped by chilling for a very short time the growth recommences at a normal rate when the tem- perature is raised; while more prolonged chilling and subsequent increase of tempera- ture is followed by a lag. The occurrence of lag cannot in general be due to the presence of inhibitory sub- stances carried over from the mother-culture (as might be concluded from Tenfold's finding that centrifugalized cultures showed decreased lag) since we note the same phenomenon in a sample of water transferred from a lake to a sample bottle. Further- more, Tenfold and Chesney show that lag in a secondary culture does not increase with the progress in the mother-culture of the logarithmic phase, and that while it does grow more marked with passage from the logarithmic phase to the phase of crisis there is no further increase with later aging of the mother-culture. Lag is therefore primarily associated with the biological condition of the cells which are transferred to a new medium (or placed under new environmental condi- tions, as when a water sample is collected). Under certain conditions this may be as- sociated with definite prior injury to the cells. Sturges (19 19) found that the develop- ment of colonies on plates seeded from sewage disinfected with copper or sulphurous acid was very much retarded (although the fact that chlorine-disinfected sewage ex- 62 THE RISE AND FALL OF BACTERIAL POPULATIONS hibits no such phenomenon suggests that the disinfectants were perhaps carried over to the plates in antiseptic concentration). Allen (1923) and others have ascribed sim- ilar slow growths of bacteria in milk after pasteurization as due to attenuation by "temperature shock." Chesney (191 6) believed this to be the main factor in the lag phenomenon in gen- eral, and he cites a striking case in which the generation time of pneumococci was slowed down in a filtrate from an old broth culture of the same organism. That the shock theory cannot be of general application, however, is made clear by the fact that similar phenomena occur in bottled-water samples. The second possibility which suggests itself is in a sense the converse of the idea that lag is due to a state of injury produced by an earlier environment. It involves the conception that, among the cells carried over from any environment A to another en- vironment B, some find themselves ill adapted to the latter and that a process of natu- ral selection must ensue until the less adapted cells are weeded out. Such a condition must apparently be assumed when the lag period is characterized by an actual de- crease in number such as occurs in samples of water, milk, or sewage, or in soU to which an antiseptic has been added. When a pure culture of a single species is transferred to a medium identical with that in which it is already living there must be something still more fundamental in- volved. We may conclude from the work of Penfold and Barber that an initial period of slow development is an essential necessity whenever bacteria pass to a new en- vironment from one in which they are not multiplying rapidly. Bacteria in active multiplication appear to be in a different biological state from bacteria in other phases of the population cycle, and it takes time to effect this change of state. We can, however, perhaps go a little further and visualize certain more concrete conceptions of what this difference in state may mean. The work of Sherman and Al- bus (1922 and 1924) indicates that cells during the early lag period are less sensitive to slightly toxic salts than are cells in the late lag and logarithmic phase, and they find that the sensitiveness to salts appears slightly before multiplication sets in at its most rapid rate. This suggests the possibility that permeability phenomena may play a part. On the whole, however, among the numerous possibilities Penfold's explanation that maximum growth presupposes the existence in the cells of intermediate bodies in the synthesis of protoplasm — intermediate bodies which diffuse out and are lost when growth is checked — seems on the whole most plausible. On such a hypothesis cells in the "resting stage" of an old culture or in a lake water or in any stable environment would be cells lacking these "intermediate bodies" while the "rejuvenated" cells of the logarithmic growth phase would be rich in them. It is also possible that such "intermediate bodies" may be transferred in the form of dead as well as of living bacterial cells, or in solution in the surrounding menstruum. This would explain the finding of Chesney that cells transferred from a mother-cul- ture during the phase of increase and washed free from the medium by centrifugation show a lag when placed in a new culture, while the cells left in the mother-culture still continue to grow at a logarithmic rate. Such a supposition would also be in accord with the conclusion of Rahn (1906) that maximal multiplication coincides with the presence of heat-stable, non-filterable substances "formed by the bacteria," and with C.-E. A. WINSLOW 63 the observation of Penfold (1914) that generation time decreases with an increase in inoculum. Rettger (1918) suggests that lag in culture media may be decreased or eliminated by supplying "satisfactory substitutes for the intermediate bodies in the form of amino acids and perhaps amines of simple composition, and also certain growth-accessory substances." 3. THE PHASE OF INCREASE When the phase of adjustment (whether this involves selection of more readily viable cells or the development within the cells of intermediate products favorable to rapid growth, or both) has been completed in a favorable medium, there next follows a period of rapid and regular increase. During this phase it has been shown by Clark and Ruehl (1919) that the average size of cell is greatly increased as compared with that which is dominant in an older culture. According to Henrici (1921, 1922, 1923, 1924), the large cells appear toward the end of the lag phase and the beginning of the phase of logarithmic increase, the average size returning to normal as the phase of in- crease proceeds. In a highly unfavorable medium, this phase will of course be entirely suppressed; but it is a very common phenomenon, by no means limited to the rich culture media of our laboratories. It was perhaps first exhaustively studied by the early water bacteriologists in the case of samples of natural waters which had been placed in a new environment by the mere fact that they had been collected in a sam- ple bottle in the laboratory. Thus Leone as early as 1886 records the following results for Munich water simply stored in flasks without the addition of any foreign material : Storage Period in Days Numbers per Cc o 5 1 100 2 10 , 500 3 67,000 4 315,000 5 Over 500 , 000 Miquel (1891) gives the striking curves reproduced in Figure 2 for a series of spring waters stored in flasks at 29°-3o° C. In the phase of logarithmic increase we are dealing with a very simple relationship due to the fact that binary fission carried on at a regular rate leads to a progressive logarithmic increase. In other words (Ledingham and Penfold, 1914) : t=K log b/B, when / = time, & = final number, and 5= initial number. The actual figures obtained for generation times under certain more or less typical conditions are indicated in Table II. The studies of multiplication in soil, feces, and bottled waters did not include counts made at sufficiently frequent intervals to be quite certain that only the phase of logarithmic growth was included, but they are cited in the table as representing the most rapid increases (under this condition) with which the writer is familiar. It appears from all the more careful work upon this subject that under the most 64 THE RISE AND FALL OF BACTERIAL POPULATIONS NUMBER OF BACTERIA IN I.C.C. 1,000,000 900^000 600,000 700,000 6oo>ooo 500,000 400,000 ^00,000 2 00,000 100,000 (\ ' »• DMUI5 — — -—--.- ST. LAURENT ♦1 • * • I » • ^, V ; It 1 1* 1 • 0: » ■ : 1 • • - • ■ 1 * ' • • 1: 1 • ■ 1 • li 1 • 1: 1 • • *' \ » • 'J ■ • • ■- 1' • >: 1 • • *' li I* 1' • • • I* !■ • • , i v • • • : 1 • 1 1 ■* 1 I ■ '' \ 1 • • '• V' '•/ ^ V» 1 \ ■: It k % 1: W\ 1*. % ■: V 1 % 1: \\ 1 \ \ 1: \^ / \ \ m ^ 1* W \ \ IJ M \ ^^ 1? \ •, ^ *: \ \ P. N •» 1 "-.. lamaaiMBHiM 1 o 10 20 30 DAYS 40 50 60 Fig. 2. — Multiplication of bacteria in bottled samples of certain spring waters (Miquel [Frankland, 1894]). C.-E. A. WINSLOW 65 favorable conditions of medium and temperature the cells of bacteria may divide once in 17-20 minutes, while under less favorable conditions the rate of multiplication may be slackened to any desired degree. TABLE II Typical Generation Times during the Phase of Logarithmic Increase Observer Conn, 1918 Jordan, 1926 Frankland, 1894 Whipple, 1901 Harrison and Vanderleck, 1909. . Buchner, Langard and Riedlin 1887 , Barker, 1908 Type of Organism Soil bacteria Bad. coli Water bacteria Water bacteria Bact. coli V. cholerae Bact. coll Medium Manured soil 20 Feces 20 Bottled sample 20 Bottled sample 24 Milk 37 Broth 37 Broth 37 Temperature, "C Generation Time, Minutes 840 720 196 IIS 40 19-40 17 The most obvious of the environmental conditions which determine the genera- tion time — and hence the K in the curve for rate of logarithmic increase — is the tem- perature (see Ward, 1895). The results of certain of the most exhaustive studies on this point are cited in Table III. ^ TABLE III Generation Time at Various Temperatures in Minutes Observer . Organism . Medium. . Whipple Water bacteria Bottled sample Harrison and Vanderleck Bad. coli Milk Buchanan Bad. coli Broth Lane-Claypon Bact. coli Broth Temperature, ° C. 864 15- 17- 20. 25- 30- 31- 34- 35- 37- 40. 42. 720 '386' 4S0 143 '56 45 180 76 43 29 17 52 32 28 19 If we plot the logarithms of these generation times against temperature, we obtain a series of straight lines, as was pointed out by Lane-Claypon (1909). The increase in rate of growth is approximately doubled for a 10° C. rise in temperature, a relationship which Snyder (1908, 1911) has shown is as generally characteristic of biological proc- esses as of chemical reactions. We may next consider briefly some of the other factors which determine the rate of logarithmic increase — a problem by no means easily elucidated. It is easy enough to understand why bacteria increase when inoculated into a rich, sterile culture me- 66 THE RISE AND FALL OF BACTERIAL POPULATIONS dium but that bacteria in more or less stable equilibrium in the water of a spring or well should increase many thousand fold (see Fig. 2) merely because a portion of the water in which they are living is placed in a sample bottle (even without any in- crease in temperature, as Whipple's data show) is far more difficult to explain. And the fact reminds us how subtle are the factors which constitute a bacterial environ- ment. Similarly, it is of interest to note the observation of Jordan (1926) that fresh hu- man feces (containing already 75,000,000 bacteria per gram) show an enormous fur- ther increase on storage, sometimes reaching several hundred times the original figure after a few days. This increase is mainly due to multiplication of Bad. coli and occurs at 10° and 20° as well as at 37°C. It is suggested by Jordan that the multiplication may be due to the loss of specific inhibitory influences present in the lower intestine. Miquel attributed the rapid growth in a new medium to the absence of toxic products of prior bacterial growth and found that boiling destroyed the "toxicity" of TABLE IV Bacterial Content of Normal and of Toluene-treated Soil at Various Temperatures Temperature, °C 5- 1 20. 30- 40. 50- Bacteria, Millions per Gram Untreated Soil 5 Days 27 Days 58 Days Toluened Soil 5 Days 8 50 43 12 2 27 Days 27 30 24 4 58 Days 28 30 31 6 water rendered unsuitable for growth by such earlier development. When we transfer from an old culture to a new sterile tube of the same medium in the laboratory, this factor must play a major part, since, as I shall point out in discussing the phase of crisis, growth is often undoubtedly checked in old cultures by the accumulation of acids or other toxic products. Cohen and Clark (191 9) found for Bad. coli in broth a generation time of 27 minutes at pH 5.0 while at pH 8.9 the time increased to 46 min- utes and in the acid range at pH 4.6 there was a decrease instead of an increase. For the multiplication following collection of a water sample such an explanation seems, however, clearly inadmissible, and the favorable effect of boiling is probably due chiefly to the alteration which heat produces in certain of the foodstuffs which are present. The work of Russell and Hutchinson (1913), Hutchinson and MacLennan (1914), and Buddin (1914) from the Rothamstead Experiment Station and the similar studies of Truffant and Bezssanoff (1922) in France have given us some important data in re- gard to the multiplication of bacteria in soils. They found that the treatment of a soil by moderate heat or by mild antiseptics caused a marked secondary increase in bac- terial numbers and crop fertility. Thus Table IV (from Russell and Hutchinson, C.-E. A. WINSLOW 67 1913) shows that in an untreated soil bacterial numbers remained constant, unaffected by temperature variations between 5° and 40° C. ; while in a soil to which a slight amount of toluene had been added marked increases occurred, at 20° and 30° and, more slowly, at 5°-i2° C. Table V (from Hutchinson and MacLennan, 1914) is of special interest as demon- strating the influence of lime upon a highly acid soil. It will be noted that o.i per cent CaO produced a very slight stimulating action. The next three concentrations (0.2, 0.3, and 0.4 per cent) caused progressively increasing stimulation with a maximum count on the ninetieth day. A strength of 0.5 per cent was somewhat less effective but its influence was more prolonged, while a concentration of i.o per cent proved toxic at first, with a later stimulation giving a higher count after the two-hundredth day than was shown by any other sample. In this particular instance the effect of the lime upon the reaction of the soil no doubt played an important part in stimulating bacterial multiplication. In general, TABLE V Bacterial Content of Acid Soil Treated with Varying Amounts of Lime Days Control -|-o.i per cent CaO . . .2 per cent CaO . . .3 per cent CaO . . .4 per cent CaO . . 0.5 per cent CaO . . -fi.o per cent CaO . . Millions of Bacteria ler Gram of Dry Soil 10 50 90 120 150 200 232 5 5 4 4 4 4 5 6 II II 9 10 4 5 23 78 82 68 73 29 19 97 170 170 132 133 106 77 151 387 906 345 178 154 87 270 281 470 260 247 180 200 2 0.2 6 7 II 176 329 310 5 3 13 63 71 77 337 however, Russell and Hutchinson attributed the effect of heat and antiseptics upon soils to the destruction of predatory protozoa. It seems quite possible that the in- fluence of the treatment upon available foodstuffs (whether derived from the bodies of protozoa or from other sources) may have been an even more important factor, as in the case of Miquel's experiments cited above. In many other instances, it is certainly to the availability of the food supply, rather than to the absence of inhibiting substances, that we must attribute the initi- ation of the logarithmic growth phase. The amount of food required by the bacteria may of course be exceedingly minute, particularly in the case of prototrophic water forms. Kohn (1906) determined the minimal nutrient requirements for certain of these types and found that they could develop in the presence of 198X 10 — '" to 198X 10 — ^^ per cent of glucose, 66X10—*^ to 66X10 — ^9 per cent ammonium phosphate. With more fastidious organisms, however, a much ampler and more diversified diet is necessary for maximal growth. Thus Tenfold and Norris (191 2) found that the maxi- mum generation time of Bait, typhosum in i per cent peptone at 37° C. was 40 minutes, but that when the peptone in the medium was reduced below 0.4 per cent the genera- tion time increased and rose quite regularly with decrease in peptone content down to 0.2 per cent. In a o.i per cent peptone medium the generation time could be cut in 68 THE RISE AND FALL OF BACTERIAL POPULATIONS half by the addition of 0.17 per cent glucose. The literature is full of comparative studies of media for water and milk analysis or for the isolation of specific organisms which bear upon this point. As an example of such studies we need only cite the recent work on media for milk analysis which has indicated the widely different results ob- tained with various brands of peptone (Shrader, 1926). Davis and Ferry (1919) note that both the growth and the toxin production of the diphtheria bacillus in a beef- infusion medium are dependent on the particular types of amino acids present and suggest that other accessory factors, perhaps of the nature of vitamines, are also es- sential. The presence of growth hormones was held to be necessary for meningococci and gonococci by Lloyd (191 6) and Cole and Lloyd (19 17); and Wildiers (1901) claimed that a hypothetical substance called "bios" was necessary for the fullest growth of yeast in a synthetic medium. Devereux and Tanner (1924) and Werkman (1927) have recently reviewed va- rious aspects of this subject and conclude that the evidence as to the influence of true vitamins or growth-promoting substances (other than those of a nutrient character) is very doubtful.' In addition to those nutrient materials which are directly essential for the up- building of bacterial protoplasm the rate of multiplication of bacteria is also governed, like many other biological processes, by the regulative action of mineral salts. This subject has been admirably reviewed by Falk (1923), and we need only point out here that Hotchkiss (1923) and others have shown that a wide variety of salts stimulate bacterial growth in low concentration and inhibit it in a higher concentration. Even such toxic salts as HgCla may stimulate growth when present in a dilution of one- millionth of a molar concentration (and inhibit it entirely in a concentration of one- hundred-thousandth molar) while NaCl and KCl stimulate in .25-M concentration and inhibit in 2-M concentration (under the conditions of the Hotchkiss study). It seems possible that the multiplication of water bacteria in a sample bottle may in part be due to the stimulant action of minute traces of salts dissolved from the glass during sterilization, since Kohn (1906) has shown that the increase is most marked in bottles of the more soluble types of glass. Finally, the dissolved gases in a medium affect bacterial multiplication in far- reaching ways which we are as yet far from comprehending. Wolffhiigel and Riedel (1886) found that multiplication of bacteria in a flask stoppered with cotton was greater than in one closed with a rubber stopper, and Whipple (1901) observed that it was greater when a bottle was only partly filled than when it was filled more nearly to the top. Curiously, however, agitation and artificial aeration seemed unfavorable to growth in WTiipple's experiments. One of the most important contributions to bacte- riology in recent years has been the demonstration by Valley and Rettger (1927) that a small percentage of carbon dioxide is essential to bacterial growth and that when this gas is entirely removed growth ceases completely. 4. THE PHASE OF CRISIS After a lapse of time, varying with the nature of the organism, the medium, and the temperature, the period of logarithmic increase draws to a close, and after an in- termediate phase of crisis a phase of decrease supervenes. This is of course a phenom- ' Cf. chapter xxxvii in this vohime. C.-E. A. WINSLOW 69 enon familiar to us in our culture media and in such substrata as soil or milk. It was noted in water by Cramer as early as 1885 in the following very clear example. Lake Zurich water stored for a period of seventy days: Storage Period, Days Numbers per Cc o 143 1 12,457 3 328,543 8 233,452 17 17.436 70 2 , 500 A much less marked but fundamentally similar cycle is given by Conn (1918) for the change of bacterial numbers in freshly manured soil. Storage Period, Days Numbers per Gram o 35, 000 , 000 I 120, 000 , 000 2 100 , 000 , 000 3 145 ,000,000 4 150, 000 , 000 6 120, 000 , 000 9 70 , 000 , 000 16 70 , 000 , 000 60 75 , 000 , 000 120 23,000,000 Jordan (1926), in one of his experiments on stored feces, noted that, taking the initial number of bacteria present as 100, the relative number rose during storage to between 2,000 and 3,000 on the fifth to the ninth day and then fell to about 1,000 on the eleventh day and to less than 400 on the twenty-first day. For colon-group or- ganisms under similar conditions he gave the following results: COLON BACILLI IN FECES STORED AT ROOM TEMPERATURES Relative Numbers Considering Initial Number as 100 Storage Period, Days Relative Numbers 3 7 , 000 5 3.730 7 3 , 600 9 2 , 400 II 930 14 530 16 24 18 26 21 24 23 14 The curves in Figure 3 plotted from averaged results given by Prescott and Baker (1904) for the numbers of colon bacilli and streptococci developing in glucose broth inoculated with polluted water and incubated at 37° C. illustrate the difference in the 70 THE RISE AND FALL OF BACTERIAL POPULATIONS rate of growth of different species in the same medium, the colon bacilH rising more rapidly at first and the streptococci becoming dominant in the later stages of the cycle. Reed and Reynolds (1916) give interesting data as to the period of maximum growth for various types of bacteria inoculated into milk in pure culture, the periods at 37° C varying from i day ior Bad. lactis-acidi and Sarcina lutea to 21 days for M. citrictis and 42 days for Oidium lad is. In media which are unfavorable the phase of crisis may set in very early. Chick (191 2) found that the usual relations as to lag period, logarithmic multiplication, and 2,ooo t B. I l»800 l,600 1,400 l,200 1,000 8 00 600 STREPTOCOCCI—— 400 200 i / ■^ / \l ^ y DAYS-*-© 7 II 16-17 23 Z7 39-40 52 63 Fig. 3. — Multiplication of Bad. coli and streptococci in glucose broth culture into which they were simultaneously inoculated. (Average of results reported by Prescott and Baker, 1904.) influence of temperature held for the growth of Bad. coli in normal rabbit serum — the only difference being that the period of increase is very brief and leads only to a doubling of bacterial numbers, after which a rapid decline sets in, due to the bacteri- cidal effect of the serum. In rich culture media the initial growth will be rapid, but here too the phase of crisis will usually set in within 24 hours as a result of the forma- tion of inhibitive waste i)roducts. In milk containing mixed cultures of many species of bacteria, the bacteria present may continue to increase for days and may reach enormous figures (several Inllions per cubic centimeter, for example, in samples held at 15.5° C. by Ayers, Cook, andClemmer, 1918). In most studies on milk bacteriology, indeed, the existence of a period of crisis and subsequent decline has not been appar- ent since observations have usually not been continued for a long enough period to C.-E. A. WINSLOW 71 pass the critical point. In the important contribution of Ayers and Johnson (1910), however, many individual samples show the beginning of a decline after 4 or 5 days even when held at 10° C. So far as the temperature effect is concerned we may note that Lane-Claypon (1909) found the end of the period of logarithmic increase for Bact. coli in broth to oc- cur after i\ hours at 37° C, after 8-8^ hours at 30° C, 12-15 hours at 25° C, and 20-24 hours at 20° C. In the work of Reed and Reynolds (1916) on the growth of pure cultures of thirteen different species of micro-organisms in milk the average period of maximum growth was 7 days at 35° C. and 20 days at 13° C. The explanation of the onset of the phase of crisis must obviously be sought in the changed composition of the medium due to the growth of the bacterial population during the phase of increase. It is clear that there is no essential and invariable life- cycle involved since the results of Penfold and of Barber indicate that transfer of cells from a culture in the early phase of increase to a new tube of the same medium yields a constant and continuous development at a logarithmic rate. The change in the medium which makes it unsuitable for further growth might theoretically be either an exhaustion of essential foodstuffs or the formation of toxic waste products, and both processes may no doubt play a part under certain conditions. In spring-water samples containing but little organic matter it seems very probable that the exhaustion of food may play a predominant role (though even Miquel stressed the conception of toxic products). In rich culture media the formation of waste products is probably the fac- tor of major importance. In milk and other sugar media the production of acid is often by itself entirely adequate to explain cessation of growth. Thus Heinemann (1915) found that a series of pathogenic bacteria were all destroyed when the acidity of milk reached 0.45 per cent and that even Bact. coli died out when the milk reached an acidity of 0.6 per cent, while Bact. coli itself increased the acidity of milk to 0.5 per cent. Cohen and Clark (1919) found the limiting pH values for growth in peptone broth to vary from 4.4 for Bact. aerogenes to 5.5 for Bact. alcaligenes; but they pointed out the extreme complexity of the phenomena involved, as shown by the fact that while fermentative activity seems to cease in a sugar medium at about the pH con- centration known to limit growth, in previously adjusted media growth in the sugar medium seems to stop at a point corresponding to concentration of the acetic acid formed rather than the pH. Furthermore, in a broth medium free from sugar growth ceases much sooner than in the sugar medium and with a total concentration of cells only one-fifth as great as that which is found in the presence of sugar. In dealing with the phase of crisis, as in the case of the phase of adjustment, it does not seem particularly profitable to refine our methods of analysis too far until more detailed experimental data are available. If we assume that the cycle of a bac- terial population starts from and returns to a stable state there are really but two fundamental processes involved, an increase from the original stable condition and a subsequent decrease to a second level, with phases of transition before the increase, between the increase and the decrease, and before the period of ultimate stability. Clearly, as a result of various factors, such as food supply, toxic products, and temper- ature, the slope of the curve during the phase of crisis may take any form from a sharp tooth to a flat plateau. 72 THE RISE AND FALL OF BACTERIAL POPULATIONS 5. THE PHASE OF DECREASE From the earliest days of bacteriology it has been noted that the decrease in bac- terial numbers under the influence of an unfavorable environment (such as is present for one reason or another on the descending side of the curve of the population cycle) followed a gradual and more or less orderly course. This was at first attributed to a process of natural selection, the surviving organisms being assumed to be of a specifi- cally more resistant character. With the work of Koch (1881), Paul and Kronig (1896), Kronig and Paul (1897), Ikeda (1897), Madsen and Nyman (1907), and Chick (1908, 1 9 10) on the action of chemical disinfectants the mortality curve was given a new in- terpretation as an expression of a more fundamental chemical phenomenon. As sum- marized by Phelps (191 1), these researches have shown that "the rate of dying, whether under the influence of heat, cold or chemical poison, is unfailingly found to follow the logarithmic curve of the velocity law, if the temperature be constant." The general slope of the mortality curve during the period of rapid decline is therefore the same as that of the curve for the increase of a bacterial population dur- ing its period of rapid multiplication. In the phase of increase the logarithm of the number of new cells formed from a single initial cell in a given time is proportional to the lapse of time ; in the phase of decrease the logarithm of the proportion of the cells present which perish in a given interval is proportional to the length of that interval. In other words, increase and decrease alike bear a direct relation to the number of cells present at the beginning of a unit period and a logarithmic relation to any time period of greater duration. The formula for the rate of decrease is, therefore, in the form used by Chick (1908), as follows: t2-h "= n where /i is the initial and (2 the final time and n^ and «. the corresponding numbers of bacteria present. Phelps, taking the elapsed time (as /) instead of the initial and final time readings (/j — ^i) and B as the initial and b the final number of bacteria, expresses the formula as: log J = Kt which is of course the formula for a monomolecular reaction.^ The results presented by Chick (1908) in regard to the regularity of the process of disinfection were very striking and seemed to justify her conclusion that "a very com- plete analogy exists between a chemical reaction and the process of disinfection, one reagent being represented by the disinfectant, and the second by the protoplasm of ' This formula is written by Falk and Winslow, o.434/v.= i// log —-^ where a = 7i, of Chick and a—x = niOi Chick because A'i= i/l \ogc ^^ and K=\/t log,o — - when C.-E. A. WINSLOW 73 the bacterium." Later Miss Chick (1910) showed that the death of bacteria when dried or exposed to sunhght, or even when killed by moderate heat in water, proceeded in general in accord with the logarithmic law. Cohen (1922) confirmed these general conclusions for the much more gradual death-curve of colon bacilli in water. In all of these latter cases, of course, the reacting agents which cause death must be within the bacterial cells themselves. This simple concept of the disinfection process has been vigorously challenged by a considerable group of workers such as Loeb and Northrop (1917), Brooks (1918), Peters (1920), and Smith (1921) who attribute the form of the mortality curve to bio- logical differences in the resistance of the individual bacterial cells. Miss Chick herself invoked this conception of individual-cell variation to explain irregularities in her mortality curves for non-spore-forming organisms such as para- t>phoid bacilli and staphylococci. When very young cultures of these organisms were used she obtained constant values of K, but with older cultures the rate of disinfection fell off in the later stages of the process. Even Cohen's curves in many instances ex- hibit a tendency to flatten out toward the end of the periods studied, and Falk and Winslow (1926) present results on the death of Bad. coli in dilute salt solutions which suggest an initial rise in the value of K with the passage of time, followed by a subsequent gradual fall. It appears certain, however, from a review of all the available literature that when a bacterial population of a reasonably homogeneous character (spores or young vege- tative cells) is subjected to an unfavorable environment — whether the unfavorable condition be a chemical disinfectant, heat, sunlight, or merely storage in a dried con- dition or in an aqueous medium where growth cannot occur — there is a period during which the death of the bacterial cells follows a fairly regular logarithmic rate. This period of regular decrease may be preceded by a brief period of slower decline, repre- senting a sort of lag or adjustment to the unfavorable environment, and seems general- ly to be followed by a final period of still slower decline. The latter may not occur when death is due to strong disinfectants such as were used by Miss Chick and did not appear in Cohen's studies because they were not prolonged for a sufficient period. It seems, however, entirely unnecessary to postulate biological variations in the cells of the bacteria to account for these deviations from the logarithmic curve. As Falk and Winslow (1926) have pointed out, it is much simpler to assume that the lethal reactions which go on in bacterial cells dying in an unfavorable environment proceed in accord with a bimolecular reaction or reactions of a still higher order rather than in accord with the monomolecular formula. There is no reason to assume that the decomposition of a single chemical compound is always the determining cause of death. It is shown in the paper cited that the values of K may often be best explained on such an assumption. Just as the lag period in bacterial growth involves the as- sumption of catenary reactions, so do the variations at the beginning and end of the curve of bacterial mortality. To obtain the values which most nearly approximate a logarithmic curve and represent the period when the lethal process most nearly sim- ulates a monomolecular reaction, Phelps has suggested that K should be determined for the middle portion of the curve — say from a reduction of 75 to one of 25 per cent. 74 THE RISE AND FALL OF BACTERIAL POPULATIONS It is a curious and interesting fact that disinfection by sodium hydroxide follows an entirely different law from that observed in other cases, K increasing progressively with the time of exposure (Levine, Buchanan and Lease, 1927). It may be of interest, in spite of the various factors involved in the problem, to consider some of the absolute values of K which have been observed in certain specific instances and to note their significance in terms of percentage reduction. At one extreme stand such results as those obtained by Jordan (1926) for the secondary reduction of Bad. coli in stored feces. These give (from the third to the twenty-third day) a K per hour of .006. The values which I have cited for the reduc- tion of the numbers of a certain strain of colon bacilli in water (Winslow and Cohen) give a A' per hour of .01 for the third to the tenth day, corresponding to a reduction in numbers of 2.5 per cent per hour. The reduction of Bad. typhosum in ice for the first three days (Sedgwick and Winslow) is of the same order {K = .02) and involves a re- duction of 5 per cent per hour. For Bad. coli dried in sand (Winslow and Abramson) K = .o6 for the ninth to the forty-eighth hour (a reduction of 13 per cent per hour). The mortality of anthrax spores exposed to 5 per cent phenol at 20° C. is about the same (Chick, 1908). Falk and Winslow's studies give a A'- value of about .1 per hour for dilute salt solutions (20 per cent per hour), and Cohen's data for acid (pH 8) give a K- value of i.o (90 per cent reduction per hour). Chick's analysis of Clark and Gage's results on the disinfectant action of sunlight give a A of 2.6, which would involve a reduction of 95 per cent per hour. The death of paratyphoid bacilli exposed to 6 per cent phenol is more rapid still, with a A of about 20, according to Chick's data (99.4 per cent reduction per hour) ; while hot water (54° C.) gives a A of over 60.0. This in- volves a reduction of 99.75 per cent per hour. The factors which govern the rate of the mortality of bacteria are essentially the same as those which govern the rate of their multiplication, though of course most of them operate in an inverse sense. When bacteria die out in water or when stored in a dried condition it is the normal katabolic processes of the cell (in the absence of com- pensating anabolism) which must control the process. It seems doubtful whether drying in itself exerts any specific harmful effect, and (if rapid and complete) it may even slow down vital processes and thus prolong life. Ficker (1898), however, found that alternate drying and moistening accelerated the lethal process. It is of interest to note that Paul, Birstein, and Reuss (1910) found the disinfection constant in drying proportional to the square root of the oxygen concentration of the atmosphere, fol- lowing the law which obtains in the slow oxidation of phosphorus. The whole subject of the longevity of micro-organisms under the influence of desiccation has been well reviewed by Giltner and Langworthy (19 16). The presence of external toxic substances may of course be one important factor in the death of bacteria in water or in soil. Thus Jordan, Russell, and Zeit (1904) found that typhoid bacilli inclosed in collodion sacs survived much longer when the sacs were suspended in pure water than when they were suspended in polluted water. In the presence of known chemical disinfectants this factor of direct toxicity is of course the determining feature. The value found for A may therefore vary within the widest possible limits, depending on the toxicity of the particular disinfectant studied; and the study of the relation between toxicity and chemical composition (as worked out, C.-E. A. WINSLOW 75 for example, by Schaffer andTilley [1927] for the various members of the alcohol and phenol group) is a fascinating one. Chick (1908) showed that within certain limits the efficiency of a chemical disin- fectant bears a logarithmic relation to its concentration, the expression log 7^ In to L'oto remaining constant in value when to and /„ are the time periods necessary for disinfec- tion corresponding to concentrations Co and C„. Furthermore, it is important to note that disinfectants not only vary in their efficiency at a given concentration but also vary in the degree to which their toxicity increases with increasing concentration. The ordinary carbolic acid coefficient (representing the ratio of the concentration of a given disinfectant to the concentration of carbolic acid which will sterilize in a given time) therefore gives a very incomplete idea of the true relationships. Thus Chick (1908) found that mercuric chloride may have a carbolic acid coefficient of 13 at one concentration and of 550 at another. Phelps (191 1 ) therefore suggests that values of K (K and K') should be deter- mined at two different concentrations (C and C). If we express the K of our previous formula (p. 72) by KC" (to allow for this concentration factor) we get log ^=KCH and log ^=K'C'H Therefore n=Iog-^ '^^^S'C From the two determinations of K we can then compute n or the concentration coefficient of the particular disinfectant studied. This figure n shows by what power of 2 the efficiency is increased if the concentration be doubled. For anthrax spores in mercuric chloride at 20° C. it is 1.08. The reaction of the medium is a special case of chemical toxicity which has been studied with particular care. Such a phenomenon occurs in nature in streams re- ceiving acid wastes, and the disinfectant action of carbon dioxide is due to the same cause (Koser and Skinner, 1922). As pointed out above, carbon dioxide, aside from its effect upon reaction, is beneficial and indeed essential to bacterial life. Winslow and Lochridge (1906) reported twenty years ago that the toxic effect of mineral acids was in large measure due to dissociated hydrogen. Cohen (1922) has given us one of the most careful recent studies of this problem, and we may cite one of his summary tables (Table VI) indicating the effect of hydrogen-ion concentration upon the death-rate of Bad. typhosum. The effect of cations other than hydrogen upon bacterial growth and death has been studied by numerous observers (see review by Falk, 1923). Those who have worked on the increase of bacterial populations in favorable media, from Richet 76 THE RISE AND FALL OF BACTERIAL POPULATIONS (1892) to Hotchkiss (1923), have found that practically all cations may show either a stimulating or an inhibiting influence, depending on their concentration, and observers TABLE VI Average Velocity Constants for the Death of Bad. lyphosnm AT Different pH Values, 20° C. PH 3-8 S-o 5.4 6.4 7.1 7.6 8.7 9.5 K 1055 00134 o.oiio 0.0138 0.0437 o. HOC 0.2134 0.2855 Relative iir*95. 5 1.2 i.o 1.5 4.8 10. o 22.4 31.4 * Taking K at pH 5.4 as unity. like Winslow and Falk (1923) who have studied the death-rate of bacteria in unfavor- able media find that in the same way small amounts of cations favor survival while Kl +1.5 +1.0 ♦0.5 -0.5 CaCU / / / NaCI **— v / 0.001 o.oi o.f 1.0 MOLAR CONCENTRATION OF SALT 1.0 Fig. 4. — Relation between viability of Bad. coli and salt concentration (Falk and Winslow, 1926). large amounts increase the mortality. Falk and Winslow (1926) pointed out the sig- nificant fact that if values of K for the effect of NaCl and CaCl2 upon Bad. coli in water be plotted against concentration one obtains a reasonably smooth curve for both salts, passing from positive values (indicating toxicity) to negative values (indi- cating a preservative effect) (see Fig. 4). It will obviously be difficult to harmonize such a phenomenon with any simple chemical assumptions. It is also important to remember that the net effect of various chemical and physi- cal factors, acting simultaneously, may be an exceedingly complex one. Thus Chick (1910) showed that very minute excesses of acid or alkali might enormously accel- erate the rate of disinfection by hot water; and the same phenomenon is of great prac- tical importance in connection with heat sterilization. On the other hand, a lethal factor may equally well be neutralized by a favorable one. Sometimes the effect is relatively simple, as in the reduction of the disinfectant power of mercuric chloride due to the presence of organic matter (Chick and Martin, 1908), a fact which makes C.-E. A. WINSLOW 77 this substance wholly unsuitable for the disinfection of feces, or in the interference of organic matter in water with the action of chlorin. Under other conditions more sub- tle reactions are involved, as in the results recently reported by Winslow and Brooke (1927). These observers found that several different types of bacteria die out almost immediately when washed free from culture medium and resuspended in distilled water. Salt and sugar solutions will not check the mortality, so we are not deahng merely with a question of osmosis; but the cells can be protected by the presence of peptone or meat extract, serving as "protective colloids" as indicated in Table VII. TABLE VII Viability of B. cereus Percentage Surviving Broth Peptone Meat Extract Ringer-Locke Solution Before centrifugation 100 56 71 100 43 53 100 51 57 After centrifugation 3 I One hour later A concentration of .005 per cent peptone or of .003 per cent meat extract will protect the cells; but a concentration of .0005 per cent peptone or of .0003 per cent meat extract fails to do so. Finally, we must consider the influence of temperature upon the course of the curve of decreasing numbers of bacteria. Since the processes of death, like those of life, are essentially chemical in nature, it is of course obvious that an increase of tem- perature will favor the lethal process when lethal factors are dominant just as it favors the growth process when growth factors are dominant. The classic work of Houston TABLE VIII Effect of Temperature on Survival or Typhoid Bacilli in Water (Houston) Temperature ° C Percentage of Typhoid Bacilli Surviving after One Week Period of Final Disappearance of Bacilli (in Weeks) 46.00 14- 00 0.07 0.04 9 7 5 4 s 10 18 (1911) on the survival of typhoid bacilli in water (illustrated in Table VIII), for ex- ample, seems at first sight puzzling, since we find an organism whose optunum for growth lies at 37° C. dying more rapidly at that temperature than in cooler waters. The explanation lies in the fact that typhoid bacilli in water lack the conditions essential for anabolism; only katabolism can go on, and katabolism is increased by a rise in temperature. The effect of high temperature in increasing the eflEiciency of chemical disinfect- ants was noted by Koch (1881) in the earliest studies of disinfection, and was first 78 THE RISE AND FALL OF BACTERIAL POPULATIONS carefully studied by Madsen and Nyman (1907), Chick (1908), and Paul (1909). These investigators found that the reaction velocity of disinfection increased with a rise in temperature, according to the formula of Arrhenius, the function T-T, log remaining constant where ti and ^2 represent times taken for disinfection and Tj, and T2 represent absolute temperatures. As the temperature at which such a reaction proceeds increases, the velocity of the reaction increases in geometrical progression. If K' and K are the constants of the reaction at the temperatures T' and T, respectively, and d is the temperature coeffi- cient (Phelps), K ^ By determining the reaction velocity of the lethal process for bacteria at two temperatures 10° C. apart, we may obtain this temperature coefhcient from the formula For anthrax spores exposed to 0.5 per cent mercuric chloride 6 = 1.17. I^i general, the mean velocity of disinfection with metallic salts increases two- to four-fold for a 10° rise in temperature (centigrade), while with other disinfectants the increase may be considerably greater. The effect of temperature upon the natural death of bacteria in water is essen- tially the same, although the absolute value of the coefficient seems to be somewhat lower. Table IX, from Cohen (1922), illustrates this phenomenon. TABLE IX Velocity Coefficients for Death of Bact. typhosum and Bad. coli at pH 3.5 at Different Teiiper.\tures Bad. typhosum Bad . coli Ratio of K FOR Bad. Temperature, ° C K Increase for 10° K Increase for 10° typhosum to K FOR Bad. coli 1. 186 1-919 2.928 5-176 0.0176 • 0373 •1654 0.6214 67 51 18 10 1.62 1-53 1-77 2.12 4-36 3 76 20 IQ 8 It will be noted that the temperature coefficient for Bact. coli is much lower than for Bact. typhosum but rises much more rapidly for a 10° C. increase. A peculiarly interesting contribution was made by Chick (1910) in the demon- stration that the death of bacteria in hot water follows the same general time relation, although of course with an enormously high time factor. In the case of Bact. typhosum the coefficient was 1.635 per 1° C. The whole phenomenon of cell death C.-E. A. WINSLOW 79 under the influence of heat is explained by Chick and Martin (1910) as a heat coagulation consisting in a reaction between protein and water which is highly accel- erated by heat. From the practical standpoint of food preservation, Bigelow (1921) has shown that the logarithmic relationship between sterilizing time and temperature holds for the destruction of both spores and vegetative cells by high heat. Phelps (191 1) has given a very valuable analysis of the variations in the effective- ness of a given disinfectant with respect to concentration, time, and temperature, and has shown how its constants can all be fixed by determining its efficiency in two dif- ferent dilutions at the same temperature and at two different temperatures in the same dilution (three determinations in all) ; the values for any other set of conditions can then be obtained from the formulas: log^ = KCH and when (as before) jB= initial number of bacteria present, 5 = final number of bacteria present, / = elapsed time, C = concentration of disinfectant. A' = velocity constant cal- culated at temperature of experiment, irr° = same at any temperature T°, Kio" — same at 20°, w = concentration exponent, and = temperature coefficient. 6. THE PHASE OF READJUSTMENT Toward the close of the phase of decrease the rate of mortality slackens and the curve passes imperceptibly into the final phase of readjustment. In the work of Chick (1908) on the effect of chemical disinfectants upon vegetative cells, and in the studies by Falk and Winslow (1926) on the death-rate of bacteria in dilute salt solu- tions, it even appears, as we have seen, that the value of K falls progressively through- out the phase of decrease (see Fig. 4) so that there is no clear distinction between the two processes. In other instances, however, it is often possible to observe two fairly distinct periods, one of rapid and one of slow decline. Thus, Winslow and Cohen (1918) in their studies of the life of Bact. coli in water observed values of K ranging from .004 to .020 for the first ten days while from the tenth to the sixtieth day the values varied only between .001 and .002. The mortality curves given by Sedgwick and Winslow (1902) for the life of ty- phoid bacilli in ice and in dry earth, by Winslow and Abramson (191 2) for colon bacilli in water, all show a falling rate of mortality toward the close of the cycle of decrease. Values for K calculated from these data are cited below, obtained by merely sub- tracting the log of the number present after a given time interval from the log of the number present at the beginning of that interval and dividing by the elapsed time. (It should be noted that in computing these values the reduction in numbers for each period are computed on the basis of the number present at the beginning of that pe- riod instead of following the usual procedure of computing K on the basis of reduction from the beginning of the whole experiment. The latter method naturally tends to obscure any differences which may occur.) The very gradual rate of decrease during the phase of readjustment may end in 8o THE RISE AND FALL OF BACTERIAL POPULATIONS either of two different ways. In a moderately favorable medium, such as water, the cycle usually ends with a new level of stability {EF in Fig. i) on which the number of bacteria may remain reasonably constant for an indefinite period. Miquel describes an experiment in which a bottle of Seine River water containing originally 4,800 bac- teria per cubic centimeter was stored for nine years and showed 220 bacteria per cubic centimeter at the end of that time. Sometimes, as is shown in Figure 2, there may be one or more secondary waves of increase before the final level of stability is reached. In his study of stored feces Jordan (1926) found that after the initial multiplica- tion and subsequent decrease a level was reached which remained more or less con- stant for long periods. The total number of bacteria present after many weeks may be as high as, or higher than, the number initially present. Bact. coli, however, ultimate- ly disappears from the fecal flora under such conditions. In a less favorable medium, TABLE X Values of K for Various Periods of Certain Mortality Curves Observer Organism Medium Temp. Period K per Hour Sedgwick-Winslow Bad. typh. Bact. typh. Bact. coli Bact. coli Dry earth Ice Dry sand Water 0° — I 20 10-20 /First 3 days \ 3-14 days ("First 3 hours ■{First 3 days [3-14 days fFirst 9 hours \ 9-48 hours [48-216 hours [First 3 days J3-10 days [10-60 days 0.04 Sedgwick-Winslow Winslow- Abramson Winslow-Cohen .004 .08 .02 .002 •03 .06 .006 .004 .on o.ooi on the other hand, the phase of readjustment ultimately ends in complete sterility, as in disinfection with high heat or strong chemicals {EF' in Fig. i). In an intermediate case — where conditions are neither sufficiently favorable to permit of the balanced growth and death which maintains a constant stable level nor sufiiciently unfavorable to lead to rapid and complete extinction — a small proportion of the original bacterial population may persist for a very long period. Thus Parkes (1903) was able to isolate typhoid bacilli from blankets soiled with feces after more than six months. Konradi (1904) reports Bact. typhosimi as surviving in water after seventeen months. Robertson (1898) isolated the same organism from moistened soil after eleven months. Studies by numerous observers on the drying of typhoid, diph- theria, and tubercle bacilli (summarized by Chapin, 191 2) have shown that all these organisms may survive drying for several months. (Table X.) Winslow and Kligler (1912) found over 51,000 colon bacilli and 42,500 acid- forming streptococci per gram of dust from city streets and 940 colon bacilli and 22,040 acid-forming streptococci per gram of house dust. Winslow and Sanjiyan (1924) conducted an extensive study of the distribution of the acid-forming strepto- cocci, presumably indices of pollution from mouth spray, on objects and surfaces of various kinds. Objects, such as eating utensils, directly exposed to mouth contan? C.-E. A. WINSLOW 8i ination showed these organisms in 62 per cent of the cases studied while objects re- cently handled (door handles, push buttons, etc.) showed them in 42 per cent of the cases. On locations such as walls six feet above the ground, the undersides of chairs and tables and the like, these organisms were isolated in 10 per cent of the cases. From the standpoint of epidemiology, it is essential to note that the pathogenic bacteria which survive for such long periods as those noted above are so few in number that their presence is of little or no practical significance for disease transmission. Houston (1908), for example, was able to isolate typhoid bacilli from water after nine weeks; but 99.9 per cent of the bacteria originally present had perished after one week. Nothing is of course more certain than the fact that transmission of disease germs oc- curs in the vast majority of instances only through the rather direct and immediate transfer of fresh body discharges. In the earlier days of bacteriology it was customary to refer to the few pathogenic bacteria which survive after long periods of time in water or earth as representing a "resistant minority." It is quite possible that in certain instances a selection of more resistant variants may in fact take place. Sedgwick and Winslow (1902) showed that individual strains of typhoid bacteria differ markedly in their ability to survive in ice; and Ayers and Johnson (191 5) found that various strains of colon bacilli show great differences in their ability to resist various pasteurization temperatures. There is, however, no direct evidence that the cells which die out toward the end of the cycle of bacterial population derived from a single strain are intrinsically more resistant than those which perish earlier; and it seems probable that the curve for such a cycle is mainly determined by a series of catenary reactions following the ordinary laws of more simple chemical processes. BIBLIOGRAPHY Allen, P. W.: J. Bad., 8, 555. 1923. Ayers, S. H., Cook, L. B., and Clemmer, P. W.: U.S. Bureau of Animal Industry, U.S. De- partment of Agriculture, Bull. 642. Ayers, S. H., and Johnson, W. T.: ibid., Bull. 126. Ayers, S. H., and Johnson, W. T.: /. Agric. Research, 3, 401. 1915. Barber, M. A.: /. Infect. Dis., 5, 379. 1908. Bigelow, W. D.: ibid., 29, 528. 1921. Brooks, S. C: J. General Physiol., i, 61. 1918. Buchanan, R. E.: J. Infect. Dis., 23, 109. 1918. Buchner, H., Langard, K., and Riedlin, G.: Centralbl.f. Bakteriol., 2, i. 1887. Buddin, W.: /. Agric. 5c., 6, 417. 1914. Burke, V., Sprague, A., and Barnes, L.: /. Infect. Dis., 36, 555. 1925. Chapin, C. V.: Sources and Modes of Infection. New York, 1912. Chesney, A. M.: /. Exper. Med., 24, 387. 1916. Chick, H.: /. Hyg., 8, 92. 1908. Chick, H.: ibid., 10, 237. 1910. Chick, H.: ibid., 12, 414. 191 2. Chick, H., and Martin, C. J.: ibid., 8, 654. 1908. Chick, H., and Martin, C. J.: /. Physiol., 40, 404. 1910. Clark, P. F., and Ruehl, W. H.: /. Bad., 4, 615. 1919. Cohen, B.: ibid., 7, 183. 1922. 82 THE RISE AND FALL OF BACTERIAL POPULATIONS Cohen, B., and Clark, W. M.: ibid., 4, 409. 1919. Cole, S. W., and Lloyd, D.: /. Path. Bad., 21, 267. 1917. Conn, H. J.: Tech. Bull. 64, N.Y. Agric. Exper. Sta. Geneva, N.Y., 1918. Coplans, M.: /. Path. Bad., 14, i. 1909. Cramer, W.: Die Wasserversorgung von Zurich und ihr Zusammenhang mil der Typhusepi- demie des Jahres 1884. Zurich, 1885. Davis, L., and Ferry, N. S.: /. Bad., 4, 217. 1919. Devereux, E. D., and Tanner, F. W.: ibid., 14, 317. 1927. Falk, I. S.: Abstr. Bad., 7, 33, 87, 133. 1923. Falk, I. S., and Winslow, C.-E. A.: J. Bad., 11, i. 1926. Ficker, M.: Ztschr. f. Hyg. u. Infektionskrankh., 29, i. 1898, Fokker, A. P.: ibid., 9, 41. 1890. Frankland, P.: Proc. Roy. Soc, London (1884-85,) 38, 379-93. 1885. Frankland, Mr. and Mrs. Percy: Micro-Organisms in Water. London, 1894. Fuller, G. W.: Ann. Rep., State Board of Health of Massachusetts for iSg4, 26, 461. 1895. Giltner, W., and Langworthy, H. V.: J. Agric. Research, 5, 927. 1916. Hadley, P. B.: /. Infect. Dis., 40, i. 1927. Harrison, F. C, and Vanderleck, J.: Rev. gen. du lait, 7, No. 15. 1909 Hehewerth, F. H.: Arch. f. Hyg., 39, 321. 1901. Heinemann, P. G.: /. Infect. Dis., 16, 479. 1915. Heinemann, P. G. : Milk. Philadelphia and London, 1919. Henrici, A. T.: Proc. Soc. Exper. Biol. &° Med., 19, 132. 1921. Henrici, A. T.: ibid., 20, 179. 1922. Henrici, A. T.: ibid., 21, 215. 1923. Henrici, A. T.: ibid., p. 343. 1924. Hotchkiss, M.: /. Bad., 8, 141. 1923. Houston, A. C: First Rept. on Research Work, Metropolitan Water Board. London, 1908. Houston, A. C: Seventh Rept. on Research Work, Metropolitan Water Board. London, 191 1. Hutchinson, H. B., and MacLennan, K.: J. Agric. Sc, 6, 302. 1914. Ikeda, K.: Ztschr. f. Hyg. u. Infektionskrankh., 25, 95. 1897. Jordan, E. O.: /. Infect. Dis., 38, 306. 1926. Jordan, E. O., Russell, H. L., and Zeit, F. R.: ibid., i, 641. 1904. Koch, R.: Mitt. a. d. Kaiserlich. Gesundheitsamte, i, i. 1881. Kohn, E.: Centralbl. f. Bakteriol., Abt. II, 15, 690. 1906. Konradi, D.: ibid., Abt. I, Orig., 36, 203. 1904. Koser, S. A., and Skinner, W. W.: /. Bad., 7, in. 1922. Kronig, B., and Paul, T.: Ztschr. f. Hyg. u. Infektionskrankh., 25, i. 1897. Lane-Claypon, J. E.: /. Hyg., 9, 239. 1909. Ledingham, J. C. G., and Penfold, W. J.: ibid., 14, 242. 1914. Leone, C: Arch.f. Hyg., 4, 168. 1886. Levine, M., Buchanan, J. H., and Lease, G.: Iowa State Coll. J. of Sci., 1, 379. 1927. Lloyd, D.: J. Path. Bad., 21, 113. 1916. Loeb, J., and Northrop, J. H.: J. Biol. Chem., 32, 103. 1917. Madsen, T., and Nyman, M.: Ztschr. f. Hyg. u. Infektionskrankh., 57, 38S. 1907. Massachusetts: Tivcnty-sixth Ann. Rept., State Board of Health of Massachusetts for 1S94, 1895. Miquel, P.: Rev. d'hyg., 9, 737. 1887. Miquel, P.: Manuel pratique d' analyse baderiologique des eaux. Paris, 1891. Muller, M.: Ztschr. f. Hyg. u. Infektionskrankh., 20, 245. 1895. C.-E. A. WINSLOW 83 Parkes, L. C: Practitioner, 71, 297. 1903. Parsons, L. B., and Sturges, W. S.: /. Bad., 14, 181, 193, 201. 1927. Paul, T.: Biochcni. Ztschr., 18, i. 1909. Paul, T., Birstein, G., and Reuss, A.: ibid., 25, 367. 1910. Paul, T., and Kronig, B.: Ztschr. f. phys. Cheniie, 21, 414. 1896. Penfold, W. J.: /. Hyg., 14, 215. 1914. Penfold, W. J., and Norris, D.: ihid., 12, 527. 1912. Peters, R. A.: J. Physiol., 54, 260. 1920. Phelps, E. B.: /. Infect. Dis., 8, 27. 1911. Prescott, S. C, and Baker, S. K.: ibid., i, 193. 1904. Prescott, S. C, and Winslow, C.-E. A.: Elements of Water Bacteriology (4th ed.). New York, 1924. Rahn, 0.: Centralbl. f. BakterioL, Abt. II, 16, 417. 1906. Rahn, O.: Tech. Bull. 5, Mich. Agric. Coll. Exper. Sta. 1910. Raju, V. G.: J. Hyg., 21, 130. 1922. Reed, H. S., and Reynolds, R. R.: Tech. Bull. 10, Va. Agric. Exper. Sta. 1916. Rettger, L. F.: /. Bad., 3, 103. 1918. Richet, C.: Compt. rend. Acad. d. sc, 114, 1494. Paris, 1892. Robertson, J.: Brit. M. J., i, 69. 1898. Russell, E. J., and Hutchinson, H. B.: J. Agric. Sc, 5, 152. 1913. Schaffer, J. M., and Tilley, F. W.: /. Bad., 14, 259. 1924. Sedgwick, W. T., and Winslow, C.-E. A.: Mem. Am. Acad. Arts &' Sc, 12, 471. 1902. Sherman, J. M., and Albus, W. R.: /. Bad., 8, 127. 1922. Sherman, J. M., and Albus, W. R.: ibid., 9, 303. 1924. Shrader, J. H.: Fifteenth Ann. Kept., Int. Assoc. Dairy b' Milk Inspectors, p. 208. 1926. Slator, A.: /. Hyg., 16, 100. 1917. Smith, J. H.: Ann. Appl. Biol., 8, 27. 1921. Snyder, C. D.: Am. J. Physiol., 22, 309. 1908. Snyder, C. D.: ibid., 28, 167. 191 1. Sturges, W. S.: /. Bad., 4, 157. 1919. Truifant, G., and Bezssanoff, N.: Science du sol, i, 3. 1922. Valley, G., and Rettger, L. F.: J. Bad., 14, loi. 1927. Ward, A. R.: Proc. Roy. Soc, 58, 265. 1895. Werkman, C. H.: /. Bad., 14, 335. 1927. Whipple, G. C: Tech. Quart., 14, 21. 1901. Wildiers, E.: La Cellule, 18, 313. 1901. Winslow, C.-E. A., and Abramson, F.: Proc. Soc Exper. Biol, b" Med., 9, 107. 1912. Winslow, C.-E. A., and Brooke, O. R.: J. Bad., 13, 235. 1927. Winslow, C.-E. A., and Cohen, B.: /. Infect. Dis., 23, 82. 1918. Winslow, C-E. A., and Falk, I. S.: /. Bad., 8, 215 and 237. 1923. Winslow, C.-E. A., and Kligler, I. J.: Am. J. Pub. Health, 2, 663. 191 2. Winslow, C.-E. A., and Lochridge, E. E.: J. Infect. Dis., 3, 547. 1906. Winslow, C.-E. A., and Sanjiyan, D. H.: /. Bad., 9, 559. 1924. Wolffhiigel, G., and Riedel, O.: Arb. a. d. Kaiserlich. Gesundheitsamte, i, 463. 1886. CHAPTER VII THE DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR PHILIP HADLEY University of Michigan INTRODUCTION Two of the most remarkable circumstances relating to the development of bac- teriology during the past half-century are: first, that bacteriologists have been so long content to conduct their experiments and to formulate their views in terms of the old monomorphic hypothesis regarding the nature of bacteria and of bacterial reproduc- tion; second, that they have been so active in evolving inadequate schemes for class- ification before they knew exactly what it was they had to classify. Looking back on the road over which we have traveled, it is impossible to estimate the loss sustained by bacteriology, especially in latter years, through the repressive and misguiding in- fluence of the strict monomorphic conceptions, or to appreciate the often serious bio- logical blunders that are to be laid at its door. In times of festival and jubilee we are accustomed to congratulate ourselves on the significant conquests of modern bacteri- ological science; and they have, it is true, been considerable. But they should have been greater; and they would have been far greater today if the science, a half- century ago, had not become impaled on a false biological conception which has never ceased to influence unfavorably both bacteriological thought and practice. And which, it may be added, even today represents a malicious dogma, accepted by tradi- tion, if not actually embraced by perhaps the majority of bacteriologists. The doctrine of monomorphism has descended to us from the early conceptions of the nature of bacteria maintained by Cohn, Koch, and others of the early school. Under its influence, in the earliest and most plastic days of the science, there were set up strict notions of "normal" bacterial cell types, "normal" colony forms, and "nor- mal" cultures. Whatever departed from the expected normality was at once relegated to the field of contaminations; or to the weird category of "involution forms," "de- generation forms," or pathological elements possessing neither viability, interest, nor significance. This monomorphic conception found its natural and fundamental sup- port in the assumed mode of reproduction characteristic of the fission-fungi. The dic- tum was then laid down that "the mode of reproduction of bacteria is by simple fission" — a view which has descended through two generations of bacteriologists and through numerous generations of textbooks, even to the year 1927. Although some early opposition to these views arose, it was probably unfortunate for the beginnings of the science that the first attempts toward modification were made by such extremists as Niigeli and his associates in the Munich group. The extreme plurimorphism which they so eagerly championed through years of bitter controversy was too radical to be accepted graciously as an antidote to strict monomorphism. Kor this reason Niigeli gained few permanent supporters; and, with the hnal collapse of his views, all views, even those 84 PHILIP HADLEY 85 espousing a more temperate plurimorphism suffered. The interpretations of the Berlin school triumphed — and to such an extent as to become later the dogma of "normal" colony and culture types that has endured, with hardly a respite, even to the present day. There did arise, however, at a later date some slight reaction to the monomorphic trend of the science. In the later eighties Gruber and his pupils were demonstrating examples of common and often curiously persistent variability in bacterial cultures. These observations were at variance to the demands of the Berlin group, although the instances concerned also fell far short of conforming to the extreme variability earlier pictured by Niigeli.' The same was true of the depictions of variability presented in the splendid series of contributions of Eisenberg.^ "Pathological variants," "involution forms," and "degeneration forms" were for a long time quite adequate to dispose of all such insignificant, though still somewhat bothersome, departures from the "normal" type. In 1906 and 1907, however, the variation- ists were fortunate enough to secure a more logical and trustworthy outlet for their explana- tions of "abnormal" forms of cells or cultures. This was found in the observations of Neisser' and of Massini-* on B. coli mutahile, whose pictures of variation they believed involved the phenomenon of mutation. In this way the De Vriesian term, together with many of its con- notations, was first introduced into bacteriology. This event was the starting-point for nu- merous observations and studies on mutating bacterial forms; and the discovery of bacterial "mutants" has continued without appreciable interruption up to the present day. Seldom, however, has the De Vriesian term been used advisedly. Its employment has merely provided a dignified and logical escape from the increasingly unacceptable "involution" hypothesis, as also from the necessity of offering any other more valid explanation of the phenomena concerned. The general result has been to bring into bacteriology many of the terms em- p'oyed in genetics — "plain variations," "impressed variations," "hereditary variations," ''clones," "biotypes," and "pure lines." But the conceptions apparently supported by the somewhat lavish use of these terms have usually lacked concreteness; and in few instances have the appellations been either appropriate or logical. For the most part, notions of bac- terial heredity among bacteriologists have been marked by extreme haziness and uncer- tainty. Beginning about 1907, however, there began to arise among the variationists two groups: first, the larger group of strict variationists who saw in their culture modifications merely a transient, but sometimes permanent (hereditary), departure from the otherwise monomor- phic type; second, the cyclical variationists, hardly numerous enough to term a group, and represented by such workers as Fiihrmann,' who believed that they could detect a certain order and direction in the culture modifications. Later supporters in this group have been rare — six only who, since the year 1907, have combatted to the best of their ability the false but always overwhelming views of bacterial type-stability. These investigators merit nam- ing at this point in our story; they are I'lihrmann, Hort, Almquist, Lohnis, Enderlein, and Mellon. To these workers particularly may be given the credit for directing the current of bacteriological thought into new channels. 'v. Nageli, C: Untcrsuchungcn iiber die nielere Filze iind ihren Beziehiing zu den Infektions- krankheiten itnd der Gcsiindhcitspjlcge. 1877. ^ See Bibliography in monograph on microbic dissociation by Hadlcy, Philip: J. Infect. Dis., 40, I. 1927. i Neisser, M.: Centralbl.f. BakterioL, Abt. I, Orig., 38, 98. 1906. '•Massini, R.: Arch.f. Ilyg., 61, 250. 1907. sFiihrmann, F.: Verli. d..ges. deiitsch. Natiirf. u. Arize, p. 278. 1906. 86 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR THE DISSOCIATIVE REACTION As I have pointed out in a previous publication/ extreme instability of bacterial types has become recognized in recent years as a commonly observed phenomenon. But its significance has been vastly underestimated and its cause or causes a matter of uncertainty. Although descriptions of bacterial "variants" and "mutants" have appeared with increasing frequency in the literature of the past thirty years, it is seldom that they have been regarded as possessing significance outside of that referable to the Darwinian or De Vriesian conceptions. It has, indeed, been only in quite recent times that the striking orderliness and persistence with which these variants are found to appear in difi'erent bacterial species and groups have enabled us to relate them to a definite law of variation, widely operative in the bacterial world. Indeed, these ob- servations have led us to the view that, within each bacterial species, there are con- stantly occurring certain transformations relating to cell morphology, colonial form, biochemical, serological, and immunological characteristics and to virulence; more- over, that these transformations are neither random variations from a "normal" type in the old Darwinian sense nor sudden saltations characteristic of mutations; but that they represent modifications which occur with a certain degree of precision in many different species when confronted with similar changes in environment. It is only within recent years that serious attempts have been made to correlate any of the different characters of the variants, such as virulence with colony form or serological reaction with cell type. Indeed, it has been the common view that such correlations were seldom possible; or, at least, not sufficiently constant to be of impor- tance. We know today, however, that such correlations are the rule rather than the exception, although they may be partly obscured at times, for a variety of reasons; moreover, that these correlations possess supreme importance for bacteriology, pathol- ogy, and medicine. The results of many observations have thus served to indicate that pure line cul- tures, of many bacterial species to say the least, are composed of cells all of which are by no means identical. From the same pure line strain may arise, depending on the manner of cultivation and on other environmental conditions, substrains possessing little resemblance either to each other or to the parent-strain. These diflferent culture types may be spoken of conveniently as "dissociated forms" or as "dissociants"; and the phenomenon involved in their production has been termed "microbic dissociation." The terms Umwandlung, Keimiimwandlimg, and Umformung (of bacterial species), employed by certain German and Swedish investigators, may be regarded as referring to the same phenomenon. Microbic dissociation thus becomes established as a new and highly significant field in the wide province of bacteriology. But it will be clear that the reactions characteristic of the dissociative phenomenon are, in a sense, superficial. Behind microbic dissociation there must exist a biological mechanism; and this mechanism, we shall come to see, concerns microbic heredity. Through this medium, therefore, dissociation is intimately related to important studies which have been conducted by a small group of investigators dealing with the so- called "life-cycles" of bacteria. ' Hadley, Philip: loc. cii. PHILIP HADLEY 87 One of the first to suggest that bacterial reproduction was characterized by phenomena more complex than those appertaining to simple fission, and something even more diversified than simple back-and-forth variation between two or three variants, was Fiihrmann' who in 1907, proposed his Eiitwicklungscydus. This conception of a cyclical development in bac- teria was further indicated in a work by Hort^ in England in 1916, by Lohnis and Smiths in the United States, and by Enderlein^ in Berlin, also in 1916. It was at this time that the latter worker introduced into bacteriological terminology the term "cyclogeny" {Cydogenie) , implying the cycle through which the microbe passes in leading up to the highest cytological state (Kulmi)ianle) and returning to its basal state (Mychil). Many of the papers by Alm- quists and Mellon^ on microbic heredity have dealt with the problem of life-cycles among bacteria; and in recent years this term, often used rather loosely to indicate certain obscure cellular transformations, has occurred in the literature with considerable frequency. While it seems probable that the time will eventually arrive when we can speak intelli- gently regarding definitely cyclical aspects of bacterial reproduction, and even though at the present moment we can often detect a certain direction in the serial transformations observed, it has seemed to me that our present knowledge of cyclical development in its details is still too slight to justify a common use of this term as describing the transformations thus far ob- served. Until more definite knowledge of the distinctly cyclical development of some one species is at hand, it is perhaps more appropriate to employ a term suggesting merely cultural transformations, often, it is true, apparently directive, but not overemphasizing the cyclical feature. For such a term, "microbic dissociation" will for the present suffice. The ultimate realities on which it depends are those involving the mechanics of microbic heredity. Micro- bic dissociation might therefore be defined as embracing those distinctly transformatory processes occurring in bacterial cultures, in vitro or in vivo, through which there arise one or more new culture forms which differ from the mother-type, and which (i) may persist for a variable time in an apparently stable state, or (2) may become transformed into still another culture type, or (3) may "revert" to the original form. The studies that bear on the problems of microbic dissociation may be grouped under two headings: (i) those random observations on culture variations which, even by the authors themselves, were not recognized at the time when the work was conducted as related to the fundamental problem of microbic heredity; (2) those later and more concise studies definitely directed upon the meaning of bacterial variation, its causes and effects. Regarding the first category, little need be said except that there exist in the bacteriological literature of the past thirty years or more numerous isolated citations which, by virtue of our present knowledge of the dissociative process, we are able to translate into the terms of microbic dis- sociation. Some of these I have brought together in a previous publication.' The second cat- egory of studies mentioned above includes those which consciously attack the problem of bacterial variation. These, in turn, are divisible into two groups, each differing from the other in its mode of approach to the fundamental problem. These involve (i) the C3^tologi- cal approach and (2) the cultural approach. In the latter group may also conveniently be included the biochemical and serological characteristics of the variants. ' Fiihrmann, F.: loc. cil. ' Hort, E. C: /. Roy. Micr. Soc, p. 11. 1926. 3 Lohnis, F. and Smith, E. R.: Jour. Agr. Res., 6, 675. 1916. See also Lohnis: ibid. 23, 401. 1923; also Men. Nal. Acad. Sc., 16, 252. 1921. ■•Enderlein, G.: Sitzungsb. ges. naturf. Frennde. Berlin, 1916. 5 Almquist, E.: Cenlralbl. f. BaklerioL, Abt. I, Orig., 60, 167. 191 1; Biologische Forschiing tiber die Bakterien. Stockholm, 1925. * See Hadley, Philip: loc. cit. ^ See ibid. 88 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR THE CYTOLOGIC AL APPROACH The cytological approach to the problem of microbic dissociation has centered mainly on the variable morphology of the bacterial cells and their nuclear appara- tus. It has followed (especially in the morphological aspects) the lines laid down in mycology at a much earlier date. Here the attempt has been made to discover, in the peculiar bacterial elements often observed in cultures, the groundwork for an inter- pretation of microbic heredity, involving modes of reproduction quite different from simple fission. Most of the earlier studies dealt with the microscopical cell changes, but without special reference to nuclear behavior. Later studies, and particularly those of Almquist, Enderlein, and Mellon, have concerned themselves as well with alterations in the nuclear apparatus. In view of the importance of the work dealing with the nuclear changes, a few words must be said regarding later conceptions of th; nuclear structure and the "chromatin" of the bacterial cell. The following represents some of the essential features of Enderlein's' view which I believe we may accept as reflecting the best knowledge now available regarding this important organelle. The nuclear unit (Mych) of the microbic cell possesses a spherical or oval form and, in the coccus, often attaches itself to the inner wall of the cell, against which it may sometimes be flattened. In cocci there is but one nuclear body while in all other forms of bacteria there are two or more. The diameter varies between o.i and 0.25 /x. It contains no chromatin and, with weak fuchsin, stains hardly any stronger than the cytoplasm of the cell. With methylene blue it may remain practically unstained. The nuclear body is observable only when the cell containing it holds but little food sub- stance in reserve. The latter commonly exists in the form of ultramicroscopic granules, the trophoconia, and this substance represents the actual "chromatic" material of the cell. It stains strongly because of its high content of nucleic acid and nucleo-proteins. The failure of a cell to stain well with methylene blue is due to the absence of the food-reserve substance. When the trophoconia bodies are abundant, their substance forms a dense aggregation about the nuclear body, and the element so formed stains readily; this is the trophosome. If only a light and thin layer of reserve substance clusters about the nucleus, this body becomes the trophosomelle, which is smaller and more delicate. Neither of these bodies (trophosome or trophosomelle) is the actual nucleus, however; they merely contain the nucleus as a central granule. Some of the granular bodies earlier described for bacteria, such as the Much gran- ules, the sporogenous granules of Ernst, and the metachromatic granules of Babes, are ac- tually trophosomes or trophosomelles. Other granules appear to be c|uite different struc- tures — sometimes the gonidia. They are all easily observable and often gram positive. If the trophosome is situated at the end of a rod form, it is a "telotrophosome"; if at other points in the cell, it is an "ascotrophosome." Moreover, to continue Enderlein's somewhat elabo- rate but necessary terminology, if a cell is free from reserve substance (trophoconia), it is an "atrophite." If it is merely poor in reserve substance, it is a "metatrophite"; if rich in reserve, it is a "pliotrophite." When the reserve substance in a cell has been used up, as in bacteria that have been starved (as in distilled water), the last remnant clings tenaciously about the nuclear body. Large amounts of food-reserve substance, which may conceal not only the nucleus but also the trophosomes, may be removed from the cell by alcohol. Under these conditions, when properly stained, it is observed that, in coccus forms, only a single point takes the stain. In rod forms, on the other hand, two or several such bodies take the stain. These are often at ' Endcdein, G.: Bak'ericn-Cydogcnie. Bcriin, 1925. PHILIP HADLEY 89 the poles of the cell and represent the true bacterial nucleus or nuclei. After cell division the heavily staining reserve substance soon appears in the daughter-cells. The first important departure from simple fission is gonidia formation. This form of reproduction was first recognized and named by Cohn' in 1872 in his study of Crenothrix, but the phenomenon was not carried over to the lower forms of bacteria. Gonidia were also recognized Ijy Lancaster in 1873, and they were noted the same year in B. lactis by Joseph Lister. They were also indicated in V . choJerae and in V . proteus [Vibrio Jiuklcr- prior) by Finkler and Prior in 1885. Out of the gonidial bodies, which were often regarded as spores, there were seen to arise extremely minute microspiral forms which, through further development, eventually attained normal size. Since these early days, gonidia have doubtless been seen many times without recognition. They have been definitely reported by Jones, ^ Lohnis,' Almcjuist," Mellon, s Enderlein,*" Tunnicliff,^ and others. Enderlein, who has been able to recognize them in many bac- terial species, regards them as the most common seed form (Fruchtform) of bacteria, being homologous with the spores of the fungi (conidia, ascospores). Morphologically and actually, according to Enderlein, they represent the true bacterial spore, which is not true of those elements usually termed "spores" by bacteriologists. Some of the so-called Much granules are regarded as gonidia, others as trophosomes. In 1870 Cohn differentiated gonidia into the large ("macrogonidia") and the small ("micro- gonidia"). In the tubercle bacillus the microgonidia are not acid fast but may be gram posi- tive. Enderlein mentions several sorts of gonidia, named according to their point of origin in the cell. If they arise at the end of a rod, they are termed "telogonidia"; if throughout the whole length of a rod, they are termed "ascogonidia." Apparently compared with such high- er forms as Crenothrix, bacteria produce only small numbers of gonidia. That the micro- gonidia may pass Chamberland filters seems to have been demonstrated by Lourens^ for the bacillus of swinepest in 1907, by Almquist' for B. typhosus in 1911, by Miehe^ for a number of bacterial species in 1923, and by Mellon^ for B. fusiformis in 1926. In other instances of the discovery of filtrable forms of bacteria there is slight basis for an opinion as to the exact nature of the filtrable unit; although, in the experiments of Fontes,^ who in 1910 was the first to demonstrate the filtrability of the tubercle bacillus, the description of his cultures suggests that the filtrable forms were microgonidia whose presence, among other small granular bodies, he was unable to recognize. The telogonidia have been recognized in spirochetes (Treponema and Leptospira) and may be concerned with the filtrability of organisms of this class, as was first demonstrated by Novy and Knapp^ for the relapsing-fever spirochete in 1906, although it also appears to be true that fine spiral forms also may pass the Berkefeld filter. According to Enderlein, the filtrable forms of bacteria comprise, not the gonidia alone, but also the gonites, next to be described. Our knowledge of the bacterial reproductive elements known as the "gonites" is limited to the results of Enderlein's'" studies, and particularly with reference to the cholera vibrio. His observations, which are highly suggestive, but which naturally de- mand extended confirmation, are presented forthwith. ' See Hadley, Philip: loc. cit. ''Enderlein, G.: loc. cil. ^ Jones, D. H.: /. Bad., 5, 325. 1920. '' Tunnicliff, R.: J . Infect. Dis., 36, 430. 1925. 3 Lohnis, F.: loc. cit. ^ See Hadley, Philip: loc. cit. '• Almquist, E.: loc. cit. ' Almquist, E.: loc. cit. 5 See Hadley, Philip : loc. cit. '» Enderlein, G.: Bakterien-Cyclogenie. Berlin, 1925. go DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR If the gonidia are maintained under conditions involving lack of nutriment, as, for in- stance, in aged cultures (thus preventing their entrance into a higher cyclostage), or if they are submitted to the influence of prolonged warming at 37° C, they become transformed into new and smaller elements, the gonites. The number of these forms increases with the gradual decrease of the gonidia. In cholera cultures left standing in the laboratory for a month or more, one finds that a great number of gonites have been produced. They are all extremely small, carry a much reduced amount of cytoplasm, and the smallest are known as the "microgonites." If the original culture is placed in sunlight, the same result occurs in a much shorter time — sometimes within a few days. The gonite is unable further to repro- duce itself as such. When cultures that have entered completely the gonite stage are trans- planted to agar, no growth occurs. Such cultures appear to be destitute of living cells. If, however, such a gonite culture is transplanted to broth, the gonites undergo further de- velopment, within a period of five to seven hours, into two new forms — namely, the spermite ($) and the oite (+). In a liquid medium copulation occurs, and the fertilized cell is capable of soon regenerating the original cell type. Here, then, we have a strict sexual form of re- production. Enderlein has followed the details especially in the cholera vibrio. With further reference to the foregoing considerations, it may be said that the re- productive significance of the gonidia in bacterial reproduction is now beyond a matter of doubt. These bodies have been observed repeatedly, and their subsequent develop- ment into the original cell type followed by several competent investigators. As for their further transformation into the gonites, and the subsequent transformation of these into the sex cells — although this phenomenon eventually may be found to occur, and to underlie a true sexual form of reproduction in the bacteria — in so important a matter one is justified in postponing a conclusion until the striking observations of Enderlein can be confirmed in the cholera vibrio and extended to other species. It may be remarked here, however, that many of Enderlein's cytological observations have al- ready been confirmed by Schumacher/ And in this case the confirmation is the more valuable since Schumacher was not acquainted with the work of Enderlein at the time of his own studies. The cytological aspects of microbic heredity have also been furthered in recent years by the valuable researches of Mellon in a series of contributions extending over many years. Among other matters of importance, Mellon's investigations have dealt especially with a mode of reproduction involving conjugation and zygospore formation, following many of the details of isogamic conjugation in higher forms. These zygospore-like bodies are probably identical with similar forms pictured less clearly by many earlier workers; perhaps with the Pettenkofer bodies described more recently by Kuhn.^ Mellon conceives that the origin of the zygospore is through the fusion of adjacent cells of a filament, sometimes indirectly by means of a peduncle. The bodies seem to be formed equally among the small and the large rodlike elements in the culture. They are often small, but in certain diphtheroids, as ob- served by Massini and by Mellon,^ and in B. diphthcriae (Park No. 8), as observed by my- self,-* they may attain a diameter of 6-7 /i. By favorable staining they usually reveal a ' Schumacher, Josef.: Centralbl.f. Bakleriol., Abt. I, Orig., 97, 81. 1926. ^Kuhn, Philaethes: Centralbl.f. Bakleriol., Abt. I, Orig., 93, 280.* 1924. See also: Arch. f. Schijfs- u. Tropen Hyg., 30, 133. 1926. 3 Massini, R.: Arch.f. Hyg., 61, 250. 1907; Mellon, R. R.: /. Bad., 2, 81. 1917. 4 Hadley, Philip: loc. cit. PHILIP HADLEY 91 cluster of nucleus-like granules. Mellon has also recorded the liberation of large numbers of minute and apparently motile granules from the "giant coccus" forms. This has also been reported by Kuhn/ who has presented beautiful micro-photographs of these cells (Petten- kofer bodies) both before and after the liberation of the minute granular bodies. From his work it appears that these small forms often fail to stain by the ordinary methods, but that the Giemsa stain is especially favorable. It may be added at this point that Kuhn sees a relation between the presence of these bodies and the ability of the culture containing them to generate the bacteriophage; and this is quite in harmony with the theory of transmissible autolysis which I have proposed in an earlier paper. Regarding the development of the zygospores, Mellon has observed that in some cases they appear to undergo a double segmentation and yield a large coccus form like that often encountered in dissociating cu tures of B. diphthcriae. In such instances Mellon regards the zygospores as transition Anlagen for the development of a new form of culture, the exact nature of which will be determined by the environment surrounding the germinating zygo- spores. Finally, with reference to the hereditary mechanism of bacteria, there should be mentioned the formation of symplastic structures such as those first described by Jones^ in 1913 and 1920 for Azotobacter, and confirmed by both Lohnis-* and Ender- lein.'' Briefly, the reaction involves the fusion of a mass of bacteria into a single group in which the cell boundaries are lost and a union of nuclear elements occurs. From such symplastic structures arise new individual cells. THE CULTURAL APPROACH As early as 1888 observations dealing with several different and more or less per- manent culture types arising from pure cultures had been described by Firtsch^ for V. proteus, and in 1895 Dyar^ gave a report on changes of a somewhat similar nature for B. lactis erythrogenes. In later years other investigators presented other instances in which striking departures arose from the long-assumed "normal" and constant type. Many of these instances we owe to the splendid researches of Eisenberg.'^ It re- mained for Baerthlein,* however, in 1918 to point out the frequent occurence of such variations, their cultural, biochemical, and — to a limited extent — serological reactions. The primary basis for Baerthlein's important study was colony variation, the signifi- cance of which had been clearly seen by Firtsch in the case of a single species, Baerth- lein showed that plating from old laboratory cultures commonly resulted in the ap- pearance of colony forms quite unlike that of the original culture. Sometimes only one or two colony variants were encountered; at other times, four or five of them. But the most important and significant feature of Baerthlein's study was his demonstra- tion that, commonly associated with colony variation, were variations in other char- acteristics — morphological, biochemical, and serological. ' Kuhn, Philaethes: loc. cit. ^ Jones, D. H.: loc. cit. 3 Lohnis, F. : loc. cit. '' Enderlein, G.: Bakterien-Cyclogenie. Berlin, 1925. 5 Firtsch, G.: Arch.f.Hyg.,8,s(>g. 1888. ^Dyar, H. G.: Ann. N. Y. Acad. Med., 8, 322. 1895. ' See Hadley, Philip: loc. cit. ^ Baerthlein, K.: ibid., 81, 369. 1918. 92 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR It is doubtful if Baerthlein appreciated the full significance of his observations. For some time at least he believed that his various culture types represented merely "mutations," such as had been described earlier (especially in secondary colony forma- tion) by Neisser,' Massini,^ Miiller,^ Tenfold,^ Thaysen/ Burri/ Eisenberg/ Leding- ham," and many others. It thus remained for Arkwright/ in 1921, to grasp more fully the significance of Baerthlein's work. Among members of the colon-typhoid-dysentery group Arkwright noted particularly two colony forms which occurred in each species with marked persistency. One was round, regular, opacjue, and characterized by a smooth, glistening surface; the other was flat, irregular, translucent, and showed a rough or sandpaper-like surface. The former was termed the "S" type (smooth), the latter the "R" type (rough). The S type culture, on aging, transformed readily into the R. The latter, however, held to its new characteristics with considerable tenacity. While the S type culture grew in broth with a homogeneous clouding, the R type gave an agglutinative or sedimentary form of growth. These two culture forms were ob- served by Arkwright in B. coli, B. typhosus, and B. dysenteriae. His splendid and far- seeing work marks the beginning of a new epoch in the study of bacterial variation. In 1921 De Kruifs also made, independently, a contribution of fundamental im- portance dealing with dissociation in the rabbit Pasteurella type. Bad. lepisepticum. In this species he observed two forms of culture, differing from each other in colony form, manner of growth in broth, serological and immunological reactions, and par- ticularly in virulence. De Kruif designated these two types "D" and "G," respectively. As we can now see, his D form was analogous to Arkwright's S, while his G form was analogous to Arkwright's R. In De Kruif's experience, while the D type culture was highly virulent for rabbits, the G form possessed little, if any, virulence. Moreover, while the D form in the killed state was of little value as an immunizing agent, the G form, living, when injected into rabbits even in small doses, produced immunity to large amounts of virulent culture. Even one dose secured these results. Since the important works of Arkwright and De Kruif in 1921, the same line of study has been carried into many other fields: to the streptococci by Cowan*; to the pneumococcus by Griffith^, also later by Reimann^ and by Amoss^; to B. typhosus and B. enteritidis b\' Ark- wright and Goyle,9 and by Goyle'" alone; to the Salmonella forms by White," and by Topley and Ayrton"; to B. cholerae suis by Orcutt"; to the cholera vibrio by Balteanu'^; to FriedLind- er's bacillus by Julianelle'^ ; and to B. coli quite recently by Dulaney.'^ De Kruif's work has ' Neisser, M.: loc. cit. ^ Massini, R.: loc. cit. ^ Hadley, Philip: loc. cit. '> Arkwright, J. A.: /. Path. &° Bad., 24, 36. 1921. sde Kruif, P.: J. E.xper. Med., 33, 773. 1921; also 35, 631. 1922. ' Cowan, Mary: Brit. J . Exper. Path., 3, 187. 1922. ^ Griffith, F.: Public Health and Medical Subjects, Ministry of Health, Rep. 18. London, 1923. * See Hadley, Philip: loc. cit. ' Arkwright, J. A., and Goyle, A. N.: Brit. J. Exper. Path., 5, 104. 1924. '0 Goyle, A. N.: /. Path, c^ Bact., 29, 149. 1926. " White, P. B.: Special Rep., Med. Research Council, No. 91. London, 1925. " Balteanu, I.: /. Path, b' Bact., 29, 251. 1926. ■3 Julianelle, L. A.: /. Exper. Med., 44, 683. 1926; also p. 735. '■* Personal communication. PHILIP HADLEY 93 been followed by Webster into lines of considerable interest. The study of Theobald Smith and Gladys Bryant' on a "mutating" form of B. coli may also be mentioned as an instance dealing with the dissociative reaction, although the authors did not emphasize the relation. Gratia at an earlier date had made somewhat similar, though less detailed, observations on the same species. The chief results of all of these studies have been to demonstrate with ever increasing clearness the new characteristics possessed by the recognized variants; also to validate many earlier observations of a similar nature, but manifestly concerned with the same phenomenon. In addition, the more recent studies, besides depicting the S and R forms, have presented evidence for the existence of the third significant culture type, the O form (intermetliate), also recognized by earlier workers, lying as a transitional form between S andR. COLONIAL, CULTURAL, AND MORPHOLOGICAL ASPECTS OF MICROBIC DISSOCIATION As has already been pointed out, microbic dissociation manifests itself in varia- tions in colony form, in cultural growth, in comparative cytology, in cell morphology, in biochemical reactions, in immunological reactions, and in virulence. In the present section we shall consider some of the details relating to the first three of these points, with the attempt to indicate that the variations and correlated characters observed are not the result of chance, but depend on certain laws governing the transformations in widely separated bacterial species.^ 1 COLONIAL ASPECTS As Firtsch^ clearly suggested by his remarkable study of variation in V. protcus in 1888, colonial variation is the most fundamental, consistent, and clearly observed phenomenon in dissociative variation. Each bacterial species possesses, not one "nor- mal" colony form, but a variety, each of which one must be able to recognize before he can affirm that he knows the "species." Each of these forms is determined by the stage of cyclogeny attained by the individual cells that comprise the colony structure. The time is now past when similarity in colony form must be taken as evidence of the close relationship of the organisms contained; or when dissimilar colony types must be regarded as indicating unrelated species. The fact of the matter is, that the de- gree of variation in colony form in one and the same species may be, and usually is, greater than the degree of variation in colony form of equivalent cyclostages of clearly distinct species. The diverse colony forms observed within pure lines of B. subtilis (Soule),4 of B. anthracis (Preisz,^ Wagner, Nungester*), of hemolytic streptococci (Cowan),'? of S.fecalis (Faith Hadley),^ of the meningococcus and gonococcus (Atkin),^ ' Smith, T., and Bryant, Gladys: /. Exper. Med., 46, 133. 1927. ^ It is impossible to consider the biochemical, serological and immunological aspects within the limits of this chapter. 3 Firtsch, G.: loc. cit. '•Soule, M. S.: Jour. Inject. Dis. 1928, No. 2. 5 Preisz, H.: Centralbl.f. Bakteriol., Abt. I, Grig., 35, 280. 1904; also 53, 510. ign. 'Nungester, W.: Proc. Soc. Exper. Biol, b' Med., 24, 959. 1927. 'Cowan, Mary: loc. cit. * Personal communication. ' Atkin, E. E.: loc. cit. 94 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR of the pneumococcus (Griffith,' Reimann,^ Amoss^), of B. diphtheriae (Corbett and Phillips),^ of Sp. finkler-prior (Firtsch)," and of V. cholerae (Eisenberg,^ BalteanuO differ from one another to such a degree that, on morphological grounds, all of the variants would be regarded as contaminations — indeed, often have been so regarded and treated accordingly. As one illustration of this, and as Soule^has already pointed out, it may be noted that the R type colony of B. subtilis on plates is almost indistin- guishable from the R type colony (Medusa-head type) of B. anthracis. On the other hand, one may conclude from the studies of Nungester^ that the S type anthrax colony is one that few investigators have ever seen, or at least recognized; and one which would commonly be taken as a contamination, so different is it from the common Medusa-head type. It is thus a rather curious fact that, while it is the S colony form of B. subtilis that has come to be regarded by bacteriologists as the "normal," it is the R type colony of B. anthracis which, during the fifty years of study that this species has received, has become established as the "normal" form of culture. In most bacterial species for which knowledge is available, the colony of the S type is smaller and more delicate than the other forms. It is round, even, usually opaque (even when young), and, in certain species such as those of the intestinal group, the streptococcus, the pneumococcus, the proteus, the pneumobacillus, and others, presents a glistening luster or "moist" appearance. In addition, a distinct fluorescent effect by transmitted light is usually seen (intestinal group, pneumobacillus, proteus, and others). The co'ony consistency is commonly soft or butyrous. Such colonies, after four or five days' growth on rich and some- what alkaline agar, often show pale or translucent, wedge-shaped invaginations where disso- ciation into the O or R types is under way. Culturing from these "blue" areas will give cul- tures of a quite different type from the original, but usually not well stabilized at this stage. Repeated culturing, accompanied by colony selection, will increase the stability of the (com- monly obtained) R form. The R type colony presents, in most species, a quite different appearance from the S form. It is usually larger, irregular in shape, uneven; when young it is thin and translucent, and reveals a distinctly rough surface, or sometimes merely a dull luster as in B. subtilis (Soule). The fluorescent effect is invariably lacking. Old colonies, however, may become as opaque as those of the S form. The consistency of the R colonies is sometimes similar to that of the S form, but often, and when they are well stabilized (as in the "extreme" R), they may be hard or even brittle, so that they may be pushed about over the agar surface. These have been observed in the pneumococcus (Griffith) and in the streptococcus (Cowan). One curious feature of the R type colonies of several species (B. diphtheriae, B. mallei, B. proteus, V. cholerae, V. proteus, B. mesentericus, meningococcus, 5. fecalis, and probably other species) is that they may take on a yellow or brown chromogenesis. Apparently a similar phenom- enon occurs in the fungus of blastomycosis (Mellon).* I have also observed brownish colo- nies arising in the dissociation of M. citrcus. Regarding the colonial features of the type O cultures, first clearly pictured by Firtsch in 1888 for V. proteus, we have less knowledge. In general, the O colonies are larger than the S, round, even, smooth, glistening, but more fleshy and convex, simulating the colonies of ' Griffith, F.: loc. cit. 2 See Hadley, Philip: loc. cit. «> Soule, M. S.: loc. cit. 3 Corbett, L., and Phillips, G.: /. Path. &' Bad., 4, 193. 1897. -• Firtsch, G.: loc. cit. ' Nungester, W.: loc. cit. 5 Balteanu, I.: loc. cit. * See Hadley, Philip: loc. cit. PHILIP HADLEY 95 B. aerogenes. They often manifest a mucoid consistency. Cultures of this sort commonly ap- pear in members of the colon-typhoid-dysentery group; also in B. anthracis, S.fecalis, and probably other species. In B. anthracis some of these colonies are slimy and readily coalesce on the agar plate. Similar colonies have been reported for various species among cultures resistant to the bacteriophage. In B. proteus the equivalent oMhe S form yields a spreading growth while the growth of the O type is restricted. In several species, such as pneumococ- cus, streptococcus, meningococcus, and gonococcus, the O type has not been described clearly. In general, it is highly unstable and may sometimes transform with great rapidity into the R. Such cultures have been termed "suicide cultures." They are very difficult to maintain on agar, and it is sometimes impossible to cultivate them in broth. It is reason- able to believe that the O colony form is at some time present in cultures of all bacterial species, but may pass unobserved. Certain members of the intermediates, such as those representing the Pettenkofer bodies of Kuhn, may play an important role in the phenom- enon of the bacteriophage, as I have pointed out elsewhere.' Although it often appears that the three chief colony types (S, O, and R), but particu- larly S and R, are clear cut in their essential features, and usually quite stable on appropriate medium, in other cases there may be observed distinct intergradations; and these may take on various aspects. For example, the S type colony may show, about its edge, either at a single point or about its entire circumference, an outcropping of the R type culture. Inter- esting examples of this have been described by Soule for B. subtilis, and observed by Nun- gester for B. anthracis; also by Faith Hadley for S. Jecalis.^ The fairly smooth and circum- scribed S colony, possessing (subtilis) the shallow fringe of filaments extending outward from the border ("bayonet-front" effect), sends out longer outgrowths which soon begin to curl under and to give a marginal appearance which simulates that of the R type anthrax colony (Soule). This outgrowth is bluish and translucent by transmitted light and rough by re- flected light. The result of this reaction is to yield an S type colony imprisoned within a ring of R type culture, the breadth of which varies with conditions which cannot be considered here. The type S anthrax colony may undergo transformations similar to those mentioned above (Nungester). Such outgrowths have been termed the "halo" or "regeneration fringe." In some cases, as in B. proteus for example, the halo may be made up of O type rather than S type culture. Indeed, there may be a series of alternate dissociations and recoveries, fol- lowing each other at intervals of a few hours, in the growth of the colony, the final effect being the production of the typical "ring growth" characteristic of B. proteus. But the O type colony also may send out such regeneration fringes; and in this case the nature of the fringe growth seems to depend on the degree of stability of the O colony. If it is in the early intermediate state, it usually forms a halo of normal S culture. If it is in the late intermediate state, it is more likely to send out a fringe of R type culture. Under these conditions, culturing from the center or from the edge of the colony will yield cultures of two different forms. The natural destiny of the type O culture is apparently to attain the R; and it usually accomplishes this in the course of time. It is often difficult to maintain the inter- mediate form in this culture state. The O form of B. proteus seems to be unusually stable, but here the R form has not been recognized with certainty. It may be added here that it has not been observed clearly that the type R culture gives regeneration fringes. It may, how- ever, as we shall see later, produce clusters of S type secondary colonies which usually appear on the free edges of growth; and these are analogous to the fringe, since it can be observed that it is by the coalescence of numerous colonies appearing at the margin of growth that the distinct fringes are produced. ' See Hadley, Philip: loc. cit. Also Arch, of Path, and Lab. Med., 1928. In press. ^ Personal communication. 96 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR Another manner in which dissociative reactions may reveal themselves in cultures is, as just intimated, the generation of secondary or "daughter-colonies" occurring in a background of primary culture. Instances of this phenomenon were given added significance in 1906 and 1907 through the observations of Neisser and Massini on B. coll mutabile, and similar observations were soon made on many forms. All these cases, manifesting the spontaneous origin of new culture types within the old culture mass, were quickly seized upon as indications of true mutations among the bacteria; and this false conception persists in the minds of many bacteriologists, even at the present day. Secondary colonies may be few or numerous ; sometimes one only, or again there may be several hundred discrete bodies. As the number increases, howexer, they blend more and more into the mass of mother culture and thus lose their colonial iden- tity — at least macroscopically. Microscopically they may still be traced as "granula- tions" of varying size, until the culture mass becomes a fine mosaic of the two or more culture elements. Sometimes the secondary colonies arise near the surface and form the well-known "papillae." Again they lie deeply imbedded in the culture, or even in the medium itself, as in the case of S.fecalis, and do not register on the contour of the primary colony. In some species (S.fecalis — Faith Hadley)' both forms of second- ary colony may be observed, and in this case they appear as entirely different culture types. The most common forms of secondary colony relate to centers of O or R type culture arising in a background of S culture. On the other hand, it appears from the older work of Preisz- and Pesch-' on B. anthracis, and from the more recent studies of Anna Dulaney on B. coli, that secondary, S type colonies may arise in R type cul- tures. In Dulaney's case they were generated particularly at the free margins of the type R colonies after a prolonged growth. Tertiary colonies arising within the second- ary have also been occasionally observed, particularly by Preisz^ for B. anthracis. The whole subject of secondary and tertiary colony formation may possess additional in- terest in its bearing upon the nature of the bacteriophage reaction, and with special reference to the homogamic theory which I have briefly outlined in a previous publi- cation.'' Here it was suggested that the resistant colonies which d'Herelle, Bordet, and others have termed "secondaries" (arising in the lytic sites on agar, or in culture fil- trates) may, in reality, be tertiary colonies arising after the disappearance of the sec- ondaries. In this case the characteristic lytic plaques would be regarded as the sites of disappearance of colonies of the secondary type, which itself might represent one of the intermediate forms of culture. For the present we may leave the subject of the secondary colonies with the con- clusion that these formations are of considerable significance as indicating that, hidden in the mass of mother culture, regardless of the type concerned, there may exist cer- tain centers where small or large groups of organisms, quite different from the mother culture in form and physiology, have arisen and where they are carrying on their inde- ' Personal communication. 'Preisz, H.: Cenlralbl. f. Bak'.eriol, Abt. I, Orig., 35, 280. 1904; also 53, 510. 1911. 3 See Hadley, Philip: loc. cil. ■* Personal communication. s Preisz, H.: loc. cit. ^ See Hadle}', Philip: loc. cil. See also: Arch, oj Path, and Lab. Med. 1928. In press. PHILIP HADLEY 97 pendent activities. When removed and purified by plating methods, they afford new forms of culture which sometimes manifest considerable permanence in their newly acquired characters. It is these forms that have erroneously been regarded as mu- tants. CHANGES IN CELL MORPHOLOGY Closely associated with the specific colony type, and in all probability determin- ing, within limits, its characteristics, are to be observed some fairly distinct morpho- logical characteristics of the cells and their organelles. Microscopic study of the type S culture, which usually passes under the appellation of "normal culture," although ex- ceptions have been noted, usually reveals a preponderance of those cell forms which are regarded as characteristic of the "species" in question. The type R culture, on the other hand, is likely to present a different picture; but this, in turn, varies with the species. In the members of the intestinal group the well-stabilized R type cell is most often coccoid, and a similar shortening is characteristic of the R forms of the diphtheria bacillus, the plague bacillus, probably of the tubercle bacillus and of several other species. In other cases, as in B. subtilis, B. anthracis, and perhaps in all the spore- formers (none of which, with the exceptions noted, has been carefully studied), the R type are much more elongated than the S type cells and are sometimes distinctly filamentous. The nature of the cell population of the O t^-pe cultures is not so clearly recog- nized. But it may be affirmed that, in comparison with the S and R, it is highly di- verse. It is particularly in cultures of the O type that one finds amassed those peculiar cell bodies which, for many years, have been termed "involution forms." They con- tain long, swollen rods, filaments (often fungoid in nature), giant coccoid bodies, apparently identical with Kuhn's Pettenkofer bodies, zygospore-like bodies, and often numerous minute granules, the exact nature of which is perhaps still in doubt, although it seems probable that some of them arise from the zygospores. The whole picture is extremely bizarre but is fairly constant for the intermediate type cultures of many bacterial species. Such forms have been produced by Kuhn and others by growth on media containing traces of lithium chloride. But it is sufficient to convince us that, under the cover of these bacterial monstrosities, are proceeding reproductive events of which we have, as yet, slight cognizance. Of one point, however, we may be assured. These peculiar forms are not "pathological" nor evidences of degeneration. They have been termed "involution forms"; but, as Mellon has suggested, "evolution forms" would be more appropriate. The details of their production and reproductive be- havior constitute at present one of the most important problems in microbic dissocia- tion. It seems possible that in their action is hidden the problem of the filtrable forms of bacteria, if not of the filtrable viruses; also perhaps the mystery of the bacterio- phage. Just as the morphology of the bacterial cell is correlated with the type of culture, so also are correlated motility, capsule formation, and perhaps spore formation. The time is past when we can state with discretion that such and such a bacterial "species" is motile, for both motility and flagellar equipment depend on the cyclostage. Up to the present time, observations seem to indicate that, if an organism shows motility, it belongs to the S type as opposed to the O or R, which are commonly non-motile. Ark- 98 DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR wright,' however, has presented certain exceptions. Of this fact we now have evidence in members of the intestinal group, in B. siihtilis, B. proteus, and several other species. Motility of a culture therefore loses all significance for species differentiation in sys- tematic bacteriology, unless we can succeed in recoghizing the cyclostage with which we are dealing. With reference to bacterial capsules, the same situation exists as for motility. So far as we know at present, the capsule is the property of the organism of the S type, the R form being destitute. With reference to the anthrax bacillus, which presents certain other anomalies, the situation is not yet clear. The correlation is, however, now recognized for B. coli, the pneumococcus, Friedlander's bacillus, M. tetragenus and some Pasteurella forms. In none of these species does the presence or absenc.' of capsules possess significance for systematic bacteriology unless we can recogniz.^ the type of culture under examination. In addition to the foregoing, microbic dissociation manifests itself in important biochemical and serological differences in the dissociated cultures; also, in a striking manner with certain problems relating to virulence and immunity. These interesting aspects of the subject cannot be discussed within the limits of the present con- tribution.^ THE INCITANTS TO MICROBIC DISSOCIATION AND THE PROBLEM OF REVERSION Although dissociative reactions must be regarded as occurring to some degree in all bacterial cultures, probably beginning in the earliest hours of colony life, and al- though in some cases they attain spontaneously such a magnitude that they attract the notice of the alert investigator, fortunately, in the study of this phenomenon, we are not dependent upon cultural material showing such spontaneous transformations. The reaction may easily be "forced" as a result of bringing to bear on the young, grow- ing culture certain extraneous influences; and the nature of these influences may be diverse. Probably the first influence to attract attention was aging, as first pointed out by Firtsch for the spirillum of Finkler-Prior in 1888. Most of Baerthlein's colony variations, as also those of Eisenberg, were consequent to aging in broth or on agar, and to the use of alkaline media. Under these conditions, one may often discover in the transition from the S to the R forms the presence of the intermediate or transitional O, as clearly depicted by Firtsch, Eisenberg, and many others. Other conditions of cultural growth favoring the reaction, with the subsequent generation of the O or R forms of culture, include the following: changes in temperature, various food substances, starvation, the physical state of the medium (solid or liquid), the presence or absence of oxygen, the presence of antiseptic substances or dyes, the reaction of the medium, the volume of the medium, microbic associations, passage through animals, the influence of various kinds of normal animal blood or tissues, normal sera or ascitic fluid, specific immune blood or sera, body excretions or secretions, and finall\' the metabolic growth products of the same or other bacterial species. To these may be added the influence of the bacteriophage which, in last analysis, is the reagent par excellence for enforcing dissociation upon the sensitive, or even on the partially resistant, culture. Indeed, only one other influence approaches it in degree or speed of action — and that is homologous immune serum, as amply demonstrated by many experiments. ' See Hadley, Philip: Joe. cit. ^ See chap, xlii PHILIP HADLEY 99 Of the influence of these various incitants, only a few brief statements may here be made. First, dissociation cannot occur unless growth occurs; cultures in a state of suspended growth, while still alive, remain fixed in the type in which growth last oc- curred. This is not necessarily true of old cultures in which growth still takes place slowly. This circumstance reminds us of the conditions limiting the action and re- generation of the bacteriophage. Dissociation, moreover, occurs most freely in liquid media, and most actively at a reaction point of pH 7,8-8,0. This, it may be noted, is also the optimum reaction for bacterial autolysis in most species. In cultures frequently transferred on favorable solid media, dissociation is more restricted and may even ap- pear to be absent. When dissociation has occurred, and the R type has once been pro- duced, this type of culture is more stable than the S, and much more stable than the 0, both on solid and in liquid media. The presence of phenol, pancreatin or lithium chloride, and other chemical substances, favors the transformation of the S type to the O type culture in some species. The influence of blood serum is of special interest. Sometimes a normal serum of cer- tain species will force the reaction. This is likely to concern the serum of animals which are not susceptible to the organism, or sera supposed to be germicidal. As mentioned above, however, the strongest dissociation-furthering power is possessed by homologous immune serum. This can be demonstrated by growing the S type culture for a few generations in broth containing about 10 per cent of the immune serum, the result being the formation of the R type culture. This has been demonstrated for the pneumococ- cus, the streptococcus, B. subtilis, Friedlander's bacillus, B. typhosus, B. paratyphosus B (SouleO, B, coli (Dulaney'), and some other forms. There is some evidence that the same reaction occurs in vivo as well as in vitro; and this suggests the possibility that the chief mechanism of protection of the bacteriotropic antibodies may repre- sent merely the enforcement in the body of a dissociation of the invading organisms, comparable to that observed in the culture tube. This possibility, which was first approached by the researches of Griffith on the dissociation of the pneumococcus in 1923, 1 have considered at greater length in an earlier publication. With reference to the effects of immune sera on the R and S culture types, it should be added here that it has been shown for B. subtilis by Soule,^ for B. coli by Dulaney, for the pneumococ- cus more recently by Avery, and for B. paratyphosus B by Soule, that, when the R type culture is grown in serum immune to this culture form, a retransformation is enforced to the original S, When one observes that the dissociative reaction can be precipitated by such di- verse substances or conditions of growth as those mentioned above, the question of the actual cause of dissociation seems to be as far removed as at the beginning. But the situation may be simplified if we can discover a common factor among all of these in- fluences. It seems safe to say that there exists such a common factor, and that it may be defined as any condition or substance that is antagonistic to the continued growth of the so-called "normal" culture type. That this factor may not be the same for all bacterial species is a view that we may well accept, but that the same influence will operate in the same manner on different members of the same group of bacteria seems well demonstrated by numerous observations. ' Personal communication, * Soule, M. S,: loc. cil. loo DISSOCIATIVE ASPECTS OF BACTERIAL BEHAVIOR Regarding the permanence of the dissociates, with their newly acquired char- acters — it has of ten been stated that they are irreversible; and, for this reason, they have often been described in the literature as mutations. This interpretation has con- cerned the O forms much less often than the R, for the former are notably unstable. Even the R types represent a series in which the degree of stability is very variable. It seems to depend, in part, upon the length of time that the R culture has been prop- agated continuously under the conditions that gave rise to it. The longer the stimulus is applied, the more stable the type seems to become. There are, however, some ex- ceptions to this. Some R type pneumococcus cultures are apparently irreversible; and the same is true of some R type Friedlander strains, according to Julianelle.' The R form of B. paratyphosus, B. typhosus, B. sicipestifer, B. dysenteriae, Bad. lepisepticum, B. diphtheriae, B. subtilis, and B. anthracis have been found highly stable, and have sometimes been reported as permanent variations. But Jordan,^ by a special method of cultivation, succeeded in causing the reversion of his R type of B. paratyphosus B; end Soule, by growth of his R type of B. subtilis in an R immune serum, was finally able to cause a return to the S type of culture. Later he was able to cause the reversion of an R type of B. paratyphosus B by similar measures. Mellon^ has emphasized the stabihty of variants of his diphtheroid forms, and I have been unable to effect the retransformation of an R type of B. pyocyaneus even after four years, although I have not tried the action of R immune serum. Apparently, the R forms of culture may remain stable for many years, but I believe there is no justification for the conclusion that they represent hereditary variations or mutations in the strict meaning of the term. It is more likely to mean that we have not yet been able to discover the adequate means for causing their return to the original form of culture. BIOLOGICAL SIGNIFICANCE OF DISSOCIATION In concluding this chapter the question arises. What is the deeper meaning of these phenomena concerned with the separation of bacterial cultures into distinct compo- nents whose nature and behavior we have now briefly reviewed? It can mean only one thing: that those living cells that we have commonly regarded in past years as among the simplest of plant forms, and characterized by a correspondingly simple re- productive apparatus, possess in reality a highly complex genetic mechanism, which enables them to reveal, in cultures, pictures of morphological and physiological di- versity with which our old and limited notions of "reproduction by simple fission" are utterly unable to deal. Although we may not yet be justified in accepting Enderlein's view of actual sexual reproduction among the bacteria, we must accept the fact that the nuclear equipment and reproductive behavior of bacteria are highly complicated matters. We can no longer doubt that the hereditary mechanism in bacterial cells makes provision for amphimixis, so long denied to these forms; nor can we hesitate in accepting phenomena of gonidia formation, zygospore formation, and perhaps a kind of budding, as common methods of bacterial reproduction. In all of these matters bacteriologists as a class have combined in denying the existence of things that they have not been willing to take the trouble to search for. ' Julianclle, L. A. : loc. cit. ^ Sec Hadley, Philip: loc. cit. ^ Mellon, R. R.: /. Med. Rcscirch, 42, 6i. 1920. PHILIP HADLEY loi The acknowledgment of the existence of definite cyclostages in the development of the bacterial culture naturally concerns our appraisal of the thing that has passed as the "normal" bacterial type. The observations already presented dealing with this subject can lead us only to the view that the older notions of normality and immu- tability of culture type have determined a highly repressive and dangerous influence on the development of bacteriology — an influence which, even at the time of writing, is still menacing the progress of the science. The present conceptions of "normal type," "normal colony," and "normal" cytology we owe to the influence of monomorphism which, even in most recent textbooks, still clings like a barnacle to modern bacteri- ology. According to its dictates, whatever departs from the "normal" must be re- garded as an "involution form," a degeneration form, a mutant, or a contamination. On the other hand, whatever form of culture the bacteriologist succeeds in causing to develop most freely in his carefully standardized media, and under other standard- ized conditions of growth which he imposes, are "normal" cultures, while aberrant forms are of little consequence. In order that there may be no lack of means for mak- ing the species recognizable, we assiduously fill out data on neatly designed descriptive charts — all dealing with the "normal" type, usually the S. Are we, then, forced to the conclusion that cultures of the and R forms are not normal cultures? It is indeed time that we revised our notions on "normality" in bacterial species. The results of many studies dealing intentionafly or unintentionally with microbic dissociation force the conclusion that there is no such thing as "normal" type in the usual meaning of the term. The O type cultures are no less normal than the S; nor the R type cultures less normal than the O. They are all normal, and can be regarded in no other light than that of isolated states or stages of the bacterial species in its progress through the cyclode. And it may be said in passing that this view may no doubt be held equally for the filtrable forms of bacteria. These have often been re- ferred to as "fragments," or as minute "fractions," of the "normal" cell, and endowed with the power of "regeneration" into the original form. We shall perhaps do well to question whether these minute living elements may not represent one or more definite units (cyclostages) in the reproductive history of the species. From this viewpoint, whether we deal with filtrable or non-filtrable forms, it is essential that we should cease to regard "normality" in the old, absolute sense, but should come to regard the characters of bacteria as related to definite stages in their development. Thus we may have a normal growth of B. typhosus on plain agar, or on phenol agar, or at 42° C, or in immune serum, or under reduced oxygen tension. The growth is normal with ref- erence to a certain condition or environment, although it is likely to differ in each of the conditions mentioned above. That "pathological" growth forms may occur can- not be doubted; but at present we are not in a position to recognize them — any more than we are in a position to recognize bacterial mutations, so called — until we have gained a fundamental knowledge of the nature, limits, and sec^uence of cyclogenic variation. CHAPTER VIII BACTERIAL ASSOCIATIONS W. L. HOLMAN University of Toronto INTRODUCTION Bacterial association has taken in recent years a much more important place in bacteriological studies than formerly. This increased interest is largely due to the greater attention which is being given to the finer metabolism of bacteria and the in- teractions which occur between the bacteria and their environment. The idea long held by many that bacteria represent the lowest forms of life and are therefore com- paratively simple in their metabolic activity has been replaced by a realization that we are dealing with just as highly specialized and complicated functional activities as in any of the so-called "higher" plants or animals. It is true that we are dealing with unicellular forms of very small size and that these as suggested by Kendall' should be considered as similar to living colloids in which surface phenomena are so important. Many of the activities in the life of the bacteria can be appreciated better if this complicated metabolism is kept in mind, and the study of mixed or double cul- tures helps in an understanding of the actual life-processes. NOMENCLATURE It is generally recognized but often forgotten that under natural conditions mixed cultures are the rule, and the earliest work on bacterial associations is to be found largely in the studies which attempted to analyze such natural phenomena. The most variable results may be obtained in these mixed cultures, and there are many factors taking part which determine the final outcome. There may be simple mixtures with no demonstrable effect of one bacterium on another, but this is uncommon since one or the other usually dominates the picture. One microbe may favor the growth and activity of another or both may be benefited by the combination. The latter condi- tion is usually spoken of as "symbiosis," but true examples of this relationship are rare. The term "metabiosis" is sometimes used where one action follows another, and is well illustrated in innumerable examples in nature. Antagonism or antibiosis is often combined with the foregoing, but is mostly employed for the occurrences where there is a clearly demonstrable harmful effect of one micro-organism on another or when a characteristic product fails to be formed or disappears in the mixed culture. Be cause of the impossibility in many cases of determining the actual processes at work these terms must be used with reservations. It is better, I believe, to use the more general word "association" for all these phenomena and "synergism," introduced into bacteriological nomenclature by Kammerer,^ for those in which definite changes are ' Kendall, A. I.: Colloid Symposium Monograph. 2, 195. 1925. ^ Kammerer, H.: Klin. Wchnschr., 2, 1153. 1923; Deutschcs Arch.f. klin. Med., 141, 31S. 1923: ibid., 145, 257. 1924; Klin. Wchnschr., z, 723. 1924. W. L. HOLMAN 103 demonstrable which indicate or suggest the combined work of two or more micro- organisms. Zoeller' has used "cumulative cultures" to express the results obtained by him in certain biological combinations. Synergism may be conveniently qualified, when one or the other result dominates, into an "antagonistic synergism" and a "be- neficent synergism." "Antagonism" alone should probably be retained for outstand- ing examples of one-sided harmful effect. GENERAL CONSIDERATIONS There are numerous examples of bacterial associations in every field of bacte- riology. In man and animals natural infection with more than one bacterium is rela- tively frequent, and the particular combinations which may occur often determine the course of the disease. There is an enormous literature on this phase of the subject but no very definite conclusions have been drawn. It would be futile to go into the prob- lems of tuberculosis and secondary infections, or those of typhoid fever, gonorrhea, influenza, and many other diseases. Certain phases of these I shall briefly discuss, but this article will deal largely with some of the outstanding phenomena studied experi- mentally, and I shall not confine myself to the pathogenic bacteria although the greater amount of work has been done on them. EARLY EXAMPLES OF ASSOCIATION Among the early observations we find the recognition by Pasteur'' of the harmful effect of "wild" yeast on the normal fermentation processes in the beer and wine in- dustries. He further noted the beneficial effect of aerobic forms which developing a scum on the surface, and using up the oxygen, favored anaerobic growth. Winogradsky^' isolated an aerobe which only fixed nitrogen from the air in the presence of other bac- teria. Burri and Stutzer^ demonstrated that horse feces split nitrate with the produc- tion of free nitrogen. He isolated from the feces B. coli communis and a strict aerobe, and these two in combination gave the same result. The B. coli could be replaced by B. typhosus, and therefore it was the strict aerobe which gave the actual gas produc- tion. Previous to this, Marshall Ward^ described a yeast and a bacterium which to- gether formed a ginger beer-like product in a saccharine fluid. ANAEROBES Nencki* reported, with a double culture of B. paralactici and B. chauvoei the for- mation from glucose of normal butyl alcohol, a substance not produced by either cul- ture alone. He believed his results might help to clarify certain difficulties in obtain- ing infections in animals with single pure cultures. Novy^ quoted Roger (1889) as the first to show that B. prodigiosus added to the bacillus of malignant edema ren- ' Zoeller, C: Compt. rend. Soc. dc bio!., 92, 435, 497, 686. 1925. ^ Pasteur, L.: Oeuvres de Pasteur (reunies par P. Vallery-Radot). Paris, 1922. 3 Winogradsky, S.: Compt. rend. Acad, de sc., 118, 353. 1894. ■* Burri, R. and Stutzer, A.: Centralbl. f. Bakteriol., I, Orig., i6, 814. 1894. sWard, Marshall: Phil. Tr. Roy. Soc, B, 187, 125. London, 1892. ^Nencki, M.: Centralbl. f. Bakteriol., I, Orig., 11, 225. 1892. 'Novy, F. G.: Ztschr.f. Hyg. u. Infektionskrankh., 17, 209. 1894. I04 BACTERIAL ASSOCIATIONS dered sublethal doses of this anaerobe fatal for rabbits, and he himself found that the injection into guinea pigs of B. proteus and his new anaerobe {B. oedcmatiens) resulted in rapid death and an enormous growth of the anaerobe in the animal body. The overgrowth was absent with pure cultures. He further was able, by adding B. proteus and other aerobes, to grow his anaerobe in the presence of air. Passing over numer- ous similar results we find Sturges' devising a method, based on bacterial association, for isolating spore-bearing anaerobes on open plates by growing them with B. coli or Staphylococcus aureus. Rhein^ used B. fecalis alcaligenes in bouillon for anaerobic growth because of the lack of saccharolytic and proteolytic activity in this aerobe. Inoculation of the mixed cultures into animals he considered practical because the aerobe is not toxic, but I believe from the work of many others that this procedure might well give faulty results. Barrieu^ noted that B. proteus and certain non-patho- genic spore-bearing aerobes found in wounds exalted, by their proteolytic activity, the virulence of pathogenic bacteria. Pringsheim^ grew Frankel's bacillus {B. welchii) with B. fecalis alcaligenes for ten transfers on agar slants and could see in the growth of the latter the opaque colonies of the anaerobe, A hquefying sarcina allowed B. welchii and B. hutyricus to grow in open tubes. After six days' growth the sarcina had disappeared from the B. butyricus culture, and he suggested this as an easy method to obtain a pure culture. Weinberg and Otelesco^ considered that many war-wound infections, looked upon as of pure anaerobic origin, may be due to an association with B. proteus since this latter organism increased the virulence of B. perfringens, V . septique, and others. Animals injected with B. sporogenes and B. proteus did not de- velop putrid lesions. This combined growth of aerobes and anaerobes on surface cul- tures I observed on a number of occasions in France while studying the bacterial flora of war wounds. Colonies picked from aerobic plates were not infrequently found to be mixed with anaerobes. Stillman and Bourn^ reported in 1920 the production of gas by 16 of 119 non- hemolytic strains of B. influenzae in i per cent dextrose agar with a little blood ex- tract. Four of 29 hemolytic strains also produced gas. Jordan and Reith^ also found gas production in certain of their strains. About four years ago, when working with cultures of a tiny anaerobe resembling B. pneumosintes^ (probably Staphylococcus parvulus of Veillon and Zuber), I mixed a culture of this anaerobe with a culture of B. influenzae and planted the mixture on blood-agar slants. A good growth of B. in- fluenzae occurred, and after five transfers I had no difficulty in recovering the gas- producing anaerobe in cooked-meat media. This anaerobe is practically always present in the oral cavity, and could easily contaminate cultures of B. influenzae. It can be ' Sturges, Jr., W. S.: Ahslr. Bad., i, 63. 1917. ^ Rhein, M.: Prcsse mcd., 27, 504. 1919. 3 Barrieu, A. R.: ibid., 28, 40. 1920. "I Pringsheim, E. G.: Ccnlralbl. f. Bakleriol., II, 51, 72. 1920. 5 Weinberg, M. and Otelesco, I.: Compl. rend. Soc. de biol., 84, 535. 1921. * Stillman, K. G., and Bourn, J. M.: /. Exper. Med., 32, 665. 1920. 7 Jordan, E. O., and Reith, A. F.: 7. Infect. Dis., 34, 239. 1924. 8 Holman, W. L.: .im. J. Ilyg., 3, 4S7. 1923. W. L. HOLMAN 105 recovered from high dilutions of the saliva, as Hall and Wing' have shown, and be- cause of its morphological resemblance to forms of B. influenzae might very well be overlooked. I would suggest this as a possible explanation of the rather infrequent ob- servation of gas production by B, influenzae. The group of B. botiilinus and the effect of associated bacteria particularly on its toxin has received considerable attention. Hall and Peterson^ found that certain acid- producing aerobes inhibited toxin production in glucose but not in non-carbohydrate media, and some of these aerobes actually destroyed toxin in glucose broth. It would appear that the acid must be in the nascent state since acid itself was inefTective. Jordan and Dack'^ found that a mixture of a large amount of B. sporogcnes with B. botulinus interfered with the development of toxin and might cause its early disap- pearance. Francillon'' studied the same problem. He found that Staphylococcus au- reus, B. coli, B. proteus vulgaris, and other bacteria permitted the growth of B. botu- linus in open tubes of plain and glucose bouillon, but the growth was never as good as under other anaerobic conditions. A moist-meat medium gave somewhat better growth. Toxin was found in the mixed cultures in bouillon and meat, the amount vary- ing with the aerobe. The B. pyrcyaneus mixture gave no toxin in the bouillon but a strong one from the meat. There was but little effect on the toxin by two weeks' con- tact with B. proteus, B. coli, or B. pyocyaneus. Dack^ reported the gradual destruction of filtered toxin by the growth of B. sporogencs and other proteolytic and non-pro- teolytic anaerobes. Passini'' found that a putrefactive anaerobe B. putrificus verrucosus destroyed B. tuberculosis in nine days. The effect of similar anaerobes on the survival of anthrax spores in dead animals has been extensively studied. Among a great many other in- teresting anaerobic and aerobic synergistic phenomena I mention a few. Ome- liansky^ studied the fixation of atmospheric nitrogen as Winogradsky^ had done years before. He noted that in the surface layers of the soil numerous organisms used the oxygen and created anaerobic conditions for the B. Clostridium pasteurianum, but in addition some of these accompanying forms also supplied carbon compounds for the anaerobe. The Azotcbacter being alkaligenic used up such products from the anaerobe as butyric acid and thus favored the synergistic process. The other aerobes may at times do harm by depriving the Azotobacter of oxygen. These two nitrogen-fixing forms, one aerobic, the other anaerobic, worked very well together. The work of Kam- merer'and his associates gave interesting examples of synergistic action. They observed that emulsions of human feces reduced pure bilirubin and mesobilirubin to urobilin but had no action on biliverdin and that the feces of herbivora did not have this action ' Hall, I. C, and Wing, H. U.: Am. J. Pub. Health, 15, 770. 1925. ^ Hall, I. C, and Peterson, E.: /. Bact., 8, 319. 1923. 3 Jordan, E. O., and Dack, G. M.: /. Infect. Dis., 35, 576. 1924. ■* Francillon, M.: Arch.f. Hyg., 95, 121. 1925. 5 Dack, G. M.: /. Infect. Dis., 38, id^. 1926. ^ Passini, F.: CenlralU.f. Bakterlol., I, 81, 447. 1926. (Ref.) ^r'* '» \. J( /*\ 7 Omeliansky, V. L.: Arch, de sc. biol., 18, i. 1915. S^ -y^\ ' Winogradsky, S.: /oc. a7. ' Kammerer, H.: /oc. a7. ' ,.^ O,' io6 BACTERIAL ASSOCIATIONS because of its active fermentation. Filtrates had no effect. These changes they be- lieved were due to a synergism between B. putrificus and certain aerobes. B. coli either helped the bilirubin production or hindered it, depending on the presence or absence of fermentable material. They further demonstrated the development of hem- atoporphyrin from blood by a similar synergism and that sugar or bile inhibited it. A particularly important instance of bacterial association was reported by Speakman and Phillips.' During the war, acetone and butyl alcohol were produced on a large scale by fermentation of cereals and carbohydrates. Serious difficulties developed in the plants, owing to the contamination of the cultures of B. granulohacter-pectin- ovorum by the aerobic bacillus B. volutans. The acetone yield, as a result of the mixed culture, dropped or disappeared and the development of lactic acid increased. This increase was due to an altered metabolism of the acetone producer so that it formed more lactic acid and less acetone. The results varied with the relative numbers of each organism present. They considered it due to an inhibitory substance from the nitrogen metabolism. In actinomycotic granules there is found a bacterium named by Klinger^ B. actinomycetum coniitans, the presence of which was confirmed by Colebrook^ in 80 per cent of his twenty cases. The significance of this associate is not known, but Colebrook suggested a possible genetic relationship. There are many other examples of anaerobic and aerobic associations in the natural metabolism of the sulphur bacteria; in silage fermentation, the heat of which was definitely shown by Hunter'' to be due to bacterial action and not to cell respiration; in sewage decom- position and cellulose destruction in which Groenewege^ believed a symbiosis occurred but stressed the action of the aerobes and Khouvine*" gave chief importance to a strict anaerobe discovered by him, B. cellulosae dissolvens (n.sp.), but pointed out that five times as much cellulose was de5tro3'ed when in association as when alone. There are other examples in the natural breakdown of organic materials of all kind 3 and in innumerable other bacterial activities in nature. THE ACroURIC GROUP (aND CERTAIN THERAPEUTIC USES) The effects of the aciduric group of bacteria on other bacteria has been closely Studied. They are usually facultative aerobes, and their action is considered as chief- ly antagonistic. Because of their use for therapeutic purposes there has collected an extensive literature brought together by Rettger and ChepHn^ and Kopeloff.* Certain points, however, may be briefly reviewed. Starting from the work of Metchnikoff with B. bulgariciis it was soon found that this organism could not be implanted in ih^ intestinal tract and B. acidophilus, a normal inhabitant, was substituted. B. bijidus ' Speakman, H. B., and Phillips, J. F.: /. Bad., 9, 183. 1924. » Klinger, R.: Centralbl.f. BaktcrioL, I, Orig., 62, 191. 1912. 3 Colebrook, L.: Brit. J. Expcr. Path., 1, 107. 1920. ••Hunter, O. W.: /. Agric. Research, 10, 75. 1917. s Groenewege, J.: reference in J .A.M. A., 76, 279. 1921. * Khouvine, Y.: Ann. de Vlnst. Pasteur, 37, 711. 1923. 7 Rettger, L. F., and Cheplin, H. A. : The Intestinal Flora with Special Reference to the Implantation of "Bacillus acidophilus." Yale University Press, 1921. * Kopeloff, N.: "Lactobacillus acidophilus." Williams & W'ilkins Co., 1926. W. L. HOLMAN 107 found in the intestines of breast-fed infants, is responsible for inhibition of the growth of other bacteria, and its acid products are thought to be mild stimulants to the bowel walls. In the vagina, B. doederleini is believed to keep the reaction acid, thus inhibit- ing the growth of contaminating bacteria. Landau' was the first to use fresh beer yeast in the treatment of leucorrhea and considered the anticatarrhal action was due to mechanical overgrowth, the using up of food material and the action of metabolic products in injuring or destroying other bacteria, neutralizing the toxins and chang- ing the reaction to acid. He suggested injecting cultures of this yeast into the bladder in cases of cystitis with alkaline urine. Since this work there have been many sug- gestions and a variety of bacteria used to obtain such biological inhibitory action. The antagonistic action of B, acidophilus has been abundantly proved against putre- factive anaerobes, B. coli, and many other bacteria. Schiller,^ with a strain of B. acidophilus from a dog, found that it rapidly destroyed and dissolved many strains of streptococci in fluid media and suggested this as a useful way to obtain bacterioly- sis of cocci. He showed that this action was not due to lactic acid since it occurred in alkaline media and filtrates from a glucose broth culture living or killed (by heat or age) allowed a good growth of the streptococcus. The harmful substance was only formed by the B. acidophilus in the presence of the streptococcus or its products. Filtrates of the mixed culture, after thirty-six hours at 37° C. (when the streptococci are killed), were as toxic as when living cultures of the bacilli were used. Strepto- coccus cultures killed by heat or age had no toxic effect on living streptococci. He considered the phenomenon an example of induced antagonism and reported other examples in a series of four articles.-' In the first he used B. mesentericus and forced on it an antagonistic action against streptococci by growing it with the latter in a medium of poor-food value. It secreted a bacteriolytic substance which digested the living bacteria as it would any other insoluble albuminous material, and the amount depended on the number of sensitive streptococci present. It also acted when the B, mesentericus had been removed by centrifugation and after evaporation and drying. It was not completely specific. Schiller further showed that yeasts can be made an- tagonistic against bacteria including B. tuber ctilos is if the medium contained sugars but lacked nitrogenous materials. They acted in the same way as the foregoing, and a more active bacteriolytic substance was secreted in the presence of more resistant forms so that the enzyme induced by B. tuberculosis was even capable of attacking beeswax. The reverse was also found. Bacteria (staphylococcus, B. typhosus, B. para- typhosus, et al.) became antagonistic to yeasts in nitrogen-free media, and the secreted cytolytic substance was similar to the foregoing but had no effect on coagulated serum or egg albumin. This method of dissolving the yeast membrane he thought might be of interest in the study of zymase. Donaldson^ during the war used a strain of B. sporogenes in the treatment of slow- ly healing war wounds. The beneficial effects he ascribed, not to direct inhibition, but to the removal by the proteolytic anaerobe of the dead tissue (the pabulum for the 'Landau, T.: Deutsche »icd. Wchnschr., 25, 171. 1899. ^Schiller, I.: Centralbl.f. Baktcriol., I, Orig., 73, 123. 1914. 3 Schiller, I.: ibid., 91, 68. 1924; 92, 124. 1924; 94, 64. 1925; 96, 54, 1925. 4 Donaldson, R.: /. Path. &° Bact., 22, 129. 1918. io8 BACTERIAL ASSOCIATIONS pathogens in the wound), and, itself producing no harmful products, it further hy- drolyzed the toxic bacterial products present or being formed. Bumm' used a durable preparation brought out by Zeissler called "neocolysin." It was made up of living, albuminolytic bacteria and gave good results in chronic purulent conditions such as osteomyelitis. The bacteria were supposed to function as in Donaldson's method and continued growing as long as there was dead tissue available. Gratia and Dath^ dis- covered an aerobic streptothrix which had a powerful destructive action on a variety of bacteria. It did not act on B. tuberculosis and showed no lipolytic enzyme. Fil- trates were equally effective, could act without free oxygen, but were somewhat variable. The active substance was better developed in old cultures and was fairly stable. The dissolved bacteria caused specific response when used for vaccination. They referred to a similar organism reported by Lieske in 1921, but his studies were confined to solid media. Rosenthal worked with an organism apparently very much like the foregoing, and found it was antagonistic to many bacteria including B. diph- iheriae. He referred to the report of Gasperini of 1890 on a similar form acting against bacteria. Rosenthal and his associates'" found that it could be implanted in the in- testines of guinea pigs and that when injected parenterally it was enterotropic. Much and Sartorius^ used a strain of B. mycoides and showed similar effects, by cul- ture and filtrates, on many bacteria and that the dissolved bacteria had not lost their antigenic properties. A very interesting study by Gratia and Rhodes^ proved that living staphylococcus could live on killed suspensions of staphylococci in saline or in saline agar made cloudy with killed staphylococci. Thus we see that bacteria can and do remetabolize their own substances or that of other bacteria, and this helps in the understanding of the antagonistic action of many forms of bacteria. They are indeed cannibalistic. DIPHTHERIA GROUP Because of the pressing problem in carriers of the diphtheria bacillus, special at- tention has been given to researches for a possible biological method, through bac- terial antagonism, which would be effective in treating these cases. Streptococci and B. diphtheriae have long been considered mutually helpful in producing severe infec- tions in the throat. There is an extensive literature on this topic. Roux and Yersin,^ in studying the problem of the return of virulence in attenuated cultures of B. diph- theriae, were successful in accomplishing this by injecting the attenuated culture along with a non-fatal dose of an erysipelas strain of streptococcus. The virulence returned, and it was retained on successive cultures. They therefore warned against the use of Streptococcus erysipelatos to combat diphtheria as had been suggested by 'Bumm: Arch.f. klin. Cliir., 138, iii. 1925. ^Gratia, A., and Dath, S.: Compt. rend. Soc. de hiol., 91, 1442. 1924; 92, 461, 1125. 1925; 93, 451. 1925; 94, 1267. 1926. 3 Rosenthal, L.: ibid., 93, 77. 1925. '•Rosenthal, L.: Hid., 94, 309, 1059, 1926; 95, 10. 1926. sMuch, H., and Sartorius, F.: Med. Klin., 20, 347. 1924. ^ Gratia, A., and Rhodes, B.: Compt. rend. Soc. de biol., 90, 640. 1924. ' Roux, E., and Yersin, A: Ann. de I'Inst. Pasteur, 4, 385. iSgo. W. L. HOLMAN 109 Babtchinski, Similar results were obtained by Barbier^ and Schreider.* Funck^ found the fact to be true but did not consider it as striking as had previous workers, and showed that the presence of the streptococci in no way affected the specific action of the diphtheria toxin. Klein^ also showed that streptococci enhanced the effect of B. diphtheriae. Arnold^ found httle or no evidence of any increased virulence in the he- molytic streptococci isolated from diphtheria throats, but that there was a decided in- crease in hemolytic streptococci during diphtheria. These strains showed limiting H-ion concentrations like pathogenic strains, but he believed this change was merely environmental. Gate, Papacostas, and Billa,'' although they found that filtrates of avirulent streptococci stimulated diphtheria-toxin production, reported that the in- creased virulence was not retained on further transfers. Zoeller^ showed it was possi- ble to produce in his cumulative cultures a diphtheria-streptococcus altero-toxin by growing a scarlet fever streptococcus in a diphtheria toxin to which had been added a little horse serum. Stovall, Scheid, and Nichols* reported that the presence of staphylococcus in mixed cultures changed the morphology of virulent B. diphtheriae so that they stained more solidly and that the non-virulent pseudo-diphtheria strains became more beaded. Streptococci had no such effect. The well-known overgrowth of B. diphtheriae by Staphylococcus aureus in cultures led many workers to try such cultures in patients following the report by Schiotz.' Among these, Lorenz and Ravenel'" had good results in nine carriers and eight clinical cases of diphtheria although nasal furuncles developed in some of them. Rolleston" found it helpful in ten carrier cases but ineffective in two cases of nasal infection. He considered it should only be used in chronic cases. There were also a number of un- favorable reports such as that of C, M. Davis'^ who reported the development of ton- sillitis following the use of the staphylococcus spray. Nicholson and Hogan'-' were encouraged by the results on nine acute cases, using sprays of B. bulgaricus and sour milk. Papacostas and Gate''' studied the question of the antagonism between the pneumobacillus of Friedlander and B. diphtheriae following the observation that clini- cal cases of such mixed infections were usually mild. Mixed cultures of these two bac- teria showed a progressive predominance of the former on serial transfers and the ' Barbier, H.: Cenlralbl. f. Bakteriol., I, Orig., 11, 382. 1892. (Ref.) ^ Schreider, M. von.: ibid., 12, 289. 1892. 3 Funck, E.: Zlschr.f. Hyg. u. Infektionskrankh., 17, 465. 1894. 1 Klein, E.: Thirty-third Ann. Rep. Loc. Gov. Bd., p. 431. 1903-4. 5 Arnold, L.: /. Lab. df Clin. Med., 8, 387, 389. 1923. ^ Gate, J., Papacostas, G., and Billa, M.: Compi. rend. Soc. de biol., 90, 500. 1924. ' Zoeller, C.: loc. cit. » Stovall, W. D., Scheid, E., and Nichols, M. S.: Am. J. Pub. Health, 13, 748. 1923, ' Schiotz, A.: see Diphtheria, p. 367. London: Medical Research Council, 1923. '"Lorenz, W. F., and Ravenel, M. P: J.A.M.A., sg, 690. 1912. " Rolleston, J. D.: Brit. J. Child. Dis., 10, 298. 1913. " Davis, C. M.: J. A. M.A., 61, 393. 1913. ••i Nicholson, S. T., and Hogan, J. F.: ibid., 62, 510. 1914. '■» Papacostas, G., and Gate, J.: Compt. rend. Soc. de biol., 85, 859, 1038. 1921. no BACTERIAL ASSOCIATIONS morphology of the latter also changed toward a more homogeneous form on staining. By the use of filtrates of each culture they could not discover any evidence that the toxin of the former was able to neutralize diphtheria toxin in vivo or in vitro. If the two are grown together, however, no toxin is formed, nor is there any if the filtrate of the pneumobacillus growth is used to grow the B. diphtheriae. They suggested the therapeutic use of filtrates. In later studies on a larger number of clinical cases Gate et al.' and Chalier, Gate, and Grandmaison^ confirmed their impressions of the usual mild course of these mixed infections. Van der Reis\ having demonstrated an antagonistic action of B. coli to B. diph- theriae, showed that it was possible, by spraying B, coli into the mouth, to have it colonize there. In nine cases it was still present after fifty-four days. A careful study of the antagonistic activity of B. coli led him to conclude that there is formed a ther- molabile, volatile, non-dialyzable, non-filterable, inhibitory substance not adsorbed by charcoal, not identical with the normal metabolic products of the colon bacillus, but that it may be a special toxic product. It was tried in acute cases of diphtheria by means of sprays of B. coli and particles of B. coli agar with the result that the B. diphtheriae disappeared more quickly than in controls. Carriers could also be rapidly freed of their bacilli. On the other hand, Pesch and Zschocke,'' although confirming the crowding out of the B. diphtheriae by B. coli in cultures, were unsuccessful in treat- ing nasal carriers because the B. coli would not grow in the nose. Bloomfield^ failed in his attempts to implant Friedlander's bacillus from carriers to non-carriers, and even a foreign strain of the bacillus failed to establish itself in the throat of a carrier of another strain. Pringsheim* studied the inhibiting effect of a strain of B. mesenteri- cus vulgatus against a variety of bacteria but particularly against B. diphtheriae. He found that B. typhosus, B. paratyphosus A and B, B. fecalis alcaligenes, B. coli, and streptococcus were without effect on B. diphtheriae. B. pyocyaneus and an air staphy- lococcus were strongly inhibitive. Staphylococcus aureus was mildly stimulating as seen in larger colonies as was also a weakly sporing B. subtilis strain. On agar plates the effect of his B. mesentericus was to produce a circular zone of inhibition and just beyond this a ring of larger colonies. Filtered or heated cultures had no effect. Other proteolytic bacteria had no such action. It was tried on patients but the results were inconclusive. The findings of Zukerman and Minkewitsch' with B. mesentericus vul- gatus were somewhat different. The antagonism was inherent in the bacillus and was not increased by serial passage. It acted only on diphtheria and pseudo-diphtheria forms and not against a long list of other bacteria. Many other spore-bearers were either negative or but weakly active. Filtrates were very active, killing in four min- utes, and were fairly heat resistant. ' Gate, J., el al.: ibid., 86, 929. 1922. ' Chalier, J., Gate, J., and Grandmaison, L.: Paris med., 61, 205. 1926. 3 Van der Reiss: Miinchen. mcd. Wchnschr., 68, 235, 1921; Zlschr. f. d. ges. expcr. Med., 30, 1922. -• Pesch, K., and Zschocke, O.: Miinchen. med. Wchnschr., 69, 1276. 1922. sBloomfield, A. L.: Johns Hopkins Hasp. Bull., 32, 10. 1921. ^ Pringsheim, E. G. : loc. cil. 'Zukerman, I., and Minkewitsch, I.: Centralbl.f. Bakkriol., I, 80, 483. 1925-26. (Ref.) W. L. HOLM AN iii PNEUMOCOCCUS The pneumococcus is usually considered a rather delicate organism in culture media, but apparently it may have a striking antagonistic effect on the staphylo- coccus. Gromakowsky' discovered in eight sputum cultures a coccus which had a definite restraining action on the staphylococcus. This coccus resembled the pneumo- coccus in morphology, but he considered it different. Mixed with eight different strains of staphylococci and after twenty-four hours' incubation, transfers to agar gave no growth of the staphylococcus. It was irregular in action and also was an- tagonistic to streptococci from abscesses. Ahvisatos,^ working with twenty-eight strains of well-identified pneumococci and three strains of Staphylococcus albus, no- ticed, when the forms were mixed, interesting phenomena on ascites agar plates. Curious clear zones appeared about the colonies of the pneumococci, and the edges of the staphylococcus colonies were irregular and suggested the action of bacteriophage. These zones varied in size, and if enough pneumococci had been added no growth of staphylococcus occurred. He never found mixed colonies. Neither the virulence nor the agglutinating type of the pneumococcus was related to the extent of the phenom- ena. There was no demonstrable change in the cultures of either of the bacteria after these contacts. Living, growing pneumococci were necessary and filtrates were nega- tive. Eight strains of hemolytic, five of viridans, and one of mucosus streptococci gave negative results. In these cases mixed colonies were frequent, and he suggested that this characteristic might be used to differentiate pneumococci from closely sim- ilar streptococci. COLON-TYPHOm GROUP The antagonistic effect of soil bacteria against pathogenic forms has been exten- sively studied. The early work of Frost^ is important and includes the literature to that date. Limitation of space forbids a further discussion of this interesting subject. Fecal bacteriology, particularly of the colon-typhoid group, is replete with examples of supposed antagonism. It has long been held that the presence of slow lactose fer- menting B. coli, so frequently observed in stool examinations, is due to this phenom- enon (von Jeney"), and Henningson^ gave examples of inhibition of gas production and proposed the name B. coli anaerogenes for these. PrelP and many others have studied such defective strains. Nissle,^ having observed an inhibitory action in cer- tain stools seeded with B. typhosus, studied the antagonistic index of the B. coli to B. typhosus with various strains of the former. The difference seemed correlated with lactic acid production. The active coli strains also were inhibitory to other coli strains. He therefore gave these active cultures in capsules to persons carrying in- efficient B. coli strains and reported good results. R. P. Smith* found that B. coli ' Gromakowsky, D.: ibid., Orig., 32, 272. 1902. ^ Alivisatos, G. P.: ibid., 94, 66. 1925. 3 Frost, W. D.: J. Infect. Dis., 1, 599. 1904. * von Jeney, A.: Ztschr.f. Hyg. u. Infektionskrankh., 100, 47. 1923. sHenningson, B.: ibid., 74, 253. 1913. ^Prell, H.: CentralU.f. BakterioL, I, Orig., 80, 225. 1917. 7 Nissle, R.: Deutsche mcd. Wchnschr., 42, 1181. 1916. * Smith, R. P.: J. Path. b° Boot., 26, 122. 1923. 112 BACTERIAL ASSOCIATIONS strains from carrier cases were more active against stock cultures of B. typhosus than were stock cultures of B. coli. Unfortunately, he did not test the B. typhosus from the carrier with its own B. coli, but the evidence suggested an inhibition because of the difficulty he had in obtaining the B. typhosus in these cases. Vignati' described the reverse phenomenon in which fresh, actively growing cultures of B. typhosus inhibited the growth of B. coli, older cultures not being antagonistic. He explained the facts on Bail's theory of the spatial needs of each bacterium. Lisbonne and Carrere,^ by a method suggesting that of Schiller,^ forced a bacteriophage to develop by the antag- onistic action of B. coli against the Shiga bacillus. They found at the end of a series of passages that an active and transmissible lytic principle was developed by what they call a "vitiation" in the metabolism of the Shiga bacillus. B. proteus X19 gave identical results. They considered that this is what occurs in the intestines where an-' tagonistic conditions are always present. They later showed that this principle was not carried by the B. coli since the same strain was tested by Beckerich and Hauduroy who suggested such an explanation, and was not found to be lysogenic. It was def- initely the result of microbial interactions. Fabry also obtained a principle of the same kind through the antagonistic stimulus of a Staphylococcus albus on B. coli which also acted on the Shiga bacillus. Bordet^ reported a similar discovery with four pri- marily non-lytic strains of B. coli in which the lytic principle appeared spontaneously and was increased by passage. Gratia^ studied an example of antagonism between two races of B. coli as Nissle^ had shown from another point of view. Filtrates of B. coli V. inhibited B. coli and caused an agglutinative culture of the latter in fluid media. The same results were obtained with living cultures on agar, and in both cases the secondary colonies were resistant to the action of B. coli V, It resembled the Gratia principle but was not regenerated by the B. coli 0, being lost by the third passage, and did not act in high dilutions as bacteriophage does. On agar plates the area about the growth of B. coli V. was inhibitive to the growth of B. coli 4> but not to B. coli V, It was therefore not a vaccination of the medium. It was very resistant to storage, chloroform, and high temperatures (100° C, for thirty minutes). Bordet*^ has carefully analyzed the various interactions between the bacteria giving the re- sults that Lisbonne and Carrere' reported, but as this is encroaching on the problem of bacteriophage which is to be presented elsewhere'" in this book, I need go no further. THE THEOBALD AND D. E. SMITH PHENOMENON A most interesting example of the inhibitory effect of bacteria in association was reported in 1920 by Theobald Smith and D. E. Smith." They found that B. para- ■ Vignati, J.: Compt. rend. Soc. de bioL, 94, 209. 1926. * Lisbonne, M., and Carrere, L.: ibid., 86, 569. 1922; 87, ion. 1922; 90, 265. 1924. 3 Schiller, I.: loc. cit. ■» Fabry, P.: ibid., 87, 369. 1922; 90, 109. 1924. sBordet, J.: ibid., go, g6. 1924. * Gratia, A.: ibid., 93, 1040. 1925. ' Nissle, R.: loc. cit. * Bordet, J.: Com pi. rend. Soc. dc bioL, 93, 1054. 1925. 'Lisbonne, M., and Carrere, L.: loc. cit. '"Chapter xl. " Smith, T., and D. E.: /. General Physiol., 3, 21. 1920. W. L. HOLM AN 113 typhosus B, after it had grown in lactose bouillon for four to six days, prevented the development of gas by B. coli when this was added. Members of the closely related hog-cholera group had no such action in the given time, but after eighteen days' growth they also inhibited the gas production for the B. coli added at this Time. A fuller analysis of this phenomenon is found in an article by Holman and Meekison.' Besson and De Lavergne^ confirmed the results of T. and D. E. Smith and found that B. aertrycki gave the reactions of the hog-cholera group. Brutsaert^ found the phenom- enon most inconstant and variable even in repeated tests of the same strain of bacillus. A hog-cholera type-agglutinating culture inhibited, but as a rule members of this group did not. Moreover, the phenomenon failed if, instead of lactose bouillon, a lactose peptone water were used. He found it too irregular for use in classification. Von Jeney" in studying this question used a bouillon previously freed from glucose by a twenty-four hour growth of beer yeast. (T. and D. E. Smiths presumably used B. coli for this purpose.) Both of these procedures may have an important bearing on the results since the effect of these preliminary cultures may be very great as is seen in many of the articles reviewed above and in the studies of Robertson^ on food ac- cessory factors in bacterial growth. Although such media may not interfere with gas production by the B. coli per se, it may have an effect on the combined metabolism. Von Jeney' investigated the subject very fully. He found five strains of B. para- typhosus B among twenty-six studied which increased the B. coli gas production. Also plates from the mixtures at times gave pure B. coli. The strains of the B. paratypho- siis, isolated from the mixture, did not always give the same results on retest. B, typhosus also inhibited. He searched for any evidence of bacteriophage action, but was only able to discover suggestions of such and no continuous passage was possible. Kauffmann* carried the work further and used besides human strains, as von Jeney had done, a large number of animal strains. He tested thirty different cultures of B. coli and found in pure culture that their gas production was most variable, ranging between o and 100 per cent in twenty-four hours, and for no known reason. He also used yeast-treated media. Some B. coli strains grown from the same stools as strains of B. paratyphosiis showed delayed gas production on glucose, but this was not the rule. On the other side he used thirty human B. paratyphosus B strains, Gaertner's bacillus, B. typhosus, and others and a further group of fifteen strains from animals. Besides the regular tests he used a number of heterologous and homologous combina- tions with the cultures from individual stools. A great irregularity was found through- out. After passage, certain strains of the B. paratyphosus increased in inhibitory powers, but the B. coli did not become more sensitive by such passage. There was, however, no regularity. The animal strains gave the same kind of results as the hu- man. Certain "pseudo-unstable" forms were sometimes seen on plates. In summing ' Holman, W. L., and Meekison, D. M.: /. Infect. Dis., 39, 145. 1926. ^ Besson and de Lavergne: Compl. rend. Soc. de biol., 86, 357. 1922. 3 Brutsaert, P.: ibid., 88, 306. 1923. ■* von Jeney, A.: loc. cit. s Smith, T., and D. E.: loc. oil. ^Robertson, R. C: /. Infect. Dis., 34, 395. 1924; 35, 311; 1924. ' von Jeney, A. : loc. cit. * Kauffmann, F.: Zlschr.f. Ilyg. u. Infeklionskrankh., 102, 68. 1924. 114 BACTERIAL ASSOCIATIONS up these divergent and variable results, I feel that they tend to confirm the inter- pretation we have given. The results will depend on many factors such as the rela- tive ability of each bacillus to attack the salts of the organic acids, the effect of al- kaline reactions, and others. THE HEMOGLOBINOPHILIC GROUP The association of bacteria with the B. influenzae group has received unusual at- tention. The discussion of this part of the subject, even briefly, would take all my available space. I must merely observe that it has been studied from the earliest days of the discovery of the B. influenzae and gives striking illustrations of the importance of the subject. Many bacteria help the growth of B. influenzae on media, otherwise inhibitory, such as human blood agar, but they have little or no effect on properly heated blood agar. These beneficent bacteria are most variable in their characters, and include many listed as highly antagonistic such as B. pyocyaneus, an organism around whose antagonistic activity there has collected a comprehensive literature. B. influenzae will grow on hemoglobin-free media in association with many bacteria, although Putnam and Gay' and others were unsuccessful in doing this, and the im- portance of this and the possible bearing it may have in infection have been repeated- ly stressed. Eggerth^ was able to grow B. influenzae in plain broth within a collodion sac immersed in cultures of staphylococcus, streptococcus, or pneumococcus and sug- gested this method as useful for studying symbiosis of bacteria while keeping each in pure culture. It would be of great interest to learn if toxin production may be permanently increased by certain of these combinations. Certainly the virulence is readily raised by mixed injections, as Yanagisawa,-5 Hudson" and others have shown. I will not attempt to review the literature because this has been done by me^ and by Kristensen.^ I do not believe there has been anything new on the fundamental ideas of bacterial associations with B. influenzae since those reports. I cannot leave the subject of influenza without mentioning the importance OHtsky and Gates,^ in a long series of articles, have placed on the relationship between the B. pneumosintes and secondary infections. I may say that their controls were quite inadequate to justify the conclusion that this organism is more potent in encouraging secondary infections than a host of others. B. ANTHRACIS In anthrax it has been repeatedly shown that combined injections frequently prevent infection. I have found that guinea pigs did not die after large injections of washings from soil contaminated months before by B. anthracis in the slaughterin;^ of ' Putnam, J. J., and Gay, D. M.: /. Med. Research, 42, i. 1920. ' Eggerth, A. H.: /. Biol. Chem., 48, 203. 1921. 3 Yanagisawa, S.: Kilasato Arch. E.vper. Med., 3, 85. 1919. ■I Hudson, N. P.: /. Infect. Dis., 34, 54. 1924. sHolman, W. L.: Studies on Epidemic Influenza, p. 161. University of Pittsburgh, 1919. '' Kristensen, M.: IlaemoglohinopltUic Bacteria. Copenhagen, 1922. (In English.) 'Olitsky, P. K, and Gates, F.L.: J. A. M. A., 74, 1497. 1920; 78, 1020. 1922; 81, 744, 21 19. 1923; /. Exper. Med., 33, 125, 361, 373, 713. 1921; 34, i. 1921; 35, i, 553, 813. 1922; 36, 501, 6S5. 1922; 37. 303- 1923- W. L. HOLM AN 115 a diseased cow, but gave typical results after injection of the cultures isolated from the soil. I have collected the literature on this subject, but it will not be included here. The explanation of the results rests largely on the fact that the anthrax spores are phagocyted by the leukocytes attracted to the site of injection by the other bacteria before they start developing and then are destroyed or eliminated. A most important discussion on the principles of such infections may be found in an article by Bail.' GAS SYNERGISM Holman and Meekison^ reported certain findings in gas production by bacterial synergism and have reviewed the literature on this phase of bacterial association and attempted to show that inhibition and stimulation are both based on the combined metabolism of the bacteria in the mixtures and that the same bacterium may use various methods in acting on the substances offered, depending on the environment. Sears and Putnam-' and Castellani^ have given many instances of its occurrence. Castellani has further discussed the whole subject of close association of different species and particularly its importance in the causation of certain diseases and their symptoms. He does not, however, review the literature. ALTERATION BY ASSOCIATION Dissociation of bacteria has been gradually attracting more and more attention, and since bacterial association is so important to appreciate the analysis of the re- sults obtained, a word or two may be added here. I have already given a number of examples of important alterations in the biochemical activities of certain bacteria living in association with others. Sometimes such changes were lasting on transfer, at other times fleeting. Rosenow^ claimed that he was able to change a hemolytic streptococcus to the viridans type by growth in symbiosis with B. subtilis. This P was unable to confirm, and I have reviewed the literature on the longevity of strepto- cocci in symbiosis and have shown the many chances of error from mixed cultures, particularly with closely similar forms, Pneumococci may live in intimate contact with non-hemolytic streptococci for long periods, and the demonstration by Alivi- satos' of the occurrence of mixed colonies of streptococci with staphylococci gives ad- ditional weight to the importance of such sources of error. The more sensitive organism will die out first, and retests may give quite different results from those found with the mixture. This danger was also emphasized by us^ for the gram-negative group of aerobic intestinal bacteria. Nevertheless, bacteria are living, reactive beings and as such are subject to alterations of many kinds. Bail' has shown how important this is in the infectiousness of bacteria (the change to the so-called "animal form"), and 'Bail, O.: Ztschr. des. ges. Expcr. Med., 50, 11. 1926. ^Holman, W. L., and Meekison, D. M.: loc. cit. 3 Sears, H. J., and Putnam, J. J.: /. Infect. Dis., 32, 270. 1923. ■• Castellani, A.: Brit. M. J., 2, 734. 1925; Proc. Soc. E.xper. Biol. & Med., 23, 481. 1926; J.A.M.A., 87, 15. 1926. 5 Rosenow, E. C: /. Infect. Dis., 14, i. 1914. * Holman, W. L.: ibid., 15, 293. 1914. . * Holman, W. L., and Meekison, D. M.: loc. cit. ' Alivisatos, G. P.: loc. cit. » Bail, O.: loc. cit. ii6 BACTERIAL ASSOCIATIONS similar adaptive alterations are to be expected in the growth in mixed cultures. How fundamental these changes may be is not as yet determined, and the study of dis- sociation should help us in our knowledge of the possible range which may occur in the bacteria as they are grouped by our present-day rather crude methods of classifi- cation. Bacteria may be forced to metabolize substances in a different way under certain environmental conditions than they would under other conditions, and this induced, but not necessarily new, function may become fairly well fixed by repetitions as a characteristic of the organism. In looking over the examples already given there are many instances to be found of such occurrences apparently brought out by bacterial association. LommeP has given an important instance of this. By growing a non- saccharose fermenting B. coli with B. typhosus, B. paratyphosiis, or the Shiga bacillus after some twenty passages it took on the function of actively attacking saccharose. She does not say how long this induced activity continued under non-associative con- ditions, as only a plate and agar slant intervened between the tests. She has, however, shown that certain B. coli lost their ability to ferment lactose by continued growth on malachite-green media and that this loss remained constant for some, but not for all strains after fifty-six transfers on plain agar. OTHER APPLICATIONS Passing by numerous other well-known examples of bacterial association such as that of B. fusijormis and Spirocheta vincenti, which Rukawischnikoff^ and others look upon as only stages in the growth cycle, and many mixed infections occurring in man and animals, I would give the interesting use made of this phenomenon by WoU- man.-' He used B. coli as an indicator to determine proteolysis by bacteria previously grown in horse serum, egg albumin, and similar substances through its ability, after such primary growths, to produce indol. He thus tested B. anthracis, B. subtilis, Staphylococcus aureus, and B. putrificus and later^ determined the proteolytic activity of streptococci by this method. Thompson^ was enabled by a symbiotic method with B. proteus to isolate an anaerobic B. acwe-like organism from cultures of B. tuber- culosis and suggested its relationship with the latter. I believe it might well have been present as a contaminant and brought to light by this technique. DISCUSSION Before closing I would call attention to the necessity of determining more care- fully than has been done the intimate metabolism of the bacteria we are studying and the environmental requirements necessary for the manifestation of their manifold characteristics, before any attempt is made to explain the phenomena I have re- viewed, those of bacterial association, or the active metabolism shown by the bac- teriophage. It is well known that d'Herelle^ considers the bacteriophage a living, sub- ' Lommel, J.: Compt. rend. Soc. de bioL, 95, 711, 714. 1926. ' Rukawischnikoff, E.: Ccntralbl.f. BaktcrioL, I, Orig., 100, 218. 1526. ^ WoUman, E.: ConipL rend. Soc. de bioL, 82, 1263. 1919. ^Wollman, E.: ibid., 87, 1138. 1922. 5 Thompson, E. T.: Lancet, 2, 1S6. 1920. •^d'Herelle, F.: The Bacteriophage. 1922; Immunily in Natural Infectious Disease. 1924; The Bacteriophage and Its Behavior. 1926. Translated by G. H. Smith. WilHams & Wilkins. W. L. HOLMAN 117 microscopic form, a strict parasite of bacteria, and therefore the most striking example of microbic association. This is discussed in other chapters of this book. There have been two main explanations offered in the interpretation of most of the phenomena of bacterial association which I have mentioned. The first is the effect of changes in reaction. Usually one of two organisms in a mixture may produce un- favorable H-ion concentration for the continued metabolism of the other organism, and indeed the latter bacterium may be killed. At other times these reaction changes may only alter the degree or kind of metabolism taking place, or the change may be of benefit to one or both. The other explanation is the production of enzymes of various kinds which directly affect the second organism in a mixture. This is undoubtedly a prime factor in many of the examples I have cited. A distinct advance has been made toward other explanations for the facts given above by M'Leod and Gordon' in grouping bacteria under their relative sensitivity to, and power to produce, hydrogen peroxide and a corresponding catalase. Certainly, if we take their table, with due consideration for variations in different strains we shall find a rather satisfactory explanation for many of the antagonistic and beneficial re- sults of bacterial associations. Burnet^ has analyzed many of these relationships, and his contributions have further made clear their application to our study. Bacteria sensitive to hydrogen peroxide will be inhibited by the presence of a strong hydrogen peroxide producer, and an organism with a well developed catalase production will assist another which forms much hydrogen peroxide to which it is itself sensitive. Anaerobes are very sensitive to this substance, and therefore a variety of aerobes producing catalase will benefit them in this respect as well as do those strongly aerobic forms which help the anaerobic conditions, A number of workers have stressed the role of carbon dioxide in the inhibition phe- nomena of bacterial cultures. Sierakowski and Zajdel' were able to show, by sealing cul- tures of various bacteria, that the H-ion concentration alone did not account for the growth inhibition, but they believed it due to the retention in the tubes of carbon diox- ide. Valley and Rettger-" showed that increasing amounts of CO2 raise the acidity and lessen the oxygen tension, and that the complete absence of CO2 in the atmosphere stops the growth of many bacteria. Other bacteria such as B. acidophilus were bene- fited by an increase in the atmospheric CO2. I^ have shown that anaerobes will form surface colonies on solid media in experiments in which their own gas production dis- places the fluid medium in inverted tubes. This may be a factor in wound infections. A further valuable contribution to our knowledge of these mutual relationships is to be found in the work of Gordon and M'Leod^ on the inhibition of growth by some amino acids. They found that the effect differed markedly as tested on different bac- teria, B. coli and staphylococcus were not at all affected while other more delicate ' M'Leod, J. W., and Gordon, J.: /. Palh. cr Bad., 26, 326. 1923. ^ Burnet, F. M.: Aiislnilian J. Exprr. Biol, iy M. Sc, 11, 65, 77. 1925; J. Path, b' Bad., 30, 21. 1927. ^ Sierakowski, S., and Zajdel, R.: Compl. rciid. Sac. dc hioL, 90, 1108. 1924. '•'Valley, G., and Rettger, L. F.: Ahstr. Bad., 9,344. 1925. sHolman, W. L.: Illinois M. J., 35, 289. 36, 10. 1919. ^ Gordon, J., and M'Leod, J. W.: /. Path, of Bad., 29, 13. 1926. ii8 BACTERIAL ASSOCIATIONS bacteria were. Tryptophane, as an example, is most toxic and affects the widest variety of bacteria. Indol, a deaminization product of tryptophane, may account for the tox- icity of tryptophane since it is more toxic than carboUc acid. Serum prevents to a de- gree these inhibitory actions. Others are beneficial, such as taurine, aspartic acid, and alanine. It is readily seen that we have here an additional explanation for certain " of the phenomena being considered. Various bacteria will produce amino acids harm- ful or beneficial to others, and on these products will depend the effect on the asso- ciated bacteria in proportion to the relative sensitivity of the latter. The marked in- hibitory effect of certain proteolytic bacteria on agar plates against the nearby col- onies of other bacteria and the stimulating effect at some distance may well be due to differences in diffusibility of the products formed. There are also, of course, direct actions of proteolytic enzymes on the associated bacteria in certain cases as already suggested, and the lack of agreement in many of the examples quoted above may well be due to different factors having been at work. There is a pressing need to correlate the wealth of available material on the various products formed by many bacteria and the effect of these on associated forms. The work of Koser' and many others on the utilization of the salts of organic acids by bacteria makes clear other groups of phenomena and assists in the explanation of changes in H-ion concentrations as these occur in the reversed reaction of single forms and in bacterial associations. The rel- ative rapidity of growth of two forms, the continual alteration in their activity and sensitivity, the adaptability of bacteria to the form of food material offered at dif- ferent stages and under different conditions as aerobic or anaerobic, the changed metabolism under acid and alkaline influences, and a host of other factors determine the resultant metabolic products of mixed cultures. The analysis of these factors helps in understanding the metabolic processes involved. CONCLUSION Bacterial association occurs under natural conditions, and it plays an important part in many infections. At one time certain resistant but relatively harmless forms may ward off the body defenses and allow a more sensitive microbe to become es- tablished. At other times the reverse may occur and antagonistic bacteria may, and no doubt frequently do, prevent numerous infections. We have in bacterial associa- tion, then, a means of studying many natural phenomena and, as has been indicated, we touch on many fields of bacteriological study. In the routine bacteriological diag- nosis it must be always in mind that there are antagonisms in our media and methods for preventing them; that pure cultures are essential, and that curious results occur from mixed ones, often quite different from those with either culture alone; that the beneficent associations are to be found and that it must be realized that bacteria com- ing from varying environments may have been under inhibitory or stimulating in- fluences which alter the results obtained in our test tubes. In artificial animal in- jections and in natural human and animal infections and diseases bacterial associa- tions as seen in lowered or raised virulence or pathogenicity; the presence of secondary infections from the animal itself leading to faulty conclusions; the changes in bacterial flora as the conditions alter, as seen in war wounds and intestinal infections; and many ' Koser, S. A.: /. Bad., 8, 493. 1923. W. L. HOLMAN 119 similar considerations must be thought of to avoid error in interpretation of results. In the study of bacteriophage and bacterial dissociation the same considerations are needed. I would finally urge, after reading Claude Bernard's Introduction to the Study of Experimental Medicine^ and in attempting to get working hypotheses for the phe- nomena of bacterial association, that more and more attention be paid to the physi- ology of bacteria as reactive, living beings with as complicated metabolisms as our own, and that the study of their pathology, if such we may call it, requires this pre- liminary knowledge of the normal limits of their physiological activities, alone and together, in the test tube and in the animal body. Thus we may be able to understand better many phenomena which at present cause confusion, and may better appreci- ate the basic principles in many of the biological activities of bacteria. ' Bernard, Claude: op. cit. Translated by H. C. Greene. Macmillan Co., 1927. CHAPTER IX CLASSIFICATION OF BACTERIA ROGER G. PERKINS Western Reserve University Bacteriological literature is crowded with classifications of bacteria of every sort, prepared by thoughtful workers, with painstaking labor. The literature is also crowd- ed with criticisms of these classifications for reasons which seem good to each author, though at times he is alone in his opinion. Therefore all I dare attempt is a summary of the principles under which these classifications and these honest criticisms have been made, and to present them as far as possible in their relations to one another and to the problem as a whole. Two main points of view are represented, quite distinct, although closely related. The ordinary student of bacteriology is chiefly concerned with the grouping of bac- terial forms to establish their general relations, involving the possibility of keys which shall enable him to place any new discovery in its approximate place. The taxono- mist, the specialist in bacterial classification, concerns himself further with the selection of the correct names for the divisions, and the evaluation of the proper sequence of order, family, genus, species, and variety, with an eye to the future. Stiles (1927), in a recent address, spoke of taxonomy as the grammar of the science and emphasized its essential value. He comments on the present status that "it has been the excep- tion — not the rule — that pupils who study zoology have been taught the grammar of the technical language they are called upon to hear, read, write and speak." This is no less true in bacteriology, and the student who does more than skim the subject of classification outside his own particular interests is rare. This is unfortunate, but perhaps if the grammar were less chaotic it would have more students. Taxonomists, not only in bacteriology but in general zoology and general botany, live in two camps : those who believe that for many reasons all names referring to in- dividuals, varieties, species, etc., should follow the Linnaean law of priority, laid down in 1 751; while the other camp holds that inasmuch as Linnaeus knew only what was known in 1751, and since so much totally new information regarding classification is available, we should no longer be restrained by the dead hand, but should be free to express ourselves. Stiles (1927) presents sharply his idea of these alternatives. First let every [zoologist] adopt any technical name he wishes, or second let us all a^ree to follow the Linnaean law of priority. The first alternative is subjective and leads to confusion, the second is objective and makes for uniformity in all objective cases .... In general I would evaluate a failure to apply the law of priority as the second most important formal factor in nomcnclatoriul confusion. And ])crhaps llic extreme in the other camp is Enderlein, with a com]i]ete new classifi- cation, and indeed a com|)lete new language. Neither side, of course, refuses lo admit new titles, new definitions, and new grou[)ings, nor does it liesitate to accept the iacl that our knowledge is progressive rather than fixed, l)ut the rules of the game differ. 120 ROGER G. PERKINS 121 On the whole it seems as though, in spite of its defects, the work of the last twenty years has established sufficient recognized groups, especially of the higher ranks, to enable us, with the appreciation that bacteriology is a young subject, to develop these rationally, without any complete upset. At present there are certainly sufficient shades of difference between the extremes to accommodate anyone. Another important controversy, more marked perhaps among the adherents to Linnean priority, centers around the question whether a name should be descriptive, giving some indication as to character or the group or individual, or whether this is un- important, and the name, if historically correct, should be retained even if wrong or confusing. Enlows stated in 1920: It is to be hoped that the many inadequately defined genera here listed may serve as glowing examples of errors to be avoided by future contributors. A plea is made, too, for the introduction of generic names which are descriptive, since many names of this sort define, and in a way, classify. Proper names converted by the addition of -iiica, -cUa, etc., are very alluring because of the acknowledgment of the debt we owe our leaders, but they are not de- scriptive terms, and offer no aid whatever to any system of classification. On the other hand, the Committee of the Society of American Bacteriologists stated in 1917 that "the name need not be appropriate, it need only be stable. It is an ar- bitrary description." Stiles stated in 1905: "It is essential to recall that names are not definitions; they are merely handles by which objects are known." These last indicate the majority opinion, save in the more independent classifications, such as Orla-Jensen (1921), Enderlein (1925), and others, where the attempt at descriptive titles is prominent. In connection with Enlows' {loc. cit.) remarks about the eponymic groupings, it is interesting to note that in medicine there is a growing tendency to- ward correctly descriptive names for diseases, with corresponding abandonment of proper names. It seems as though neither the apologists nor the higher critics are satisfactory or satisfied, nor is it surprising in a science in formation. The essential contention ap- pears to center about the proper, relative proportion of a conservatism which objects to the removal of well-known signposts, partly on the basis that their familiarity more than overbalances their misdirections, and a liberalism which wishes to accept only such names and descriptions as fit our present knowledge. What shall be the mixture and who shall mix it? It is stated with much force of logic that constant change in names and in qualifi- cations is confusing, and that even now the bacteriologist has to have at least one key, if not more, at his elbow when he reads his literature, and that historical articles will soon be quite unusable, a mere hieroglyphic literature, understandable only by a few. It is true also that if in the last thirty years or less the discoveries have been such as to invalidate old classifications, what warrant have we that the next thirty years may not repeat the process? Further advance in technicjue may reveal details of form yet unknown to us, may crystallize our information, and may yet uncover unsuspected biological relations. On the other hand, it is clear that at present our known details of morphology of any degree of constancy are numbered, and that chemical and phys- ical differentiations have become more and more stabilized within certain group lim- its. Although it may be true that new information may and probably will necessitate 122 CLASSIFICATION OF BACTERIA changes in another quarter-century and even in a decade, it is likely that the new in- formation will not be morphological. When acknowledged experts disagree widely on so complex a subject, an ordinary bacteriologist like myself must avoid as far as possible technical taxonomies end ex- tensive references to authority, of which there is a sufficiency so arranged that any diligent reader can make his own evaluations. To the general reader the main lack is in summaries of opinions which make it possible to omit the individual technical articles. The recent classifications and keys due to the industry of Bergey (1923), Buchan- an (1916-25), and the Committee of the Society of American Bacteriologists (191 7- 20) with the critical summaries of taxonomic authority by Buchanan (loc, cU,), En- lows (loc. cit.), and others make the entire history of the development of classification readily available. One should note also that not the least service of such attempts at complete classification of a series of organisms, concerning which the authors them- selves are the first to admit our information deficient, is their courage in presenting their plans and ideas for criticism. Unless broad plans of this sort are accessible, re- vision is impossible, since most of us are more particularly concerned with some re- stricted portion of the whole. It is far easier to criticize than to construct, but only out of a combination of construction and constructive criticism can we hope to reach stability. At first the whole affair looks extraordinarily discouraging. Each attempt at im- provement meets with a volley of objections (Hall, 1927), and such attempts as seek to break away from conventions of taxonomy have few adherents. Closer study, how- ever, offers more hope. One finds that there is a great deal of acceptance of main groups, and that most of the trouble comes from disagreement as to the rank of these, and from persons studying a comparatively small group and believing that their ar- rangement is the only proper one. To my mind the first essential, as brought forward by many, and ignored by as many more, is that bacteria in comparison with higher plants and with animals are notably unstable, and that such conformity to type as we see in pure cultures of Plym- outh Rock chickens, or white rats, or Lima beans is not to be expected. The more we study them, the less stable appear the characters on which we base much of our classification. If zoologists find, as they do, that complex multicellular organisms are altered by environment to a point where, had they not been followed through, they would be thought different genera, and the botanists present similar evidence, how much more may we expect environmental modifications of a temporary or permanent character among bacteria? One must admit, of course, that unicellular organisms which divide by simple fission carry heredity in a manner very different from that of more complex forms, each half being supposedly identical with the other; but recent work in variation and in mutations in single-cell cultures has forced a modi- fication of those ideas. There is no a priori reason why a colony of anthrax might not undergo in nature conditions similar to those by which we modify it artificially. Evi- dence that the citrate-using B. coli preceded or followed the non-citrate-using is not yet conclusive. We are told that the development of terminal flagella on a coccus tends to change it to a rod form, and we find that an organism may be changed from ROGER G. PERKINS 123 a gram+to gram — by the use of suitable chemicals. Winslow quotes Wolf as re- porting "a considerable number of temporary modifications and some permanent in- heritable ones stimulated by exposing bacteria to the action of chemicals. White and dark-red strains were thus produced from a normal B. prodigiosus, the resulting modi- fications breeding in each case true to their new type." Winslow calls these changes "impressed variations," and further on says: The fact that all cells are potentially reproductive removes any bar against the inherit- ance of acquired characteristics. Again, the absence of sexual reproduction must operate to preserve variations which arise from within or without .... with fission as the normal mode of reproduction every variation which can arise can be handed on unchanged; .... there are sharp limits to the variability even of the bacteria, and for practical purposes we find the larger groups quite constant in their general properties .... in part at least I am inclined to believe that this is due to the direct or selective action of similar environmental conditions. Since we are dealing not only with variables, but with variables in a group of or- ganisms susceptible to permanent and hereditary change, we must select as most im- portant those characters which as nearly as possible approach constants, and which seem least susceptible to these permanent changes. One method of approach is well exemplified by the work of Winslow (1906) in his study of the Coccaceae, and the work of Hucker (1924) in a similar study. Believing that improvement might result from a statistical analysis of a variety of characters in the hope of determining cor- related groups, these extensive and painstaking studies were undertaken. The fact that the authors are often in marked disagreement shows that the method of pro- cedure, while suggestive, is as yet unsatisfactory. We must not forget that the actual number of bacteria described is but a small fraction of those which exist. This is of course true in botany and zoology as well. Stiles (1927) states that there are hundreds of thousands, possibly millions, of genera and species still to be given technical baptismal certificates. Aldrich (1927) quotes Horn as saying; "Whoever as an entomologist looks into the future knows full well that we are steering into a shoreless sea, no matter whether he estimates the total num- ber of insect species at three, ten or fifteen millions. In the near future any beginner will be greyheaded before he has caught up with what is already known." Although we have not quite reached this status with bacteria, partly because the details of struc- ture and other characteristics are so much less complex, it is already practically im- possible for anyone to be an expert in all the lines of bacteriology, and especially in their finer taxonomic relations. The student in medicine interests himself in the non- pathogenic forms only in their relation to the pathogenic (many early classifications, such as Fliigge, were of pathogens only), and even neglects the organisms relating to those diseases of animals that are not transferable to man, to say nothing of those which cause disease in plants, while the veterinary and the plant bacteriologists are similarly exclusive. It is only very recently that the workers on filterable viruses have appreciated that the botanist, the zoologist, and the medical biologist are working on an identical problem and that in their combined effort may lie the key to success. Students of the non-pathogenic organisms naturally tend to the study and iso- lation of those groups which have economic relations with soil values, with fermen- tations, with decompositions, and consider all others as merely annoying contami- 124 CLASSIFICATION OF BACTERIA nations. Yet new perfections in technique constantly cast new organisms on the shores of the bacterial sea, where they become accessible. Workers with the anaerobic group tell us that it is c^uite possible to describe totally new organisms at the rate of several a week, and it is only when someone gets interested in a special problem that we get a list of the organisms concerned. It is clearly impossible to foresee new discoveries beyond what may be safely prophesied from analogy, and the best we can do is to make and preserve a scheme which we believe will act as a framework on which to hang not only the groups we have, but those which may turn up from time to time. It seems clear, then, that in common with students of other biological groups we have reached only a small fraction of the varieties which make up the mass, and that it is logical to believe that the changes in our knowledge and beliefs which have fol- lowed the lights of informative changes in technique are only an earnest of what is to follow. Bacteriology, as we now understand it, is less than fifty years old, and inas- much as bacteria are either plants or animals, it fell heir to all the taxonomic litera- ture relating to both. The discovery of the principles of pure-culture study resulted in such a sudden burst of investigation that it was a lost month in which a new organ- ism was not described, catalogued, and laid away, very frequently in the wrong grave. With this introduction we can enter upon a discussion of the criteria used in past and present times to establish the various groups from order to variety with an at- tempt at a critical summary of modern opinion, BASES OF CLASSIFICATION In the minds of many, perhaps most persons, the actual definition of the words used in taxonomy are vague, and some definitions may be helpful. Inasmuch as the "genus" is the center, so to speak, the grouping which brings together a collection of species, and itself forms the base for larger groupings, let us begin with it. Agassiz de- fined genera as "most closely allied groups of [animals] differing .... simply in the ultimate structural peculiarities of some of their parts." The Century Dictionary (iQoi) speaks of it as a classificatory group ranking next above the species, containing a group of species (sometimes a single species), possessing certain structural characters differing from those of any others. The value assigned to a genus is wholly arbitrary, that is, it is entirely a matter of opinion or current usage what characters shall be considered generic .... a genus has no natural, much less necessary, definition, its meaning being at best a matter of expert opinion, and the same is true of the species, family, order, class, etc. A genus of the animal kingdom in the time of Linnaeus was a group of species approximately equivalent to a modern family, some- times even to an order. Stiles {loc. cit.) defines it as "a taxonomic complex of specimens grouped for the mo- ment (according to our subjective and never absolutely perfect knowledge) around a genotype." These definitions emphasize that it is impossible to lay down rules which will hold future experts, at least beyond a certain general plan, and also show why it is that in so many cases the desire for revision starts with the study of a genus, and later in- volves its position in connection with other genera. Perhaps one of the most important points in the description of genera is the selec- ROGER G. PERKINS 125 tion of the genot>'pe. If this is properly done, the personal equation involved in the selection of various group characters for inclusion is checked and crystallized by the selected type example. This has long been theoretically necessary, but has been much neglected. Buchanan {loc. cit.) and Bergey {loc. cit.) have been the most consistent, not only in their demands for this, but in actually supplying the types. There are five main headings which influence classification. Beginning with the earliest, "Morphology," we have added "Chemistry and Physiology," "Evolution," "Habitat," and "Immunology." The most successful work has been accomplished with the first two. MORPHOLOGY In the beginnings of biological classification, this was the only basis, the alpha and omega, and until discoveries multiplied was quite satisfactory. But as more and more individuals were discovered, groups became very large and needed further or differ- ent subdivisons. Under the head "Morphology" we generally admit not only form and arrangement, including organelles, capsules, and spores, but also tinctorial dis- tinctions such as gram stain, acid fastness, and granule formation, though of course these might also be considered chemical. But even with these admissions the number of distinctive characters is limited. Moreover, as noted earlier, even form is unstable, and subject to temporary or permanent modification which may readily become in- herited. But whatever may be the changes in morphology under extraordinary conditions, we must still admit that under ordinary conditions, or under readily obtainable stand- ard laboratory conditions, a rod form remains a rod form, a sphere continues as a sphere a spiral form clings to its spirals. Moreover, a coccus with the habit of dividing in certain planes tends to retain this habit with some obstinacy, and a rod form which after division tends to retain two or more individuals in a chain may be reasonably expected to continue this activity. What constants may we add to form and planes of fission? Obviously, the most conspicuous are motility and spore formation. Migula (1897-1900) went so far as to make a major division on the basis of mo- tility, speaking of the motile form as "bacillus," the non-motile as "bacterium." Al- though this was rather widely accepted for a time, it finds little support at present. Leaving aside the question as to whether motility is a higher or lower divisional point, there is marked variation in this character, to such a degree that insistence upon it will make wide separations of closely allied strains. In this group, moreover, not only is the actual motility a differentiating feature, but inasmuch as in the Eubaderiales (Buchanan) motility is dependent on flagella, the arrangements of these may also be important. Orla- Jensen's two orders are based on such arrangement, separating those with flagella at the end from those with flagella all around. Breed and others (191S) state: In passing, it is of interest to notice that there is a close analogy between the generally recognized groups of bacteria and those of protozoa. Thus the cephalotrichic and peritrichic bacteria find their analogies respectively in the flagellates and ciliates. This analogy goes further than a mere resemblance in the arrangements of organs of locomotion ; for the ciliates and peritrichic bacteria are both highly specialized grc ups, while both flagellates and cephalo- trichic bacteria contain all gradations between primitive forms and highly specialized human parasites. 126 CLASSIFICATION OF BACTERIA I must confess, however, that on tabulating the one group against the other, save for the fact that more of the peritricheae are pathogenic, the evidence of superiority ap- pears inconclusive. It does not seem to me that our knowledge of bio-chemistry is sufficient to show that the power to utilize or break down certain chemical compounds proves more or less "advancement," and the analogies with the protozoa, while un- questionably fascinating, seem to me to stop at that point. Without plunging too deeply into controversy, one may say that the division on the basis of spore formation is logical, as showing a major activity, and is widely ac- cepted, though the resultant group is variously placed. Some (Table I) accept this character as a basis for families under the Euhacteriales , others prefer tribal divisions under the family of rod forms, others as divisions of even lower rank. Classifications such as Orla-Jensen's (1921), fundamentally based on physiology and chemistry, are much less interested in spore formation. How far should the differentiation by morphology carry? It seems to me that this basis can certainly carry us as far as genus, and Hall (1927) goes further, saying: 'T submit that morphologic criteria should enable us to identify the genus, and I be- lieve that the definition of orders, families and genera should be based solely upon morphologic data." With this sentiment many but by no means all agree. CHEMISTRY AND PHYSIOLOGY The most extensive changes have been due to attempts to use the activities of the bacteria rather than their form and arrangement as differentiating factors. The changes have ranged from a practically complete substitution, such as Orla-Jensen's (after the original flagellar division), to studies of small fractions of the problem. The general tendency of taxonomists is to emphasize morphology in the larger groupings and to reserve biological activities for smaller collections. Pigment formation, prob- ably the earliest observed of the definite chemical activities, has been graded all the way from family to variety, sometimes in connection with other group characters, sometimes practically alone. No very adequate reason has been adduced why groups of chromogenic organisms with different metabolic activities should be classed by their pigments rather than distributed according to their other characteristics. It is true that we have a very moderately studied collection of pigment-formers with little else by which to classify them and that this procedure may be temporarily convenient. But why the power to use fixed oxygen rather than free oxygen, or to break down sug- ars, or to form complex toxins or proteolytic ferments is not as remarkable as the power to form pigments, has never been demonstrated to my satisfaction. Hucker {loc. cit.) states in regard to the cocci: It seems evident that pigment production by the mass-forming cocci is a very important character for use in classification. However, due to its general lack of correlation with other characters, it does not appear that the group should be divided into genera with differences in chromogenesis as the chief diagnostic feature. On the other hand it seems that pigment production is sufficiently constant to warrant its use as a character of importance in differ- entiating species, and when so used should be interpreted along broad lines rather than to attempt to make fine distinctions in shades of color. In the same way the "nitro-" bacteria, placed as high as a family by some, a sub- family or tribe by others, seem to be getting more attention than they deserve. As ROGER G. PERKINS 127 will be seen later, even the arguments for early historical precedence are not generally accepted. In other words, are not most of these chemical and physiological characters even less definite and stable than morphology, and should not their use begin where the criteria of morphology alone are inadequate? It has, of course, been frequently brought out that characters important in one group are unimportant in another, as, for instance, fermentations in the cocci and in the bacilli, or character of division in the same groups. It is probably impossible to formulate as a rule whether a given character should always define species or confine itself to varieties. HABITAT AS A DIFFERENTIAL POINT: PATHOGENICITY "Pathogenicity may be taken as a type of those powers of the organism which are easily and profoundly modified by external conditions." This quotation from Wins- low in 1906 seems incontestable, yet later he uses "habitat" in the separation of his genera in the Coccaceae. This seems rather inconsistent even though he insists upon groups of characters rather than individual ones. Here, again, there is marked dis- agreement, as Hucker differs from Winslow in a similar paper, as follows: Due to the fact that the micrococci are found in such a wide variety of sources, and due to their ability of adaptation to various environments, the use of habitat alone as a char- acter in separating the general group is precluded. It is true also that no other character will definitely correlate with the source of the different types and bear out any conclusions that different natural groups of micrococci can be secured from different habitats. It would seem that if this applies to this group, other groups in which the same ob- jections hold true would be similarly affected. It has always seemed to me that the selection of pathogenicity as a prime factor of division is only part of the pride of the human race in its superiority, a pride which has from time to time admitted some of the lower animals under the wider cloak of animate, moving life. In the consideration of bacteria, however, why should one metabolic activity be superior to another? We believe that pathogenicity is more or less of an accident, that bacteria are not toxic in order to be pathogenic, but pathogenic because they happen to be toxic. Because some by-product of digestion when applied to a mucous membrane causes death and breaking down of the local cells, whereas other products of digestion can break them down only after cell death, may be a matter of degree rather than character. Non-bacterial poisons may have the same effect, but are not classified on this basis, but rather on the general character of their possible chemical combinations. Should we not therefore consider the chemical activ- ities of bacteria, due to metabolic products with various chemical characters, rather on the basis of these characters alone than on some special reaction, which is, as Wins- low (1905) calls to our attention, readily modifiable by environment? Breed, Conn, and Baker {loc. cit.) criticize the Society of American Bacteriologists' Committee (1917) in that "too great weight has been placed on pathogenicity," attacking special- ly Hemophilus, Pasteurella, and Erwinia. It seems to me dangerous in a general clas- sification to say that a group is generally, or usually, or essentially parasitic, in view of the well-known fact, already recalled, that our knowledge of bacteria is confined to a very small fraction of the total in existence, and since scarcely any groups which contain pathogens do not include forms closely related but without pathogenicity. 128 CLASSIFICATION OF BACTERIA Can we determine habitat by a limited series of findings? Any organism that requires oxygen, moisture, warmth, and broken-down organic matter may develop on the surface of the body and remain there for various periods of time. It will also survive or develop anywhere else under similar conditions, but would not be called parasitic if found on a filthy blanket, HABITAT OTHER THAN PATHOGENIC The same general criticisms are applicable. It is unquestionably valuable to know that most of a group have been isolated from water or soil or mucous membranes, but the connotation to the average reader is that there is some inherent relation be- tween the organisms and the particular environment. And this may or may not be the case. This is a dififerent question from that of taxonomic names, and whether such a definition is misleading or not is a matter of sharp controversy. CHRONOLOGICAL CLASSIFICATIONS Orla-Jensen (Joe. cit.) and Kligler (1913) have presented more or less elaborate classifications on this basis, and a large part of the former's grouping, especially that relating to the nitro-bacteria, has been taken over by the Committee of the Society of American Bacteriologists. Breed, Conn, and Baker {loc. cit.) make a well-consid- ered attack on both these systems, emphasizing that we are now dealing with end- processes of evolution and not the primary forms except in so far as environmental conditions have remained more or less permanent. (It is likely that conditions even in water are not now identical with those of the period of origin of bacteria.) They quote the Committee in their definition of the Nitro-bacteriaceae, and state that if we ac- cept this family, we indorse the theory that its members are modern representatives of the primordial bacteria. They object to this, and indicate that the theory, while interesting, is without adequate proof. (Buchanan [1918] attacks Kligler's paper even more sharply.) The most logical reason adduced for family rank is the close relation of rods and spheres in this group and the inconvenience of separation. It seems to me also that in this question of primordial relations as a basis for di- vision, too little notice has been taken of the well-recognized fact that characters may be lost as well as acquired. An organism once able to utilize and synthesize simple ma- terials may, under conditions of parasitism or even symbiosis, lose those powers, and in the vast numbers of generations of bacteria it is not far fetched to consider the possibility that strains with certain powers may lose part of them under changed en- vironment, and under subsecjuent changes may either regain what was lost, acquire new powers, or any combination of these two changes. Orla-Jensen shows that it is quite probable that actual changes in form from sphere to rod may have taken place, and botanical and zoological alterations due to environment under short-time ex- perimental conditions are well known. Conclusions drawn from the conditions of light, heat, available food, etc., in those geological periods during which bacteria de- veloped are based on disputable evidence, and must be considered cautiously. IMMUNOLOGY This division of scientific work is still in its infancy, and its application to classifi- cation of bacteria has been unsuccessful, save in the differentiation of varieties, and ROGER G. PERKINS 129 rarely in confirmation of species which are already shown to be in close relation. As an aid to differentiation, for such groups as the pneumococci, its value is high. THE PROBLEM OF A NEW CLASSIFICATION With all these difBculties of a classification based on a variable combination of morphology and chemistry, what are the possibilities of a complete revision? I con- fess it seems to me that unless someone is sufificiently omniscient to foresee the new developments, and to select characteristics which will remain stable for the next hun- dred years there is little hope for such a classification. Man is conservative and would demand proofs of omniscience and prophecy which would be hard to offer. The boldest now in view is that of Enderlein (1925), who believes that morphology is ab- solutely the only important criterion, especially in its development. He decries mono- morphism, mutations, and monocytism, and goes into extensive details as to the minute anatomy of the bacterial cell, with nucleus, sexual and asexual division, re- consideration of the spore, etc. The entire vocabulary is different, so that no com- parison can ])e made. PRESENT STATUS OF BACTERIAL CLASSIFICATION Reviewing the present status of bacterial classification, what hope does it offer for the future? There is practical unanimity in accepting the class Schizomycetes as first brought forward by Naegeli in 1857. Vuillemin gave a good description and Buchanan (1925) modified it somewhat. Vuil'.emin stated: They are simple organisms, formed of a single element without septa and unbranched. The element is circumscribed by a rigid vegetable-like membrane, elastic, but not contractile, sometimes also with a capsule. It may undergo plasmolysis or plasmoptysis. The protoplasm is less differentiated than that of most cells; the chromatin particles do not form an individual nucleus of a permanent type. Division is amitotic. Some forms are motile with flagella which traverse the membrane at points characteristic of the species (polar or diffuse). They are not broader than 5 ix when not in bunches or unimpregnated with metal or colloidal coloring material. The resting stage may be either an arthrospore resulting from a simple modifica- tion of the membrane or an endospore. In some cases the spore-bearing element retains its form or is modified passively by the enlargement of the spore, in other cases it is a specialized element for spore production. Only the latter type of sporulation is satisfactory for generic characters. The amitotic division usually occurs by a pinching transversely with rapid sepa- ration of two individuals. These may remain united into families, in chains, la3'ers or packets as determined by the successive planes of deviation. He emphasizes that all forms showing contractility should be placed in the protozoa, that the myxobacteria constitute a distinct group, and that forms like the tubercle bacillus which show branching should be placed in the mold group Microsiphones. Buchanan used the following characterization: Typically unicellular plants, cells usually small and relatively primitive in organization. The cells are of many shapes, spherical, cylindrical, spiral or filamentous; cells often united into groups, families or filaments; occasionally in the latter showing some differentiation among the cells, simulating the organization seen in some of the blue-green, filamentous algae. No sexual reproduction known. Multiplication typically by cell fission. Endospores are formed by some species of the Eubacteriales [see below], gonidia (conidia, arthrospores) I30 CLASSIFICATION OF BACTERIA by some of the filamentous forms. Chlorophyll is produced by none of the bacteria (with the possible exception of a single genus). Many forms produce pigments of other types. The cells may be motile by means of fiagella; some of the forms intergrading with the protozoa are flexous, a few filamentous forms (as Beggiatoa) show an oscillating movement similar to that of certain of the blue-green algae (as Oscillatoria) . The Committee used the following description: Minute, one-celled, chlorophyll-free, colorless, rarely violet-red or green-colored plants, which typically multiply by dividing in one, two or three directions of space, the cells thus formed sometimes remaining united into filamentous, flat, or cubical aggregates. Capsule or sheath composed in the main of protein matter. The cell plasma generally homogeneous without a nucleus. Sexual reproduction absent. In many species resting bodies are pro- duced, either endospores or gonidia. Cells may be motile by means of flagella. Under the Schizomycetes a number of orders have been proposed, in some of which there is general agreement and in some less. In the discussion following, except where a definite published group is referred to, I shall attempt to avoid the technical terms, using groups, forms, kinds, etc., as well as the common term "bacteria" or "cocci" to indicate rods or spheres. Among the groups generally known as "higher bacteria" and less important to the medical bacteriologist there is a great deal of agreement. Under the Schizomycetes the groups of the sulphur {thio-) bacteria, the sheathed (chlamydo-) bacteria, and the pseudoplasmodial (myxo-) bacteria are fairly well ac- knowledged and contain much the same material the world over, though the rank is not always the same. This leaves us the "true bacteria" {Eubacterlales [Buchanan]) with the border-line groups of "actino-" and spiral forms. Taking up first the border-line groups of thread forms and spiral forms, the sum- mary of their history leading to the present status is about as follows: Actinomyces — In 1877 Harz described the thread fungi in "lumpy jaw" of cattle as "actinomyces" and started the group on its quarrelsome path. Rivolta in i8/3 preferred "discomyces," and various observers (Blanchard [1895], Brumpt [1910], and Merrill and Wade [1919]) agree with him. Streptothrix (Cohn) is accepted by others, but there is apparently reason to believe this term invalid (Buchanan, p. 497), though it still has plenty of adherents. Study of the group brought out the fact that there are two distinct methods of reproduction within the group, and Trevisan in 1899 sug- gested the name Nocardia for those forming spores, as distinguished from the non- spore-forming ''actinomyces,'" a term accepted by Wright in 1904. Buchanan (p. 405) regards it as a synonym. Thus we have four names, each more or less ably supported as the ten pages devoted to the subject by Buchanan indicate. The Committee adopts Actinomycetales as an order, as do also Buchanan and Bergey, while Breed, Conn, and Baker consider them as a family — Actinomycelaceae. Whatever name we prefer and select, new arguments arise as to the contents of the group (see Table I). Some authors under the prefix "actino-" include the thread fungi and also the tubercle-bacillus group and the diphtheria group, others include the same series under the prefix "myco-," while others separate the thread fungi from the "myco-bacteria," placing these last directly among the non-spore-forming rods. The modern tendency, since Lehmann and Neumann, seems to be to place tuberculosis I ROGER G. PERKINS 131 and its associates with diphtheria and its associates and to consider them both as re- lated to the thread fungi which have true branching. It is also the prevailing practice where Nocardia is used to confine this to the special group noted above. It is to be hoped that a better agreement may develop in this group. Spiral fonns. — There has naturally been much contest as to the placing of these. One school places all spiral forms, from the cholera organism to the treponema and its associates, in one group, subdivided into: (c) simpler forms long known to us as the cholera group; and {b) the more "protozoan-like" (if that means anything), including treponema, leptospira, spirochaeta, etc. Another school places this second group in a completely separate division, leaving the others in Spirillaceae {q.v. infra). Some believe the "protozoan-like" group to be intermediate between bacteria and protozoa but it is interesting to find recent textbooks on protozoa omitting it entirely (Hegner and Taliaferro, Craig). (See Table I for details). In general, the first of the two groups corresponds to Spirillaceae Migula (1894) emended Committee, So- ciety American Bacteriologists (1917), with the following description: "Cells elon- gate, more or less spirally curved. Cell division always transverse, never longitudinal. Cells non-flexuous. Usually without endospores. As a rule motile by means of polar flagella, sometimes non-motile. Typically water forms, though some species are in- testinal parasites." The second corresponds in the same way with Spirochaetaceae (Swellengrebel) described by the Committee as follows: "Free living or parasitic spirilliform organisms with or without flagella, with undulating or rigid spiral twists. Reproduction by transverse division and by 'coccoid bodies,' the equivalent of spores." This brings us to the group of true bacteria (Eubaderiales , Buchanan) (see Table I). This vast group of simplified forms, aerobic and anaerobic, spherical and rod shaped, pigment formers or non-pigment formers, fermenters or non-fermenters, mo- tile or non-motile, gram positive or negative, spore-formers or non-spore-formers, and with many other positive and negative characters, has been divided and subdivided on the basis of combinations of these until there is practically no theory which may not find substantiation in published work. The minimum number of divisions under this head is three: the spherical forms, the rod forms, and the spiral forms. Spherical forms. — This group was recorded as Coccaceae by Zopf in 1884 and is the most widely accepted of all, including all spherical forms of whatever arrange- ment, with certain minor and not undisputed exceptions. In spite of possibilities that in the development of bacteria there may be a ten- dency for cocci, especially when there are one or more flagella, to elongate, it seems agreed by most observers that the spherical forms are worthy of a grouping of their own, and the rod forms likewise. If one selects habitat, or fermentation, or color, or motility, or pathogenicity as prime factors, this grouping will at once disappear. Some observers (Orla- Jensen) believe that the chain-cocci and the chain-rods are to be con- sidered together, but their arguments, though interesting, are necessarily based on theoretical grounds, and, as noted elsewhere, our actual, objective knowledge of the evolution of bacteria from the original, whatever it was, is mostly speculative. Whether the modern work on variation will reach a point from which we may argue 1.^2 CLASSIFICATION OF BACTERIA o "-- fi; a < ^ 52:^ S S^ 5 !5y^ i; -^ -* ra — 3^K -C 3 -« ~ s c Ko I'e "H ° 3-c^ ^"2 in -►^ „^ > ■0 • - u c ^ a a< CI) c •^■^ ^ u. It 33 s a D 3 rt M 'C '& a ~ a *-< a '■" a S a ii Si -J a tj ^ >. ^=E < _ 1 OJ 'J ^ ^2 cf a Q - "i |-aSI £8 E S 03 a cl C c a; "ti c: a ,^j a 0) a C a a -0 a c S 1% a< ,^ S c u a^ c III d u "a in a "^ a "^ 2 ft. S 0. 4, 3 rt u to a £ u _a a OJ Q. a ;„ a a s a jj jj " t- u Sen ■pEg 3 u 2 -a ^cco >. n u || §- s M u "i- u C >,J5-^ tf-S5 U a"" g (£0 IS ffi c u ^ £ c OJ a a T3 3 =S D. a. 2 E,2 -§1 1 a 3 a &1 c B 4J u 3 60 1 .2 a g = s ^i2 2'a tcco J3 3 c C 5 , aM to-a3 3 a a CO Z 0,0 IS K c „ 3 c a CO <^ 3 g , a 0— a Lehman and Ne' mann i8g6 Chester 1897 Beneckc 191 2 Castellani-Chai- mers 1919 u oi 0. 3 u g hi ■£.^ en a .a ii a PQ ^^UJE.S E S 5 j;-^ , i2 a^^ 00 _a 5j Ji2 z. S M .5 a 1 it! c c 3 -^ g " „ " M —r rt «l T3 a u '3> rt.2 K - E.2 4) ., ^ S 2 Sternber Migula I Fischer 1 Hein 191 Engler i Meyer i Lohnis i Vuillemi ^■5 a a J! ^'C a b^J S c S 33'? So mm i:i ri ■ S C fc CO c 'o.S 0- c C r^ c. OJ 3 ■U n, .0 a >. g-E oj a §1 V O' ' — ' rt S '-' a " ' til OJ u ^ a " -c a ^^.a ii C tf. ajS 00 CO rt -o bo c W) X ;i^ :3 E a en £ P .. E 3 ■0 c L- c E *a .y; s X! s a 1^ J3 3 1-1 "B -0 0. 3 -fi < a CO 'a CO CO z P^ s £ ROGER G. PERKINS 133 backward is an open question, and in the meantime we find in general that, at least under ordinary conditions, in the laboratory of nature rather than in that of the specialized scientist, the coccus remains coccus. There are two qualifications to the acceptance of a single group for all spherical forms. Some desire to place the so-called ''primitive" nitro group together, both the spheres and the rods, on the basis of sim- ilar physiological characters regardless of form. In a similar manner there are those who believe that organisms of the lactococcus type should be placed with those of the lactobacillus type. In the first report of the Society of American Bacteriologists Com- mittee in 191 7, Lactobacillaceae was recorded as a family, and in the final report it was placed as a tribe under Baderiaceae. Further subdivisions of the family have been made in various ways. Winslow, Bergey, Castellani and Chalmers among recent workers made a primary division on the basis of parasitism, following this by morphology. The Society of American Bac- teriologists follows Winslow closely, but many writers feel that the establishment of habitat, or its subdivision parasitism, is likely to be misleading and does not produce natural groups. Hucker {loc. cit.) considers pigment as a species rather than a generic distinction, and sees no reason for the retention of the red pigment formers as a genus among thp cocci, the rest having already been generally abandoned in this group. In general, all the chemical and physiological characters are best used in the differentiation of species and varieties. Rod forms. — There remains the large group of rod forms about whose classifica- tion there is a more or less consistent disagreement. As will be noted by the chart, the tendency of foreign writers is to group all of these under one head, sometimes with one name, sometimes with another, though Orla- Jensen has made much wider sepa- rations, from which one of the American expansions has come. This is the group of supposedly "primitive" organisms, mentioned in this paper as the "nitro"- forms, accepted with more or less modification by Buchanan, the Committee, and Bergey and criticized by others, as noted earlier in the paper. An additional family, the Pseudo- monadaceae, including pyocyaneus and allied forms, is found as such only in the Society of American Bacteriologists and in Breed's comments (1917), elsewhere hav- ing a maximum rank of genus. Another American expansion is the elevation to fami- ly rank of both the spore-formers and the non-spore-formers, as Baderiaceae and Ba- cillaccae, a proper division, though there is argument as to the rank which should be given these groups. Buchanan and Castellani, in agreement with most of the Euro- pean authors, prefer subfamily rank. The variety of further divisions is unending, and, as noted earlier, far more work has been done on species and variety than on genus and higher ranks. It is almost impossible to summarize within any reasonable space the variety of opinions. Primary difi"erentiations, after or before relation to spore formation, are made on the basis of pigment formation, habitat, morphology, and physiology. Bergey, in his eleven tribes of non-spore-formers, names two on the basis of pigment formation, three on parasitism, five on cultural characteristics, and one on morphology. Buchanan makes two divisions of the same group by morphology, with a second division by physiological needs and further subdivisions on different bases for different groups. Castellani and Chalmers divide their family of Bacillaceae 134 CLASSIFICATION OF BACTERIA into ten tribes, primarily on the cultural characters, or physiological activities. This is sufficient to show clearly how little bacteriologists have been able to agree on evalua- tion of the actual relations of these various characters. I SUMMARY OF "eUBACTERIALES" 1 Within the Eubacteriales the final report of the Committee forms the largest rium- ber of families, and may consequently be used as a basis. The accompanying chart shows the general relations and the variations in content of most of the European and American classifications, except those which depart so far from the prevailing methods as not to be readily comparable. It will be at once noted that the American group is more detailed. One may summarize the chart somewhat as follows: The "spherical" group is generally accepted. The "spiral" group is generally accepted, but the content varies, some writers including both the spirals typified by the cholera organism, and those of the "spiro- chaete" type, ochers placing the latter group separately, usually in an order by itself. The "cylindrical" or rod group is the most varied. Under it, however, certain groups are usually put together, such as the tuberculosis-diphtheria combination, unless this is placed with actinomyces. The "spore-formers" and "non-spore-formers" are usually differentiated, but the rank varies. The "nitro" -bacteria are generally kept together though again with different ranks and occasionally placed with the pyocyaneus-fluorescens group. The "pigment formers" are also kept more or less together. In the various classifications such characters as motility, flagella arrangement, gram stain, fermentation, proteolysis, etc., are mostly well down the line. REFERENCES No attempt has been made to develop or extend a list. Any student of classifica- tion must necessarily consult Buchanan, who gives a full bibliography. This is supple- mented by Enlows as concerns genera, and the various articles by Winslow and his associates add enough references to occupy the student for some time. Save when otherwise specified, classifications recorded by these writers have been accepted and are referred to in the published articles. Aldrich, J. M.: "The Limitations of Taxonomy," Science, 65, 381-8.15. 1927. Bergey, D. H., et al.: Manual of Determinative Bacteriology, 1923. Breed, R. S., Conn, H. J., and Baker, J. C: "Comments on the Evolution and Classification of Bacteria," /. Bad., 3, 445-59. 1918. Breed, R. S., and Conn, H. J. : "Nomenclature of Actinomycetaceae," ibid., 4, 585-602. 1919; I ihid (Addenda), 5, 489-90. 1920. Buchanan, R. E.: General Systematic Bacteriology. 1925; "Nomenclature of the Coccaceae/' /. Infect. Dis., 17, 528 41. 1915; "Studies in Nomenclature and Classification of Bac- teria," ibid., 1-3. 1916-18; "The Evolution of Bacteria," Science, 47, 320-24. 1918. Castellani, Aldo, and Chalmers, A. J.: Manual of Tropical Medicine. 1919; Ann. dc I'lnst. Pasteur, 34, 600-621. 1920. Chester, F. O: Manual of Determinative Bacteriology. 1901. Enderlein, G.: Bakterien-Cyclogenie. Berlin and Leipzig, 1924. I ROGER G. PERKINS 135 Enlows, E. M. A.: "Generic Names of Bacteria." Hyg. Lab. Bull. 121. Felt, E. P.: "Nomenclatural Efficiency," Science, 65, 489-Qi. 1927. Hall, I. C: "Some Fallacious Tendencies in Bacteriologic Taxonomy," /. Bad., 13, 245-53. 1927. Hitchcock, A. S.: "How the Taxonomists May Utilize the Instructional Committee on Nomenclature." Science, 65, 412-15. 1927. Hucker, G. J.: "Studies on the Coccaceae," Tech. Bull. 99-103. Geneva, N.Y.: New York State Agricultural Experiment Station, 1924. Kligler, I. J.: "A Systematic Study of the Coccaceae in the Collection of the Museum of Natural History," J. Infect. Dis., 12, 432. 1913. Lehmann, K. B., and Neumann, R.: English translation of second German edition by G. H. Weaver. Merrill, E. D., and Wade, H. W.: "Validity of the Name Discomyces, etc.," Philippine J. Sc, 14, 55-69. 1919- Migula, W.: System dcr Baktcrien. 1887-1900. Orla- Jensen, S.: "Hauptlinien des natiirlichen bakterien Systems," Centralbl. f. Bakterio . Abt. II, 22, 305-46. 1909; "The Main Lines of the Natural Bacterial System," /. Bad., 6, 263-73. 1921. Stiles. C. W.: "International Code of Zoological Nomenclatures as Applied to Medicine." Hyg. Lab. Bull. 24; "Underlying Factors in the Confusion in Zoological Nomenclature," Science, 65, 195-99. 1927. Winslow, C.-E. A., d al. (Committee of the Society of American Bacteriologists on Character- ization and Classification of Bacterial Types.) "The Families and Genera of Bacteria" (Preliminary Report), /. Bad., 5, 191-229. Baltimore, 1917; "The Families and Genera of Bacteria" (Final Report of the Committee of the Society of American Bac- teriologists on Characterization and Classification of Bacterial Types), ibid. Baltimore, 1920. Winslow, C.-E. A., and Rogers, Anne F.: "A Revision of the Coccaceae," Science (N.S.) 21, 669-72. 1905; "A Statistical Study of Generic Characters in the Coccaceae," J. Inject. Dis., 3, 485-46. 1906. Winslow, C.-E. A., and Winslow, Anne Rogers: The Systematic Relationships of the Coccaceae. CHAPTER X "T > f^ f [} Jd P r c N > (<^) ATOMS, IONS, SALTS, AND SURFACES WILLIAM D. HARKINS University of Chicago ATOMS, IONS, AND SALTS THE ARRANGEMENT OE ATOMS IN SOLIDS Several different types of evidence indicate that solid substances are in general built up from minute building blocks called "atoms," whose diameters vary slightly with the element involved, but are of the order of a hundred-miUionth of an inch. The atoms attract one another in a way which is suggestive of the action of small magnets; it is believed that the forces involved are largely electrical but partly magnet- ic. In any crystal the atoms or mole- cules are arranged in a definite pattern or lattice, and this pattern largely de- termines the outer form of the crystal. Thus in rock salt (Fig. la) sodium and chlorine occupy alternate corners of the cubes of the lattice, and each atom of chlorine is surrounded by six atoms of sodium and each atom of sodium by six atoms of chlorine. Calculations based upon the density of rock salt, its molecular weight and the number of molecules in the molecular weight (6.06 X lo-^), indicate that the distance between the center of any sodium and any adjacent chlorine atom is 2.81 hundred-millionths of a centimeter, or 2.81 A units.' Crystals have 32 types of outer form and 14 types of atomic pattern which may be arranged in 230 different ways. THE COMPOSITION AND STRUCTURE OF ATOMS All known material substances are considered as built up from 92 simple forms of matter known as the "chemical elements." Of these 90 have been discovered and 2, with element numbers 85 and 87, have not been found. Each of these elements was formerly supposed to consist of only one kind of atom of a definite atomic weight, but in recent years it has been found that an element may consist of from one to eleven (probably even more) atomic species which are almost identical in their chemical ' A glossary of symbols and terms is given on pages 176 and 177. 136 ;^ y y ■7> ^ y ^ ^ D y c E ^ y /" % y B ^ A ^ ^ <^ A , K^ (6) (O Fig. I. — {a) Space lattice of sodium chloride, (6) face-centered, and (c) body-centered cubic lat- tice. Fig. 2. — Tni electrons (C. T. R. Wilson). Note that the tracks are dotted and irregular P'iG. 3a. — ( Fig. 2,l> shows how to find the track of a positive electron in this figure.) Tracks of positive helium atoms, and a track of the positive electron (proton or nucleus [H+] of the hydrogen atom). This photograph illustrates the synthesis of an atom, since a helium atom strikes a nitrogen atom and forms a (heavier) o.xygen atom and a hydrogen atom. (Photograph by Harkins and Shadduck.) WILLIAM D. HARKINS 137 behavior. Since all of the species which constitute an element are given only one place in the periodic system of the chemists, they are called "isotopes" (iso = "sa.me," to pe = "place"). The lightest-known atoms are those of hydrogen with a mass of i.66Xio~^-'gm., but the relative or atomic weight is taken as 1.0078 in order that the atomic weight of oxygen may be exactly the whole number 16. The mass of a projectile may be determined by the diilculty of deflecting it from its path when moving at any definite speed, since the momentum (mv) is proportional to the mass at a given velocity. Certain negatively charged particles are thus de- flected about two thousand times more easily than hydrogen atoms. These particles are designated as "negative electrons," and their charge is exactly ecjual in magnitude, within the limits of error of the most exact work, to that carried by positively charged hydrogen in the electrolysis of an acid in water. The tracks of negative electrons in air are shown in Figure 2. It may be noted that these tracks are very irregular, while the tracks given by hydrogen or other atoms are straight. Thus, this is true of the atom tracks shown in Figure 3. The dim track in the right-hand view is that of posi- tive hydrogen, which is the lightest positively charged particle known, so it is considered as the positive elec- tron, often called the "proton." All uncharged atoms are built up from equal num- bers of protons and electrons. In the hydrogen atom there is one proton, which contains almost all of the mass of the atom and acts as its central sun or nucleus, and one electron which is supposed, in the Bohr theory, to move in an orbit around the proton somewhat as the moon moves around the earth. It is supposed that the atomic weight of an atomic species gives the number of protons in its nucleus and also the total number of elec- trons in the atom. The nucleus of the helium atom consists of four protons and two electrons so that there is a net positive charge of two on the nucleus and there are, therefore, two negative planetary electrons in the outer part of the atom. Helium is the second element in the chemical periodic table, so its atomic number is 2. It may be noted that the atomic number expresses also the positive charge on the nucleus and the number of outer planetary electrons. Fig. 3?;. — Shows two views of the synthesis and disinte- gration of atoms given in Fig. 3(1. The track marked H rep- resents the track of a positive electron or proton, shown at the left-hand side of the right- hand view of Fig. s(^. The angles are the same as those in the photograph. MASS, ENERGY, AND FREQUENCY OF VIBRATION Mass and energy may be considered as the same but measured in different units. Thus I gm. of mass is equivalent to i times the square of the velocity of light (c = 3X 10" cm. a second, soc^ = 9Xio"'), or 9X10'" ergs. Thus a i-gm. weight (mass = i gm.) is 9X 10^" ergs of energy. In 191 5 Harkins and Wilson showed that, according to the special relativity the- 138 ATOMS, IONS, SALTS, AND SURFACES ory of Einstein, the conversion of hydrogen into helium should liberate an enormous amount of energy. The reaction may be written: 4H^He 4.0312-gm. mass-^4.ooo-gm. mass+o.o3i2-gm. mass in the form of radiation 0.0312-gm. mass is equivalent to 0.0312X9X10^° ergs = 2.81X1C ergs = 6.71 X 10" calories In more ordinary terms, the conversion of i lb. of hydrogen into helium should give off as much energy as radiation — which could be transformed into heat — as would be given by the burning of 10,000 tons of coal. The frequency of light- vibrations is very great. According to the quantum theory, if this frequency is multiplied by a con- stant //, the quantum constant, the energy of one quantum of the radiation Q is obtained, E = hv. Conversely, the energy quantity divided by h gives the frequency of the vibration, which is the Q velocity of light (c) divided by the wave-length (X). From these relations x-v we find that if four hydrogen atoms ^^ were to be converted into one helium, Q all of the particles having small veloci- ties, the wave-length emitted would be 0.00046 A, or this would be of the type of the cosmic radiation which falls upon the earth from outside. The reaction Fig. 4 -Neutral sodium atom (diagrammatic probably occurs in several steps, but a O representation in a plane: the structure is un- ■ r ^u 1 ^\, v.^ 1 i. u ^.^^^^^^,i part of the wave-lengths emitted should be of this general order of magnitude. known). IONS Atoms, or molecules which are not electrically neutral, are called "ions," so ions contain either a larger or a smaller number of electrons than of protons. The non- nuclear, or planetary, electrons of an atom are classified in sets. Thus in the sodium atom there are supposed to be three of these sets : the innermost with two, the next with eight, and the third or outermost with one electron. These numerical relations are represented by Figure 4. The chemical properties of an element are supposed to depend to a large extent, but not wholly, upon the number of electrons in the outer set. Thus the atoms of the alkalies, lithium, sodium, potassium, rubidium, and caesium, are all supposed to have one electron in the outer set. They ionize, i.e., lose this outer electron, more easily than any other elements. All of them react violently with water, and in all of their other chemical, and in most of their physical, properties act almost alike. With helium the outer set of electrons is complete when two electrons are present, but with heavier elements completeness in this set seems to be reached only when eight electrons are present. WILLIAM D. HARKINS 139 Sodium, magnesium, and aluminium have, respectively, one, two, and three outer electrons. Such atoms lose electrons to form positive ions. If, however, the outer set of electrons is more than half complete in the neutral atom, there is a much greater tendency to pick up electrons than to lose them, so such atoms usually form negative ions. If the three elements mentioned above are in the gaseous form and the sodium atom loses one (Na+), the magnesium two (Mg++), and aluminium three electrons (A1+++), then ten planetary electrons are present in each of these ions (Fig. 5). This is the number of planetary electrons present in the neutral neon (Ne) atom. Since it is supposed that the arrangement of the electrons is largely conditioned by their number, it is supposed that Ne, Na+, Mg++, and A1+++ have the same structure ex- cept that the electrons are bound somewhat more tightly as the charge on the nucleus changes from 10+ for neon to 13+ for aluminium. That the idea of a common struc- ture is justified is indicated by the fact that in so far as they have been investigated the spectra of these ions upon further dissociation is like that of neon. O o o o o o P^ o o 00 GO Neutral neon atom Triply positive aluminium ion (A1+ + +) Fig. 5. — The composition of Ne, Na+, Mg++, or A1+++. The nuclear charges are 10+, 11+, 12+, and 13+, respectively. IONS IN SOLID SALTS In rock salt the mean distance from the center of a sodium atom to that of any of the six adjacent chlorine atoms is, as has been stated, 2.81 A (2.81X10"* cm.). The fact that the analysis of the structure of such a crystal by X-ray methods has given no evidence that the sodium atom is more firmly attached to any one of these atoms than to the five others has led to the hypothesis that no molecules exist in such a crystal, and that the chlorine atoms are attracted to the sodium atom because the former are charged with a negative (Cl~) and the latter with a positive (Na+) electrical charge. Thus the assumption is that the crystal is built up from individual positive and nega- tive ions held together by the attraction between charges of like sign. The attraction between the ions of unlike sign of charge is assumed to follow Coulomb's law that at- traction or repulsion between electrified particles varies as the inverse of the square of the distance between them: F= 6162 (i) I40 ATOMS, IONS, SALTS, AND SURFACES Madelung and Born find that if Coulomb's law is assumed to hold for the attractive forces, it is necessary, in order to accord with the known values of the compressibility of solid salts, to assume that there is in addition a repulsive force between the ions which varies as about the inverse tenth power: ylO According to this type of theory, solid salts consist entirely of ions, and in this sense the salt is already completely ionized. THE IONIZATION OF SALTS IN AQUEOUS SOLUTIONS The dielectric constant {D) of ordinary solid salts is about 5, while that of water at ordinary temperatures is about 80. Since the attraction between charged particles (equation |i]) varies inversely as the dielectric constant, the attractive forces between ions of op])osite sign of charge should be very much less in water than in solid salt. If, therefore, it is assumed that the solid salt is completely ionized, it would be unreason- able to assume that the same salts are less than completely ionized when dissolved in water. The idea that salts in aqueous solution are completely ionized was suggested by Sutherland in 1907. The theory has been put in more definite form by Bjerrum, by Milner, and by Debye and Hiickel. Their fundamental idea is that the electrical at- tractions between ions of unlike sign, and repulsion between those of like sign, give rise to the following effect: on the average any positive ion will be immediately sur- rounded by more negative than positive ions, while any negative ion will be surrounded by more positive than negative ions. The most important result is that when the solu- tion is diluted the separation of the ions involves the expenditure of energy.^ ' Debye and Hiickel have calculated this electrical internal energy by assuming that the charges on each ion are concentrated at a point, and that the distribution of these points is determined by probability. They use the probability relation of Boltzmann, developed in connection with the kinetic theory of gases, together with the equation of Poisson derived from the laws of electrostatics, includ- ing Coulomb's law. Suppose that we have an ion of valence + n and charge + nE. In any concentric shell of thick- ness dr, the potential is P and the density of the electric charge is p. The average kinetic energy of the molecules at a temperature Tis 3/2 kT. The Boltzmann constant k is equal to the gas constant 7? divided by the number of molecules in a gram molecule {6.o6Xio^'5). According to Boltzmann's principle, if the molecules are distributed in a field of force, such as in an electrical field, the distribution will be such that the number of molecules, instead of being equal -E to N, the number present in the absence of the field of force, will be equal to Ne *^ . Thus the number present is modified by a factor in which the base of Naperian logarithms is raised by a power equal to the potential energy of the molecule divided by two-thirds of its mean kinetic energ>'. The equation of Poisson applies to the variation of the potential P around a point when it is distributed with spherical symmetry. It may be expressed as follows: r^ dr \ dr ) ~ dr' The electrical density is represented by p. 2 dP _ 47rp '^~r'dr'~ D' WILLIAM D. HARKINS 141 LAW OF MASS ACTION Suppose that two substances, A and B, react to form the substances E and F, and that the reaction has proceeded until equilibrium has been attained. The chemical re- action may be expressed: aA+bB = cE+fF. (i) It was found by Guldberg and Waage that in such a case a definite law, known as the "mass law," determines the condition of the system at equilibrium. If the substances are gases this may be expressed Pe-Pf j^ / X Pa-Pb in which Kp is a constant. In the case of the simple dissociation A^B^C, (3) the mass law becomes -^ '^P' (4) Pa ^ In the ordinary development of the mass law it is assumed that the gas law {pv = NRT) is true, so the mass law fails to hoM in any case in which the gas law is invalid. The gas law may be written p^K^ = CRT. (S) V If this is substituted in (4) the following expression is obtained: ^^ = ^ (6) C^ RT ' ^ ' but since the temperature is constant and R is the gas constant, Kp/RT is a constant, or ^^ = K,. (J) ^A DEVELOPMENT OF THE LAW OF MASS ACTION The law of mass action may be developed very simply by a consideration of the amount of work necessary to compress a gas reversibly, i.e., in such a way that the same amount of work may be regained when the gas expands. The amount of work done when a force (F) acts through a distance (S) is W = F.S. (i) If a gas expands in a cylinder provided with a piston, the force (F) is equal to the pressure (p) times the area (A) of the piston, so W=pAS=p-Av. (2) 142 ATOMS, IONS, SALTS, AND SURFACES If the increase of volume (A v) is infinitesimal, it is designated by dv, so dW=p-dv. (3) But by the gas law P —, (4) so dW=NRT - . (S) The work (W) done by the reversible expansion of the gas from a volume to a volume V2 is W= I 'dW = NRT\ '^ = NRTln^. (6) Since, however, the volume of a perfect gas varies inversely as its pressure (/>), W = NRTln^. (7) P2 Consider a chemical reaction aA+bB%eE+fF , in which all of the substances are in the gaseous state. Let the initial pressures of the gases A and B be p^ and p^ and the final pressures of E and F be p^ and /?/. Assume that a large box contains all of these four gases in equilibrium with one another at pressures pA, pB, pE, and pp- Let the gases A and B at pressures p^ and p'g be con- tained in two cylinders provided with pistons. The first step in the process is to change the pressure /?j to pA- The work done by a gas in such a process equals the number of mols (c) of gas times RT times the loga- rithm of the initial pressure divided by the final pressure, or P'a W, = aRTln^. (8) Pa By the use of a well-known characteristic of logarithms, this becomes W. = RTln^. (9) Pa The similar equation for the gas B is W2=RTln^. (10) Pb The gases A and B are now at pressures equal to their partial pressures in the large equilibrium box. Suppose that the cylinders which contain these gases are set on I WILLIAM D. HARKINS 143 the equilibrium box and that at the bottom of each cylinder there is a membrane permeable only to the gas in the cylinder (Fig. 6). Force the a mols of the gas A and the b mols of the gas B slowly into the box, and as rapidly as these react take out the e mols of the gas E and the/ mols of the gas F which are formed. Since work must be done upon the gases .1 and B to force them into the box, the work they do is negative, or Wi = p■^v==-aRt , (11) which is obtained from equation (2) by substituting in it the gas law p.Av = NRT iN=-a) . 1 I 1 1 Gas A Gas E Gas F Gas B Equilibrium Mixture of A, B, E, and F Fig. 6 For the gas B W,= -hRt. Since the gases E and F come out of the box, they do work, and Ws=^eRT, Wt=JRT . (12) (13) (14) The gases E and F are now at pressures pE and pp. Let them now be changed to their final state in which the pressures are p^ and pp: W^ = RTln-^^, Pe (is) W%=RTln-^ P± P'/ (16) The total work (W) is the sum of these eight quantities of work, or p^ pf p'ep'f W^RT In ^-RT In Y^M^+f -^-b) RT . ^A^B ^A ^E (17) 144 ATOMS, IONS, SALTS, AND SURFACES Now, e-\-J—a—b is simply the increase (A n) in the number of molecules when the given reaction takes place, so P%P-i Et^F W = RTln'^^-RTln (Term i) (Term 2) P'^P'J (18) (Term 3) If the reaction takes place at a constant temperature, term 2 is a constant, since the pressures designated by primes are fixed values. Term 3 is also constant, sine • An, the increase of the number of mols of gas, is fixed by the reaction. The work done in a series of reversible changes depends only on the initial anrl final states, and is therefore a fixed quantity for the given reaction. Equation (18) may now be written: K, = RTln KPi or RT P%pfn 4^5 pU'^"'^"' ■ Ki — K2 — K^ = K^ , Therefore pipV K, (19) (20) (21) which is the law of mass action. This equation gives the mass law in a general form. For a reaction in which only i mol of each substance is involved, it takes the form A+BZE+F , PeXPf Pa^Pi K Thus, according to the mass law, the product of the partial pressures in the equi- librium mixture of the substances formed in a chemical reaction, divided by the prod- uct of the partial pressures of the substances from which they are formed, is equal to a constant at any given temperature. If more than i mol of any of the substances is involved in the reaction, the corresponding partial pressure must be raised to a power equal to the number of mols. ESCAPING TENDENCY OR ACTIVITY According to the mass law, the effect of any substance present upon the chemical equilibrium is proportional to its concentration. Thus, the activity (a) of a substance may be expressed by its concentration, or CbCc = K (8) WILLIAM D. HARKINS 145 While the activity of a perfect gas, or a substance which in solution obeys the per- fect-gas law, is expressed by its concentration, this is not the case if the gas law does not hold. The activity may then be defined as that quantity which, when substituted for the concentration of a substance in the mass-law equation, expresses its effect in determining the equilibrium. If a small quantity of iodine is shaken with water and carbon disulphide at 25° C. until equilibrium is attained, it is found that a unit volume of carbon disulphide con- tains six hundred times more iodine than the same volume of water. Since there is equilibrium, the tendency of iodine to escape from the water is the same as that from carbon disulphide, i.e., the vapor pressure of iodine should be the same over the two solutions, and this is found to be true. The identity of vapor pressures of the iodine indicates that the activities are equal. If it is considered that the activity of the iodine in the carbon disulphide is equal to its concentration, then the activity in water, which is the same, must be six hundred times the concentration of the iodine in the water. Thus carbon disulphide is a better solvent than water for iodine since it can hold six hundred times as much of the latter and still give no more activity to the iodine.' 1. When the activity of a constituent is the same in two difTerent phases the con- stituent will not increase its concentration in one phase at the expense of the other un- less energy is supplied to effect the transfer. 2. If the activity of a constituent is greater in one phase than another the con- stituent (if transferred at all) will pass from the phase in which it has the greater into the one in which it has the lesser activity. THE ACTIVITY COEFFICIENT OF ELECTROLYTES A solution of common salt which contains 58.5 gm. of sodium chloride to 1,000 gm, of water is considered to be i molal in concentration. If m represents the molality of a solution, its activity coefficient (a) is defined as the ratio of its activity to its mo- lality, or a a = — ; so a = ma . m The activity coefficient for a salt in its extremely dilute solution is i.oooo by defi- nition. At a molality of o.oi the activity coefficient for sodium chloride in its aqueous solution is 0.922, while at o.im it is 0.798. Now the activity coefficient plays the same ' The escaping tendency may be expressed also in terms of the "fugacity" (/), a term introduced by G. N. Lewis. The fugacity of an ideal gas is equal to its pressure. Any gas is practically ideal at low pressures. At higher pressures its fugacity is the geometric mean of the actual pressure {P) of the gas and the ideal pressure {Pi) calculated from the gas law, so that ^ Pi The activity of a constituent may now be defined as its relative fugacity (/) as compared with its fugacity in some standard state (/J, or 146 ATOMS, IONS, SALTS, AND SURFACES part in the newer work on the ionization of salts in solution as that of the degree of ionization in the older theory. Thus the degree of ionization of sodium chloride in its 0.1m aqueous solution was given as 0.86, while the activity coefhcient as given above is 0.798. The activity coefficient is sometimes designated as the "thermodynamic de- gree of dissociation." Table I gives the activity coefficients for sodium and potassium chlorides and a few salts of higher types. It may be noted that as the product of the valence of the ions of the salt increases the activity coefficient decreases. TABLE I Activity Coefficients of Salts in Aqueous Solution .4t 25° C, as Calculated from the Lowering of the Freezing-Point Molality m Activity «NaCl Coefficient "KCl "Bad. SSo Benzoic 60 Ortho Hydroxy benzoic i ,020 Chlor benzoic 1,320 Nitro benzoic 6, 160 Bases Ammonium hydroxide 18. Methyl ammonium hydroxide . . 500 . Phenyl ammonium hydroxide. . 0.0004 HYDROGEN-ION CONCENTRATION AND pH Suppose that a primary cell consists of an electrolyte with two hydrogen elec- trodes. At the electrode on the left-hand side the pressure of hydrogen is 10 atm. (atmospheres), and on the right-hand side i atm. The cell is M+IIz (10 atm.) , Electrolyte with //+ ions , M + II2 (i atm.) . Meta Para 87 29 15s 90 345 396 WILLIAM D. HARKINS 149 The change of state is Left — (10 atm.)-^ n+ (at concn. x) , 2 Therefore, the total change is Right H+ (at concn. x)-^ — ^ (i atm.) 2 ^(10 atm.) -^ — ^(latm.) 2 2 The electromotive force ((5) of the cell is an energy quantity and may therefore be calculated as the equivalent of the maximum work which would be obtained if § mol of hydrogen gas expands from a pressure of 10 atm. to that of i atm.' Now the amount of work done by a force F in acting through a space 5 is W^FS . But since force equals pressure times area (A), W^pAS^pAv, a A V represents the increase of volume. Thus for a small amount of work dW^pdv , but since dW^NRT'^, V or by integration between the limits V2 and Vi , W = NRT In ^-- = NRT In ^ . This gives the amount of mechanical work which is equal to the change of electrical energy (S'DfJ, if '^l is the number of equivalents of electricity transferred. '^, the value of one equivalent of electricity, is equal to 96,500 coulombs. So ^m=NRTln^, p2 or ^NRlli^^ log t, ^i 96,500 p2 ' ' The small amount of work involved in the change of volume of the liquid is neglected for the sake of simplicity. I50 ATOMS, IONS, SALTS, AND SURFACES Pi In the special case given above, pi = io and p2 = i, and log t^=i, the number of P^ mols of hydrogen (N) transferred is \ and the number of charges transferred (91) is I, so 96,500 If the temperature is 25° C, then r= 270+ 25 = 298° K., and (g = °-°59i5 volts = 0.02957 volts. A cell may contain two aqueous solutions with diliferent hydrogen-ion concen- trations, such as M+H2 {p atm.), H+Cl- {nh molal), H+Cl- (w. molal), M+fl^, {p atm.) . Now if I Faraday of positive electricity is passed from left to right, the following changes occur: Left Electrode IT — ip atm.) -^ H+ (;«, molal) , 2 Liquid Junction T^jj + )H+ (nil molal) -^ ^(/7 + ) ^'^ (^2 molal) , T(^Qi^^Cl~ [niz molal) -^ T^ (Ci-) CZ+Cmi molal) , Right Electrode U+ (w. molal) — [p atm.) . 2 If the changes at the electrode are alone considered it is found that as much hy- drogen {H2/2) appears at p atm. as disappears at that pressure, so this change involves no work, and therefore no electromotive force. If the liquid junction is neglected for the moment, the total remaining change is H+{tn2 molal) -> H+ (nii molal) and @£ = -sT?r- ^« — (Approximate) , or ^E = -^^r^ In - (Exact) . Here iV/9^ = i. For the liquid junction potential, K r,. , RT , Wi I ^ RT , Ml g nii iS nh rj. , RT m2 . rj. RT , W2 = -T{H+) -^ In \-T(Ci-) -^ In ~ , or since WILLIAM D. HARKINS 151 Tci-+Tn + = i, T(ci-)-{i — Tci-) = {2Tci- — i), .*. © = @£+@L = 2r(a-) -^ In —^ ; 5 Wi or more exactly: S = 2r(c/-) -^ In— . 15 fli By the use of such concentration cells it is possible to find the hydrogen-ion ac- tivity in one solution, provided its activity in some other solution with the same anion is known. If the anion is not the same, difficulties arise in the calculation of the liquid- Hquid junction potential. The results of part of the research by biologists upon hydrogen-ion concentration have been based on the assumption that two different liquid-liquid junctions give the same potential, and can therefore be canceled, which is in general not true. Since the electromotive force, and therefore the maximum work or free energy, varies as the logarithm of the activity (or inexactly as the concentration) ratio, the hydrogen-ion activity or concentration is often expressed as the logarithm. Thus in pure water at 25° C. the hydrogen-ion concentration is considered to be io~' molal, or the logarithm is— 7. This is often expressed according to the system of Sorensen as pH = 7. If, for example, pH = 5.2, the hydrogen-ion concentration is lo""^"^ molal, and the solution is on the acidic side of the neutral point (Cfl + = o.63 lo"^ mols per liter). THE SOLUBILITY PRODUCT Suppose that we have two saturated solutions (i and 2) of thallium chloride sep- arated by a partition which consists of a single crystal of the salt. Let solution i be on the left and solution 2 on the right. Tl++Cl-^{TICI (solid)5r/++a- . (i) The mass-law relation is ari +Xaa-= flsoiid Tici =aTi+Xaa- = K . , (2) This is known as the "solubility product." In the case cited, the solid salt of the mid- dle phase has a constant activity; but even if other salts, such as TI+R~ or NaNOj, are present, the relations between the activities in the right- and left-hand phases may be expressed: -77— =-7^' \3) aTi+ aci- 152 ATOMS, IONS, SALTS, AND SURFACES THE MEMBRANE EQUILIBRIUM Let the solid salt be replaced by a membrane, and let one of the salts in the left- hand solution consist of one ion {Na'^), which is, and one (i?~), which is not, diffusible through the membrane. The equilibrium relations are represented below: Equilibrium Na+ Cl+ a" a" Na+ Cl- R- QR- If both solutions are considered infinite in volume, then the transfer of i mol of sodium chloride from left to right will not disturb the equihbrium. If i mol is thus transferred, the decrease of free energy ( — A/^) is aifa+ aci~ But the characteristic of an equilibrium process is that the change of free energy (— AF) is zero, so aNa+ aci~ or aNa+ aci- Ijut this is identical with (3), the relation which expresses ratios obtained from the solubility product. Thus, to this extent the membrane equilibrium is identical with the equilibrium with a solid. Equation (6) may be written iiNa + X aci - = axa + X aci , (7) which is the same as the solubility-product relation between the two solutions (2) but differs in that no solid of constant activity is concerned in the equilibrium. In the solubility product the product of the activities of the ions of the saturating salt, in either of the solutions on the two sides of the solid salt, equals the activity of the solid salt which is constant. In the membrane equilibrium the same product is equal to the activity of the salt in the membrane between the two solutions, but this activity is variable. In order to calculate the cciuilibrium in any special case, the activities under the conditions of the membrane equilibrium must be known. An attempt was made by Donnan to solve this problem without a knowledge of these activities, but there is no conclusive evidence to indicate that such a solution corresponds with the actual equi- WILLIAM D. HARKINS 153 librium. However, the method of treatment for a simple case is presented below, where the initial and equilibrium conditions are represented. Initial Na+ ci- Na+ Cl~ 2C c R- C C c Equilibrium Na+ ci- Na+ ci- 2C-X c-x R- C OX c+x Here A' represents the number of mols of NaCI transferred from left to right. If X happens to be negative, the equations will reveal that this is the case. The membrane product relation, written in the inexact form in which the con- centration (C) is substituted for the activity, is {2C-X){C-X) = {C+Xy-, or 20-2,CX+X' = 0^-2CX+X' , so X = iC . Equilibrium Na+ ci- Na+ Cl- liC ic R- C liC • liC se is outlined below: Initial Na+ a- R- Na+ Cl- iiC C loC C C (xiC-x) (C-x) = {C+xy, iiO-i2Cx+x^ = 0-\-2Cx-irx\ Na+ ci- }C Equilibrium R- loC Na+ Cl- ifC MEMBRANE "POTENTIAL" The equations for calculating the electromotive force for a liquid-liquid junction are well known in the case in which different concentrations of the same salt are pres- 154 ATOMS, IONS, SALTS, AND SURFACES ent on the two sides of the junction. The same equations have been applied without change when a membrane has been supposed to be present. Thus, with the cell given below Na+ Cl- aNa+ aci- Na+ CI- R- III aNa oci aR~ let I Faraday of positive electricity be passed from left to right. The decrease of free energy is -AF=TNa+ RT In ^^+Tcr RT In ^ = {2TNa+-i) RT In ^ . (8) aNa+ act- aNa+ Since the number of equivalents of sodium and chlorine ions which cross the boundary are given by their transference numbers {T!fa+ and Tci-) the decrease of free energy is also equal to the electrical work done, or -AF=(S9^5 (9) or, since in the special case given above the number of equivalents of electricity (^l) transferred is one, ^==(2TNa+-i)^ln^, (10) ^ aNa + which is the equation commonly given for the membrane potential. In general, (^ = iTc-TA)^ln^. F a+ Up to the present time no trustworthy experimental verification of these equa- tions has been obtained from any membrane equilibrium, since in the tests of these relations thus far the assumption has been made that other liquid-liquid junction potentials involved are negligible, without proving that this is true. SURFACES AND SURFACE ENERGY The importance of the energy stored up in the surfaces of bodies and in the inter- faces between the particles of the bodies is due in part to the influence which the sur- face tension exerts upon the form of the bodies and the particles. However, the effect of the surfaces upon the chemical composition and action, and upon the electrical phenomena, are of even greater moment. Interfaces are of particular significance in living organisms, since the motion of an organism as a whole is evidently brought about by transformation of one kind or another of the interfacial energy resident in it. The term "surface" unfortunately im- plies the entire absence of a third dimension in space, that of thickness, but physical surfaces and interfaces, sometimes designated as "phase boundaries," although they are exceedingly thin, commonly have a thickness as great as the sum of the diameters of several atoms, or a distance of the order of a millionth of a millimeter (10 A), which is by no means negligible. At many interfaces films or membranes collect, and these WILLIAM D. HARKINS 155 are of particular importance in biological systems, particularly in the human body itself. If a cubic centimeter of water is sprayed into spherical droplets o.oi ix (100 A) in diameter, the area of the surfaces thus formed is 600 sq.m. or approximately one- eighth of an acre. The free surface energy {yy^A) for this area at ordinary tempera- tures is about 2.2X10^ ergs or 10.5 calories, while the total surface energy {H) is larger and equal to 16.6 calories. This is one-third as large as the total energy of heat vibration of all of the molecules in the water. Any system in which the area of the surfaces (interfaces) becomes large enough so that the surface energy is appreciable in comparison with the energy of (heat) vibra- tion of the molecules of the disperse phase is considered as a "colloid." It is commonly observed that water drops on a hot stove or on very dry dust assume a spherical shape, which is the form assumed by a balloon surrounded by a uni- form elastic membrane. This suggests that every liquid is surrounded by an elastic film, the tension of which causes the surface to contract to the smallest possible area for the volume of the liquid, provided other forces (such as gravitation) do not act to change the form. If a faucet with a narrow orifice is turned on very slightly, a drop may be seen to form, and hang for some time, after which it drops very suddenly. The drop is supported before it falls by the vertical component of the surface tension. If a capillary tube is dipped into water, the liquid inside the tube rises higher than that outside. Here, also, the film of liquid on the inside wall of the tube exerts an upward pull. If a camel's-hair brush is dipped into water, the hairs remain spread apart as if they were dry and in the air, but when the wet brush is pulled out of the water, the pull of the surface tension of the water binds all of the hairs compactly together. A soap film stretched on a wire frame such as that shown in Figure 7 has two sur- faces. If the distance AB is | cm., then this lower wire is in contact with ^ cm. of the film or I cm. of the surface. The pull on the film as measured by the weight of the wire and the weights suspended from it at W gives the surface tension of the surface per unit length. This may be expressed in dynes. If the wire is pulled downward i cm., then the surface increases in area by i sq.cm., so the work done is force times area equals 7X1=7 ergs. This energy may again appear as work when the film contracts to its original position, so it possesses the characteristics of free energy. Thus 72.8 dynes per centimeter is the surface tension of water at 20°, and 72.8 ergs per square centimeter is the free surface energy of water at this temperature. Fig. 7. — Maxwell frame for the determination of the surface tension of a soap film. TENSILE STRENGTH AND TENSILE ENERGY There are a number of phenomena which indicate that the forces between ad- jacent molecules in soUds and liquids are very high. Tensile-strength tests on bars of 156 ATOMS, IONS, SALTS, AND SURFACES steel show that it is necessary to apply a force of 100,000 lb. to rupture a bar i sq.in. in cross-section. If it were possible to carry out a tensile-strength test in an ideal way such that the bar (of i sq.cm. cross-section; see Fig. 12) would not be deformed before the break occurs, and so that the rupture would give two plane surfaces at right angles to the longitudinal axis of the bar, then the energy used would be equal to twice the free surface energy (27) per square centimeter at the temperature of the test. This is true because all that occurs in such an ideal rupture is the formation of a new surface on the steel of 2-sq.cm. area. This is equal numerically to twice the surface tension of steel per centimeter. The work necessary thus to pull apart a bar of unit cross-section may be designated as the work of cohesion {Wc). Wc = 2y. (i) If an endeavor is made to apply such a tensile-strength test to a bar of liquid, it is found that certain experimental difficulties arise. Nevertheless, the numerical value of the work of cohesion is known with considerable accuracy in such a case, since it may be obtained from the surface tension of the liquid. The surface tension of water at 20° is 72.8 dynes per centimeter, so its work of co- hesion is 145.6 ergs per square centimeter. This small value may seem to indicate a small tensile strength (force of cohesion) in water, but just the opposite is true since the distance to which molecular attraction remains appreciable is very small, and is only of the order of molecular dimensions. Furthermore, it decreases as a moderately high power of the distance. Suppose that the summation of this rapidly decreasing force is equivalent to the action of a constant force through io~^ cm. Then the force of cohesion would be ■ _ or 1 .4s6X 10'° dynes = 1 .48X lo^ gm. per square centimeter , 10 * or about 14,000 atm. The theory of van der Waals indicates a value of about 11,000 atm., while other methods of calculation usually give between 10,000 and 15,000. LATENT HEAT OF A SURFACE According to the rule of Le Chatelier, if the state of a system is changed, the sys- tem alters in such a way as to oppose a resistance to that change. Thus if the solubil- ity of a salt increases with the temperature, the last amount of salt which dissolves to saturate the solution produces a cooling, since this cooling lowers the solubility, and thus opposes the solution of the salt. Now, since the surface tension decreases with rise of temperature (Fig. 8), a surface must cool if it is expanded, since by cooling the surface tension is increased, and this opposes an extra resistance to the further extension. That heat should be used up in the formation of a surface is to be expected on other grounds. In the vaporization of a liquid the kinetic energy of molecular vibra- tion of the molecules of the liquid, which determines the temperature, is partly con- verted into molecular potential energy, i.e., the molecular energy of motion is utilized in the separation of each molecule from its neighbors and against the attraction which they exert. Now since a molecule which is in the interior of a liquid must move into WILLIAM D. HARKINS 157 the surface against the attraction of the surrounding molecules, as a part of its migra- tion into the vapor phase, it seems probable that in surface formation as well as in vaporization molecular kinetic energy would be utilized and transformed into poten- tial energy of the surface. That heat is actually used in the formation of the surface is shown by the thermodynamic equation of Clapeyron, which gives the latent heat (/) of the surface as 87 l=-T hT Fig. S. — The free surface energy (or surface tension) of organic liquids. This equals one-half the tensile work (or work of cohesion Wc) per square centimeter. Here I gives the amount of molecular kinetic energy which is transformed into molecu- lar energy of position when i sq.cm. of surface is formed. TOTAL SURFACE ENERGY The total energy Qi) of a surface is equal to the sum of the free energy (7) and the latent heat (/), h=y-\-l=y-T dy dT' In the formation of a surface a part of the energy must be supplied in the form of work in order to give rise to the free surface energy, and a part comes from the kinetic 158 ATOMS, IONS, SALTS, AND SURFACES Air energy which the molecules themselves possess. Thus, if a person extends a surface by doing work upon it, the liquid will also contribute its share to the total energy. The Clapeyron equation tells us that the temperature, i.e., the wealth of the molecules in kinetic energy, is an important factor in determining the extent of this contribution. If a surface is to be formed on a definite liquid at a definite temperature, a definite amount of energy must be contributed and converted into potential energy. THE ORIENTATION OF MOLECULES IN SURFACES The ordinary observation of large-scale objects, such as logs or ships, as they lie upon the surface of a body of water, indicates that these objects exhibit a character- istic orientation with respect to the surface. Thus logs, when not too closely crowded together, lie flat upon the water, i.e., the longitudinal axis is parallel to the surface. However, if one end of each log is loaded with a mass of iron or brass of the proper weight, it floats upon the surface and the longi- tudinal axis becomes vertical. If there is just a suffi- cient number of logs, the surface becomes covered with a single layer of vertical logs with their sides more or less in contact, while with any greater number, bunches of logs are found raised above the common level in certain places. If the number is smaller, a part of the surface remains uncovered. These phenomena may be illustrated by the use of a large number of cylindrical sticks of wood 3 mm. in diameter and 14 cm. long, weighted by a small cylinder of brass placed at one end. These are thrown upon the surface of the water in a large glass cylinder. This is represented in a diagram- matic way in Figure 9. If one of the vertical sticks is taken from the water, the brass weight removed, the stick dropped upon a vacant space upon a water sur- face, it at once assumes a horizontal position, thus exhibiting another type of orientation. It is well known that the molecules or ions which make up a crystalline solid are arranged in an orderly way. A certain type of orderly array of very long and highly symmetrical molecules is found also in certain liquids, which are said to contain liquid crystals. Ordinary liquids are often supposed to be characterized by a complete disorder in the arrangement of their molecules, but it is probable that this disorder has been overemphasized. For example, in organic liquids of the type of acetic acid, the molecule of which consists of the polar carboxyl group, and the "non-polar" methyl group, there is some evidence of molecular association, presumably a type of orienta- tion in which two or more polar groups come close together in the pure liquid as they do in benzene' (Fig. 11, upper part). The theory that the molecules in the surface of a ' The number of molecules which unite in this manner must be large in some groups, as they give definite X-ray patterns. The grouping may be more analogous to that given by the bristles of two brushes which are set with bristles together; this is called the "cybotactic state." -- j^ Water r^ Fig. 9. — Orientation of mole- cules of an alcohol (or an organic acid) at the surface of its aqueous solution. WILLIAM D. HARKINS 159 liquid are oriented in a characteristic fashion is of comparatively recent origin. It seems peculiar that the birth of so obvious a conception should have been so long de- layed, but it is probable that this is due to the general habit of considering molecules as spherical, even when their formulas are highly elongated. In such cases the former conception was that it would roll itself up into a sphere. It is obvious that even in a dissymmetrical field of force, such as may be assumed to exist at the surface of a liquid, a molecule which is a perfectly sym- metrical sphere could exhibit no orientation. However, even in a uniform gravitational field a perfect sphere may orient itself Fig. 10.— Illustrates provided its mass is not uniformly distributed, as will be seen if the orientation of a a sphere is weighted on one side, so that even if all molecules weighted sphere under were spherical, molecular orientation would not be at all impos- theintiuence o sible (Fig. 10). ^""'^- SOLUBILITY AND MOLECULAR ORIENTATION IN SURFACES The cohesion in liquid ethane, H H 1 I H— C— C— H , I I H H is extremely low, so low that liquid ethane cannot exist at ordinary temperatures. The introduction of one oxygen atom gives rise to ethyl alcohol in which the cohesion is equivalent to a pressure of 3,000 atm. The hydrocarbon molecule (ethane) is non- polar, but the hydroxyl group ( — OH) of the alcohol is polar. The attraction between two such polar groups is very much greater than that between two non-polar groups or that between a polar and a non-polar group. The solubility of an organic acid, an alcohol, or an amine in water is due to its polar group which is greatly attracted by the water. The solubility of such a substance in hexane is, on the other hand, due to the presence of the hydrocarbon groups. Thus, all saturated hydrocarbons are practically insoluble in water. The old rule is: Similia similibus solvimtur ("Like dissolves like"). If, now, we have a two-phase system consisting of water below and hexane above, and add butyric acid, H H H I I I H— C— C— C— C— 0— H , I I I II H H H O the hydrocarbon end, which we may designate by CZZl , is soluble in the hexane, but not in the water, and the carboxyl group O is soluble in water but not in the hexane. However, the carboxyl group will drag some molecules of the butyric acid into the water, while the hydrocarbon group will drag others into the oil. At the interface between the water and the oil, however, the hydrocarbon of the i6o ATOMS, IONS, SALTS, AND SURFACES molecule may dissolve in the oil and the polar group in the water. Thus, each end of the molecule is highly soluble in the liquid toward which it turns. If this is true: (a) butyric acid should be very much more soluble in the interface than in either the water or the benzene; {b) the butyric acid molecules should be oriented with their polar ends toward the water and their non-polar ends toward the hexane. (Fig. ii). _ — Water Phase —-^ __^. GENERAL STATEMENT OF THE THEORY OF ORIENTATION OF MOLECULES IN SURFACES The following statement, written in 1916, gives in concise form the general funda- mental principles of the orientation theory: I. The molecules in the surfaces of liquids seem to be oriented, and in such a way that the least active or least polar groups are oriented toward the vapor phase. The general law for surfaces seems to be as follows : // we suppose the structure of the stirface of a liquid to be at first the same as that of the interior of the liquid, then the actual surface is always formed by the orienta- tion of the least active portion of the molecule toward the vapor phase, and at any surface or INTERFACE THE CHANGE WHICH OCCURS IS SUCH AS TO MAKE THE TRANSITION TO THE ADJACENT PHASE LESS ABRUPT. This last Statement ex- presses a general law, of which the adsorption law is only a special case. If the molecules are monatomic, and s^onmetrical, then the orienta- tion will consist in a displacement of the electromagnetic fields of the atom. This molec- ular orientation sets up what is commonly called a ''double electrical layer" at the sur- faces of liquids and also of solids. This law, if applied to special cases, indi- cates for a few pure liquids the following orientation: In water the hydrogen atoms turn toward the vapor phase and the oxygen atoms toward the liquid. With organic paraffin derivatives the CH3 groups turn outward, and the more active groups, such as NO. CN, COOH, COOM, COOR, NH„ NHCH3, NCS, COR, CHO, I, OH, or groups which contain N, S, O, I, or double bonds, turn toward the interior of the liquid. If any of these organic compounds are dissolved in water, their orientation in the water surface is the same as that just given, with the active groups inward. At interfaces between two pure liquids the molecules turn so that their like parts come together in conformity with the general law. With solutions, the solute mole- cules orient so that the ends of the molecules toward the liquid A are as much like A as possible, and the ends toward B are as much like B as possible. So at interfaces be- tween organic liquids and water, for example, the organic radical sets toward the or- ganic liquid, and the polar group toward the water. Fig. II. — A two-phase system of water and benzol which contains butyric acid. The greatest concentration of the acid is at the interface. The acid is more or less as- sociated in the benzol. WILLIAM D. HARKINS i6i 2, If at an interface the transition from a liquid A to the liquid B is made by a saturated film of solute molecules which we may call A-B, i.e., they have one end like A and the other like B, then the free surface energy is greatly reduced. For example, with water and benzene with sodium oleate as the solute, the free energy falls as low as 2 ergs per square centimeter. 3. If the solvent is polar, such as water, then solutes will in general be positively adsorbed in the surface if they are less polar than water (or if a part of the molecule of the substance is less polar than water), and the least polar end of the molecule will be turned outward. Solutes more polar than water are negatively adsorbed. EVIDENCE FOR ORIENTATION OF MOLECULES IN SURFACES AND INTERFACES I. Evidence from the energy of rupture. — The orientation theory indi- cates that if a bar of liquid octyl alcohol (Fig. 12) were to be pulled apart, one of the steps in the process would be for the molecules to orient where the break is to occur (Fig. 13) in such a way that this will become the weakest part of the bar. Evidently this means that the final break will occur between the non-polar ends of the molecules. If octane (CsHis) is ruptured, the work done for a bar of i sq.cm. cross-section (Wc) is 43.5 ergs. When octyl alcohol — - - :Alcphol-_ - z-i Fig. 12. — Bar of liquid of unit cross- section. is pulled apart, additional energy must be utilized in orienting the molecules, so it is not surprising that the work of rupture (Wc) is slightly higher (55.0). If, however, the octyl alcohol is to be pulled away from water, and the molecules of the alcohol are oriented in the interface, then t/ie final break must come between the polar hydroxyl ( — OH) groups of the alcohol and the polar molecides of water (Fig. 14): therefore (the orientation theory predicts that in this case) the work of rupture (work of adhesion, Wa) should be high. The experimental results show that this prediction of the theory is justified, since the work of adhesion is found to be 92 ergs, or 60 per cent higher than the work required to rupture the alcohol. Even more remarkable is the fact that this is 1 11 per cent higher than the work required to rupture octane, and 1 10 per cent higher than the amount of work necessary to separate octane from water. The extremely remarkable nature of these results is evident when it is considered that the molecule of octane contains 26 atoms, while that of octyl alcohol contains these same 26 atoms and only one more, an atom of oxygen which gives the polar nature to the molecule. Thus an increase of less than 4 per cent in the number D Alcohol Fig. 13. — Represents the orientation of the molecules which occurs if a bar of alcohol is pulled apart. l62 ATOMS, IONS, SALTS, AND SURFACES of atoms increases the work of attraction for water by i lo per cent, which is suffi- cient evidence that the oxygen of the alcohol must be oriented toward the surface of the water. If the alcohol surface is pulled from the water surface at the interface between the two the interface disappears and a water {A ) surface and an alcohol {B) surface ap- pear. The work done is aided by the free energy of the surface which disappears, and hindered by those which appear, so WA=yA-]-yB—yAB . Values of the interfacial tension (t^b) for a number of liquids are plotted in Figure 15, and for the work of adhesion in Figure 16. THE SPREADING OF ONE LIQUm ON THE SURFACE OF ANOTHER Most organic liquids will spread on water, but water spreads on almost no organic liquids. It is easy to show that spreading or non-spreading is determined by the work of adhesion {Wa) between the liquids and the work of cohesion (Wc) for the upper liquid S=Wa-Wc(B). If S is positive, the liquid B will spread on the surface of A;ilS is negative, it will not spread. The presence of polar groups in the organic liquid is not essential for spreading, since hexane and octane, as well as benzene, spread on water. Not only organic liquids, but water as well, spread on a clean surface of mercury: Fig. 14. — Octyl alcohol over water. Illus- trates the oreintation of the alcohol mole- cules at the interface. S = yA-(y -{-y ) . 2. Evidence for orientation of molecules in surfaces of pure liquids; comparison of energy of surf ace formation with heat of vaporization. — If a liquid consists of molecules with one end polar and the other end non-polar, the energy required to lift the non- polar end (the "light" end from the standpoint of electrical forces) into the surface is much less than for the polar end, so the orientation theory predicts that in the outer layer of molecules the non-polar groups will be at the surface. However, if such a molecule passes into the vapor state, the polar end of the molecule must be separated from the liquid, and thistwill require a relatively large amount of energy. Therefore, according to the theory, the energy per molecule of surface formation (/?) should be small as compared with the energy of vaporization (X). If the molecule is symmetri- WILLIAM D. HARKINS 163 cal, then h/\ should be much larger, since there is no "light" end which can be lifted into the surface. In entire confirmation of these ideas, h/\ is very small (0.18) for the lower al- cohols which have highly unsymmetrical molecules, and much larger (0.50-0.60) for highly symmetrical molecules, such as those of oxygen, nitrogen, or mercury. These values are those found at a corresponding temperature of 0.7 (T = o.'jTc), but the same general relations are followed at other temperatures. /o £0 JO 40 so Fig. 15. — The free interfacial energy (or interfacial tension) between organic liquids and water This is the only evidence thus far found which indicates strongly that the mole- cules in the surfaces of pure liquids are oriented. 3 . Evidence for orientation in relations of monomolecular films. — If a long-chain fatty acid, such as stearic acid, is dissolved in hexane it spreads on water to form a dilute film I molecule thick. This may be compressed between the movable barrier (back of Fig. 17) and the floating barrier attached to a film balance (front of Fig. 17). When the film is compressed until it is tightly packed, but still monomolecular, the area per molecule is 19.3 A units of area (19.3 A^ = 19.3X10"'^ sq.cm.) (Fig. 18). The square root of this area, 4.4 A, gives an idea of the diameter of the area occupied by the 164 ATOMS, IONS, SALTS, AND SURFACES molecule. This area is found to be nearly the same for long-chain compounds of from 16 to 30 carbon atoms long, which indicates that the molecules in the film are oriented. The thickness of such a film, calculated on the basis of the idea that its density is that s& 'f2- 3^ Fig. 16.— Adhesional work, ergs per square centimeter, between organic liquids and water. (The names of the substances represented by the curves are given at the right while the names given in the middle of the diagram represent substances for which the values are given at 20° only.) of the pure organic substance, indicates that each CH2 group adds about 1.4 A to the thickness of the film, while X-ray measurements on the solid substance give a mean value of about 1.15 A. FILMS ABSORBED FROM LIQUIDS The best evidence at present available indicates that the films on water of butyric acid, amyl amine, octyl alcohol, phenol, resorcinol, and all analogous substances are WILLIAM D. HARKINS 165 essentially monomolecular at sufficient concentrations of the solutions. With long- chain (10 carbon atoms) compounds, only dilute aqueous solutions are obtained, so the time necessary for the diffusion of sufficient organic substance into the surface to Fig. 17. — Perspective drawing and photograph of filmometer (design of B. B. Freud, a modi- fication of the filmometer of N. K. Adam). i66 ATOMS, IONS, SALTS, AND SURFACES give a monomolecular film which is in equilibrium with the solution is considerable — more than thirty minutes in the case of decylic acid (lo carbon atoms) (see Fig. 19). Fig. 18. — Areas per molecule (X-axis) for monomolecular films on water. The F-axis represents the "force of compression" which is measured by the film balance, and is equal to the surface tension of water minus the surface tension of a water surface covered by a film of the organic substance. The curves for palmitic acid, and for stearic acid alone, are of the more usual t>'pe. These films are under high pressures. Under low pressures the curves are similar to the p, v curves for gases. For com- parison, two curves for polymolecular films of phenanthrene and triphenjdmethylcyanide are given. The areas per molecule for these two substances are from 2 to 6 A, which is too small an area for a monomolecular film. The line for palmitic acid extrapolated to zero pressure gives 20.2 A, and for stearic acid on water alone the two lines give 19.1 and 19.2 A of area. Laurie acid forms a dilute monomolecular film. WILLIAM D. HARKINS 167 The equation of Gibbs, which gives the number of gram molecules (u) absorbed per square centimeter of surface formed, may be expressed: I 87 RT d In a .08 < Fig. 19. — Effect of time on the drop weight (surface tension) of decylic acid, 0.0015 N Form. ; Acetic Prop/ on I c 'ric Va/en CoprdfC Hepti/(c NONl/i Decy/ c -a -1 ■ WatS/- LoK of Concentration Fig. 20. — Adsorption curves for fatty acids i68 ATOMS, IONS, SALTS, AND SURFACES a In Cj , but the correction is less than lo per 4 3 Z ^^ ^ lie, ^-^.^^ KCI c^ci^ The difficulty in the application of this equation is that activities in solution are known for almost no organic substances. Figure 20 shows how the surface tension of solutions of organic acids varies with the logarithm of the concentration The slope of k-t — is slightly greater than ^r-. — - o In a o-'o d In C cent in the case of the condensed monomolecular film of butyric acid. The nearly iden- tical slopes of the 7, log C, curves indicate that the adsorption is nearly independent of the number of carbon atoms in the molecule for the tightly packed mono- molecular films. The calculations made thus far indicate that the area per molecule in the film for these soluble sub- stances is of the same order as that for the longer mole- cules which are insoluble. Data on the surface and interfacial tensions indi- cate that between water and benzene, or between water and hexane, the num- ber of butyric acid mole- cules in unit area of the film is exactly the same, within the limits of experimental error, as between water and vapor. This is additional evidence that the number of molecules in a condensed film depends mainly on the size of the molecules and their orientation. MEAN THICKNESS OF THE WATER FILM ON SALT SOLUTIONS Water is positively absorbed on aqueous salt solutions, i.e., the surface contains much less salt than the solution. It is possible to calculate a mean thickness for the water film if it is assumed that the film consists of pure water, and that just below the film the concentration of the solution is the same as farther inside. The remarkable relation which emerges from such calculations is that in very dilute solutions the calculated film thickness is almost exactly the cube root of the volume occupied in water by a water molecule (3.1 A). Figure 21 plots the thickness of the water film as a function of the concentration of the salt. The water film between water and benzene is found also to have practically the same thickness at the same concentration as between water and air. Thus, either between aqueous solution and vapors, or between the aqueous phase and a non-polar liquid, the water film is monomolecular in dilute solutions, and even thinner in concentrated solutions. Both between salt solution and vapor and between salt solution and benzene the surface tension increases as a linear function of the mo- lality of the salt in the solution. Fig. 21.— Strom units). Mols of salt per i,ooo-gm. water ■Thickness of the water film on salt solutions (Ang- WILLIAM D. HARKINS 169 ELECTRICAL CHARACTERISTICS OF EMULSIONS AND SUSPENSIONS If an emulsion or a suspension is placed between a positive and a negative elec- trode, it is fcund that the particles move (with respect to the water) toward one of the two electrodes, and that in general all of the particles of the same material in the same medium move at the same speed, whether they are large or small. This phenomenon of the movement of small particles in the electric field is known as "cataphoresis." It may be considered that this is analogous to the conductance of electricity by the ions of a salt in electrolysis. If equal parts of hexane and an aqueous solution of a sodium oleate soap are mixed together by shaking or by stirring with an egg-beater, it is commonly found that the droplets vary from less than 0.2 /x to about 10 tx in diameter, with the largest number of drops at a diameter of 1-1.5 m. Determinations of the amount of soap absorbed indicate that each droplet of oil in a stable emulsion is surrounded by a monomolecular film of soap. Since soap is part- ly hydrolyzed, the film should consist of molecules of sodium oleate and of oleic acid. The total number of oleate molecules in the film around a droplet of a diameter of i /u is of the order of 10,000,000 or 15,000,000. According to the orientation theory, the hydrocarbon groups of the soap are turned toward the oil and the polar groups with their positive sodium ions toward the water. It is to be expected that such a droplet ivill act like a highly polyvalent salt mole- cule, and that sodium ions will diffuse off into the solution, leaving a negative charge on the droplet. The question now arises, To what extent is the droplet ionized? Since, however, the different sodium ions diffuse to different distances, the ionization should be expressed as a relation concerning the distribution. However, it is easy to calculate a mean or effective ionization by the use of the law of Stokes for the motion of a spherical droplet in a viscous medium. In this way it is found that the velocity of such a particle (4 /j, per second per volt per centimeter) corresponds to a negative charge of 2,430 electronic charges on a par- ticle I (X in diameter, i.e., if all of the 10,000,000 or 15,000,000 molecules of the soap except the 2,430 were completely un-ionized, and if the 2,430 molecules of sodium oleate were so completely ionized that the 2,430 Na+ ions are at an infinite distance, then the oil droplet should have a negative charge equal to that of 2,430 univalent negative ions. The equation is j^T_ 6Trrr] v e A in which e is the charge on the electron, N is the number of charges, 77 is the viscosity V of the solution, and ^ is the mobility or the velocity for unit potential gradient. The potential of a charged sphere is , Ne , dirri V

obtained by the use of the equation of Stokes is always 3/2 the fictitious zeta (f) potential usually given in books on colloid chemistry. According to the relations given above: (i) the effective ionization (A^) of a col- loidal particle varies directly as its radius. (2) The effective ionization per unit area A^\ — ) varies inversely as the radius of the particle, and therefore directly as the curva- ture of the surface. (3) The potential for the particle is independent of the radius. Therefore the potential is the same for all spherical particles of the same material in any certain solution. (4) The effective ionization (N) and the potential () depend upon the nature of the particle and upon the nature of the medium in which it is suspended. The Helmholtz-Lamb equation for the velocity in cataphoresis is ) from 69 to 55.5 millivolts). TABLE V Lowering Effect of Salts upon the Cataphoretic Mobility (y), the Zeta Potential (f), or the Phi Potential (0) for Droplets of Oil in Water No. of Milliequivalents of Salt Electrolyte Required to Lower f from 46 to 37 Millivolts (or ^ from 6g to 55.5 Millivolts) KCl 24 BaCU o. 90 AICI3 03 ThCl4 0.02 The same relationship concerning the valence of the ion of opposite charge is ap- parent in the flocculation values. Thus, for the negatively charged arsenic trisulphide TABLE VI Flocculation Values; Number of Milliequivalents of Salt per Liter Required to Flocculate the Arsenic Trisulphide Sol c 1. Ajj J Flocculation Value Milliequivalents Salt Added ^f g^H pg^ Liter Monovalent Cations LiCl 58 NaCl 51 KCl 50 KNO3 SO ^K.S04 63 HCI 31 Divalent Cations MgS04/2 1.62 MgC]2/2 1.44 CaCl2/2 1.30 ZnCl2/2 1.36 Trivalent Cations AICI3/3 0.279 Al(N03)3/3 285 Ce(N03)3/3 0-24 Tetravalent Cation Th(N03)4 0.36 sol the number of millimols per liter of salt required to cause flocculation decreases very rapidly with the valence of the positive ion (Table VI). The work thus far done upon the carrying down of ions by precipitates shows that with a negatively charged sol flocculation is produced by about the same number of equivalents of one positive ion as another (Table VII). This indicates that inor- ganic ions of high charge (valence) are much more highly absorbed than those of low charge (valence). WILLIAM D. HARKINS TABLE VII Flocculation Values 173 Ion In Milliequivalents per Liter Milliequivalents of Cation Absorbed by 25 Gm. AsjSj K..... (Aoneline) SO 2-5 0. I 1. 2 I. 2 I. 2 I. 2 0.27 2.05 1.8^ (New fuchsin) Ca Sr Ba uo. 1.90 2.50 2. 05 215 2. 20 Ce 1.72 STABILITY OF AN EMULSION AND THE MONOMOLECULAR FILM An emulsion produced from equal volumes of water and oil by the use of sodium oleate (a soap) as an emulsifying agent is found to be stable for a period of years if a o.i-molal solution of soap is used, but changes greatly in a few days if produced by Fig. 22. — Shows how the change from the soap of a monovalent metal, such as sodium, to that of a bivalent metal, such as calcium, may change the emulsion from one of oil droplets in water to an emulsion of water droplets in oil. 0.005-molal soap. In the latter case not enough soap is present to form a condensed mono- niolecidar film around the particles of oil, so the droplets of oil unite in order to de- crease the interfacial area and thus increase the concentration of the soap in the film. With o.i-molal soap a condensed fikn of approximately monomolecular thickness is present, and this gives a considerable degree of stability to the emulsion. If the soap is the salt of a univalent metal, the droplets will be of oil, with water outside; but with bi- or trivalent metals several hydrocarbon chains are present for each single positive ion, and (Fig. 22) the droplets are of water, with oil outside. 174 ATOMS, IONS, SALTS, AND SURFACES THE DETERMINATION OF SURFACE AND INTERFACIAL TENSION The use of what is often called the "drop-number method" of determining the surface or interfacial tension is not only a waste of time but fills the literature of bi- ology with worse than useless data, since they are so extremely deceptive. Drop-weight method. — The capillary-height and the drop-weight methods are the most accurate now known. The drop- weight method is in general much the more suitable of the two for biological investigations. It depends upon the fact that if a drop of liquid is allowed to fall from a horizontal circular disk (end of a glass capillary tube) with sufficient slowness, the weight of the drop is a definite function of the surface tension, the radius of the tip, and the volume of the drop which falls, so W = Mg = 2 -nryf , where/ stands for the value of the function. Thus 2-Kr] or if V— 2 7r/' F=-' 7 = M^F. r To calculate the surface tension: (i) The volume (F) of the drop is its mass in grams divided by its density ( ^ = ^ ) • (2) Multiply F by - , in which r is the radius V . of the circular tip. (3) Find — in Table VIII, and note the corresponding value of F. (4) Divide g (in dynes per sec.^ = 980.3 at Chicago) by r^ {r in centimeters), and multiply by M in grams, and by F. The result gives the surface tension in dynes per centimeter. In the determination of interfacial tension the volume of the drop is measured di- rectly. The weight of the drop as it hangs in a second liquid is V {d^—d^, in which d^ is the density of the heavier and d^ that of the lighter liquid. The liquid which is dropped should always be the aqueous phase, so if the oil is the heavier the tip should face upward. Ring method for determination 0] surface tension. — The pull on a ring just being de- tached from the surface of a liquid does not give the surface tension, but the pull must be multiplied by a factor F, as given by Young and Cheng and the writer in a recent paper in Science. The outline of the method is as follows: 1. Determine the total pull in grams (M) necessary just to detach a circular ring of diameter R, of circular wire of diameter r, from the surface of the liquid by the use of a chainomatic balance or a torsion balance, such as that of Du Nouy. 2. The surface tension is given by the equation 3. Obtain the value of F, the correction factor, from Figure 23. F is plotted on the Ri F-axis. To do this find the value of vf > the cube of the radius of the ring divided by WILLIAM D. HARKINS 175 M the volume of liquid (F) upheld by the ring. V= — , or the volume of liquid is equal to the weight given by the balance, divided by the density of the liquid. 4. After finding the value of ..j on the A'-axis, find a point on one of the curves TABLE VIII Drop-Weight Surf ace-Tension Corrections (Factor for Multiplication = F), Based on the Value 72. 75 as the Surface Tension of Wate