o= X- 5= tor cr : =r : r^ so ^ CD s a THE BACTERIOPHAGE AND ITS BEHAVIOR The Bacteriophage AND ITS BEHAVIOR BY F. D'HERELLE, M.D. Directeur du Service hacteriologique du Conseil Sanitaire, Maritime el Quarantenaire d'Egypte TRANSLATED BY GEORGE H. SMITH, PH.D. Associate Professor of Bacteriology and Immunology, School of Medicine, Yale University PUBLISHED BY THE WILLIAMS & WILKINS COMPANY BALTIMORE, MD., U. S. A. 1926 Copyright, 1926 THE WILLIAMS & WILKINS COMPANY Made in United States of America Published March, 1926 all rights reserved COMPOSED AND PRINTED AT THE WAVERLY PRESS FOB The Williams & Wilkins Company Baltimohe, Md., U. S. a. PREFACE The preliminary notes upon the phenomenon of bacteriophagy, beginning with those published in 1917, attracted the attention of scientists to but a moderate degree, despite the fact that the facts presented appeared to be contrary to all of that which had been de- scribed up to that time. Only after the publication of the first collected work in 1921 did investigators on all sides really undertake the study of this phenomenon, as interesting as it is complex. Four years have elapsed since then and during this time more than six hundred memoirs and notes have appeared, to what purpose the reader of the following pages will see. The present text is divided into three parts. It is evident, a priori, that by virtue of the principle that "there can be no effect without a cause" the phenomenon of bacteriophagy is induced by a "something" — by a "principle." This is necessarily true whatever may be the hypothesis advanced as to the state or nature of the causative agency. A study of bacteriophagy in vitro is the first aspect of the problem which we will consider. In reality, this is simply a study of the re- spective behaviors of the unknown "principle, "the cause of the phenom- enon, and of the bacterium which is subject to the phenomenon. Before studying the nature of a principle we must know something of how that principle behaves. From the broader point of view all of the studies carried out on the phenomenon of bacteriophagy itself have confirmed my first observa- tions. Thanks to the experiments of a great many authors certain details of the process have been more clearly defi^ned, and it may be said that today the phenomenon itself is well understood. There remains, however, still another side to the question, a phase which opens a field of the greatest theoretical and practical importance, namely, the problems associated with the resistant forms of bacteria, with the infravisible forms in particular. What are the properties of this "principle" which causes the phenom- enon? What is its physical state? And more important yet, what is its biological state? These are the things that it is important for us to discover. These are the points which it is particularly important to bring to the attention of the trained scientific mind. CONTENTS INTRODUCTION I, Historical 1 The Phenomenon of Bacteriophagy 1 Bacterioclysis: The Twort Phenomenon 13 II. Terminology and Technic 18 Terminology 18 Technical Procedures 21 Ultrafiltration 24 Preparation of ultrafilters 26 Assembling the ultrafilter 29 Denitrification 30 Sterilization 32 PART I. THE PHENOMENON OF BACTERIOPHAGY Chapter I BACTERIOPHAGY IN A FLUID MEDIUM 1. Isolation of the Bacteriophagous Principle 37 Ubiquity of the bacteriophage 37 Methods of isolation 38 Technic of isolation 39 2. Serial Action 40 3. Environmental Conditions Favoring Bacteriophagy 41 General conditions 41 Reaction of the medium 43 4. Effect of the Condition of the Bacterium 46 5. Effects of the Relative Concentrations of Bacteriophage and Bacteria.. 49 Limits of bacteriophagy 51 Limits of activity of the bacteriophage principle 55 6. Influence of Physical Conditions 58 Action of heat 58 Pressure conditions 63 Viscosity of the medium 63 7. Effect of the Chemical Conditions of the Medium 65 Colloids 65 Dyestuffs 67 Electrolytes 68 Sugars 70 Salts 70 Body fluids and other substances 71 Antiseptics 71 IX j^3l X CONTENTS 8. Phenomena Correlative with Bacteriophagy 73 Acceleration of bacterial growth 73 Agglutination 73 Resume 74 Chapter II THE BACTERIOPHAGE CORPUSCLE 1. Bacteriophagy upon Solid Media 76 2. The Bacteriophage Corpuscle 82 3. The Plaque: A Colony of Bacteriophage Corpuscles 87 4. Conditions Essential for Plaque Formation 88 5. The Characters of Plaques 92 6. Enumeration of Bacteriophage Corpuscles 96 Resume 99 Chapter III THE mechanism OF BACTERIOPHAGY 1. The Corpuscle: Obligatory Bacteriophage 101 2. Fixation of the Bacteriophage Corpuscle 104 3. Penetration of the Corpuscle into the Bacterium 112 4. Multiplication of the Bacteriophage Corpuscle 115 The course of multiplication 116 Influence of temperature upon multiplication 123 Multiplication as affected by the state of the bacteria 124 Multiplication in relation to the number of bacteria bacteriophaged. . 125 Influence of the conditions of the medium 126 Cause of the arrest of multiplication 127 5. Bacteriophagy under the Microscope 130 Resume 135 Chapter IV THE VIRULENCE OF THE BACTERIOPHAGE 1. Variation in the Activity of Bacteriophage Corpuscles 137 2. Evaluation of the Virulence of a Bacteriophage 141 3. Pure Races of the Bacteriophage 155 4. Variability in the Virulence of the Bacteriophage 159 5. Increases in Virulence 160 6. Attenuation of Virulence 165 7. Homogeneous and Heterogeneous Bacterial Species 168 8. Multiple Virulences of the Bacteriophage 170 9. Persistence of Virulence 171 10. The Mechanism of the Persistence of Virulence 173 11. The Acquisition of Virulence 175 12. Bacteriophagy in Mixed Cultures 178 Resume 179 CONTENTS XI Chapter V RESISTANCE OF THE BACTERIA 1. Secondary Cultures 182 2. The Origin of Secondary Cultures : 188 3. Variability in Acquired Resistance 191 4. The Acquisition of Resistance 194 5. The Behavior of the Bacteriophage in Secondary Cultures 196 6. The Loss of Resistance 203 7. The Bacteria of Secondary Cultures 206 8. Mixed Cultures 208 9. The Cause of Secondary Cultures 215 10. The Resistant Bacterium 217 11. Ultrabacteria 226 12. Natural Mixed Cultures 230 13. The Purification of Natural Mixed Cultures 236 14. Isolation of the Bacteriophage from Naturally Mixed Cultures 237 R4sum6 238 Chapter VI SPECIES OF BACTERIA SUSCEPTIBLE TO BACTERIOPHAGY 1 . Homogeneous Species 242 1. B. dysenteriae Shiga 242 2. B. dysenteriae Pliss 243 3. B. dysenteriae Flexner 244 4. Pseudo-dysentery organisms 244 5. Bacilli of fowl typhoid 245 6. Pasteurella bovis 246 7. B. pestis 247 2. Heterogeneous Species 248 1 . B. typhosus 248 2. B. paratyphosus A 251 3. B. paratyphosus B 254 4. B. suipest'ifer 254 5. B. enteritidis 254 6. B. txjphi-murium 254 7. B. coll 255 8. The Pneumobacillus of Friedlander 256 9. The Bacillus of Flacherie 256 10. B. proteus 257 11. The Bacillus of Swine Fever 258 12. B. diphtheriae 258 13. Nodule Bacteria of Plants 259 14. B. suhtilis 261 15. Vibrio cholerae 261 16. The Staphylococci 264 XU CONTENTS 17. The Enterococcus 266 18. Streptococcus pyogenes 266 3. Phenomena Simulating Bacteriophagy 266 B. anthracis 266 B. pyocyaneus -. 267 Resume 269 PART II. THE BACTERIOPHAGE Chapter I THE BEHAVIOR OF THE BACTERIOPHAGE TOWARD DIFFERENT AGENTS 1. The Physical State of the Bacteriophage 273 Filtrability 274 Diffusibility 277 Volatility 277 Sedimentation 280 Nature of the "Substance" of the bacteriophage corpuscle 281 2. Conservation of the Bacteriophage Corpuscle 283 3. Flocculation of Corpuscles 287 4. Adsorption of Bacteriophage Corpuscles 288 5. Effects of Irradiation 291 6. Effect of Temperature 292 7. Action of Inorganic Chemical Substances 300 8. Action of Organic Compounds 302 9. Variability in the Resistance of the Bacteriophage 306 Resume 307 Chapter II HYPOTHESES CONCERNING THE NATURE OF THE BACTERIOPHAGE 1. Possible Hypotheses , 309 I. Hypothesis of a chemical principle foreign to the bacterium 309 II. Hypothesis of a principle derived from the bacterium 311 A. Hypothesis of an abnormal inert principle 311 B. Hypothesis of a living abnormal principle derived from the bacterium 315 C. Hypothesis of a normal autolysin 316 D. Hypothesis of a living principle, normally present in the bacterium 325 III. The Bacteriophage is a Living Being, Foreign to the Bacterium. . 326 Resume 327 Chapter III THE NATURE OF THE BACTERIOPHAGE 1. Statement of the Problem 329 2. The Criteria of Life 330 3. The Autonomy of the Bacteriophage Corpuscle 333 CONTENTS XIU 4. The Power of Assimilation of the Bacteriophage Corpuscle 341 5. The Power of Adaptation of the Bacteriophage Corpuscle 341 6. The Faculty of Multiplication of the Bacteriophage Corpuscle 349 7. Variability of the Bacteriophage 350 8. The Bacteriophage Corpuscle: A Living Ultravirus 354 Resume 356 Chapter IV THE UNICITY OP THE BACTERIOPHAGE PROTOBB 1. The Bacteriophage Protobe 358 2. Assimilation of the Protobes 360 3. The Concept of Species Among the Microbes and the Protobes 365 4. The Unicity of the Species Protohios hacteriophagus 366 5. The Mode of Action of the Bacteriophage 369 6. Consequences Resulting from the Living Nature of the Bacteriophage. . 374 Resum6 376 PART IIL THE BEHAVIOR OF THE BACTERIOPHAGE PROTOBE Chapter I THE BACTERIOPHAGE AS AN ANTIGEN 1. Inoculation of the Bacteriophage 381 2. Antibacteriophagic Sera 383 3. The Course of the Action of the Antibacteriophagic Serum 386 4. Variability in the Behavior of the Bacteriophage 387 5. The Bacteriophage Antigen 391 6. The Nature of the Antibacteriophagic Property 392 7. Complexity of the Antibacteriophagic Serum 397 8. The Anti-antibacteriophagic Serum 398 9. The Action of Antibacterial Sera 400 10. The Phenomenon of Antiphylaxis 403 11. The Opsonic Action of Bacteriophage Suspensions 408 Resume 414 Chapter II THE ubiquity OF THE BACTERIOPHAGE 1. The Bacteriophage in the Intestinal Tract 416 2. The Bacteriophage in Healthy Man 418 3. The Bacteriophage in Animals 424 4. The Bacteriophage in the Horse 426 5. The Bacteriophage in the Chicken and in the Goose 429 6. The Bacteriophage as Found in a Number of Different Species 430 7. The Virulence of the Bacteriophage in the Normal Animal 431 8. The Bacteriophage in the External Environment 435 Resume 436 XIV CONTENTS Chapter III THE BEHAVIOR OF THE BACTERIOPHAGE IN DISEASE 1. Variations in the Virulence of the Bacteriophage 437 2. The Bacteriophage in Bacillary Dysentery 439 3. The Bacteriophage in Typhoid Fever 451 4. The Bacteriophage in Avian Typhosis 479 5. The Bacteriophage in Staphylococcus and Streptococcus Infections .... 483 6. The Bacteriophage in Colon Bacillus Infections 483 7. The Bacteriophage in Typhus Exanthematicus 485 8. The Bacteriophage in Plants 487 Resume 488 Chapter IV THE BEHAVIOR OF THE BACTERIOPHAGE IN EPIDEMICS 1. Avian Typhosis 490 2. Hemorrhagic Septicemia of the Buffalo (Barbone) 497 3. Bubonic Plague 503 4. Bacillary Dysentery 506 5. Flacherie of the Silk Worm 507 R6sum6 507 Chapter V IMMUNIZATION WITH BACTERIOPHAGE SUSPENSIONS 1. The Problem of Prophylactic Immunization 509 2. Immunization against Avian Typhosis 510 3. Immunization against Barbone 520 4. Immunization against Bacillary Dysentery 533 Resume 538 Chapter VI SPECIFIC therapy WITH BACTERIOPHAGE SUSPENSIONS 1. The Specific Therapy of Bacillary Dysentery 540 2. Bacteriophage Therapy in Different Intestinal Disturbances 549 3. Bacteriophage Therapy in Typhoid and the Paratyphoid Fevers 549 4. Bacteriophage Therapy of Colon Bacillus Infections 553 5. Bacteriophage Therapy of Staphylococcus Infections 558 6. Bacteriophage Therapy of Infected Wounds 564 7. Bacteriophage Therapy of Streptococcus Infections 567 8. Bacteriophage Therapy of Bubonic Plague 567 9. Aphthous Fever 576 Resume 577 Conclusion 577 Bibliography 579 Index 609 INTRODUCTION I. Historical THE PHENOMENON OF BACTERIOPHAGY Before considering the historical development of our knowledge concerning the phenomenon of bacteriophagy, let us consider the cir- cumstances which offered a veiled suggestion that such a phenomenon existed, although none of the facts pertaining to the reaction were at that time known. Let us consider the initial observation which served as the starting point for the study of the phenomenon of bacteriophagy. During the course of the investigation of a disease, bacterial in origin, affecting locusts, first noted in Mexico in 1909 where whole swarms of the insects succumbed to the infection* several new facts were observed. The disease, as it occurred naturally was primarily a septicemic condition, accompanied by intestinal disturbances, as re- vealed by the profuse diarrhea seen in the infected insects. The pathogenic microorganism, Coccobacillus acridiorum, was present in the intestinal fluids in great abundance. Inasmuch as these locusts constitute an insect pest, causing enor- mous destruction of crops in many tropical regions, and indeed in some sub-tropical zones, it seemed that it might be possible and distinctly advantageous to artificially implant this natural epizootic among the colonies made up of larval forms and thus destroy the harmful insects in large numbers. To this end, the virulence of the coccobacillus was enhanced by successive passages through locusts in the laboratory. Large quantities of a fluid culture medium were inoculated with the bacterium and this material, after incubation, was distributed from place to place among the masses of insects, still in the larval state. Under such conditions the disease developed rapidly. f * d'Herelle— Sur une epizootie de nature bacterienne sevissant sur les Sau- terelles en Mexique. Compt. rend. Acad, sci., 1911, 152, 1413. t Consult on this subject: d'Herelle— Le Coccobacille des Sauterelles. Ann. Inst. Pasteur, 1914, 28, 280; 387. Beguet, M. — Deuxieme campagne contre les sauterelles Stauronotus maroc- canus Thun. en Alg^rie au moyen du "Coccobacillus acridiorum" d'Herelle. Ana, Inst. Pasteur, 1915, 29, 520. 1 2 THE BACTERIOPHAGE AND ITS BEHAVIOR As has been said, the preparation of cultures for the mass infection of the colonies of insects involved the isolation of the virulent cocco- bacillus from the intestinal contents of locusts with an experimental laboratory infection. And in this procedure certain observations were made which now offer the point of greatest interest. On several occa- sions the culture tubes used for isolation, or for transplanting the cul- tures, yielded colonies which were of indented irregular contour, or, in the midst of a group of confluent colonies there were at times areas entirely free of growth. These cultural irregularities were sufficiently _ pronounced to arouse my curiosity, and to explain them, and the phenomenon leading to their formation, an hypothesis was advanced, one which proved to be entirely false. Indeed, it must be added that for a very long time this hypothesis led me to perform many useless experiments. In accord with this hypothesis it seemed that the coccobacillus could only be an "associated" organism, the true pathogenic agent neces- sarily being an ultramicroscopic organism, which, occasionally reaching the agar, inhibited the growth of the associated bacterium. This hypothesis appeared the more natural in view of the admirable work of de Schweinitz and Dorset upon hog cholera. But, after having demonstrated that the filtrate by itseK contained nothing virulent for the locust the hypothesis was modified in accordance with the concept that the disease required that two agents, visible and invisible, be simultaneously present. Throughout a long series of investigations attempts were made to determine if this hypothesis could be reconciled with the observed facts Beguet, M. — Campagne d'exp^rimentation de la methode biologique centre Ic Schistocerca peregrina en Algerie de Decembre 1914 a Juillet 1915, et en particular dans la region de Barika (Departement de Constatine). Ann. Inst. Pasteur, 1916, SO, 225. Velu, H., and Bouin, A. — Essai de destruction du Schistocerca peregrina au Maroc par le "Coccobacillus acridiorum" du d'Herelle. Ann. Inst. Pasteur, 1916, 30, 389. Velu, H. — Deuxieme campagne d'experimentation de la methode d'Herelle au Maroc contre Schistocerca peregrina Olivier. Ann. Inst. Pasteur, 1917, 81, 277. Revista del Ministerio de Industrias del Uruguay, 1918. Revista del Ministerio de Obras publicos de Venezuela, Suppl., 1913. Cheyssial, A. — Experimentation de la methode de d'Herelle en Guin^e fran- Qaise pour la destruction des acridiens. Bull. Soc. path, exot., Par., 1922, 15, 762. INTRODUCTION 6 in some of the human diseases, particularly bacillary dysentery and typhoid fever. But here again, always under the domination of a false hypothesis, the studies were made upon intestinal contents of patients in whom the disease was at its height. To this end filtrates were prepared from a suspension of fecal material and these filtrates were combined with suspensions of the bacterium, B. typhosus or B. dysenteriae as the case might be, and these were next inoculated into laboratory animals, hoping thereby to induce symptoms resembUng those seen in the human subject during the disease. At the same time, an agar culture medium was inoculated with the filtrate-bacterial suspension mixture in an attempt to reproduce the cultural "abnor- malities" noted with the coccobacilli from the locusts. It is true that this procedure occasionally revealed cultural irregu- larities, but the phenomenon was very inconstant and this fact pre- vented a solution of the question. One day, in reexamining my experimental data my attention was attracted to the fact that when such cultural irregularities appeared it was never at the beginning of the disease but always when filtrates were used which were prepared from fecal material collected during convalescence. I then resolved, and logically this is where I should have commenced, to examine the fecal discharges of individual patients systematically, from the onset of the disease up to the time when convalescence was established. In August, 1916, an adult with a severe bacillary dysentery (Shiga) was under treatment in the Pasteur Hospital. Each day about 10 drops of the stool were collected and placed in a tube of bouillon. After incu- bation over night the suspension was filtered through a Chamberland candle. Into some bouillon, previously inoculated with Shiga bacilli, about 10 drops of this filtrate were placed, and the material was re- turned to the incubator at 37°C. Throughout the duration of the disease, all of the tubes, prepared each day in the same manner, gave normal cultures of B. dysenteriae. One day, the tube prepared the day before remained sterile. Investiga- tion showed that the patient gave evidence of notable improvement, and, as appeared later, this was shortly followed by definite con- valescence. To the bouillon thus inoculated and containing filtrate, and which had remained to all appearances sterile, a suspension of Shiga bacilli derived from a fresh agar culture was added to yield a marked turbidity. This tube was placed in the incubator. After about 10 hours it was again clear. r: 4 THE BACTERIOPHAGE AND ITS BEHAVIOR This, of course, made it at once apparent that my first hypothesis was of necessity false, the truth of the matter being that the fecal material used in preparing the filtrate contained something which dis- solved the dysentery bacilli. Nevertheless, my first hypothesis had one virtue, since, as it had led me for such a long time to consider the question of a virus pathogenic for the man or the animal it offered the suggestion that the dissolving principle might be a virus pathogenic for the bacterium. And because of this last hypothesis the following experiment was devised and carried out. A drop of a dissolved culture was added to a fresh bouillon culture of Shiga bacilli. About 15 hours later the bouillon was clear, all of the bacilli originally present had been dissolved. Thus, several succes- sive passages were effected in the same way, employing each time a drop of the culture previously dissolved added to a fresh culture of Shiga bacilli. In this repetition of the process, instead of becoming weaker the activity became more and more pronounced, that is, the disappearance of the bacilli was effected with greater and greater rapidity. This time the guiding idea appeared to be correct. The principle present in the intestinal contents became regenerated at the expense of the Shiga bacilli. It behaved like a filtrable organism, parasitic of bacteria. The next step was an attempt to reproduce the cultural irregular- ities upon a solid medium. To do this, a very small amount, about 0.0001 cc. of one of the dissolved cultures was added to a young broth culture of Shiga bacilli and the mixture was subcultured immediately to an agar slant. Similarly, subcultures were made after incubation for 1, 2, and 3 hours, a drop from the inoculated tube being used in the transfers. After incubation of these agar subcultures the growth revealed some of the "abnormal" appearances which had formerly per- plexed me. But this time, the characteristics of these abnormalities were so outspoken that their significance could not be overlooked. In the first tube the surface of the agar was well-covered with a normal layer of B. dysenteriae, except that in the midst of the culture there were two little islands, two "plaques," perfectly circular in form, where the agar was bare, entirely free of all traces of culture. The second agar tube, planted 1 hour after the original combination was made, revealed 6 of these plaques. In the third, prepared after the dissolving principle had acted for 2 hours, there were about 100 of the plaques. The fourth tube remained without any evidence of bacillary growth. INTRODUCTION This, then, gave a new proof that the dissolving principle actually- regenerated in the course of the action. Further, it demonstrated that the principle was condensed in the form of active particles. It is to this principle that I have given the name Bacteriophage; the phenomenon of bacterial solution caused by it being termed Bac- teriophagy. With this simple statement of what is meant by the "phenomenon of bacteriophagy" in its broader aspects, let us see if the literature pub- lished prior to my papers on the subject contains data such as might be construed as dealing with the same phenomenon. In the first work summarizing my communications on the subject^"^* mention was made particularly of a paper by Hankin dealing with a bactericidal property of the water of the Jumna and of the Ganges rivers. Since the text, "The Bacteriophage, Its Role in Immunity," was published various authors have made a study of the literature and many have sought to discover, in the many studies conducted upon the subject of "bacteriolysis," facts which bear upon the question of bacteriophagy. Let us state immediately, and we will return to this question, that the term "lysis," which should always have been appHed strictly in its sense of "a dissolution," has lost in biological usage all significance, and is in fact applied to phenomena which are without any effect upon the vitalitij of the bacterium regarded as undergoing a "bacteriolysis," This is the first source of error, as we shall see. The second cause of error is resident in a faulty logic on the part of those who have examined these earher communications. Bacteriolysis, even true bacteriolysis, resulting in a dissolution of the bacterial cell, is not a phenomenon with but a single cause. Bacteriolysis is a syndrome, one might say, which can be provoked by different causes or diverse agents. A number of varieties of bacteria undergo dissolution when placed in water saturated with ether. May one speak here of bacteriophagy? The pneumococcus, like some other fragile bacteria, is dissolved within a few days if it is allowed to remain in the liquid culture medium where it has developed. Is this bacteriophagy? Certainly not. For, in neither case is the dissolution accompanied by the very special charac- teristics which delimit bacteriophagy. Bacteriophagy, as we will see in the course of this text, is a phe- nomenon presenting very distinctive characteristics, such as permit it * Referring to the bibliographic material appended to the text. 6 THE BACTERIOPHAGE AND ITS BEHAVIOR to be clearly defined. To attempt to reconcile "bacteriolysis" and "bacteriophagy," making them identical and co-extensive is a scientific absurdity. Yet this is precisely what some authors, fortunately now becoming fewer in number, seem to have thought they must do. In a lengthy review of the literature dealing with the bacteriophage Otto and Munter^^"* go into the historical aspect of the subject,* citing all of the more important contributions upon the question of bac- teriolysis in general, beginning with that of Kruse and Pansinif upon the autolysis of the pneumococcus. But bacteriolysis and bacteriophagy are by no means synonomous. Bacteriolysis, the general syndrome, is but an episode in bacteriophagy. It is not the event which distinctly characterizes the phenomenon. Bac- teriophagy is certainly not involved in the autolysis of B. pyocyaneiis, as observed by Emmerich and Low,| nor in the autolysis of B. anthracis, as noted by Gamaleia§ and studied by Malfitano-H The mechanism of the process here is not related to bacteriophagy any more than is the bacteriolysis of this same anthrax bacillus when subjected to the serum of certain animals. There is a similar lack of relationship as regards the inhibitory effects of old normal cultures upon the development of certain bacteria, as first reported by Eijk- * Mention may be made of the fact that in presenting this historical review Otto and Munter did not seek to introduce any question of priority. Indeed, their paper is entitled "Bacteriophagy," bearing the sub-title "d'Herelle's Phenome- non," a fact in itself significant. Otto and Munter, like the other German investi- gators, have, in my opinion been perfectly logical in this respect. My sole criti- cism of the historical part of the review of Otto and Munter is one of a purely scientific nature; a criticism concerning the abuse of generalizations. t Kruse, W., and Pansini, S. — Untersuchungen iiber den Diplococcus pneu- moniae und verwandte Streptokokken. Zeitschr. f. Hyg. u. Infektionskrankh., 1892, 11, 279. X Emmerich, R., and Low, O. — Bakteriolytische Enzyme als Ursache der erworbenen Immunitat und die Heilung von Infektionskrankheiten durch diesel- ben. Zeitschr. f. Hyg. u. Infektionskrankh., 1899, SI, 1. § Gamaleia. — Bakteriolysine-bakterienzerstorenden Fermente. Abst. in: — Centralbl. f. Bakt., I. Orig., 1899, 26, 661. II Malfitano, G. — La bacteriolyse de la bacteridie charbonneuse. Compt. rend. Acad, sci., 1900, 131, 295. Malfitano, G. and Strada, F. — Evaluation du pouvoir prot6olytique des bac- teridies du charbon. Compt. rend. Soc. biol., 1905, 59, 118; — Des influences qui peuvent faire varier le pouvoir prot6olytique des liquides en contact avec des bact^ridies du charbon. Compt. rend. Soc. biol., 1905, 59, 120; — Influence de I'a^ration des cultures sur le pouvoir proteolytique des bacteridies charbonneuse. Compt. rend. Soc. biol., 1905, 69, 197. INTRODUCTION 7 man,* and later by Conradi and Kurpjuweit,t Rahn,t and Faltin,§ to cite only the more important studies. All of these demonstrations of the bacteriolytic process can be re- peated with the greatest ease, and none of them show the shghtest resemblance to bacteriophagy. To even suggest a relationship is im- possible. Indeed, it is but fair to state that none of the authors men- tioned have endeavored to establish such a relationship. Recently Eijkman has conducted some experiments upon bacteriophagy and he states 1 1 that he can not see any possible connection between the phenomenon which he formerly studied and that of bacteriophagy. More recently Hajos^ by the advice of Koranyi, has considered this question again, more particularly to determine if there is any rela- tion between the inhibitory action of filtrates of old cultures and the phenomenon of bacteriophagy. His conclusions are identical with those of Eijkman,^ — the phenomenon of inhibition has no demonstrable connection with that of bacteriophagy. These former studies are mentioned here simply because of the fact that certain authors (Pico^^^) have attempted to correlate all of the phenomena in which bacteriolysis represents a phase, establishing thus a certain degree of confusion, and because they are mentioned in the historical discussion of Otto and Munter. There are, on the other hand, some experimental observations which may possibly be interpreted as revealing the intervention of the bac- teriophage, but even here it is impossible to definitely affirm this, since some of the findings reported by the authors seem to oppose such a correlation. Hankin** stated that the water of certain rivers of India possesses * Eijkman, C. — Ueber thermolabile Stoffwechselprodukte als Ursache der natiirlichen Wachstumshemmung der Mikroorganismen. Centralbl. f. Bakt., I. Orig., 1904, 37, 436. t Conradi, H., and Kurpjuweit, O. — Ueber spontane Wachstumshemmung der Bakterien infolge Selbstvergiftung. Munchen. med. Wchnschr., 1905, 1761. J Rahn, O. — Ueber den Einfluss der Stoffwechselprodukte auf das Wachstum der Bakterien. Centralbl. f. Bakt., I. Orig., 1906, 16, 417; 609. § Faltin, R.- — Studien iiber Hetero- und Isantagonismus, mit besonderer Beriicksichtigung der Verhaltnisse bei infektiosen Erkrankungen der Harnwege. Centralbl. f. Bakt., I. Orig., 1908, 46, 6; 109; 222. II Reunion annuelle de la Societie des Bacteriologues Neerlandais, 1923. U Haj6s, K. — Beitrage zur Frage der wachstumshemmenden Wirkung von Bouillonkulturen. Centralbl. f. Bakt., I. Orig., 1922, 88, 583. ** Hankin, E. — L'action bactericide des eaux de la Jumna et du Gange. Ann. Inst. Pasteur, 1896, 10, 511. 8 THE BACTERIOPHAGE AND ITS BEHAVIOR an extremely marked antiseptic action for bacteria in general, and for the cholera vibrio in particular. Thus, the water of the Jumna, as it left the town of Agra, contained more than 100,000 bacteria per cubic centimeter, while some 5 kilometers further down the bacterial count was reduced to but 90 to 100 organisms. Dealing more particularly with the cholera vibrio, his laboratory findings gave the results presented in the following table. In this table the first line shows the effect of the Jumna river water after filtration. The figures of the second line represent the action of the same filtered water after boiling. In both instances, the water had been inoculated with a culture of V. cholerae, and the rate of action is shown by the bacterial counts made after different intervals. TIME hour 1 hour 2 hours 3 hours 4 hours 25 hours 49 hours Filtered, unhealed water 2,500 5,000 1,500 4,000 1,000 6,000 500 10,000 6,000 10,000 Filtered, boiled water. . 36,000 The germicidal action of the water of these rivers could always be detected, but it was not uniform in degree. It is of interest that it is to this antiseptic action that Hankin attrib- utes the fact that he never was able to demonstrate that the ingestion of the water of these rivers was responsible for the development of a single case of cholera. Certainly these rivers were never the carriers of epidemics; cholera always spreads from down-stream upwards. Hankin showed that the antiseptic principle was destroyed by boil- ing, and, from his experiments, he deduced that it was volatile. Flu^^" denies that the bacteriophage was the agency responsible for this germicidal action, since Hankin had shown that a volatile sub- stance was involved. It is certain that if this statement of Hankin is correct, it by itself suffices to prove that the bacteriophage was not involved. As a matter of fact the experiment on volatihzation offers abundant chance for error, and we shall see that this error has con- tributed to the results of some investigators who have considered the question of the volatile nature of the bacteriophage. The error Hes in the fact that when distillation is carried out at low temperatures, without special precautions, materials are carried over into the dis- tillate, leading to the erroneous conclusion that a volatilization has INTRODUCTION 9 taken place, when in reality it has not occurred and does not occur when the experiment is properly conducted. It should be the duty of some bacteriologist in India to repeat the studies of Hankin, For if the antiseptic substance present in the water of these rivers is actually volatile the action can not be bac- teriophagic in nature. While, on the other hand, if these waters repro- duce the phenomenon with aU of its characteristic features, the bac- tericidal action observed by Hankin must necessarily be referred to bacteriophagy. There is another communication* where it is possible that the bac- teriophage may not have been foreign to the results, — results which stimulated certain somewhat sarcastic remarks on the part of Metch- nikoff. It must be admitted that before the era of the bacteriophage, these results might well excite wonder, and, indeed, certain recom- mendations of the authors of the memoir in question may, with reason, seem strange. For they state that bacteriologists should not attempt a confirmation of their experiments, most probably because they them- selves could not interpret the phenomena observed and were unable to repeat them. However that may be, here is the passage in the work of Metch- nikofff which refers to these investigations. ". . . . Emmerich and Low attribute acquired immunity to particular substances which they call "nuclease-immunoproteidine." In accordance with their supposition the bacterial products which are liberated within the body during the period of vaccination, the nuclease, combines with the protein sub- stances of the blood and of the organs, yielding the substance designated by these authors by this very complex name. In their last publication Emmerich and Low go so far as to describe a method for the production of this substance outside of the body, by causing beef blood, or better yet, ground-up spleen, to act upon the nuclease produced by the bacteria in old cultures. t They attribute to this the property of dissolving diverse bacteria, of vaccinating against, and of curing, many infectious diseases. But these authors do not state whether this substance, so very remarkable, is identical or analogous to the antibacterial ferments com- * Emmerich, R., and Low, O. — Die klinstliche Darstellung der immunisierenden Substanzen (Nucleasen-Immunproteidine) und ihre Verwendung zur Therapie der Infektionskrankheiten und zur Schutzimpfung an Stelle des Heilserums. Zeitschr. f. Hyg. u. Infektionskrankh., 1901, 36, 9. t Metchnikoff, E. — L'Immunite dans les Maladies Infectieuses. pp. 267-68. I These words are not underscored in the text of Metchnikoff. I have under- lined them simply to attract the attention of the reader, for purposes which will become apparent shortly. 10 THE BACTERIOPHAGE AND ITS BEHAVIOR posed as we know, of microcytase and of fixateur. It may be inferred that they believe it comparable to the alexin of Buchner, which is simply a mixture of the two substances already named. Unfortunately, all that this theory of Emmerich and Low accomplishes is to confuse the reader, and in their publications no proof of their aflBrmations is to be found. In fact, many statements which they make are at variance with well established observations. Thus, they speak of a com- plete dissolution of the bacilli of swine erysipelas within the vaccinated animals by their soluble "immuno-proteidine-erysipelase." This has never been demon- strated by them and is indeed in complete contradiction to conscientious observa- tions and to well established facts. On the other hand, they make statements in themselves contradictory. The "immuno-proteidine-pyocyanease" is a sub- stance possessing an extraordinary bactericidal power, not only for the pyo- cyaneus bacillus but also for several other bacteria, such as the organisms of anthrax, of diphtheria, of typhoid, and of plague. This substance quickly dissolves these bacteria and cures experimental diphtheria and anthrax, but at the same time it is actually subject to contamination by even the most banal organ- isms, such as B. subtilis, from which it is necessary to protect it by the addition of antiseptics. To all of these contradictions, uncertainties, and inaccuracies it is still further necessary to add the advice, actually given to bacteriologists by Emmerich and Low, that their experiments should not be repeated, for they are not to be successfully performed with ease. In this state of affairs I believe that despite the attractiveness in attributing to bacterial products a role in the elabo- ration of antibacterial substances, it is necessary to forbear from following further these authors. Did the bacteriophage play a role in these experiments of Emmerich and Low? It is indeed difficult to tell, but, if they are correct, or at least if the basis is correct, there can be no doubt that these authors have observed "something." There is hardly more than one possible explanation. One of the old cultures employed in obtaining their so-called "immuno-protein" must have been contaminated with the bacteriophage. We know, for example, that in working with about 100 different strains of the cholera vibrio Flu-o encountered one, and one only, which was contaminated with the bacteriophage. This bacteriophage caused within 3 or 4 hours the dissolution of a suspen- sion of the cholera vibrio derived from any strain whatever. Upon the basis of a single fact, accidentally observed, Emmerich and Low doubtless made generaUzations too hastily. Imbued with the theory of antibodies they were unable to comprehend what they had observed. Neither they themselves nor anyone else have been able to repeat their experiments, and because most certainly exaggerated from the standpoint of generahzation their observation has remained sterile. On the other hand, if one wishes to hold the text of these authors to strict accountabiUty and to take their statements Uterally INTRODUCTION 11 it would be easy to demonstrate that it could not have been the bac- teriophage which was involved, just as the bacteriophage could not have been the cause of the phenomenon observed by Hankin. But in the one case as in the other, making allowances for errors of com- mission and for generahzations which may have been prematurely drawn by the authors, over-enthusiastic concerning the facts acci- dentally observed, I can see a possible explanation only in the bacterio- phage. A third communication where the bacteriophage may have been the cause of the facts observed, and here the probability is somewhat greater, is that of Gildemeister.* In 1917, under the name of "Flat- tenformen" he described irregular aberrant colonies of certain bacilH (typhoid and coh). As a matter of fact it appears that what Gilde- meister observed were simply bacterial colonies contaminated naturally by the bacteriophage. I attribute the formation of these aberrant colonies to bacterial mutations in the sense of deVries. I have found only these three communications in which the facts disclosed might be explained as due to the action of the bacteriophage. In view of the violence, as it might be termed, with which bacteriophagy is often effected, it is indeed strange that it has not more often at- tracted attention forcibly, especially in view of the ubiquity of the bacteriophagous principle. I beUeve this can be explained in only one way. There must have been many bacteriologists who have witnessed the complete clearing of a broth culture which had been prepared with material derived from the body of a man or sick animal, or who have experienced the impossibility of subculturing to a sohd medium an organism derived from the body, or again, who have observed on a solid medium the presence of "plaques," the bare spots mentioned in preceding paragraphs. Indeed, this is certain, since during the past few years many bacteriologists have told me that they have encoun- tered such things. In 1919, prior to my departure for Indo-China, that is, before I had discovered that resistance to the plague bacillus, in man and in the rat, was due to the presence in these animals of a bacteriophage viru- lent for this bacillus, Nageotte told me that in the course of a con- versation with Haffkine upon the subject of my studies, the latter told him that he had observed repeatedly that bouillon tubes inoculated * Gildemeister, E. — Weitere Mitteilungen iiber Variabilitatserscheinungen bei Bakterien, die bereits bei ihrer Isolierung aus dem Organismus zu beobachten sind. Centralbl. f. Bakt., I. Orig., 1916/17, 79, 49. 12 THE BACTERIOPHAGE AND ITS BEHAVIOR with the contents of a plague bubo, after becoming immediately turbid through the development of B. pestis, became within the space of a few hours absolutely cleared. The phenomenon was known in his laboratory by the name of "B. pestis suicide." Unquestionably it was due to the bacteriophage; in these particular cases B. pestis and the bacteriophage being co-existent in the bubo developing toward recovery. Pinoy has informed me that at the beginning of the late war, being in Morocco engaged in the preparation of anti-typhoid vaccine, he isolated a strain of B. paratyphosus A which presented definite plaques, comparable in all respects to those which I have shown to be produced by the bacteriophage. Also during the war, while stationed at Tiflis where he had charge of the sanitary control of the water of the Koura river, Ehava observed the following phenomenon. The water under examination was added to a peptone-water medium. After incubation for a few hours a speci- men removed from near the surface of the medium showed micro- scopically an abundance of vibrios with a normal form. Transfers to agar gave a light dull layer of growth which was microscopically com- posed of a culture of the vibrios. Some 12 hours later, from both the peptone water and the agar all trace of the vibrios had disappeared. This observation was made several times, always giving a similar result and it was impossible to obtain a culture of a vibrio which had once commenced to develop and then disappeared a few hours later. This phenomenon was inexphcable up to the time when he noted the first communications dealing with the bacteriophage. It is, therefore, certain that a large number of bacteriologists have accidentally demonstrated that such a strange phenomenon may take place in a sohd or a liquid culture. But, because of the impossibihty of reproducing the phenomenon at will, they have been unable to pursue the study and they have not ventured to publish such things since they appeared to be at variance with all known facts. It is, indeed, simply to the strangeness of the phenomenon of bac- teriophagy, to its character of being what might be called paradoxical that I owe the chance, so rare in contemporary science, of having been able to perfect the study of such an extremely complex phenomenon, to have had time to investigate its effects in nature, chiefly from the point of view of the cure of infectious disease, to study their experi- mental reproduction, even to make to this end a voyage and a year's stay in Indo-China, there to more readily observe human and animal INTRODUCTION 13 contagious diseases, and all of this before anyone even attempted to prove that the phenomenon itseK was real, a labor which might have required a half hour's time. This is the more unusual since I had published in some ten communications the facts observed and the re- sults obtained. They must have considered the author as a dreamer; indeed, some have since admitted that this was the case. BACTERIOCLYSIS : THE TWORT PHENOMENON In 1915, almost two years before my first communication upon the subject of bacteriophagy, Twort described a phenomenon* which possesses a character in common with that which I have described, namely, it is reproducible in series. Aside from this common charac- ter, it offers other characteristics, not merely different but which preclude all possibility of identity, for the characteristics of the two phenomena are mutually exclusive. But inasmuch as some authors have tried, despite this, to attribute the two phenomena to a single cause, quite without any experimental demonstration it is true, it seems necessary to consider this subject at some length. First, let me present that part of Twort's paper which describes the phenomenon which he observed. The transcription is literal. Some interesting results, however, were obtained with cultivations from glycerinated calf vaccinia. Inoculated agar tubes, after 24 hours at 37°C., often showed watery-looking areas, and in cultures that grew micrococci it was found that some of these colonies could not be subcultured, but if kept they became glassy and transparent. On examination of these glassy areas nothing but minute granules, staining reddish with Giemsa, could be seen. Further experiments showed that if a colony of the white micrococcus that had started to become trans- parent was plated out instead of being subcultured as a streak then the micrococci grew, and a pure streak culture from certain of these colonies could be obtained. On the other hand, if the plate cultures (made by inoculating the condensation water of a series of tubes and floating this over the surface of the medium) were left, the colonies, especially in the first dilution, soon started to turn transparent, and the micrococci were replaced by fine granules. This action, unlike an ordi- nary degenerative process, started from the edge of the colonies, and further experiments showed that when a pure culture of the white or the yellow micro- coccus isolated from vaccinia is touched with a small portion of one of the glassy colonies, the growth at the point touched soon starts to become transparent or glassy, and this gradually spreads over the whole growth, sometimes killing out all the micrococci and replacing these by fine granules. Experiments showed * Twort, F. W.— An Investigation on the Nature of Ultramicroscopic Viruses. Lancet, 1915, ii, 1241. 14 THE BACTERIOPHAGE AND ITS BEHAVIOR that the action is more rapid and complete with vigorous-growing young cultures than with old ones, and there is very little action on dead cultures or on young cultures that have been killed by heating at 60°C. Anaerobia does not favor the action. The transparent material when diluted (one in a million) with water or saline was found to pass the finest porcelain filters (Pasteur-Chamberland F. and B. and Doulton White) with ease, and one drop of the filtrate pipetted over an agar tube was sufficient to make that tube unsuitable for the growth of the micro- coccus. That is, if the micrococcus was inoculated down the tube as a streak, this would start to grow, but would soon become dotted with transparent points which would rapidly extend over the whole growth. The number of points from which this starts depends upon the dilution of the transparent material, and in some cases it is so active that the growth is stopped and turned transparent al- most directly it starts. This condition or disease of the micrococcus when trans- mitted to pure cultures of the micrococcus can be conveyed to fresh cultures for an indefinite number of generations; but the transparent material will not grow by itself on any medium. If in an infected tube small areas of micrococci are left, and this usually happens when the micrococcus has grown well before be- coming infected, these areas will start to grow again and extend over the trans- parent portions, which shows that the action of the transparent material is stopped or hindered in an overgrown tube; but it is not dead, for if a minute portion is transferred to another young culture of the micrococcus it soon starts to dissolve up the micrococci again. Although the transparent material shows no evidence of growth when placed on a fresh agar tube without micrococci it will retain its powers of activity for over six months. It also retains its activity when made into an emulsion and heated to 52°C., but when heated to 60°C. for an hour it appears to be destroyed. It has some action, but very much less, on Staphy- lococcus aureus and albus isolated from boils of man, and it appears to have no action on members of the coli group or on streptococci, tubercle bacilli, yeasts, etc. Such is the description given by Twort of the phenomenon which he observed. The first remark, extremely important, is that there is no lysis, no dissolution of the bacteria. The final result of the transformation as described by Twort is a vitreous or transparent substance, formed of fine granules which take a red tint when stained with Giemsa. There is a fragmentation of the cocci, a phenomenon of bacterioclysis. In the phenomenon of bacteriophagy a complete dissolution of the bacterial cell takes place. There is no residue. But since Twort says nothing in any of his papers, neither in that of December, 1915, nor in that of 1922^°^ of what happens when he adds his "transparent material" to a broth suspension of staphylococci, it is not possible to establish a comparison. It is necessary, there- fore, to restrict our comparison to the characteristics of the two phe- nomena as manifested on agar. INTRODUCTION 15 Twort states that when a drop of a filtrate containing his active principle is spread upon the surface of an agar slant and this tube is next inoculated with a normal culture of the staphylococcus, a normal growth of the staphylococcus commences to develop. Then it under- goes a vitreous transformation, the change having its inception at certain points, to later spread throughout the whole extent of the bac- terial layer. If the principle is but slightly diluted the glassy trans- formation occurs at the same time that the growth takes place. In the phenomenon of bacteriophagy, if one spreads upon an agar slant a drop of filtrate containing a little of the bacteriophage principle, and if one then inoculates the surface by spreading over it a suspension of the staphylococcus, one obtains a culture for the most part absolutely normal in appearance, but here and there small islands, circular plaques, are found, where the agar is bare without any trace of growth in any form whatever. These plaques undergo no change, even after several days. They never invade the surrounding culture, nor are they ever covered by the bacterial growth. All about these plaques, the cul- ture, retaining its normal appearance, is formed of cocci preserv- ing their normal microscopic form. When one spreads upon the agar a filtrate containing a large quantity of the bacteriophage, and when one next seeds it with a normal culture of the staphylococcus, the agar surface, after incubation, remains naked, free of all evidences of bacterial growth or of anything visible. If, says Twort, there remain in an infected tube some small regions where the culture is normal, these micrococci develop and invade the areas covered by the transparent material. I have already stated that in bacteriophagy the plaques, isolated or confluent, where the agar is bare, remain unchanged and are not re- covered by the normal surrounding culture, and this is true even if there be but a single plaque upon the entire surface of a culture, and even if this plaque is very small, having a diameter of but a fraction of a milHmeter. Twort states that if a normal culture of susceptible staphylococci is touched with a trace of the "transparent material" derived from a vitreous colony, the culture at the touched point becomes transparent, and the transformation next extends over the entire culture, the micrococci being replaced by granules. If one touches an agar culture of staphylococci, young or old, in one or in many places, with a fluid containing the bacteriophage, or even if one touches a normal culture with a platinum wire previously 16 THE BACTERIOPHAGE AND ITS BEHAVIOR touched to the surface of a plaque or to the periphery of the plaque, and then places the culture at any temperature whatever — ^room or incubator — ^one never observes any transformation. For any length of time whatever the culture as such retains its normal appearance. Microscopic examination shows, except at the point touched, that in all other portions of the culture the micrococci retain their normal form and are never transformed into granules. Indeed, granule forma- tion occurs nowhere. As is evident, the phenomenon observed by Twort and the phe- nomenon of bacteriophagy present two entirely different aspects. In the first there is the transformation of the bacteria into fine granules, a "breaking down" according to Twort himself, that is, a bacteriocly- sis. While when bacteriophagy takes place there is a total dissolution of the bacterial cells, leaving no solid residue visible under any mag- nification of the microscope. Gratia^^^'^^^ beheved that he had proof of the similarity of the two phenomena when he stated that he had isolated from vaccinal pulp a principle causing the phenomenon of bacteriophagy with all of its characteristic manifestations, such as I have described them. As a matter of fact, Gratia proved precisely the opposite, that is, he showed that the two phenomena are necessarily different. One might admit a priori that the phenomenon of bacteriophagy might be manifested under different aspects in accord with the bacterium against which it is directed, that is to say, that it might be able to effect the detailed process as I have described it when it takes place in a culture of B. dysenteriae, B. typhosus, B. coli, B. pestis, B. proteus, Pasteurella, Vibrios, etc., and that it might occur under the form described by Twort when acting upon cocci. But Gratia has demonstrated that this is precisely what is not the case, but that on the contrary, under the influence of the bacteriophage, the staphylococcus undergoes a typical bacteriophagy, identical in all respects to that of other bacteria. The sole conclusion to be drawn from the work of Gratia is that staphylococci (and undoubtedly other bacteria as well) are capable of presenting two "diseased states;" the one consisting of a fragmenta- tion showing the characters described by Twort, the other expressing itself by a total dissolution. And for the latter, the distinctive mani- festations are not simply different from those of the first, but quite exclusive. In the vaccinal lymph may be found, accidentally, the one or the other of the "principles" which cause these phenomena. There is, indeed, nothing impossible in the idea that they may co-exist in a INTRODUCTION 17 single specimen. Quite commonly two principles, the causes of dif- ferent phenomena, may be found together in the same medium. I have isolated from vaccinal pulp at two different times cocci pre- senting the phenomenon described by Fleming.* May one say that this phenomenon is bacteriophagy, simply because the susceptible coccus occurs in vaccinal lymph? Incidentally, those authors who have likened the phenomenon of bacterioclysis of Twort to the phenomenon of bacteriophagy have re- stricted themselves to affirmations only, without offering any supporting proof whatever. I am certain that, with their attention being attrac- ted to this point, should they wish to solve the question they will only have to read the paper by Twort on one hand, and on the other perform a few experiments upon the bacteriophagy of the staphylococcus, and for this a suitable bacteriophage is available to everyone. I believe that all will be in accord with me in the view that if the prin- ciple discovered by Twort and the bacteriophage are identical, when both are acting upon the same bacterium, the staphylococcus, and under identical conditions as to medium and temperature, they ought to incite identical phenomena. If these phenomena are different, as they actually are, it can only be because the principles differ. When these authors have themselves made these observations, I trust that they will be willing to distinguish between the two phe- nomena, and to employ the term "phenomenon of Twort," or better the "phenomenon of bacterioclysis" to the bacterial fragmentation presenting the characteristics described by Twort, and the term "phenomenon of bacteriophagy" to the dissolution of bacteria present- ing the characteristics which I first described. In concluding, it may be remarked that from the very beginning I have considered the arguments which I have here mentioned and which show the dissimilarity of the two phenomena.^^r These considera- tions were further developed in the book "The Bacteriophage, Its Role in Immunity." They were again repeated at the meeting of the British Medical Association in 1922, a meeting attended by Twort also, who confined himself to simply repeating the statements of his paper of 1915,®"® affirming that the two phenomena were similar, but without discussing, or even aUuding to, the arguments which I had advanced in opposition to his point of view. Again I returned to the * Fleming, A., and Allison, V. D. — Further observations on a bacteriolytic element found in tissues and secretions. Proc. Roy. Soc, Lond., 1922/23, 5^6, 142. 18 THE BACTERIOPHAGE AND ITS BEHAVIOR question at the Scarborough Congress of the Institute of State Medi- cine in 1923.^" Twort was again present but he failed to discuss the arguments which I have advanced. It is difficult to avoid the conclusion that if Twort refrains from such a discussion it is simply because the facts reveahng the dis- similarity of the two phenomena are indisputable. II. Terminology and Technic TERMINOLOGY Lack of precision in terminology is, in all branches of knowledge, a permanent cause of confusion. In biology it is the basis of the most unfortunate errors. I have shown elsewhere* that the science of immunity has been held back for more than twenty years by the equivocation created in the subject by the term ''lysis," which as a matter of fact has entirely lost its real significance. As it is, so to speak, impossible to re-establish the true etymological meaning to a word when it has once undergone deflection, and as I desire on the other hand to avoid all misunderstand- ing, I shall not employ the word "lysis" but will use the word "dis- solution," or "to dissolve," and it is necessary to take these terms in their strict sense of "the passage of a solid body into a soluble state, without residue, macroscopically or microscopically visible." I have apphed the term "bacteriophagy" to the phenomenon, in reahty very complex as we will see, which consists essentially in a "dissolution" of bacteria through the operation of a principle which I have termed "bacteriophage." A bacterial suspension, or a culture in a Hquid medium, in which complete bacteriophagy has taken place becomes a perfectly clear medium, all of the bacterial cells being dissolved. In it, under the highest magnification, either in the fresh state or after staining, will be found neither microorganisms nor granules. Since the name "Bacteriophage" has been criticized, I may again state that obviously I have not used the suffix "phage" in its strict etymological sense of "to eat," but in that of "developing at the ex- pense of," a sense which it bears very frequently in scientific nomen- clature. Several examples of this might be cited, but I will only men- tion one, to which I will have occasion to return in the course of this * Immunity in Natural Infectious Disease; Williams & Wilkins Co., Bait., 1924. INTRODUCTION 19 discussion. Dangeard* has described a Chitridinea, belonging to the group of Oomycetes, which parasitizes and develops in the nucleus of Ameba verrucosa Ehr. He has termed this Nucleophaga amebae. The word "phage" has exactly the same meaning in the two cases. Up to the present time we have considered the term "sterile" as implying the freedom of a medium from visible or cultivable bacteria. Nevertheless a medium termed "sterile" is contaminated if it contains an ultravirus, whether this virus be the bacteriophage or the virus of rabies, of vaccinia, or of avian plague, even though the highest magni- fications of the microscope fail to reveal the living agent or our arti- ficial media fail to yield a growth. For it is only necessary to place this material in contact with a susceptible Hving being to demonstrate that "a something" is present which can be cultivated in vivo. Con- sequently, although such a medium is termed "sterile" it is not so in reality. In the discussion to follow the term "sterile" will be em- ployed in its usual sense, and we will designate as "ultrasterile" a medium which contains neither visible microorganisms nor an actually demonstrable ultravirus. I underscore the word actually for it is quite possible that we may sometime discover that there is no organic medium, natural or artificial, which is ultrasterile, at least, until after it has been subjected to an adequate amount of heat or treated with appropriate antiseptics. Only thoSe ultraviruses which exercise a definite pathogenic effect upon another living being are demonstrable at the present time. Among the saprophytes only those can be de- tected which produce a demonstrable chemical transformation.! When one introduces a trace of bacterial culture into or upon a medium one says that this medium is "inoculated." Evidently the same word might be employed to designate the introduction of a trace of liquid containing the bacteriophage into a medium. It would be necessary to say "I inoculate bouillon with such and such a bacterium and again inoculate it with the bacteriophage," and such a statement in certain cases at least could readily lead to confusion. I shaU employ then, the term "inoculate" in its usual sense, meaning the introduction of a bacteriophage into a medium, and when bacteria are introduced into a medium the terms used will be "plant," "implant," or "seed." Thus, I may say "I implant a medium with such and such a bacterium and then "inoculate" it with the bacteriophage." * Dangeard, P. A— Le Botaniste, 1894/95, No. 4, 199; 248. t With reference to this subject see Immunity in Natural Infectious Disease, Williams & Wilkins Co., Baltimore, 1924. 20 THE BACTERIOPHAGE AND ITS BEHAVIOR By the words "normal suspension" as applied to bacteria should be understood ''a suspension containing 250,000,000 bacteria per cubic centimeter" prepared from a young agar culture of the bacterium. In the course of this text I will often have occasion to speak of "a strain" of such and such a bacterium and likewise of "strains" of the bacteriophage. In order to avoid repetition and possible confusion I will make use of the word "strain" in its usual sense as appHed to bacteria, and in treating of the bacteriophage the word "race" will be used. In order to avoid needless circumlocution, in designating the origin of a bacteriophage I shall precede the word bacteriophage by the name of the bacterium which this bacteriophage parasitizes within the body or from which it has been isolated, or against which it manifests its virulence. For example, a "Shiga-bacteriophage" is one which was originally isolated from a case of dysentery due to B. dysenteriae Shiga or it is a bacteriophage virulent for this organism. A "Staphylo- bacteriophage" was isolated from a lesion caused by the staphylococcus. A "Cholera-bacteriophage" was originally derived from a case of cholera. A "Plague-bacteriophage" was derived from a convalescent from plague or from an animal which had resisted this disease. Or, failing this immediate du-ect connection through origin, they may be races of the bacteriophage virulent for the staphylococcus, for Vibrio cliolerae, or for B. pestis. This scheme will be followed in speaking of aU races of bacteriophage. I devote these few paragraphs to the matter of terminology in order to facihtate the discussion of the facts to be presented, and, I hope, to aid in their comprehension. It is impossible to be too careful in treating a subject of such extreme complexity, where we have to con- sider always the simultaneous actions and reactions of two Hving beings, and often of three, when we include the evolution of the bacteriophage in nature. And the subject is still further comphcated in that it deals with rudimentary beings; with those whose adaptations to the conditions of the moment are rapidly effected. This fact already dominates the study of bacteria, since from our point of view as human beings, that which interests us most in these other beings is their "virulence" and this is inherently variable. But this faculty of adaptation, one of the principal characteristics of life, is even yet more marked in the bac- teriophage, since this is an elementary living being. INTRODUCTION 21 TECHNICAL PROCEDURES* Every bacteriological laboratory possesses the materials necessary to conduct experiments upon bacteriophagy. I will only mention the apparatus needful for the isolation of the bacteriophage but I believe it pertinent to discuss the procedures usually employed in biology to effect filtration.f Under ordinary circumstances the bacteriophage passes through all of the usual filter candles, including those made of porcelain, of in- fusorial earth, of asbestos, etc. Thus, it is possible to use candles of all types, but simply because of economy, in view of the large number of filtrations which it is necessary to make, it is preferable to employ candles which can be sterihzed and repeatedly used. Chamberland filters are of this type. A small candle, with an outside length of 7 to 8 cm. may be obtained, which is particularly convenient for test filtrations carried out with small quantities of fluid. It is thus possible to filter the contents of a tube of bouillon containing about 10 cc. and to recover 7 to 8 cc. of filtrate. Immediately after the filtration is completed, before the liquid saturating the filter has dried, I would suggest that the filter be boiled in water, as in a casserole, a procedure which is adequate to kill the pathogenic organisms which were present in the fluid subjected to filtration (in the case of spore-producing pathogens it is obviously necessary to subject the filters to autoclaving at 120°C.). After boiling for about 10 minutes the candles can be removed from the boihng water with forceps and placed in the incubator at 37°C. to dry. When a number of used filters have been collected they can be sterihzed by heat, care being taken that the temperature does not go sufficiently high to damage the material (as the enamel) of which the filter is made. The small amounts of organic material present in the filters after boiling, provided the boihng process is carried out before the filters dry after the filtration, are thus consumed and the filter is restored to a condition comparable to a new candle. By proceeding in this manner it is possible to work for several years, carrying out from 6 to 8 filtrations daily, with 4 or 5 dozen filters, without having to supple- ment the supply. I emphasize this question a Httle because a number * This section of the text is of interest only to those engaged in experimental work upon the subject of bacteriophagy. t On this subject, of such extreme importance from the point of view of the study of all ultraviruses, consult the text already mentioned — Immunity in Natural Infectious Disease. 22 THE BACTERIOPHAGE AND ITS BEHAVIOR of students located in countries where the rate of exchange is un- favorable have told me that it was impossible for them to make a study of bacteriophagy because of the price of filter candles. We will see in a moment, however, that even this difficulty can be circumvented. With regard to porosity the most useful candles are those which correspond to the Chamberland L3 filters, which are impermeable to Fig. 1. Filter Assembled According to the Method of Martin all bacteria. But let me state once more and finally, that a candle capable of heat sterilization and repeated use is perfectly suitable no matter what the make. I simply mention the Chamberland as indi- cating a type and because they are the filters which I have used here- tofore. Another question having a certain practical importance is the arrange- INTRODUCTION 23 ment of the filtering candle. The best, certainly that which com- bines the greatest assurance of sterility with the highest degree of simplicity, is the scheme of Martin (fig. 1). This arrangement makes use of a cylinder with a neck, of a separating funnel and of a tube to receive the filtrate. The candle is mounted in the tube in advance by rolhng a narrow band of non-absorbent cotton* about the varnished portion of the candle in such a manner that a plug is formed which fits firmly to the ground rim at the neck of the tube. In this way at one time a number of candles may be prepared and sterilized by dry heat. For the filtration, upon the long tube of the funnel are placed, first, a large rubber stopper which fits into the neck of the cylinder, and then, a small stopper, also of rubber, which is introduced tightly into the opening of the candle. The apparatus being assembled, the fluid to be filtered is poured into the funnel, and in the cylinder a moderate vacuum (a few centimeters of mercury is adequate) is estabhshed. When the filtration is complete, the large stopper is removed, lifting out in this way the candle adapted to its tube, and the funnel is re- moved. It only remains then to collect the filtrate with a pipette. If sufficient tubes are available, and if it is desired to preserve the filtrate, it is possible, instead of transferring the filtrate with a pipette, to remove the candle and to insert a sterile cotton plug withdrawn from a large test-tube. I have tried a great many types of filtering apparatus, but of all of those which have been recommended, that of Martin is the most con- venient and the most trustworthy. I have effected thousands of filtra- tions without a single filtrate being contaminated. The apparatus for assembling this type of filter is made in three sizes, the smallest utilizing the candles mentioned above, and serving well for the filtra- tion of quantities of from 10 to 15 cc, the middle size is the usual laboratory size, and is adapted to the recovery of 40 to 50 cc. of filtrate, and the largest makes use of the same candles as the latter but is adapted to tubes having a capacity of about 150 cc. This last serves particularly well for the preparation of relatively large quantities of bacteriophage suspension intended for use in the treatment of patients. * It is strange that a great many laboratories employ absorbent cotton for all purposes, even for plugging culture tubes. This is certainly illogical, since this cotton, quite true to its name, is hydrophile, i.e., it absorbs water. It is pref- erable to use a hydrophobe cotton, as a non-absorbent cotton, which has not undergone a special treatment converting it to the condition where it absorbs water. 24 THE BACTERIOPHAGE AND ITS BEHAVIOR ULTRAFILTRATION I have stated above that the porous candle usually allows the passage of the bacteriophage. When a liquid contains but a very few bac- teriophage corpuscles these may, indeed, be absorbed by the candle and consequently the filtrate becomes ultrasterUe, As we will see, the bacteriophage corpuscle, like all ultraviruses, possesses the general properties of colloids. Indeed, this is a charac- teristic of all living things. For a long time the physical chemists have recognized that the porous candle can not be used for the filtration of colloids because of their adsorptive property which causes them to retain the substance fixed to the porous material. Even colloids, the micellae of which possess dimensions infinitely smaller than the pores of the candles, may not pass through. As regards ultraviruses we know that for a long time bacteriologists have argued the question of the filtrability of the agents of rabies, of vaccinia, and of variola. And this difference of opinion has been quite legit- imate, for, when filtered through porous candles the passage of the virus is but inconstantly observed. Under these circumstances there is always the possibility that when passage takes place it may have been because of a defective filtering apparatus. But if, instead of the candle an ultrafilter is employed the filtration experiment is in- variably successful, even though the pores are, indeed, very con- siderably smaller. The reason for this is simple. Adsorption, very marked with porcelain, infusorial earth, asbestos, etc., and in general, with all of the mineral substances of which filter candles are com- posed, is reduced to a minimum with the ultrafilter membranes. Theoretically, only ultrafilters should be employed in microbiology. But, as I have said, in working with the bacteriophage, at least for ordinary studies, the convenient filter candle may be used. For special investigations such as seeking for the bacteriophage in body tissues, fluids, or products, it is essential to resort to ultrafiltration. Since the matter of ultrafiltration seems to be, in general, rather poorly understood by bacteriologists, it may be well to describe the procedure which is, not only the simplest, but also the most satis- factory in all respects. I have tried all of the methods which have been proposed, and have decided that the method best suited to the work is that of the collodion sac. J. C. Martin was the first to apply ultrafiltration, employing a sihca jelly or gelatin. Shortly afterward Roux and Sahmbeni devised the INTRODUCTION 25 collodion sacs such as have been employed since for the introduction of bacterial cultures into the peritoneal cavity of laboratory animals. Borrel next utiHzed them for the filtration of toxins. But Malfitano should receive the credit for having applied them in 1904 to the sys- tematic study of colloids. The method to be described is that devised by him. It is preferred to the method of Bechhold (or those of other investigators) devised two years later, which Ukewise makes use of collodion membranes, but in which the apparatus is far more com- plicated and an assurance of aseptic technic is lacking. Collodion is a solution of nitrated cotton in a mixture of abso- lute alcohol and sulfuric ether.* The viscous Hquid which results, spread out in a thin layer, becomes gradually impoverished in ether, and then in alcohol, as they evaporate, leaving a layer of nitrocellu- lose, homogenous and relatively resistant. At the beginning of the drying the ether evaporates much more rapidly than does the alcohol, and, as the nitrocellulose is insoluble in alcohol alone a gel of sufficient toughness is obtained at a certain stage of the desiccation. At this moment it is necessary to interrupt the drying by plunging the membrane into water, for if drying becomes complete, the membrane is impermeable. Spongy membranes are thus obtained, permeated by an infinite number of ramifying invisible, or microscopic, pores, the latter being, according to the composition of the collodion employed, from some hundred-thousandths of a milUmeter to one or two millionths of a millimeter in diameter. The greater the amount of alcohol present in the collodion and the lower the content in nitrate of cotton, the larger the pores, and con- sequently the filters are the more porous and allow a more rapid filtra- tion. It can be seen from this that to obtain all possible degrees of porosity it is only necessary to vary the relative proportions of the three elements which enter into the composition of the collodion. The most open membranes still retain bacteria with certainty and readily allow ultra viruses to pass through; the tightest retain both. Membranes prepared from a collodion with the following formula permit the passage of the ultra viruses: Alcohol, 96 per cent 500 cc. Ether, 65 per cent 500 cc. Nitrate of cotton 20 grams * The officinal collodion containing castor oil can not be utilized for it gives an absolutely impermeable film. As a general rule, to insure success, one must even prepare the collodion. 26 THE BACTERIOPHAGE AND ITS BEHAVIOR As an indication of penetrability, a sac prepared from collodion of this composition will allow 55 cc. of water per hour per square decimeter to pass through when under a pressure of 50 cm. of water (Duclaux). A very dense membrane, impermeable for ultraviruses of all kinds, is obtained by preparing a collodion according to the following: Alcohol, 96 per cent 250 cc. Ether, 65 per cent 750 cc. Nitrate of cotton 50 With a membrane prepared from this collodion a square decimeter filters only 5 cc. of water per hour under a pressure of 1 meter of water (Duclaux) . By varying the relative proportions of the constituents it is possible to have in reserve a series of collodions suitable for preparing mem- branes appropriate, as to porosity, to performing an experiment of any type, whatever its nature. Preparation of ultrafiUers For the purpose of conducting investigations which must be carried out aseptically, as is always the case in microbiological work, the sole * The following may be stated as practical suggestions. In order to obtain a quick and complete dissolution of the cotton it is necessary to insure the absorp- tion of the alcohol by first saturating the cotton with a small portion of the alcohol, then add the ether little by little and when the mass has become trans- parent, add the rest of the alcohol. Another rather important point is that membranes prepared from a freshly made collodion are less homogeneous, and less tough, than are those made from a "ripened" collodion. It is wise, there- fore, to pour the freshly made up collodion into a tightly stoppered flask and to keep it for a week in the incubator at 37°C. Once ripened the collodion may be used indefinitely, provided it is properly preserved, at a low temperature by preference and in tightly stoppered containers. I have not found these recom- mendations definitely stated anywhere, yet they are "tricks" well-known in some laboratories and of considerable importance. While on the subject of "tricks" I may mention one other. It is sometimes very difficult to obtain membranes free of air bubbles, yet these can very readily be avoided by placing the flask of collodion, still stoppered, in the incubator at 37° for a few hours before it is to be used. When the mold which is dipped into the collodion to form the membrane has a temperature higher than that of the liquid (and this is usually the case, because it is necessary to carefully dry the mold with a soft cloth before immersing it in the collodion, and during this pro- cedure the temperature of the hands heats the mold), the ether evaporating from contact with the walls forms bubbles. This does not occur if the temperature of the collodion is higher than that of the mold. INTRODUCTION 27 practical method of assembling the ultrafilter is that which was first described by Malfitano. The ultrafilter, which has the form of a sac, is adjusted to the end of a glass tube of the same diameter. It is first necessary to prepare a mold. Unless one has in view some special investigation necessitating a large quantity of ultrafiltrate (and then it is necessary to select a mold of appropriate size) filter sacs with a diameter of 15 mm. and a height of from 6 to 8 cm. are to be preferred. To make the mold a test-tube of the appropriate size is selected and by means of a blow-pipe a bulbous enlargement is A B Fig. 2. A, Mold for Preparing the Ultrafilter; B, the Celloidin Sac Adjusted to Supporting Tube a, glass supporting tube; b, ligature; c, celloidin sac formed near the middle of the tube (fig. 2). The mold being perfectly clean and thoroughly dry, and at a temperature slightly lower than that of the collodion (see the note on the preceding page), is plunged ver- tically into the collodion up to the point where the surface of the liquid reaches the middle of the bulb. It is then withdrawn very slowly and at a uniform rate.* * It is well to have a watch available so that the same length of time can always be used for the different procedures, thus permitting the sacs to be comparable as regards porosity. The greater the viscosity of the collodion the more slowly should the mold be removed, otherwise membranes are obtained which are too thick. As more specifically indicating the procedure the following may be of 28 THE BACTERIOPHAGE AND ITS BEHAVIOR Immediately after removal from the collodion the mold should be held horizontally and should be rotated continually until the gel attains the requisite consistency. If the evaporation has not been sufficient the sac is hkely to tear during removal from the mold; if drying has proceeded too far, the sac is liable to wrinkle and is removed from the mold with difficulty. With a Httle experience one is able to sense the opportune moment; if one employs the time intervals indi- cated in the preceding note a membrane of the requisite consistency is obtained after about a minute and a half. At this moment, plunge the mold into the collodion a second time, observing the same pre- cautions as in the first immersion, and withdrawing at the same speed. Allow it to dry somewhat, holding it horizontally while continuously rotating it between the fingers. With the second layer permit the evaporation to continue for a sfightly longer time, about two minutes.* When the membrane thus prepared is of the requisite consistency, plunge the mold into water for a few seconds, then, separating with the finger nail the upper margin of the sac from the middle portion of the bulb, under a stream of running water, take hold of the loosened portion and turn it back on itself fike the finger of a glove. The ex- terior surface of the membrane when the sac was upon the mold thus be- comes, after removal, the interior. Place the sac in distilled water, or, if it is not to be assembled for use at once and if it is desired to pre- pare a supply of sacs ready for use, they should be immersed in an aqueous 20 per cent alcohol. f The reader must not assume from this detailed description that the preparation of ultrafilters is difficult. After a few minutes' practice one "gets the knack" if one has the least manual dexterity. For sacs with a diameter greater than 4 or 5 cm., that is, for sacs interest. For the more fluid collodions the removal of the mold should require 10 to 15 seconds, for the more dense collodions this period should be extended to 30 to 45 seconds. * Naturally the period of drying varies with the surrounding temperature, the presence or absence of air currents, etc. , since anything which hastens evaporation shortens the time. In any case, the first sac made provides an index for further procedure. If the sac tears in the process of removal the period of drying must be extended; if it is wrinkled, stiff, and is detached with difficulty, drying has gone too far. t If the proposed investigation is to demand a great many filtrations, it is well to prepare at one time enough sacs to meet all needs. In the first place this will save time, and in the second place, and this is of particular importance, it will tend to give greater uniformity in the series of sacs to be employed in the study. INTRODUCTION 29 with a large capacity such as may be needed for special studies requir- ing a large amount of filtrate, the dipping method is not suitable. It is then necessary to proceed in the following manner. Prepare a suitable glass mold as has been indicated above, and attach this mold, horizontally, to a shaft connected with a small motor. The centering should be as perfect as possible and the speed of rotation should be from one to two turns per second, according to the size of the mold. With the mold rotating, pour a fine stream of collodion upon the mold, beginning with the bulbous enlargement (the open end of the sac) and proceeding toward the closed end of the sac. Per- mit it to dry for an appropriate time, pour on some more collodion to give a second layer, repeat a third time, and even a fourth, thus in- creasing the toughness of the sac, which, being large, is proportionally fragile. Remove the sac as with the smaller sacs. The larger the sac, the more difficult is its construction. Assembling the ultrafilter Select a glass tube exactly* the same diameter as the exterior of the mold, and some 20 to 25 cm. (or more in certain cases) in length. Long tubes facihtate filtration, for in filling this supporting tube with the fluid to be filtered sufficient pressure is obtained on the membrane to effect filtration quickly, at least this is the case when using membranes sufficiently porous to allow the passage of ultraviruses. Draw the mouth of the sac upon the supporting tube, to a height of from 1.5 to 2 cm. Tie the sac to the tube with a fine and strong string, introducing between the sac and the Hgature a band of parch- ment paper to avoid tearing the sac (fig. 2-B). At no time during the manipulation should the sac be allowed to dry, for with drying nitrocellulose membranes become impermeable, even if they are again immediately moistened. It is, however, rela- tively easy to keep them moist with water. With the sac thus attached to its supporting tube, take a tube of large diameter and hah fill it with distilled water. Also fill the adjusted sac with distilled water in such a way that the level of the water is some 4 to 5 cm. above the hgature. Place the sac within the large tube and suspend it there at the desired height (the level of the water being the same in both tubes) by means of a strip of cotton rolled tightly about the supporting tube in such a way as to form a plug for the large tube. * It is well to measure the diameter exactly, with calipers. 30 THE BACTERIOPHAGE AND ITS BEHAVIOR Plug the opening of the small tube with cotton, as one would a culture tube (fig. 3). If it is not essential to make the filtration aseptically, the ultrafilter is then ready for use. If sterihty is requisite, the filter must be steriKzed, either as it is or after denitrification. <» Fig. 3. The Ultrafilter Ready for Sterilization Denitrification This detailed description of the preparation of ultrafilters is here inserted because ultrafiltration must shortly become a common pro- cedure in all experiments deaUng with ultraviruses, the group of beings to which the bacteriophage belongs. That the description of the INTRODUCTION 31 preparation may be complete, the method of denitrification, as it has been termed by Jacques Duclaux, should be included. Membranes prepared from collodion are simply a gel of nitrocellu- lose. Such membranes possess very low adsorptive capacities, but this property can be diminished still further by decreasing the thick- d.. Fig. 4. Denitrified Ultrafilter Arranged for Drying a, celloidin sac; b, ligature; c, supporting glass tube; d, column of water; e, rubber stopper;/, pinch-cock, ness of the membrane, and this is what happens when the nitrocellulose is transformed into pure cellulose by denitrification. At the same time this procedure increases the toughness of the membrane. For denitrification, mount the sac upon its supporting tube, adjusting it as has been described, but instead of immersing it in distilled water, substitute a 25 per cent solution of ammonium hydrosulfide in dis- 32 THE BACTERIOPHAGE AND ITS BEHAVIOR tilled water. Place in a water-bath at SO^C. for 45 minutes. Next wash the sac, outside and in, with a 10 per cent solution of ammonia, taking considerable care for at this stage the sac is very fragile. Finally- wash with distnied water. It is then necessary to completely dry the membrane, maintaining a positive air pressure upon the inside to prevent the sac from becoming deformed. The following has been found to be the most practical method. A rubber stopper (with one hole) which will fit the opening in the supporting tube is provided with a short glass tube, to the latter being attached a piece of rubber tubing provided with a good screw clamp (fig. 4). Pour a few cubic centimeters of water into the sac and insert the stopper equipped with its tube and clamp. Blow through the rubber tube to distend the sac, taking care not to rupture the still fragile membrane. Close the clamp. When attached to a support, the sac uppermost, the few cubic centimeters of water placed in the sac will make an hydrauUc seal and prevent the escape of air. When dried, a denitrified sac will keep indefinitely. For use, it is placed in a large tube* in distilled water, after the manner indicated for sacs which have not been denitrified. Sterilization Such is the description of the preparation and assembhng of sacs for ultrafiltration as given in texts designed for physical chemists. For us, as microbiologists, the procedure can not stop here, for we must work in an aseptic manner, that is, the ultrafilters must be sterilized. But, it is practically impossible to steriUze a collodion ultrafilter by moist heat in an autoclave. f The sac becomes deformed, the mem- brane is rendered opaque, and porosity is modified. It is possible, as Duclaux has shown, to effect denitrification and once this is accompHshed an ultrafilter can be sterilized by steam in the autoclave. Nevertheless, the deformation of the membrane, although very much less than with the original collodion membrane, is still very considerable. After many trials I have adopted the following method, which has * A more minute description of the details of ultrafiltration will be found in text-books dealing specifically with colloidal chemistry. t It is easy to see the enormous errors attending the use of unsterilized or poorly sterilized ultrafilters,^" yet of all the proposed types of ultrafilters, the only ones that can be effectively sterilized are those designed by Malfitano, the preparation of which is here described. INTRODUCTION 33 proved to be entirely satisfactory, since the ultrafilter undergoes no change, the membrane remains perfectly transparent, and sterihty is assured. Adjust the filtering sac as was directed in the section on "assembling," but instead of fiUing it, and the outside tube, with distilled water, use 80 per cent alcohol. On the other hand, in a small autoclave, replace the water by 90 per cent alcohol. Connect the air escape valve of the autoclave to a condenser by means of a rubber tube (simply to prevent the volatilized alcohol from escaping into the laboratory at the beginning of the procedure). Place the ultrafilters, mounted in alcohol, as indicated above, in the autoclave, as for an ordinary steriliza- tion, and start the autoclave as would ordinarily be done. In brief, the sterihzation is accomplished in a vapor of alcohol. When all of the air has been drawn off (this is shown by the fact that the alcohol will commence to distill over and with cooling will condense drop by drop in the condenser) close the exhaust valve, and regulate the source of heat in such a way that the autoclave will have a pressure of i to J of a kilogram per square centimeter. The sterihzation being thus effected in hydrated alcohol, the temperature attained in this way is sufficient. Maintain this pressure for 30 minutes, allowing the auto- clave to remain closed until after coohng is completed. This method of sterilization can be applied equally well, whether the collodion ultrafilters are normal (nitrocellulose) or denitrified (pure cellulose) . Except for particular investigations this method of sterihza- tion renders denitrification unnecessary. If, as I would advise, one prepares at one time enough ultrafilters for a whole series of experiments they can all be sterilized at one time, and preserved in alcohol just as they come from the autoclave. At the time of use, empty the alcohol out aseptically, both from the interior of the sac and from the exterior tube, and replace it with distilled water. Allow the water to remain for 10 to 15 minutes, and then empty this out. Then introduce the fluid to be filtered into the sac (fig. 5). With membranes of medium porosity, that is, those most commonly used, allowing ultraviruses to pass with certainty and just as surely retaining the smallest of the bacteria (Asterococcus of pleuropneu- monia, for example), some ten cubic centimeters of ultrafiltrate can be obtained in a few hours (during a night), by simply filling the glass tube supporting the sac with the liquid to be filtered. The shght pressure thus exerted by the liquid in the tube and sac (15 to 20 cc, or even more if desired, since the volume can be increased by simply 34 THE BACTERIOPHAGE AND ITS BEHAVIOR adjusting the sac to a longer tube) is adequate to accomplish the ultrafiltration unless the membrane is too dense. If it is necessary to use very dense ultrafilters (for example, when it is desired to retain the ultra virus and thus to obtain an "ultrasterile" ultrafiltrate, as is essential in carrying out studies upon the secretory products of the ultra viruses) pressure must be apphed. For such purposes it is wise to prepare sacs, not with two layers of collodion, but with 4 or 5, in- creasing thus the toughness of the membrane. They should be deni- trified, and assembled as has been outlined, except that in the place Fig. 5. The Ultrafilter Assembled as (A) an Ultrafilter and (B) a DiALYZiNG Sac of the large tube into which they are suspended, they should be intro- duced into a tube with a side neck. It is also obvious that it is then necessary to adapt the tube bearing the sac to the side-necked tube by means of a perforated rubber stopper. To the reader all of these manipulations may appear compHcated, but as a matter of fact, a little experience will show that the preparation of ultrafilters, and ultrafiltration itself, are but simple procedures. PARTI THE PHENOMENON OF BACTERIOPHAGY CHAPTER I Bacteriophagy in a Fluid Medium 1. isolation of the bacteriophagous principle Ubiquity of the bacteriophage The description of the fundamental experiment, as presented in the early pages of the Introduction, demonstrates that the phenomenon of bacteriophagy consists essentially in the dissolution of the bacterial cell.* This is accomplished through the action of a "principle" which passes through porcelain filters, and even, as we shall see, through ultrafilters whose pores are large enough to permit the passage of par- ticles with a diameter as great as 30 miUicrons.f Quite naturally the first question to arise is that of the isolation of this dissolving principle, that is, how can it be obtained in a pure state, in the bacteriological sense of the word. Where is it to be found in nature? What types of material must be examined to obtain it? It is everywhere present, one might say. Up to the present time it has been shown to be present not only in the intestinal contents of the normal man and of healthy anunals (d'Herelle^^'')| but par- ticularly in those who are convalescent from a bacterial infection {d'B.eTe\\e^^°'^^*''^^"), in the urine of these convalescents (d'Herelle^^"), in theu' blood (d'Herelle^^^), in pus (d'Herelle^-^, in river water (Dumas' ^^), and in cultivated soil (Dumas^^^). Being, in fact, a con- stant inhabitant of the intestinal tract it may be encountered in every- thing which may be contaminated by fecal material. Its constant presence in the intestine suggests indeed, a priori, the thought that certain bacterial strains may occasionally be found "con- taminated" by a bacteriophage, and in fact, Otto and Munter^^^ j^^ve * The word "cell" is used here simply because it is sanctioned by usage. As a matter of fact, it is very doubtful if a bacterium can be formed of a cell, in the strict sense of the word. This question is discussed in the next to the last chapter of "Immunity in Natural Infectious Disease." t A millicron is one one-millionth of a millimeter, or one one-thousandth of a micron, i.e., Iju/z. J Only the original papers are here cited. In a later chapter dealing in greater detail with the distribution of the bacteriophage we will review more extensively the findings of those who have investigated this question. 37 38 THE BACTERIOPHAGE AND ITS BEHAVIOR isolated it from such cultures. Let us hasten to state, however, and we will return to this important point, that such contaminated cultures are rare. Methods of isolation The bacteriophagous principle is encountered but rarely in a sub- stance which, m the usual sense of the word, is sterile. Usually it is necessary to isolate it from a medium in which bacteria are also present. Two methods may be followed to this end. Since the bacteriophage will pass through porcelain filters and through ultrafilters, the material under examination may be suspended in water or in bouillon and sub- jected to filtration through porcelain or ultrafilters (d'Herelle^^°). The bacteria are held back by the filter; the bacteriophage passes through and is thus found in a pure state in the filtrate.* The bac- teriophage resists temperatures of at least 60°C. Thus, if it is pres- ent in a liquid medium containing non-spore-forming bacteria it is only necessary to heat at 58°C. for a time sufficiently long to kill the bacteria. The bacteriophage will then be found in a pure state (Bordet and Ciuca^"). The bacteriophage will resist three successive heatings made at this temperature (58°C.) and consequently it is possible to isolate it by this means, even if the mixture contains spore-forming organisms, since they will be ehminated by the method of fractional steriHzation (Tomaselli^^^) . I have satisfied myseK that the bacterio- phage may be isolated from feces by this method. In studying the influence of physical agents upon the bacteriophage we will see that temperatures, even below 60°C., may cause some attenuation. It is therefore advisable to utihze filtration wherever possible and to resort to isolation through heating only in particular cases and as a last resort. * This is true, naturally, only when the material does not contain, in addition to the bacteriophage and the bacteria, some other filtrable organism, such as the viruses of vaccinia, of rabies, of herpes, etc. In cases of this type a separation by filtration or ultrafiltration can not be effected, since, as we shall see, all ultra- viruses, the bacteriophage included, possess comparable, or possibly identical, dimensions. This is the case, certainly, for the ultraviruses mentioned above, as has been shown by Levaditi.*'^ Where there is an associated ultravirus separa- tion by heating may be employed, at least in those cases where the associated ultravirus is less resistant than the bacteriophage. In the opposite case (if, for example, the mixture contains an ultravirus of the mosaic group) separation can be accomplished by making several successive passages in the presence of a susceptible bacterium. Here, only the bacteriophage will multiply, and at the end of a few passages the associated ultravirus will have been eliminated by dilution. BACTERIOPHAGY IN A FLUID MEDIUM 39 Technic of isolation The isolation of the bacteriophage may be undertaken under one or another of the following circumstances: 1. The bacteriophage may be sought in a sterile fluid, for example, in normal blood or in an organic fluid collected aseptically. With such no treatment is necessary. 2. The material to be examined may be a clear, but not sterile, liquid. With this, filtration will eliminate the bacteria while the bacteriophage passes through into the filtrate. 3. The material may show a homogeneous turbidity; as a bacterial culture. Here, direct filtration results in an early occlusion of the pores of the filter candle. Thus, it is desirable to resort to a prelimi- nary filtration. The following method of treatment is most satisfactory. Provide a funnel with a folded filter paper sufficiently large to receive at one time the entire volume to be filtered. Fill the filter with water to which has been added a small amount of infusorial earth. When the water has passed through, the paper is left coated with a thin layer of the infusorial earth, thus rendering the paper less permeable. Through this the material to be examined is filtered prior to filtration through the candle. 4. The material may be a fluid holding in suspension organic particles, or it may be matter more or less solid in nature. This is the type of substance most frequently examined; such as fecal material, more or less fluid, pasty, or solid; or excreta admixed to a greater or less degree with earth, organic debris, etc. In such a case it is necessary to dis- integrate as completely as possible the material to be examined. To effect such a disintegration the most simple procedure consists in carefully suspending the material in bouiUlon, about 5 grams to 50 cc. of the medium, and incubating this suspension at 37°C. for from twelve to eighteen hours. The bacterial fermentations which ensue, because of the diverse organisms introduced into the medium, lead to a suffi- cient disintegration. Upon removal from the incubator the material may be treated, as indicated above, by filtration through infusorial earth and a candle. If the material under examination contains the bacteriophage and has been subjected to filtration, it will be found in the filtrate. We will see that the bacteriophage possesses an activity manifested against a wide variety of bacterial species; without doubt against all. We will also see that against a given bacterium this activity is very 40 THE BACTERIOPHAGE AND ITS BEHAVIOR variable. Races of the bacteriophage may be isolated which are ex- tremely active, causing within a few hours a total dissolution of all of the bacteria contained in a culture or in a rather turbid suspension. On the other hand, other races may not cause any detectable dissolu- tion, and it is only by spreading the mixtures of bacteriophage and bacterium upon an agar medium that their presence can be disclosed. More will be said upon this point in Chapter IV and those following. Let us leave, for the moment, the study of these slightly active races and consider in these first three chapters only the typical phe- nomenon of bacteriophagy, that is, the phenomenon leading to a total dissolution of the bacteria of a young culture or a suspension of hving organisms. Whatever may be the bacterial species involved, under the action of an active bacteriophage the phenomenon of bacteriophagy manifests itself always in the same manner. But the diverse races of the bacteriophage, active upon different bacterial species, are more or less frequent in nature and more or less easy to disclose. The bacteriophage which it is always easy to pro- cure, in whatever place it may be found, is that which causes bacteri- ophagy of Shiga dysentery bacilli, and it is for this reason that, in the majority of the experiments to be recorded, I will take as types this race of the bacteriophage and this bacterium. One may isolate, with certainty, a race of the bacteriophage always very active against dysen- tery bacilli from the excreta of a convalescent from bacillary dysentery, often, indeed, from the fecal discharges in a case of any acute intestinal disease. Furthermore, such races are to be found in the excreta of the majority of horses and domestic fowls, even when in a normal state of health. 2. SERIAL ACTION The basic experiment, as recorded in the Introduction, has shown that the bacteriophagous principle reveals itseK through bringing about the dissolution of bacteria. But, and this is a distinctive pecu- liarity of this action, it is only in proportion as this dissolution is effected that the bacteriophagous principle, which is the cause of it, reproduces itself ^ — multiplies . To demonstrate this phenomenon of serial activity a small quantity of bacteria are removed from a young agar slant culture and suspended in bouillon in such a concentration as to produce an obvious turbidity. A platinum inoculating needle is then dipped in a "bacteriophage fluid" BACTERIOPHAGY IN A FLUID MEDIUM 41 and the minute quantity adhering to the wire is inoculated into the prepared bacterial suspension. Within a few hours the suspension is clear; all of the bacteria have disappeared. To all appearances they have dissolved in the bouillon, just as sugar becomes dissolved in water. At this time, a bacterial suspension, prepared as was the first, is made and the tip of the platinum wire is immersed in the clear fluid which originally was the first suspension, and thus a minute quantity is transferred to the second turbid suspension. Again after a few hours, this second suspension will in turn have become Hmpid. A needle dipped in this second cleared suspension inoculated into a third results in a repetition of the process. In each successive suspension the bacteria become dissolved, and in this way it is possible to continue "serial passages" of the bacteriophage principle as long as may be desired. After some thousands of passages comparable to those described above the last suspension, once it has become clear, represents a "bacterio- phage fluid" just as active as that originally employed to cause bac- terial dissolution in the first passage. By this procedure I have main- tained for almost ten years several particularly active bacteriophage races, certain of them having undergone several thousand passages at the expense of the appropriate susceptible bacterium. The phenomenon of bacteriophagy is then, in reahty, a double phenomenon. A dissolution of the bacterial cells takes place, and, in the course of this dissolution, the bacteriophage principle regenerates, reproduces itself. It is unnecessary to give here experimental protocols supporting these statements. No one has questioned them, and, as a matter of fact, this entire text is simply an exposition of the manner and the results of this reproductive capacity of the bacteriophage. 3. ENVIRONMENTAL CONDITIONS FAVORING BACTERIOPHAGY General conditions Of the many culture media devised up to the present time none possess the composition requisite to the multiplication of the bacterio- phage. The sine qua non for multipKcation of the bacteriophage principle, thus permitting a dissolution of the bacteria, in itself a direct result of this multiplication, is the medium provided by living bacteria. It is further necessary, but this is evident a priori, that the bacteriophage principle which one opposes to this bacterium be active against the latter. 42 THE BACTERIOPHAGE AND ITS BEHAVIOR These two basic conditions being satisfied, it may be said that, as a general rule, the most favorable medimn, that in which bacteriophagy will take place in the most perfect fashion, is that which, because of its composition, provides best for the development of the particular type of bacteria selected to undergo the dissolution. This is not strange, for it is not in fact in the medium itseK that the bacteriophage acts and multipHes, it is within the bacteria themselves. The interior of the bacterial cell is the true and sole medium for the multiplication of the bacteriophage (d'Herelle^^"). This is by no means equivalent to saying that the composition of the medium is of no consequence, for all conditions which modify the state of the bacteria are reflected in the phenomenon, one of whose manifestations and indeed the most obvious one, is the dissolution of these bacteria. For example, it is shown by many experiments that the "critical period" in the life history of the bacterial cell in the presence of the bacteriophage is the moment of its division. But it must not be assumed that only those bacteria in the process of division are subject to attack. It is then, because of its influence upon the development of the bacteria that the composition of the medium has a reflected effect upon the phenomenon of bacteriophagy. All of the experiments presented in the first edition of this text* were carried out, except where stated to the contrary, with cultures or suspensions of bacteria prepared with the ordinary bouillon used in the Vaccine Laboratories of the Pasteur Institute. This is the so-called Martin's bouillon, made by mixing in equal parts a beef infusion (400 grams per liter) and a peptone solution, prepared in the laboratory by acid autodigestion at 50°C. of pig stomach (200 grams of minced gas- tric mucosa per liter). The adjustment of the reaction was effected by the old method, using phenolphthalein as indicator, the final reac- tion being -6 to -8, which corresponds very closely to a pH of 7.6 to 7.8. Preliminary control experiments had shown me that such a degree of alkahnity was best suited to the reaction (d'Herelle^^^), as the following indicates. Peptone water (containing 25 grams of Chassaing peptone and 5 grams of NaCl per Uter) is neutralized to phenolphthalein. The medium is then frankly alkaline to litmus. It is then distributed into tubes, 10 cc. to each. Hydrochloric acid is added to each tube in dilu- tions to form an increasing degree of acidity. All of the tubes are * The Bacteriophage; Its Role in Immunity. Williams & Wilkins Co., Balti- more, 1922. BACTERIOPHAGY IN A FLUID MEDIUM 43 planted with a concentrated suspension of Shiga bacilh, sufficient being added to give a normal suspension of 250 million per cubic centi- meter. Finally, each tube is inoculated with 0.001 cc. of the bacterio- phage fluid. After 24 hours the appearance of the suspensions shows a certain correlation to the reaction of the medium. The results are given in table 1. At the beginning of my studies I stated that bacteriophagy could take place in an alkaline physiological salt solution^'^'^-^''^ Expressed in this manner, it may be that this statement may not be accurate, al- though assuredly under these conditions a very definite, sometimes com- plete, clearing of the medium may be observed. Others (Davison ,i^^ Kabelik,'^^) have also observed this and have attributed, as I had REACTION TO APPEARANCE OF THE SUSPENSION TUBE PHENOLPHTHALEIN AFTER 24 HOURS 1 Very slight clouding 2 -2 Very slight clouding 3 -4 Limpid 4 -6 Limpid 5 -8 Limpid 6 -10 Limpid 7 -12 Limpid 8 -14 Slight turbidity 9 -16 Turbid 10 -18 Turbid 11 -20 . Turbid 12 -22 Turbid done, the bacterial dissolution occurring in saline under the influence of the bacteriophage, to a typical bacteriophagy. We will return to this phenomenon later and consider the correct interpretation. Maitland^2 j^^g^ however, shown that bacteriophagy may take place in a medium very poor in food materials, such as physiological saline containing but 1 per cent of bouillon. I have substantiated this; under such conditions bacteriophagy undoubtedly occurs. Reaction of the medium Unquestionably, alkalinity of the medium affords the most favorable reaction for accompHshing the phenomenon of bacteriophagy. But if an attempt is made to define more precisely the exact degree of 44 THE BACTERIOPHAGE AND ITS BEHAVIOR alkalinity that provides optimum conditions it becomes apparent at once that the conditions obtaining in one experiment are optimum conditions only for the bacterial species and the particular race of bacteriophage with which the experiment is performed. If another species of bacteria and a bacteriophage derived from another source are utihzed the best conditions for effecting the phenomenon may be quite different from those of the first experiment. Situations analogous to this will be observed repeatedly in the course of this study, and the reason for such a lack of fixed relationships is very obvious. Although in physics, or in chemistry, it is always possible to definitely fix the conditions of an experiment, to record these condi- tions by giving them invariable numerical expression, and to make the experiment entirely without regard to the past history of the chemical substances entering into the reaction, this is not possible in biology. Here, the past, the inheritance of the beings involved, constitutes a factor of prime importance. Moreover, this factor is always difficult, often impossible, to evaluate in advance. This is why two experi- ments in bacteriophagy, carried out under identical conditions, may yield different results, simply because in the two experiments the bacteria are of different species, or simply of different strains. Bacteri- ophagy is a disease of bacteria, and the aphorism, so true, "there are not diseases, there are patients," meaning that a single disease may have varied manifestations according to the individual affected, is just as true whether the patient be a man, or whether it be a bacterium. In the last analysis, it is of course true, that disease represents the sum total of a series of purely chemical reactions, but whereas in chemistry a reaction can be predicted in its most minute details be- cause of restricted conditions, all determinable in advance, in biology generally, and certainly in the case with which we are particularly concerned, the number of factors is so great, the majority of these factors being absolutely indeterminable, that it is impossible to predict the optimal conditions for a reaction. Only the general nature of the reaction can be foretold. This concept must always be held in mind throughout the study of the phenomena caused by the bacteriophage. A great many authors have neglected these fundamental principles, with the result that many conflicting reports, with their attendant arguments, have appeared, each author assuming that he could generahze from his individual results. As a matter of fact, these basic facts are all-important, and generalization is particularly hazardous. BACTERIOPHAGY IN A FLUID MEDIUM 45 To return to the subject under consideration, that of the effect of the reaction of a medium upon bacteriophagy, we will see that through a process of adaptation it is possible to so alter the bacteriophage that the processes of dissolution will take place in an acid medium. A further discussion of this is reserved for a later section. It is mentioned here simply to show the complexity of the conditions contributing to bacteriophagy and to emphasize the impossibility of stating in a defi- nite manner conditions such as will provide for optimum activity regardless of the race of bacteriophage involved. It is certain, never- theless, that in general an acid medium is but poorly suited to bac- teriophagy. With the great majority of races of the bacteriophage the phenomenon does not take place readily when the reaction of the medium is acid (d'Herelle^^^). Scheidegger^^^ states that B. coli grows normally in a bouillon with a pH of 4.5, even though the bacteriophage is present. But under such conditions the principle is not destroyed, for when such a medium is rendered slightly alkahne, bacteriophagy occurs. While working with B. coli, investigating the process of bacteriophagy occurring under particular conditions (when a very small number of bacteria (simple seeding) in a peptone bouillon medium were mixed with an indeterminate, but very great (10 drops) quantity of a filtrate containing a but slightly active bacteriophage), Gratia-*^ noted that the degree of alkalinity most favorable was found in the neighborhood of pH 8.5. He states, however, that the phenomenon takes place in a slightly acid (pH 6.8) medium. Scheidegger^^ also records bacteri- ophagy in a medium of pH 6.5. These findings, together with other observations, show that the optimal reaction is not a constant, varying not only with the bacterial species and the race of the bacteriophage concerned, but differing also with the nature of the medium. In testing a Flexner-bacteriophage, acting upon its homologous bacterium, da Costa Cruz^^^ showed that bacteriophagy could take place in Martin's bouillon with a reaction of pH 6.5, but that to obtain a reaction of equal intensity in peptone water it was necessary to adjust the reaction to pH 8.5, Using a Shiga-bacteriophage in conjunction with its homologous organism, I have shown that in a peptone water (2.5 per cent), con- taining 0.5 per cent of salt, with the pH at 7.5, a total dissolution of a normal bacterial suspension takes place in 14 hours at 37°C. At pH 8.0 the dissolution is complete in 9 hours, and at pH 7.0 dissolution is only partial. 46 THE BACTERIOPHAGE AND ITS BEHAVIOR A correlation of all of these findings shows, therefore, that the opti- mum pH varies (o) with the bacterial species involved, (6), with the race of the bacteriophage reacting upon it, and (c) with the composition of the medium in which the reaction takes place. Nevertheless, it is possible to say that, in general, a slight alkalinity of the medium pro- vides a favorable condition. Hence, unless one has in view some special investigation demanding particular conditions, the best medium for routine purposes is the ordinary peptone bouillon "with a reaction of pH 7.8. Bacteriophagy will take place in synthetic media provided these permit the growth of the bacteria against which the bacteriophage is to act. Here the conditions as regards alkalinity are the same as in bouillon. The formula for such a medium, suited to the growth of B. coli, as well as to some, but not all, strains of B. dysenteriae, is: Water 100 cc. Sodium chloride 0.5 gram Potassium phosphate 0.1 gram Asparagine 0.5 gram The reaction to be adjusted in accord with the experiment to be performed. Another medium, suggested by Gratia^^^ for B coli is: Water 1000 cc. Glycerin 30.0 cc. Sodium chloride 5.0 grams Calcium chloride 0.1 gram Magnesium sulfate 0.2 gram Dipotassium phosphate 2.0 grams Ammonium lactate 12.0 grams The reaction adjusted to pH 7.4. 4. EFFECT OF THE CONDITION OF THE BACTERIUM If we take, then, 10 cc. of this beef bouillon, containing peptone and salt, adjusted to pH 7.8, and suspend some dysentery baciUi of the Shiga-Kruse type removed from an agar slant which had been planted some 18 to 24 hours previously, to provide a suspension containing approximately 250 million per cubic centimeter, and further, inocu- late this suspension with a trace (0.001 cc, for example) of a filtrate containing a bacteriophage very active against B. dysenteriae, we will find, after incubation at 37°C., that within a few hours, from 4 to 24, BACTERIOPHAGY IN A FLUID MEDIUM 47 according to the activity of the race of bacteriophage involved, all of the bacteria are dissolved, and the medium has again become hmpid. Instead of selecting Shiga-Kruse bacilli, we could have prepared in the same way a suspension from a bacterium of another species, and combined it with a race of bacteriophage of sufficiently high activ- ity against this bacterium. The final result would have been the same; a complete dissolution of the bacterial cells and a clarification of the medium. But such a complete dissolution does not take place under all con- ditions; the state of the bacterium exposed to the dissolving principle is of significance. Instead of taking a suspension prepared from a young freshly grown culture we may inoculate the bacteriophage into a fifteen-day old broth culture. A clearing of the medium, a partial dissolution, results, but a certain degree of turbidity remains. Never- theless, it is possible to continue to use such a medium, making as many passages as may be desired. Some tube of the series when planted on agar or in bouillon will remain sterile, and a drop of this tube in- oculated into a suspension of young bacilli will cause a perfect dissolu- tion. In the old culture, then, the bacteriophage multiplies normally, although dissolution does not take place, at least, the solution is not complete. What is the explanation of this reaction? To answer this it is sufficient to compare the results of counting the total number of bacilli existing in an old culture (this can be done by the method of counting cells) with the results secured by counting the viable or- ganisms only (done by the plating method). For a confirmation of this type, a Shiga culture in Martin's bouillon is made, incubated for 14 hours, and allowed to stand at laboratory temperature for 15 days. The total count of bacillary bodies will be about 625 milhons; that of the viable bacilli, that is, those capable of yielding colonies when transferred to agar will be about 2 milhons, in each half cubic centi- meter of culture. Now, as we have seen, the bacteriophage is able to develop at the expense of Hving bacteria only, these being the ones which are dissolved. In the old suspension which we have mentioned, in which there is only about one organism in three hundred which is capable of being dissolved, it can readily be comprehended that if the dissolution of a suspension be taken as a criterion, the bacteriophage appears to be without action. As a matter of fact, it is not necessary to resort to old cultures to find dead bacteria, for even in fresh bouillon cultures dead organisms wiU be found after as short a time as 24 hours. In a broth culture 48 THE BACTERIOPHAGE AND ITS BEHAVIOR of Shiga bacilli, after only 24 hours of incubation about one-third of the organisms present are incapable of producing colonies when planted on agar. If, on the other hand, an agar slant culture is utihzed, al- most all of the bacteria are living after 24 hours at 37°C. A 24-hour bouillon culture will, then, remain shghtly turbid when the bacterio- phagic process is accomplished, while a suspension made in broth from a young agar culture containing the same number of bacteria will be perfectly limpid when the dissolution is achieved. In this last case all of the bacteria were living and susceptible to the attack of the bacterio- phage. It is for this reason that it is preferable to effect bacteriophagy in a suspension of bacteria rather than directly in a bouillon culture. Certain bacteria give a homogeneous growth in a young culture in bouillon but when taken from agar they can be suspended only with difficulty. B. pestis is such an organism. When working with such bacteria it is preferable to have the bacteriophage act on a broth culture in the following manner. A bouillon tube is hghtly seeded with the bacterium. When the culture has clouded, the bacteriophage active for this bacterial strain is introduced and at the same time the culture is diluted with an equal volume of sterile medium. This dilution should be made before the bacteriophage has had time to multiply sufficiently to parasitize an appreciable number of bacteria. Thus, the bacterial culture at the time of "departure" will consist almost entirely of young bacilli, readily subject to attack. The following experiment demonstrates clearly that the products of bacterial growth as found in an old culture, products which, as is well-known, inhibit the development of bacteria (as in the so-called "vaccinated" media) are without effect upon the phenomenon of dissolution. Two cultures of B. dysenteriae Shiga, the one aged 15 days, the other, 18 hours are centrifugaHzed. The sediment from the first culture is suspended in the supernatant fluid of the second, and the sediment of the second culture is combined with the supernatant fluid of the first. Both suspensions thus formed are inoculated with a drop of a bacteriophage filtrate. The suspension consisting of "old" bacilli and "young" medium remains turbid; that of "young" bacilli and "old" medium becomes perfectly clear after 7 hours. But, although the products of bacterial metabolism are not inhibitory for the process of dissolution, the products of dissolution, as we will see, exert quite a different action. These products are the result of the activity of the bacteriophagic process, and, as such, they impede its activity. BACTERIOPHAGY IN A FLUID MEDIUM 49 Although, as has been stated above, bacteriophagy will not take place with dead bacteria, this does not mean that it is essential that the bacteria be young. Various authors (Kuttner^^^ first, and later Bordet and Ciuca®^) have suggested that bacteriophagy can only be effected when the bacterium is in process of division. That the moment of division represents the most critical period for the bacterium, that bacteriophagy takes place much more actively when the bacteriophage acts on young bacteria, was stated among the very first of my reports. But it is none the less true that old bacteria, certainly no longer dividing, can undergo bacteriophagic dissolution, as has been shown in the experiments presented above. Moreover, this fact has been confirmed by Maitland,*^2 working with organisms of the typhoid-dysentery group, and more recently by collaborators of Bordet (Gratia and Rhodes^^^). Gratia combined a Staphylo-bacteriophage of a very high potenc}^, using an extreme dilution of the filtrate, with a suspension of Staphylococcus aureus, and observed, under these conditions, that bac- terial dissolution commenced only after about a week. The dissolution was, nevertheless, complete. He concluded quite rationally that bacteriophagy may take place with bacteria which are no longer reproducing.* From these facts it may be deduced, in brief, that whatever the bacterial species, bacteriophagy may take place with a bacterium of any age, provided it be alive and normal. Yet, although all hving unaltered bacteria are susceptible, the critical moment, the period when the bacterium is most vulnerable, is the moment of division. 5. EFFECTS OF THE RELATIVE CONCENTRATIONS OF BACTERIO- PHAGE AND BACTERIA Let US now consider the variable characteristics attending the phe- nomenon of bacteriophagy when it occurs under different concentration * Gratia and Rhodes even add that the reaction occurs with bacteria in process of disintegration, since microscopic examination showed that after a week modi- fied staphylococci were present. That these degenerating staphylococci had been dissolved is certain, since the medium ultimately became completely clari- fied, but to assume that the dissolution of these altered bacteria was brought about directly, by the bacteriophage itself, is another question. This possibility we will have occasion to treat at some length when we consider the mode of action of the bacteriophage. Here, let us simply say that the dissolution of modified bacteria, and dead cells as well, appears to be effected through the action of substances which become disseminated in the medium during the course of bacteriophagy. 50 THE BACTERIOPHAGE AND ITS BEHAVIOR relationships, such as may be provided by varying within a given medium the proportions of the two antagonists involved, the bacterio- phage principle and the bacterium to which it is opposed. It must be recalled that in these first chapters we are dealing only with extremely active bacteriophage principles, that is to say, with those capable of completely and 'permanently dissolving all of the bacteria present in a medium, even though only an infinitely small quantity of the filtrate containing the bacteriophage principle (a millionth of a cubic centimeter, or less) is introduced into a definitely turbid sus- pension of bacteria. Into a constant quantity of medium, or peptone broth with a pH of 7.8, we may introduce, either a very few bacteria (as about one to each centimeter) or a very great number (several biUions). On the other hand, whatever the number of bacteria, we may inoculate the suspension with a very minute quantity of a filtrate containing the bacteriophage principle, as 0.000,000,000,1, or IQ-^^ cc* or with a con- siderable quantity, as 1 cc, or, indeed, with any intermediate amount. Many of the experiments which I have published dealing with the effect of the relative concentrations in the medium of bacteria and bac- teriophage in their bearing upon the nature and course of the phenom- enon of bacteriophagy, have been repeated by many investigators, and in all cases their observations have been comparable to mine. It could hardly be otherwise; the facts are so clear-cut that doubt is impossible. As for the deductions inspired by these facts, questions bearing upon the mode of regeneration of the active principle, for * Throughout the text the logarithmic notation, the most convenient for ex- pressing such values, will be utilized. Despite the fact that this notation has been rather widely employed in recent publications, it may be unfamiliar to some. The following table of equivalents is therefore provided. 10-1 =0.1 cc. (a tenth of a cubic centimeter) 10-2 — 0.01 cc. (a hundredth of a cubic centimeter), etc. 10-3 = 0.001 cc. 10-" = 0.000,1 cc. 10-5 = 0.000,01 cc. 10-« = 0.000,001 cc. 10-^ = 0.000,000,1 cc. 10-8 = 0.000,000,01 cc. 10-9 = 0.000,000,001 cc. 10-1" = 0.000,000,000,1 cc. (a ten-billionth part of a cubic centimeter) In brief, the negative exponent represents the number of figures at the right of the decimal point. It is likewise the characteristic of the log of the decimal fractional number expressing the quantity of liquid. BACTERIOPHAGY IN A FLUID MEDIUM 51 example, discussion must be reserved for a later chapter. For the time being let us simply treat of the macroscopic appearance of the phenomenon; let us describe only those things that can actually be seen. It may be well, however, to consider at once the significance of the distinction made by certain authors between what they term a "proc- ess of inhibition" and a "process of bacteriolysis." This distinction is based upon whether the bacteriophage principle is inoculated into a medium simply seeded with susceptible bacteria or whether it is introduced into a cloudy suspension of these bacteria. Such a differen- tiation implies that in the first case growth does not occur, the bacterio- phage seems to "inhibit" multiplication, while in the second case the turbid medium becomes perfectly clear after the complete dissolution of the bacterial bodies. As a matter of fact, it is difficult to conceive how the two cases can possibly be considered as distinct. For whatever may be the actual number of bacteria present in a medium the phenomenon is exactly the same, the course of the reaction is the same, and the end result, a complete dissolution of the bacteria present, is the same. It is very obvious that if the number of bacteria is so small that the medium appears clear from the beginning (as in a simple seeding) the medium will show no change, it will remain clear, for the few bacteria present will be dissolved. If, on the contrary, because of the enormous num- ber of bacteria present, the medium was clouded or turbid at the out- set, that is, at the time when the bacteriophage was inoculated, it becomes limpid only when all of the bacteria have been dissolved. In one case, just as in the other, the same phenomenon has taken place, the action of the bacteriophage has been of the same nature. A con- sideration of the different cases which we will present will leave no doubt upon this point; a basic and vahd distinction between an "in- hibition" and a "bacteriolysis" is impossible. Under both conditions as to quantity the phenomenon is qualitatively the same. Limits of baderiophagy Let us repeat once more that all of the experiments presented in this first chapter deal with the typical phenomenon of bacteriophagy, that is, with that which takes place through the intervention of an extremely active bacteriophage. To enter upon a study of bacteriophagy using races of the principle having but shght activity is simply to wiUfuUy invite difficulties, both in experimental procedure and in interpretation. 52 THE BACTERIOPHAGE AND ITS BEHAVIOR Having in mind the effects of variations in the relative concentra- tions of the factors involved in bacteriophagy, we may first consider the maximum concentration of bacteria permitting the return of the medium to a Hmpid state, in other words, what the maxunal quantity of bacterial cells is which is capable of being dissolved in a given quan- tity of fluid. The following experiments show the situation as regards B. dysen- teria Shiga. Comparable results have been secured with Flexner and INITIAL NUMBER OF BACTERIA PER QUANTITY OF BACTERIOPHAGE APPEARANCE OF THE MEDIUM AFTER FILTRATE CUBIC CENTIMETER INOCULATED 24 hours 48 hours 5,000 million CC. 0.1 Turbid 2,000 million 0.1 Cloudy 1,000 million 0.1 Slightly cloudy 500 million 0.1 Clear 1,000 million 0.01 Cloudy (150) Cloudy (100) 900 million 0.01 Cloudy (150) Cloudy (100) 800 million 0.01 Cloudy (100) Cloudy (50) 700 million 0.01 Cloudy (50) Clear 600 million 0.01 Cloudy (50) Clear 500 million 0.01 Clear Clear 400 million 0.01 Clear Clear 300 million 0.01 Clear Clear 200 million 0.01 Clear Clear 100 million 0.01 Clear Clear 50 million 0.01 Clear Clear 25 million 0.01 Clear Clear 10 million 0.01 Clear Clear 5 million 0.01 Clear Clear 1 million 0.01 Clear Clear with Hiss strains. The experiments presented in tables 2 and 3 are conducted in 10 cc. of a salt peptone bouillon at a pH of 7.8. The figures in parentheses indicate the opacity of the cloud in the medium, expressed in milhons of bacteria per cubic centimeter, as determined by comparison with titrated control suspensions. The incubation is carried out at 37°C. The last three tubes, because of the small quantity of bacteria suspended in the medium, were clear at the beginning of the experi- ment. They remained so of necessity. BACTEEIOPHAGY IN A FLUID MEDIUM 53 When this experiment was repeated, under certain modifications, such as varying the quantity of bacteriophage filtrate within the Hmits between 1 and 0.001 cc, the results were identical, despite the fact that different strains of B. chjsenteriae were used. Three different races of Shiga-bacteriophage, all of maximal activity, have likewise given results practically identical. Results of the same nature as those recorded above are shown in the next experiment (table 3) in which a highly active Staphylo-bac- teriophage acts upon Staphylococcus aureus. Here again, incubation is at 37°C. TABLE 3 NUMBER OF STAPHYLOCOCCI QUANTITY OF BACTERIOPHAGE INOCULATED APPEARANCE OF THE MEDIUM AFTER PER CUBIC CENTIMETER 24 hours 48 hours 1,000 million CC. 0.05 Cloudy Cloudy (100) 750 million 0.05 Cloudy Cloudy (50) 500 million 0.05 Cloudy (200) Clear 250 million 0.05 Cloudy (100) Clear 100 million 0.05 Cloudy (50) Clear 50 million 0.05 Clear Clear 25 million 0.05 Clear Clear 10 million 0.05 Clear Clear 1 million 0.05 Clear Clear 100 thousand 0.05 Clear Clear 10 thousand 0.05 Clear Clear 1 thousand 0.05 Clear Clear 1 hundred 0.05 Clear Clear ten 0.05 Clear Clear The suspensions containing less than 10 million staphylococci per cubic centimeter were clear from the beginning of the experiment, and they remained so indefinitely. After a month of standing at room temperature the tubes were returned to the incubator for 48 hours. No change in appearance resulted ; the aspect remained as when original- ly removed from the incubator, the first two were cloudy, all of the others were clear. The maximum number of bacteria capable of being dissolved in the medium is the same, whether the total number is introduced at one time at the beginning of the experiment, or whether the organisms are added in several fractions. This is shown by the following experiment, performed with B. dysenteriae. A salt peptone bouillon medium, ad- justed to pH 7.8 is used. Incubation at 37°C. 54 THE BACTERIOPHAGE AND ITS BEHAVIOR A suspension of 250 million of bacilli per cubic centimeter is inocu- lated with 0.0001 cc. of a bacteriophage filtrate. After 14 hours the dissolution of the bacteria is complete, the medium being clear. At this time, a concentrated suspension of young bacilH is added to this clear medium in such a way as to restore the titre to 250 million per cubic centimeter. Seven hours later the medium is again limpid, all of the bacteria having been dissolved. Another addition of a new quantity of concentrated suspension is made, again yielding a turbidity cor- responding to 250 million bacteria to each cubic centimeter. This time, after 48 hours, the medium is still sHghtly cloudy. To this medium, not entirely clear, a further addition of concentrated bacterial suspension is made, restoring the turbidity to the equivalent of 250 milhon bacteria per cubic centimeter. Eight days later the medium has cleared somewhat, but it is still definitely cloudy. How- ever, plantings made from it upon agar and into bouillon remain sterile. As is seen, whether the bacteria were present in the medium from the beginning, or whether they were introduced by fractions in the course of the action, the dissolution was complete only for quantities below approximately 700 millions per cubic centimeter of medium. Later we will have more to say about the cause which operates to hinder dissolution of more than a certain number of bacteria per unit volume of fluid. These experiments reveal the fact that the bacteriophage principle is able to effect a complete dissolution of the bacterial cells in suspen- sion in a medium propitious for bacteriophagy when the medium con- tains from one to 700 million bacterial cells per cubic centimeter. Within certain very wide hmits the quantity of bacteriophage filtrate necessary to inoculate to cause this dissolution is a matter of no moment; the course of the action and the final result of the phenomenon is the same, whether the amount inoculated is 1 cc. or 0.001, to 10 cc. of medium. The total number of bacteria which may undergo a complete dis- solution under the action of a very active bacteriophage appears to vary with different species of bacteria. As has been seen above, the maximum is about 700 milUon for B. dysenteriae, whether it be Shiga, Flexner, or Hiss, and for the staphylococcus, whether it be albus, aureus, or citreus. With B. coli, B. typhosus, and B. paratyphosus A and B, a complete dissolution has been obtained with quantities of 350 million per cubic centimeter of medium but not of higher concen- BACTERIOPHAGY IN A FLUID MEDIUM 55 trations. For B. gallinarum and the different Pasteurella organisms, the maximum titre is the same. With B. pestis, it has been impossible to go above a limit of 200 million per cubic centimeter. But here we must bear in mind that with B. dysenteriae and the staphylococcus, except for the extremelj^ active races of the bacteriophage, the maxi- mum number capable of complete dissolution is only 350 to 400 million per cubic centimeter, and it is quite possible that for B. coli, B. typhosus B. pestis, etc. a complete dissolution of more concentrated suspensions might be obtained with races of the bacteriophage still more active than those which I have isolated and worked with up to the present time. In bacteriophagy all is relative; all depends upon the aptitudes, the qualities, of the race of bacteriophage with which one is working. Too many authors seem to forget this. Limits of activity of the bacteriophage principle What are the maximum and minimum quantities of the bacterio- phage with which bacteriophagy can be effected? The phenomenon takes place if bacteria are suspended in an undi- luted bacteriophage filtrate. It is unnecessary to support this state- ment by citing experiments, particularly since those described upon the preceding pages demonstrate this adequately. They show that it is possible to add a new quantity of bacteria to a medium resulting from a complete dissolution of bacteria and that they are in turn entirely dissolved. Experiments upon the lower limit are more interesting. Let me say once more that the condition portrayed here applies only to races of the bacteriophage having a high potency.* * Although to some, these repetitions may appear quite uncalled for, experience shows that they are necessary. For many workers, carrying out their experiments under conditions other than those which I have indicated ha\e obtained results differing from mine (it could hardly be otherwise) and have, upon this basis, felt warranted in contradicting my experimental findings. For example, and this example is selected from among many others, several investigators, early in their studies, have affirmed that the dissolution of bacteria is never complete, a con- clusion reached simply because of the fact that they used a bacteriophage of but little activity. It is true that they have later recognized that a total dissolution occurs, a fact which today is unanimously accepted. But despite the fact that these authors have later revised their conclusions, it would have been somewhat more logical to have worked first under the conditions as I described them. This would have rendered unnecessary a subsequent retraction. 56 THE BACTERIOPHAGE AND ITS BEHAVIOR We may consider first the reaction as it develops in suspensions of low bacterial content, for example, in a suspension containing 1 million Shiga bacilli per cubic centimeter. Let us inoculate a series of tubes, each containing 10 cc. of such a suspension, with decreasing quantities of the Shiga-bacteriophage as indicated in table 4, The results, ex- pressed by the macroscopic appearance of the tubes at different inter- vals will be as indicated. The figures within the parentheses indicate the opacity of the cloud, by comparison with control tubes containing titrated suspensions of formolized Shiga bacilH. Further experiment showed that tube 11 did not contain any of the bacteriophage principle. QUANTITY OP APPEARANCE AFTER INOCULATED 4 hours 5 houra 6 hours 18 hours 1 CC. 10-1 Clear Clear Clear Clear 2 10-2 Clear Clear Clear Clear 3 10-3 Clear (?) Clear Clear 4 10-4 (?) (25) (?) Clear 5 10-6 (?) (25) (25) Clear 6 10- « (?) (50) (25) Clear 7 10-7 (?) (75) (50) Clear 8 10-8 (?) (75) (75) Clear 9 10-3 (?) (75) (100) Clear 10 10-10 (?) (75) (100) Clear 11 10-" (?) (75) (100) Cloudy (?) = clouding doubtful; if any, very slight. Let us repeat this experiment, under the same conditions, except that we will combine the same series of dilutions of the bacteriophage filtrate with a suspension containing 100 million bacilli per cubic centi- meter, that is to say, with a definitely cloudy suspension rather than with one having but 1 million organisms per cubic centimeter. As will be seen, the ultimate results are entirely comparable to those obtained with the less concentrated bacterial suspension (table 5). The following experiment (table 6) shows that the general course of the process of bacteriophagy is the same if it is performed with a Staphylo-bacteriophage in decreasing amounts combined with a sus- pension of the staphylococcus. Here, each tube contains 10 cc. of a suspension of Staphylococcus aureus in a salt-peptone bouillon, adjusted to pH 7.8. Incubation is at 32°C. BACTERIOPHAGY IN A FLUID MEDIUM 57 The experiments presented above reveal the following facts : The final result of the phenomenon, that is to say, the total dissolu- tion of the bacterial cells present in the suspension, does not depend upon the quantity of the bacteriophage filtrate inoculated into the suspension (d'Herelle^^°). We shall see, however, that although TABLE 5 TUBE QUANTITY OF BACTERIO- APPEARANCE AFTER INOCULATED 4 hours 5 hours 6 hours 24 hours 36 hours 1 CC. 10-1 (100) (75) Clear Clear Clear 2 10-2 (125) (100) (25) Clear Clear 3 10-3 (125) (125) (25) Clear Clear 4 10-" (125) (125) (100) Clear Clear 5 10-5 (125) (125) (125) Clear Clear 6 10-5 (125) (150) (150) Clear Clear 7 10-7 (125) (200) (175) Clear Clear 8 10-8 (125) (200) (200) Clear Clear 9 10-9 (125) (200) (250) Clear Clear 10 10-10 (125) (200) (300) (100) Clear 11 10-" (125) (150) (175) Cloudy Cloudy QUANTITY OF APPEARANCE OF THE SUSPENSIONS AFTER BACTERIOPHAGE INOCULATED 6 hours 18 hours 24 hours 60 hours 1 CC. 10-2 (125) Clear Clear Clear 2 10- -» (150) (25) Clear Clear 3 10- « (150) (100) (25) Clear 4 10-8 (150) (250) (200) Clear 5 10-10 (150) (350) (350) Clear 6 10-" (150) (350) (350) Clear 7 10-12 (125) (250) (300) Cloudy* * Tests showed that this tube did not contain any of the bacteriophage principle. quantity is a neghgible factor, the quality of the bacteriophage is the most important feature of the phenomenon. The only influence exerted by the quantity of the bacteriophage principle inoculated is in the time required for the completion of the phenomenon (Kuttner;^^* d'Herelle^^^). 58 THE BACTERIOPHAGE AND ITS BEHAVIOR Between the smallest quantity of the bacteriophage filtrate capable of provoking bacteriophagy and the amount immediately below this a partial effect is not obtained. The action is complete or there is no activity at all (d'Herelle^i"). The smallest quantity of bacteriophage filtrate, of a maximum activity, capable of causing bacteriophagy in a suspension of dysen- tery bacilh, is in the neighborhood of a ten-billionth of a cubic centi- meter (d'Herelle^^"). Against the staphylococcus, the smallest quantity to be active is even less; in the experiment given above, about one hundred-biUionth of a cubic centimeter, that is, about 10 cubic micra (d'Herelle). Ellis has stated, ^^^ although he presents no protocols to demonstrate the fact, that if two bacterial suspensions are inoculated with the bacteriophage, one with a large quantity, the other with but a small amount, bacteriophagy will not take place equally in both, that is, dissolution will be only partial in the first, and complete in the second case. Even more recently Gohs has reported the following results obtained with a bacteriophage filtrate which had been preserved for a year. When the quantity of filtrate used varied between ^ ^ ^ ^ (^ ^ and 1 0,000,000 ^^ ^ *^i*0P bacteriophagy was complete; while when the quantity of filtrate used was from 5 drops to nTi^o ^^ ^ drop the bacteria remained alive. It is indeed difficult to reconcile results of this type with the many experiments which I have made; experiments in which the filtrates have varied in age from those used immediately after preparation to those which had been held for several months and even several years, and in which all of the other conditions contribu- tory to the process of dissolution have been varied in all directions. It would seem that experimental error, something such as an inter- changing of tubes, must have occurred. 6. INFLUENCE OF PHYSICAL CONDITIONS Action of heat As a general rule it may be said that bacteriophagy may always take place at that temperature which is the most favorable for the development of the bacterium subjected to the action of the bacterio- phage. This is not equivalent, however, to saying that this temperature is that at which the phenomenon is effected at the greatest rate. More- over, the phenomenon may occur at temperatures very remote from the optimum growth temperature of the bacterium. BACTERIOPHAGY IN A FLUID MEDIUM 59 We may here state what has been demonstrated in this connection up to the present time for different bacteria when subjected to the action of homologous races of the bacteriophage. B. typhosus. Bacteriophagy takes place at temperatures up to 41°C. ; it is even more active at this temperature than at 37° (Kuttner^^^). The phenomenon does not take place at 45°C. (Kuttner^®^). B. dysenteriae. A complete dissolution of dysentery bacilli takes place at temperatures between 8° and 41°C. (d'Herelle^^O, but the time required for the end result to be attained is very variable. Three tubes, each containing a normal suspension of Shiga bacilli* are inoculated with 10~^ cc. of a filtrate containing the bacteriophage. These tubes are placed, one at 8°C., one at 22°, and the third at 37°. Subsequent examination shows that in the suspension held at 8° the bacteria have been completely dissolved after 16 days. In the one held at 22° dissolution is complete after 25 hours. In the one kept at 37° the dissolution is complete after 13 hours, B. pestis. Two races of the bacteriophage have been tested against a single strain of B. pestis. In both cases the maximum temperature limit for complete dissolution was 41°, with the optimum temperature at 37°C . Staphylococcus. Against a single strain of Staphylococcus aureus two different races of staphylococcus bacteriophage were tested; the first, race H, caused a complete dissolution at temperatures up to 43°C., the other, race B, only up to 41°. The optimum for the first was at about 32°, for the second at about 3G°C. B. coli. Doerr and Griiningeri^^ have reported that bacteriophagy of B. coli does not take place at 43°, despite the fact that the strain of B. coli used by them developed at this temperature. It seemed, indeed, from their experiments that the bacteriophage, acting under such conditions of temperature, totally disappeared from the medium at the end of 5 to 7 hours. In attempting to verify this experiment, the results of which appeared to be rather unusual, I obtained entirely different results. Details of these experiments follow. The B. coli, strain H, was isolated from a case of cystitis. The bacteriophage, * When, in the description of an experiment the volume of a suspension is not stated, it should always be understood to be 10 cc. In the same way, in order to avoid repetitions such as would make the explanation unnecessarily long, unless indicated to the contrarj^, the culture medium is a beef bouillon (400 grams of muscle extracted per liter) with 1 per cent of peptone and 0.8 per cent of salt. The reaction is adjusted to a pH of 7.8. 60 THE BACTERIOPHAGE AND ITS BEHAVIOR race c, was isolated from the stools of a patient convalescent from Asiatic cholera. The medium was prepared with Liebig's meat ex- tract, containing 0.5 per cent of salt and 1 per cent of peptone. The reaction was adjusted to pH 7.6. I. The water-bath was regulated to maintain a temperature of 45°C. Ten cubic centimeters of a suspension of B. coli, 100 million per cubic centimeter, were inoculated with 0.02 cc. of bacteriophage filtrate. After 1 hour the opacity was equivalent to 100 millions per cubic centimeter. After 2 hours the opacity was equal to 75 millions. After 3 hours the medium was clear; dissolution was complete. Control 1 ; the same suspension, but uninoculated with the bacterio- phage. After 1, 2, and 3 hours at the same temperature of 45°C. there was no change. The opacity of the supension remained the same as at the beginning. Control 2; bouillon simply seeded with B. coli. After 3 hours at 45°C. the medium was clear; throughout a period of 3 hours at this temperature there was no obvious development. II. The water-bath was regulated to maintain a temperature of 46°C. The conditions were the same as for the preceding; 10 cc. of a suspension of B. coli, 150 million per cc, inoculated with 0.05 cc. of bacteriophage filtrate (a filtrate of the suspension dissolved at 45° in the preceding experiment). After 1 hour the opacity was equal to 150 milhons per cubic centimeter. After 2 hours the opacity was the same. After 3 hours the opacity was equal to 100 million. After 3| hours the opacity corresponded to 50 million. After 4 hours the medium was clear; dissolution was complete. Control 1; bouillon simply seeded with B. coli, without the addi- tion of the bacteriophage. No development occurred within 4 hours. The medium remained as it was at the beginning of the experiment, immediately after seeding. Control 2; suspension of 150 millions per cubic centimeter, without bacteriophage. The turbidity was the same after 1, 2, 3, and 4 hours as at the beginning. The culture showed no evidence of bacterial multiplication. III. The water-bath was regulated to maintain a temperature of 47°C. The procedure was the same as that given above. BACTERIOPHAGT IN A FLUID MEDIUM 61 After 6 hours the turbidity was the same as at the beginning. Bac- teriophagy had not taken place. But the bacteriophage was not destroyed, for a drop of this suspension spread upon agar failed to yield a growth after incubation. Nor were the bacilli killed, for the inocula- tion of a drop of the control suspension, uninoculated with the bacterio- phage, yielded a growth of B. coli. These experiments, therefore, justify the conclusion that under the conditions under which they were performed bacteriophagy is effected up to temperatures of 46°C., and that at this temperature it is very rapid indeed, even more rapid than at 37°C. It is difficult to explain the results obtained by Doerr and Griininger. But one possibihty suggests itseK, namely, that their results were due to the unintentional use of an acid bouillon as culture medium, or, which would amount to the same thing, to the use of a medium containing sugar and rendered acid by the fermentation caused by the colon bacilli. This possibility is suggested to explain the destruction of the bacteriophage at this temperature. As for the absence of bacteri- ophagy at this temperature (43°C.) it is readily explained by the fact that the temperature limit varies with the race of bacteriophage em- ployed. For example, in so far as B. coli is concerned, using the same strain as that which was employed in the experiments previously cited, but using a bacteriophage of a different race (isolated from the feces of a convalescent from typhoid fever) the dissolution of the bacilli was not complete at 42°; at 43° it did not take place at all. In bacteriophagy it is vain to undertake to estabhsh rigid rules (there are some who even speak of "laws") fixing with an air of finality the conditions of the phenomenon. What is true for one race of the bacteriophage is not necessarily true for another. 'T have isolated several hundred strains of this bactericidal "principle," and I have not yet found two which were absolutely identical." These words appeared in one of my first publications,^'^^ and it is indeed unfortunate that those who have since worked with the phenomenon have been unable to comprehend their true meaning, since had they done so they would have refrained from stating many "rules" which further investigation has not confirmed. The single method to follow, — indeed, the sole logical procedure if one wishes to determine as exactly as possible any one of the conditions of bacteriophagy, — is to execute a large number of experiments, utiliz- ing different bacterial species and even different strains of the same species, combining each one of them with several races of the bacterio- 62 THE BACTERIOPHAGE AND ITS BEHAVIOR phage. The results thus obtained will show the extreme limits within which the phenomenon can occur, and it will show at the same time how vain it is to attempt, from a single experiment, to fix the hmiting conditions or the optimal conditions for the reaction, since these vary for each race of the bacteriophage which acts, and for each bacterial strain subjected to its action. As far as temperature relationships are concerned, experiment demon- strates that the minimal temperature permitting a typical bacteri- ophagy, that is to say, the lowest temperature at which the final result is a complete dissolution of all of the bacterial cells contained in a suspension subjected to test, is found at about 8°C. (with B. dysen- teriae, d'Herelle'^^). The maximum temperature observed up to the present time is 46°C. (with B. coli, d'Herelle). This by no means implies that subsequent experiments will not extend these limits. I have specifically stated that these limits are those which are com- patible with a total dissolution of the bacterial cells. Above and below these Hmits bacteriophagy may still be effected, but the dissolution is only partial. Having available a race of bacteriophage, active against B. dysenteriae, whose upper limit for total dissolution was 41°C., it has been possible to effect several serial passages at 44°, in spite of the fact that at this temperature a dissolution of the bacteria could not be detected macroscopically. But despite this apparent lack of activity bacteriophagy took place, since the active principle, the bacteriophage, regenerated itself, and this in itseK affords proof that destruction had not taken place. As regards the optimal temperature, that is to say, that at which the phenomenon manifests itself most quickly and most completely, basing my statements upon the results of my experiments with B. dysenteriae and B. typhosus, I had concluded that the temperature most favorable for bacteriophagy was that which was likewise most favorable for the development of the bacteria. The results of many subsequent experiments, performed with a variety of bacterial species, indicate that this conclusion may have been too restricted. As a general rule, the statement is certainly true for the bacteriophagy of B. dysenteriae and B. typhosus, but it is not equally true for all other species of bacteria. The optimum growth temperature for several different strains of the staphylococcus with which I have worked has been found to be at about 37 to 38°C. With three different races of the bacteriophage the reaction was effected with these strains in a perfect manner at between 32 and 34°C., with another race of bacteriophage complete dissolution occurred only at 37°C. BACTERIOPHAGY IN A FLUID MEDIUM 63 Several of my laboratory strains of B. pestis have an optimum growth temperature of 32 to 33°C. Two races of the bacteriophage acted upon these cultures most vigorously at temperatures of 37 to 38°C. With B. coli, we have seen that, at least with certain races of the bacteriophage, an extremely rapid dissolution occurs at a temperature of 46°C., under conditions where the development of the colon bacilli did not take place, or at least where it was so slow that it could not be detected in control suspensions not containing the bacteriophage. Pressure conditions According to experiments which I have carried out with B. dysen- teriae, it would appear that bacteriophagy occurs as actively in vacuo as under normal atmospheric pressure. ''^^ Various authors, Wollstein in particular, have reached this same conclusion. Viscosity of the medium j)QgPj.i78 jjg^g stated that in a bouillon medium the addition of an adequate amount of gelatin prevents dissolution of the bacteria by the bacteriophage. The experiment upon which he based this con- clusion was performed with B. coli. In collaboration with Berger,^^' Doerr reconsiders this statement, modifying considerably the rigor of his first conclusions. He observes that it is necessary that the medium contain a certain quantity of gelatin in order to prevent the dissolution of the bacteria, and that the dissolution is the less as the concentration of the gelatin is increased. Agar acts in the same way. On the other hand, the greater the concentration of the bacteriophage present, the higher must be the content in gelatin to prevent the dis- solution. But despite this inhibitory effect the bacteriophage is still capable of acting upon the bacteria even in media with very high concentrations of gelatin, for, as he showed, the principle reproduced in such a medium. Consequently bacteriophagy must have taken place. In a series of independent investigations Nakamura has studied the effect of substances having properties analogous to those of gela- tin4fi8,469 jjg showed that gum tragacanth, or salep, exerted an in- fluence upon bacteriophagy comparable to that of gelatin. The sugars also, but to a less degree, interfered with the process. It is significant that he observed that all races of the bacteriophage do not behave in the same way. With some the activity is but slightly modi- 64 THE BACTERIOPHAGE AND ITS BEHAVIOR fied by the introduction of gelatin into the medium; with others, on the contrary, the same medium prevents multiphcation completely. Brutsaert^^" has confirmed this observation. Again and again we are confronted by the same conclusion: the most important factor in bacteriophagy is the quality of the bacterio- phage under consideration. A final explanation for the inhibitory action of gelatin has been provided by Hauduroy.^^^ He states, "It is solely the high viscosity of the medium containing gelatin which interferes with the production of the phenomenon." He showed, in fact, while working with several substances, such as the gums and egg albumin, having the property of augmenting viscosity, that the inhibition is a direct expression of the viscosity, quite unrelated to the chemical nature of the viscous sub- stance. To attribute this inhibition of bacteriophagy to anything other than a reaction between colloids, allied to other reactions of this type, is impossible. In no case is the bacteriophage destroyed. After a period of contact of any duration it is only necessary to dilute the medium with bouillon, thus diminishing its viscosity, to permit the inception of the process of dissolution. Certain experiments which I have made, confirming fully these conclusions of Hauduroy, may be mentioned. To 10 cc. of a peptone bouillon containing 0.8 per cent salt, is added 20 per cent of gelatin. The pH is then adjusted to 7.8. With the medium at a temperature of 35°C., Staphylococcus aureus is introduced to give a concentration of about 200 miUion organisms per cubic centi- meter. It is then inoculated with 0.05 cc. of a highly active bacterio- phage filtrate, that is, with one capable of regularly causing a total and yeimanent dissolution of a normal suspension in bouillon. After incu- bation for 6 days at 35°C. the medium was perfectly limpid, dissolu- tion was complete. In a control mixture in plain bouillon, without gelatin, dissolution was completed in 24 hours. The conclusion is obvious. When added in a sufficient quantity to a medium in which bacteriophagy should be effected, gelatin completely inhibits the action of races of the bacteriophage of low activity, while for more potent races the process is retarded, and the number of bacteria dis- solved is the greater as the bacteriophage is the more powerful.* With * With the acquisition of a resistance by a portion of the bacteria, an acquisi- tion favored by the delay associated with the viscosity of the medium, we are not now concerned, yet in this connection the retardation is of considerable signifi- cance. This feature of the reaction will receive further attention when we deal with the behavior of the bacterium toward the bacteriophage. BACTERIOPHAGY IN A FLUID MEDIUIVI 65 races possessing a maximum activity all of the bacteria are destroyed and dissolved. The role of gelatin, in the last analysis, consists solely in altering the rate of the phenomenon, that is, in extending the dura- tion of the action. All neutral substances, which possess the property of augmenting viscosity, affect the phenomenon of bacteriophagy as does gelatin, and the intensity of this effect is in direct proportion to the viscosity of the medium. In the particular case under consideration the course of the phenom- enon is regulated by variations in two factors, the degree of activity of the bacteriophage involved, and the viscosity of the medium. 7. EFFECT OF THE CHEMICAL CONDITIONS OF THE MEDIUM Colloids We have seen that gelatin, and this is also true for the gums and for egg albumin, have of themselves no effect upon bacteriophagy. It is only when such substances are added to the medium in a quantity sufficient to significantly augment the viscosity that they exert an effect, and even then this effect is simply a retardation of the action. Certainly this is the case when a potent bacteriophage is used. It is of interest, in comparison with the above, to observe the effect exerted by a colloid which does not modify the viscositj^ of the medium to which it is added and whose action, if any, must be due to properties inherent in the colloid itself. For this purpose I have chosen colloidal silver (collargol, Clin, as provided in ampoules for purposes of injection). The following experiment indicates the nature of the effects with such a colloid. To 8 cc. of Liebig's extract bouillon, made up with 1 per cent of peptone and 0.8 per cent of salt (reaction pH 7.6) collargol is added. Three tubes are prepared. The mixtures are implanted with a heavy suspension of Staphylococcus aureus derived from a young culture, the final concentration being 100 milhon organisms per cubic centimeter. Each of the suspensions is then inoculated with 0.02 cc. of a Staphylo-bacteriophage, and the tubes are incubated at 32°C. The appearance after 24 hours is as follows : TUBE NUMBER AMOUNT OF COLLARGOL ADDED APPEARANCE AFTER 24 HOURS 1 2 3 CC. 0.5 1.0 2.0 Clear Clear Clear 66 THE BACTERIOPHAGE AND ITS BEHAVIOR Control tubes, lacking the bacteriophage filtrate, show that the staphy- lococcus develops normally in bouillon containing these quantities of collargol. Under such conditions bacteriophagy proceeded normally, as rapidly and in as complete a manner, as in plain bouillon, even though the colloidal silver added (2 cc.) was sufficient to give the medium such a deep brown color that it was impossible to see and read print through the layer of fluid. As an incidental finding, the following observation is interesting and is in some degree significant. In the control tubes, containing the same quantities of collargol and the same suspension of staphylococci, but without the bacteriophage, after incubation for 24 hours the col- loidal silver was completely fiocculated, forming a black precipitate on the bottom of the tube. In the suspensions inoculated with the bacteriophage no flocculation took 'place, the liquid remained perfectly clear after bacteriophagy, presenting the same aspect as a tube of sterile bouillon to which a comparable amount of collargol was added. This experiment has been repeated with B. dysenteriae with the same result. In another series of experiments I have used the dry colloidal silver of Heyden, bearing the trade name "Collargolum steril." To 10 cc. of culture medium (the same as that used above) 5 mgm. of the dry collargolum are added, and then enough of a thick suspension, prepared from a young agar growth, of cocci to yield a count of 250 million per cubic centimeter. Finally, 0.05 cc. of a Staphylo-bacteriophage filtrate is added. After 36 hours bacteriophagy is complete; the medium is absolutely clear and of a deep red-brown color. A drop of this spread upon agar yields no growth. If performed with B. dysenteriae this experiment gives the same result. As a check on these results, it is found that B. dysenteriae, and the staphylococcus as well, grow perfectly well in bouillon containing colloidal silver in the amount employed in the above experiment. The statement is very frequently made that colloidal silver is a power- ful antiseptic. It would seem to be a fair question to ask how experi- ments should be conducted to demonstrate this fact. Unquestionably, several authors have published experiments tending to show that the bacteriophage is destroyed by the presence of negative colloids, of colloidal silver in particular, but, as a matter of fact, far from being destroyed, the bacteriophage develops normally in the presence of this substance. BACTERIOPHAGY IN A FLUID MEDIUM 67 May not the erroneous conclusions reached by these authors be due to the fact that they have used commercial solutions of colloidal silver to which some antiseptic agent had been added, and have they not attributed to colloidal silver an effect which was due to some agent of whose presence they were unaware? Obviously, we can not tell. But in any case, a considerable number of experiments warrant the statement that pure colloidal silver (Collargolum siccum, Heyden,* for example) exercises no antiseptic action upon the colon-typhoid- dysentery group of bacilli or upon the staphylococci, and that in its presence bacteriophagy occurs normally.f Colloidal suKur, another electro-negative colloid, has also been tested. In its presence bacteriophagy takes place in an absolutely normal fashion. Indeed, with this colloid it would seem that bacteri- ophagy is, if anything, stimulated, as has been observed by Otto and Munter.494J In brief, then, negative colloids, and these are the only ones which can be tested, since bacteriophagy will not take place in an acid medium, cause, of themselves, no effect upon the phenomenon of bacteriophagy. Dyestuffs Five-hundredths of a cubic centimeter of a Staphylo-bacteriophage is added to 10 cc. of a suspension (100 million cocci per cubic centi- meter) of Staphylococcus aureus. To such mixtures the dyestuffs, as indicated in table 7, are then added. Control tubes are prepared, the bouillon containing the same quanti- ties of the dyestuffs, and are simply seeded with the staphylococcus. These tubes demonstrate the^ possibility for growth in the dye-con- taining medium. To all of these tubes which showed bacteriophagy a second implan- tation of a concentrated suspension of staphylococci was made, yield- * A finely granular colloidal silver, yielding in water a very stable red pseudo- solution. t These results suggest grave doubts upon the validity of the experiments showing the so-called "oligodynamic" action of metals, of silver in particular. If silver in its colloidal form is not active there is the more reason to suspect that it may be inert when in its usual form. t We will see later that "secondary cultures" develop when the bacteriophage used is not of a maximum potency. It is significant that in the presence of colloidal sulfur secondary cultures are difficult to obtain, even though the bacteriophage used does not prevent their formation in normal bouillon. 68 THE BACTERIOPHAGE AND ITS BEHAVIOR ing 200 million cocci per cubic centimeter. Again bacteriophagy was complete in all tubes after 24 hours. From these results it is clear that when the substance, like fuchsin, has an antiseptic action bacteriophagy does not take place; if the dye is lacking in harmful effects upon the bacterium bacteriophagy is normal. Of itself, the dyestuff, like colloids, interferes in no way with the phenomenon (d'Herelle). DYESTUFF QUANTITY ADDED APPEARANCE AFTER 24 HOURS SEEDED CONTROL TUBE CC. 0.05* 0.05t 0.05t 0.05* 0.05* Clear Clear Turbid Clear Clear Growth Methylene blue (Grubler) Growth Fuchsin (Grubler) No growth Neutral red (Grubler) Growth Acid fuchsin (Grubler) Growth * A saturated aqueous solution, t A saturated alcoholic solution. TUBE NUMBER B. DYSENTERIAE APPEARANCE CONTAINING 8 CC. BACTERIOPHAGE ADDED FLEXNER, 24 HOUR AFTER 24 HOURS OF MEDIUM CULTURE AT 37°C. 10/14 1 (control) CC. 0.05 Very cloudy 10/14 2 0.05 0.05 Clear 10/15 3 (control) 0.05 (from tube 1) Very cloudy 10/15 4 0.05 (from tube 2) 0.05 (from tube 1) Clear 10/16 5 (control) 0.05 (from tube 3) Very cloudy 10/16 6 0.05 (from tube 4) 0.05 (from tube 3) Clear 10/17 7 (control) 0.05 (from tube 5) Very cloudy 10/17 8 0.05 (from tube 6) 0.05 (from tube 5) Clear Electrolytes In several communications da Costa Cruz has emphasized experi- ments which indicate that bacteriophagy will not take place in a medium very poor in electrolytes, a medium such as a 1 per cent Witte peptone solution in distilled water. When, however, he added various salts to this medium (NaCl, CaCl2, Na2S04) the bacteria were dis- solved.i^S'^^® Although it may be true that in such a medium, which is incidentally BACTERIOPHAGY IN A FLUID MEDIUM 69 rather unfavorable to the bacterium involved {B. chjsenteriae Flexner), a complete dissolution of concentrated bacterial suspensions does not take place, it is equally true that bacteriophagy will take place, and in a complete fashion, when the suspension is less heavy, as the following experiments show^''^ (table 8). The culture medium is a 1 per cent peptone water, having a pH of 7.0. This demonstrates that the bacteriophage is capable of multiplying in media poor in electrolytes, inasmuch as in such media bacteriophagy occurs. Brutsaert^^° arrived at the same conclusions, but, according to his observations certain races of the bacteriophage fail to develop under such conditions, while others, on the contrary, are unaffected and lead to a total dissolution of the bacterial cells present in the medium. Here again, the same observation is pertinent. It is the quality of the bacteriophage which determines the course of the phenomenon. Ciuca^'*^ has also shown that bacteriophagy occurs normally in media poor in electrolytes, provided the active principle operates upon young bacteria. His media were alkaline, pH 7.8 to 8.0. In general, then, the conclusions seem to be that in media poor in electrolytes, that is, in salts, bacteriophagy does not take place when the medium is acid. In a neutral medium (pH 7.0) bacteriophagy may or may not occur, depending upon the race of the bacteriophage concerned. In an alkaline medium, the phenomenon takes place with all races; and with the most active the dissolution of the bacteria is complete. Instead of considering the effects of deficient quantities of electro- lytes in the medium, the reverse situation offers a problem. What happens when the medium contains a large amount of salt? Bacteri- ophagy takes place normally in bouillon containing 2.5 per cent of salt (d'Herelle^-^) . Brutsaert^"^ has shown that B. coli does not grow in a medium containing more than 5 per cent of NaCl, while staphylococci still develop freely in broth having 14 per cent of salt, yet in such media he observed that bacteriophagy took place normally. According to him, bacteria cultivated in hypertonic bouillon and then placed in contact with the bacteriophage are even more readily attacked than the same bacteria grown in a medium with a normal concentration of salt. 70 THE BACTERIOPHAGE AND ITS BEHAVIOR Sugars Asheshov" has shown that with certain races of the bacteriophage (one only among those which he studied) bacteriophagy takes place more rapidly if glucose is added to the medium. With this particular race, active against B. dysenteriae Flexner, dissolution of the bacteria was complete after 4| hours in the presence of glucose, while in or- dinary bouillon it was only partial after a period of 7 hours. Further studying the reaction with this particular race he concluded that the acceleration must be due to the fact that B. dysenteriae in fermenting the glucose rendered the reaction acid. This, then, represents a particular distinctive race, with which the optimum activity occurs in a medium of increasing acidity. We will return to this observation. Seiser^^'' has suggested that the addition of glucose to the medium favors bacteriophagy in all cases, but in agreement with Asheshov, I have not found this to be true. Indeed, it appears that the situation is quite the reverse. The experiments which I have performed during the past few years have not modified my original conclusions in this respect. The addi- tion of non-fermentable sugars* to a bacterial suspension subjected to the action of the bacteriophage has no effect upon the phenomenon. In the case of fermentable sugars, if the inoculation of bacteriophage has been massive, bacteriophagy takes place normally. If the inocu- lation has been weak (or if the race of bacteriophage is of low activity) bacteriophagy is incomplete or does not occur, the result depending upon the amount inoculated. The reason for such an effect is obvious. We already know that the great majority of races of the bacteriophage are very sensitive to the action of free H ions. With a minimal inocu- lation of bacteriophage the bacteria start to grow, they attack the sugars, and the medium becomes acid before the quantity of the bacterio- phage, regenerating in the course of the action, is sufficient to effect a dissolution of the bacteria in the tmie available. In a word, the limit of acidity incompatible with the phenomenon is reached before the bacteriophage is able to become effective (d'Herelle^-^- Salts In previously reported experunents^-^ I have shown that calcium chloride interferes with the bacteriophagy of Shiga bacilli; that potas- * This holds only when the quantity of sugar added is not sufficiently high to materially modify the viscosity of the medium. We have already seen that an increase in viscosity retards bacteriophagy. BACTERIOPHAGY IN A FLUID MEDIUM 71 sium chloride retards the process; that magnesium sulfate and the phosphates of sodium and of potassium, in low concentrations, possess a stimulating action, especially with races of the bacteriophage of but weak potency. I have not observed these effects in bacteriophagy with other bacterial species. This apparently warrants the conclusion that when a salt, in small amounts, seems to exercise an inhibitory or a stimulating effect, it is because it favors or interferes with the develop- ment of the bacteria, rather than because it exerts any specific direct effect upon the process of dissolution. Body fluids and other substances When added to a bacterium-bacteriophage mixture, substances devoid of action upon the bacteria, have, in general, no effect upon the phenomenon. Thus, the process is not modified, for example, by normal serum, ascitic fluid, or urine (d'Herelle^^^). Bile is unques- tionably inhibitory. Antiseptics Kabeshima^^^'^^'' has stated that the bacteriophagy of B. dysenteriae occurs even in the presence of antiseptic substances, for example, in media containing sodium fluoride or an excess of chloroform. If these observations are correct, they are quite at variance with the results which I had obtained. At my suggestion Bablet^' has repeated the experiments of Kabeshima. From his (Bablet) experiments, very carefully performed, and carried out as were those of Kabeshima upon B. dysenteriae, it appears that in a bouillon medium containing 1 per cent of sodium fluoride, the bacteriophage principle does not regenerate for after three passages in such a medium it has completely disappeared. Under such conditions, therefore, bacteriophagy does not take place. The results were the same in the medium containing chloroform. I have shown^-^ that bacteriophagy does not take place in media saturated with essences of thyme or of cloves, even though in such media the bacteria remain alive for at least 48 hours. Wolff and Janzen®-^ have reported that bacteriophagy is not accom- plished in the presence of different antiseptics, such as optochin, eucu- pin, vusin (these three substances being derivatives of quinin), chinosol yatren, trypaflavine, rivanol, and malachite green, even if these sub- stances are added in quantities so small that the bacteria are killed only after an appreciable interval. Under these circumstances, al- though bacteriophagy is lacking, the bacteriophage is not destroyed; 72 THE BACTERIOPHAGE AND ITS BEHAVIOR it simply remains inert. Their experiments were conducted with B. typhosus, B. coll, B. dysenteriae, and the staphylococcus. That bacteriophagy does not occur in bouillon containing a quantity of methyl violet sufficient to affect the vitality of the bacteria has been reported by Tomaselli.''^* A somewhat more interesting situation is revealed by those experi- ments in which the antiseptic agent is added to the medium in amounts so small as to allow bacterial development. Certain of my experi- ments were performed for the specific purpose of determining what happens under these conditions, sodium fluoride being selected as the antiseptic. Incidentally, in this series of studies I demonstrated that, contrary to what is stated in a number of physiological text-books, life is perfectly possible in media containing 1 per cent of salt. In such media B. dysenteriae or B. coli live perfectly well. In fact, multipli- cation, in agglutinated floccules, takes place provided the organisms have undergone a preliminary adaptation by a few passages through media of increasing salt concentration. But despite the fact that the bacteria are able to develop in this solution, bacteriophagy will not take place in such a medium. A further fact revealed in these studies is that while B. coli develop freely, without a prehminary adaptation, in a bouillon containing 0.2 per cent of sodium fluoride bacteriophagy will not take place, even if the bacteriophage introduced is very active. If 10 cc. of this fluoride containing medium is seeded very lightly with B. coli and then 0.1 cc. of an extremely active bacteriophage filtrate is added, the colon bacilli develop as vigorously and as abundantly as in the same medium with- out the bacteriophage. Nevertheless, the bacteriophage is not de- stroyed; it simply remains inert (d'HereUe^''") . By this experiment I had designed to show only the fact that the bacteriophage may remain inactive under conditions which permit the development of the bacterium. Brutsaert^"" has extended this study and has sought to determine if the concentration of fluoride, compatible with the development of the bacterium, but inhibitory as regards bacteriophagy, is the same for all bacterial species. He found that the concentration limiting bacteriophagy varies for each species studied (from 0.25 to 0.05 per cent). Indeed, this might have been predicted, for although the bacteriophage remains passive it is cer- tainly not because the fluoride has an effect upon it, but because it modifies the state of the bacterium. This conclusion is supported by the fact that in bouillon containing fluoride the bacteria do not grow normally but in agglutinated clumps. BACTERIOPHAGY IN A FLUID MEDIUM 73 These observations may be summarized very simply, then, by stating that bacteriophagy is operative only with living and nonnal bacteria. 8. PHENOMENA CORRELATIVE WITH BACTERIOPHAGY Acceleration of bacterial growth We have ah-eady seen, in the experiments described, that if a sus- pension of bacteria is inoculated with a relatively small amount of bacteriophage, the bacteria develop, as revealed by the increasing turbidity, and it is only after this initial growth that the bacteria commence to dissolve. But if the experiments described above be carefully watched something else may be observed, namely, that the growth is more rapid, often very much more so, in the suspensions where bacteriophagy is to take place than in the control suspensions uninoc- ulated with the bacteriophage. It would seem that the presence of this dissolving principle applies a stimulus of some sort, as though the multiplication of the bacteria undergoes first a "speeding-up;" an acceleration of the processes of cellular division. Agglutination Very often it may be observed that dissolution of the bacteria is preceded by a very outspoken agglutination. This phenomenon is par- ticularly marked when the bacterial suspension is inoculated with a relatively large amount of bacteriophage filtrate of average potency (d'Herelle^^i). Sometimes this agglutination takes place very quickly, even within a few minutes after the bacteria come into contact with the bacterio- phage. The cause of this agglutination does not appear to reside in the bacteriophage principle itself, but in "a something" which is as- sociated with it in the filtrate. The following experiments show, in fact, that this agglutination takes place in physiological saline, and even if the bacteria are killed previously by heat. The agglutinating substance passes through colloidion membranes that are relatively open, and it resists ageing. A bacteriophage filtrate, about 6 months old, active for B. dysenteriae Shiga, was diluted to 1 : 1000 in physiological sahne and dialyzed, in a collodion sac, against physiological saline. After dialysis for 48 hours living Shiga bacilli, taken from a young agar culture, were suspended in the dialysate, yielding a suspension containing 100 milHon organisms 74 THE BACTERIOPHAGE AND ITS BEHAVIOR per cubic centimeter. The bacilli were immediately completely ag- glutinated in the form of fine floccules, clearly visible macroscopically. The same experiment was carried out with a bacteriophage active for the staphylococcus. When the dialysate was combined with organisms (Staphylococcus aureus) killed by heating for 30 minutes at 60°C. a fine agglutinate immediately formed, the clumps being readily visible macroscopically. There is present, then, in a filtrate, aside from the bacteriophage principle, which is inoperative upon killed bacteria, a substance capable of augmenting the surface tension of the bacteria, whether they be living or dead, against which the bacteriophage present in the filtrate possesses the power of bacteriophagy. RESUME Summarizing the data presented in this chapter the following state- ments can be made. There is a principle, very widely distributed in nature, normally occurring in the intestinal contents, which possesses the property of dissolving bacteria. This principle is present in a particularly active form in the intestinal canal and in the excreta during convalescence from a variety of infectious diseases (d'Herelle^^*'). The phenomenon which this principle causes, in vitro, in a liquid medium containing a suspension of bacteria susceptible to its action, consists essentially in a dissolution of the bacterial cells. This phe- nomenon of dissolution occurs only with living organisms, the medium best suited to the action being that where the bacterium under con- sideration develops best (d'Herelle''^°). Dissolution of the bacteria is accompanied not only by a regeneration of the active agent but by a very pronounced multiplication of the principle. The agency responsible for the phenomenon has been termed Bacterio- phage, the phenomenon itself is called Bacteriophagy (d'Herelle^^^) . By virtue of the fact that in the course of its action the bacteriophage principle multiplies, the dissolving action can be carried out serially for an indefinite period (d'Herelle^^"). Multiplication of the bacteriophage principle takes place whatever may be the number of bacteria present in the suspension, or in the culture into which it is inoculated (d'Herelle^-0- Although multiplication of the bacteriophage is a process qualita- tively independent of the number of bacterial cells exposed to its BACTERIOPHAGY IN A FLUID MEDIUM 75 action, the dissolution of the bacterial cells, a direct result of this multiplication, is complete only within the limits of 1 and 700 million bacteria per cubic centimeter when the bacteriophage principle in- volved possesses a maximum activity. With a greater number of bacteria per cubic centimeter the dissolution is but partial (d'Herelle^-^). The minimal quantity of bacteriophage principle necessary to ob- tain dissolution of a suspension or a bacterial culture is, in the more favorable cases, equal to a ten-billionth part of a cubic centimeter (lO-i*^ cc); sometimes even a hundred-billionth part is sufficient (d'Herelle^'i"). Bacteriophagy takes place under aerobiosis or anaerobiosis. The temperature extremes are 8 and 4G°C., but for all bacterium-bacterio- phage systems the hmits, as well as the optimum temperature are not identical. The temperature relationships vary, on the one hand, with the bacterial species, and even with the bacterial strain, and, on the other hand, with the race of bacteriophage (d'Herelle^-^). Only living and normal bacteria are suited to the phenomenon of bacteriophagy. That is to say, the phenomenon does not take place in the presence of antiseptics, when the amount present is sufficient to modify, in any way, the state of the bacteria (d'Herelle^^''). Bacteriophagy may be accompanied by accessory phenomena. The process of multiplication of the bacteria subjected to the action of the bacteriophage principle may exhibit an accelerated rate prior to the phase of dissolution. An initial phase of the reaction may be charac- terized by an agglutination. CHAPTER II The Bacteriophage Corpuscle 1. bacteriophagy upon solid media The phenomenon of bacteriophagy can be demonstrated, not only in a hquid medium, but upon a sohd medium as well, the presence of the active principle being readily revealed by the following simple procedure. Take a young culture or a rather cloudy suspension of dysentery bacilli in bouillon. Also, dilute a drop of bacteriophage fluid, that is, a suspension which has been cleared through bacteriophagy, active for the dysentery organism, in a liter of sterile physiological sahne. Inocu- late the bacterial suspension with a drop* of this dilution of bacterio- phage filtrate. The final mixture will then contain a great many bacilli and very little bacteriophage. Finally, remove a drop of this inoculated suspension and spread it over the surface of an agar slant or upon an agar plate. After incubation the agar will be covered by a layer of bacilli, but this layer presents a curious aspect, for throughout its extent there will be spots, perfectly circular in form, where the agar is bare, devoid of all apparent growth (d'Herelle,^'^'^). What do these bare areas, these ''plaques" as I have termed them, mean? In the first place it is certain that they bear some relation to the bacteriophage, for they never appear upon plates made with a nor- mal culture of any species of bacteria. But they are always apparent, and always have an identical appearance, when the agar is planted with a culture or a suspension of any bacterial species previously inoculated with a very minute quantity of a bacteriophagic filtrate active for the bacterial species involved (d'Herelle^^^) . The phenomenon is, then, not restricted to certain bacterial species. It is to be observed with any species provided the homologous dissolving principle is present. This simple prehminary experiment shows clearly that there is a causal relationship between the presence of the bacteriophage in a cul- ture or suspension and the appearance of the plaques in the layer of growth obtained by seeding such a suspension upon an agar medium. * When speaking of a drop, it is to be understood that a normal drop, 0.05 cc. is meant. 76 THE BACTERIOPHAGE CORPUSCLE 77 Let US carry the experiment farther, and see if the nature of this rela- tionship may be disclosed. Take a series of 12 tubes, each containing 9 cc. of bouillon to which a concentrated suspension of dysentery baciUi has been added to give a count of 250 million bacilli per cubic centimeter. With these 12 tubes, all containing like amounts of the same bacterial suspension proceed as follows : Tube 1. Into one of these tubes, inoculate 1 cc. of a bacteriophage liquid, one very active for the dysentery bacillus. Each cubic centi- meter of the tube will then contain 250 milHon bacilli and 0.1 cc. of bacteriophage fluid. Tube 2. Into a second tube introducje 1 cc. of the contents of tube 1, just prepared. This second tube will then contain, per cubic centi- meter, 250 million bacilli together with the tenth part of the bacterio- phage fluid transferred in the cubic centimeter of material from the first tube, that is, 0.01 cc. Tube 3. Into a third of the 12 tubes of bacillary suspension introduce 1 cc. of the contents of tube 2. This tube (no. 3) will then contain, as did the others, 250 million bacilli per cc, and the bacteriophage pres- ent in the cubic centimeter of fluid transferred from tube 2, that is, to each cc, 0.001 cc. of the bacteriophage fluid. Continue in this manner with eleven of the tubes of dysentery bacil- lus suspension, as first prepared. Each cubic centimeter of the fluid in all of these tubes will then contain 250 million bacilli per cubic centimeter, but each tube of the series will contain less of the bacterio- phage fluid, since each successive tube will contain but a tenth of the amount in the tube preceding. We will have a series of tubes, all containing both bacteria and bacteriophage; but as is indicated in table 9, the bacterial content is constant; the bacteriophage content is a variable. Immediately after preparing these successive dilutions transfer a normal drop (0.05 cc.) of each of the 12 tubes to agar, either a slant or a plate, taking care to distribute the drop evenly over the surface, in other words, spread* it in a uniform layer. Place the 12 agar cultures in the incubator at 37°C. After incubation, the following facts may be noted. The tubes upon which the specimens removed from the dilutions 10~^, 10~2, and 10~^ (tubes 1, 2, and 3) were spread show no trace of * I shall often have occasion to speak of this operation. The word "spread" implying the even distribution of the'liquid over all of the surface of the agar slant or the media of the Petri dish, will be employed. 78 THE BACTERIOPHAGE AND ITS BEHAVIOR growth whatever; they are bare, just as though they had not been planted. The tube corresponding to dilution 10~^ (tube 4) presents a few traces of B. dysenteriae growth; irregular fragments of a layer of bacterial growth being scattered over its surface. The tube corresponding to dilution 10~^ (tube 5) shows a layer of bacillary growth studded with an infinity of confluent plaques. The tube corresponding to dilution 10"*^ (tube 6) is entirely covered by culture, but scattered throughout the layer there are some 20 plaques. BACTERIAL TUBE NUMBER CONTENT PER CUBIC CENTIMETER BACTERIOPHAGE CONTENT million 1 250 + 10~i cc. of bacteriophage fluid 2 250 + 10~- cc. of bacteriophage fluid 3 250 + 10~^ cc. of bacteriophage fluid 4 250 + 10~^ cc. of bacteriophage fluid 5 250 + 10 ~5 cc. of bacteriophage fluid 6 250 + 10~'' cc. of bacteriophage fluid 7 250 + 10"'' cc. of bacteriophage fluid 8 250 + 10~8 cc. of bacteriophage fluid 9 250 + 10~^ cc. of bacteriophage fluid 10 250 + 10"'" cc. of bacteriophage fluid 11 250 + 10~" cc. of bacteriophage fluid 12 This tube, a, control contains the bacillary suspension, 250 mil- lion pel cubic centimeter, but is without bacteriophage The tube representing dilution 10~^ (tube 7) is also covered with a culture of dysentery organisms, showing but 2 plaques. The tubes corresponding to the dilutions 10~^, 10~*, 10~^°, and 10~^^ (tubes 8, 9, 10, and 11) are covered with perfectly normal growth. They present an appearance exactly comparable to that found upon the control tube (tube 12), which contained only the bacillary suspension. These cultures may be incubated for any length of time whatever, even up to the point where the medium becomes dried up, without caus- ing any change in their appearance. Cultures which originally were free of growth (tubes 1, 2, and 3) remain bare indefinitely, just as though they were sterile.* Those cultures containing plaques remain, like- * I repeat once more that in the first three chapters of this text I am dealing only with the conditions found in typical bacteriophagy, that is, with what takes THE BACTERIOPHAGE CORPUSCLE 79 wise, as they first appeared ; the plaques once formed undergo no modi- fication, they never increase in size and they are never overgrown by the surrounding bacterial culture. As for the cultures presenting a normal bacterial growth (tubes 8, 9, 10 and 11, and, of course, tube 12), they also undergo no further modi- fication and if they be further examined by subculture it is found that they will yield indefinitely normal cultures of the dysentery bacillus. If we observe the nature of the reaction which takes place in the sus- pensions themselves, that is, the tubes from which the agar slants were implanted we will find that after incubation for 24 hours bacteriophagy is complete in the tubes containing dilutions 10~^ to 10~^. After 48 hours it is also complete in tubes 6 to 10, that is, in those containing dilutions lO"" to 10-^°, while the tube with the 10"^^ dilution remains turbid. This last tube may be subcultured indefinitely with the result that the successive cultures are as normal as are those of the control tube (tube 12). This experiment has been repeated a great many times with bacteria of different species: the staphylococcus, B. pestis, B. typhosus, B. gallinarum, etc., and has always given, from the broad point of view of the principle involved, analogous results. The course of the phenome- non is always the same. The only point subject to variation is the extent of the dilution, and such a quantitative difference is, of course, compatible with what we know of bacteriophagy. For example, in working with another race of bacteriophage, also active against the dysentery bacillus, the last active dilution may be 10"^, and in this case the agar tubes corresponding to dilutions 10~^ and 10"^ will be bare. The agar tube representing the dilution 10~^ will show shreds of growth. The tube representing 10~^ will be covered by a layer of cul- ture studded with about 100 plaques, the one corresponding to 10"^ will present but 12 plaques, and those tubes corresponding to the remain- ing dilutions of the series will be covered with normal cultures of dysentery bacilli. In another experiment carried out with Staphylococcus aureus and with a race of the bacteriophage active for this organism, the last active dilution may be 10-^^ The agar tube corresponding to the dilution place with races of the bacteriophage which are extremely potent, races which cause in a liquid medium a total and permanent dissolution of a normal suspension, the medium remaining limpid indefinitely. If there are any who have not been able to isolate such a race I will gladly send them one upon request. These races will permit them to carry out all of the experiments described in this text. 80 THE BACTERIOPHAGE AND ITS BEHAVIOR 10~^ may be covered by a layer of culture eroded by confluent plaques. The agar slant prepared from the 10~^ dilution will present a layer of staphylococcus growth spotted by about 100 plaques, and the tube corresponding to 10~^ will give a culture where only about 8 plaques can be found. The sole difference appearing in the results of these experiments is that the different bacteriophage filtrates each act up to a different dilu- tion. Aside from this difference in "strength" the aspect of the phe- nomenon is always the same. A further fact revealed by these experi- ments is, and this is of extreme importance, that the number of plaques formed bears, in every experiment, a strict relationship to the quantity of bacteriophage liquid. When there are, for example, 20 plaques on the agar slant corresponding to a given dilution there will be, practically, one-tenth as many plaques on the agar tube corresponding to the dilu- tion which is ten times greater. As we know that the bacteriophage principle reproduces in the course of its action, since the phenomenon is indefinitely reproducible in series, let us take a suspension of dysentery bacilli and inoculate it with an extremely minute quantity of the bacteriophage liquid, for example, with 1 cc. of a 10"'^ dilution of a bacteriophage filtrate. Place this bacterium-bacteriophage mixture in the incubator at 37°C. and from hour to hour throughout the incubation spread one drop upon an agar slant or an agar plate. As the bacteriophage begins to multiply in the course of its action the plaques should increase in number, and in fact, after these agar subcultures have been incubated for 24 hours, we will find the following: The agar tube planted from the mixture, after incubation for but one hour, shows no plaques; it is covered by a normal growth of dysentery bacilh. The agar tube prepared one hour later, that is to say, after the trace of bacteriophage has been in contact with the bacilli for two hours, will show about a dozen plaques. The agar tube seeded after the action has progressed for 3 hours is covered by a bacillary growth through which about 100 plaques are scattered. The appearance of this agar tube is then approximately the same as that which had received a drop of the 10~^ dilution of the preceding experiment. But in the first experiment the suspension was spread upon agar immediately after the inoculation of 10~^ cc. of bacteriophage liquid, while here the 10~'^ cc. of hquid bacteriophage added to the suspension has acted during 3 hours and has, therefore, had time to multiply. The agar tube planted after the suspension had remained for 4 hours THE BACTERIOPHAGE CORPUSCLE 81 in the incubator showed only a few traces of bacterial culture, the appearance resembling the agar tube representing the 10~^ dilution of the preceding experiment. As for the agar tubes upon which a drop of the suspension was spread after the bacteriophage had acted for 5 hours or more, none presented any evidence of growth. They were sterile. In appearance these tubes were just the same as were those of the other experiment where the plantings had been derived from the suspensions inoculated with a large amount of bacteriophage liquid. It seems that the bacteriophage was so abundant that the entire surface of the agar forms but a single plaque, or, to express it more correctly, the plaques were so numerous that they touched one another leaving no room for bacterial growth. In brief, then, to summarize all of this, bacteriophagy in a liquid medium reveals itself macroscopically by a dissolution of the bacteria contained in the medium, the latter becoming as clear as sterile, unin- oculated bouillon. Upon agar bacteriophagy takes place in the same manner. The agar, at the point where the phenomenon occurs, is bare, without any sign of growth, having the same aspect as a sterile agar slant. In this condition it remains indefinitely (d'HereUe^^"-^^^). To mention all the authors who have confirmed these facts would be to enumerate, in fact, almost all who have investigated the phenome- non of bacteriophagy. These facts have not been disputed, although, as we will see, the interpretations of the facts differ very materially. These experiments show that the number of "plaques" on agar is related to the quantity of bacteriophage contained in the fluid, but they suggest, in addition, an hypothesis of extreme interest, and one worthy of test for it is burdened with matters of the greatest concern. We have seen that when a very great number of bacteria and a very small quantity of the bacteriophage principle are combined and spread upon agar immediately after the inoculation of the bacteriophage, the result, after incubation, is a culture formed by the development of the bacteria distributed upon the agar, and this is spotted with bare areas, or plaques. The small amount of bacteriophage present appears, then, to concentrate its action at particular points (d'Herelle^^°). Can the physical state of the bacteriophage be "discontinuous"? Can this principle exist, in what up to this time we have called a bacteriophage liquid or a bacteriophage filtrate, in particulate form, as corpuscles in suspension? 82 THE BACTEEIOPHAGE AND ITS BEHAVIOR 2. THE BACTERIOPHAGE CORPUSCLE The hypothesis which I have formulated in answer to the above ques- tions is easy to verify. The experiment described in the preceding section showed that bacteriophagy occurred in the bacillary suspension containing 10~^*' cc. of bacteriophage filtrate, while the phenomenon failed to take place in the suspension with the next higher dilution, 10-11. If the bacteriophage exists in corpuscles this result is at once explained. What happens would then be exactly of the same nature as that which takes place when tubes of sterile bouillon are planted with serial dilutions, ever more and more dilute, of a bacterial culture. The tube of bouillon which receives one drop of a sufficiently high dilution will be implanted with but a single bacterium, but it will yield, after incubation, a culture as abundant as that of another tube seeded with several million of the same bacteria. On the other hand, the tube which receives one drop of the next dilution, will not yield a culture, for the drop introduced did not contain even a single bacterium. Either there will be growth, or there will not be growth. And, in the same way, bacteriophagy either occurs, or it does not occur. There are no inter- mediate gradations. Let us find out if this is the case; if experiment confirms theory. Prepare again the same series of dilutions, in tens, as those described in the experiment of the preceding section, employing the same bacterio- phage filtrate, but instead of making the successive dilutions in suspen- sions of B. dysenteriae, make them in sterile bouillon. The procedure consists simply in introducing 1 cc. of bacteriophage filtrate into 9 cc. of sterile bouillon, removing 1 cc. of this first dilution and placing it into a second tube containing 9 cc, then 1 cc. of this second dilution carried over into the next tube, and so on up to the tenth dilution. This last tube will then contain 10 cc. of bouillon in which there will be 10"^ cc. of bacteriophage filtrate, and each cubic centimeter of this tenth dilution will therefore contain lO-^" cc. of the filtrate. Suspend in a flask containing 90 cc. of sterile bouillon some B. dysen- teriae removed from a young agar slant, in such a way that each cubic centimeter of the medium will contain about 100 million bacilli. Dis- tribute this 90 cc. of bacterial suspension among 10 tubes, 9 cc. to each. To each tube add 1 cc. of the bacteriophage principle previously diluted to 10"i°. We now have 10 tubes of suspension, and in each of them an added 1 cc. of a 10-i° dilution of the bacteriophage filtrate. Place the 10 tubes in the incubator at 37°C. THE BACTERIOPHAGE CORPUSCLE 83 After 48 hours, 3 of these suspensions are clear (this is the average of 7 experiments of 10 tubes each, made with the same bacteriophage fil- trate). The other 7 are turbid, and repeated control tests show that they contain normal cultures of dysentery bacilh. The result is, then, complete bacteriophagy in 3 suspensions and complete absence of bac- teriophagy in the other 7 (d'Herelle^^^). This experiment settles the question. If the bacteriophage was to be found in a state of solution in the 10~i° dilution, it is obvious that each of the 10 cc. of this dilution would have contained a tenth part of it, that is, none of the 10 cc. would have been favored. Each would have contained a like quantity, and all of the 10 suspensions, each receiv- ing one of these 10 cc, would have behaved in the same manner; they would have been the seat of a comparable phenomenon. But this is not the case, as shown by the fact that 3 suspensions undergo bacteriophagy, while 7 do not. There is, then, among the 10 cc. dis- tributed in equal amounts among the ten suspensions, 3 portions of 1 cc. each which contained the bacteriophage principle. In the other 7 it was lacking. This is an absolute proof that the bacteriophage exists in discontinuous form, that is to say, in corpuscular form.* Experiments of this type always yield the same result, regardless of the bacterial species involved, provided the bacteriophage principle be very active. The single difference that may appear among the differ- ent experiments is due solely to the fact that different filtrates do not all contain the same number of bacteriophage corpuscles per cubic centimeter. Sometimes the dilution will be 10~^, sometimes 10"^ often 10-^ or 10-^", and rarely 10~i\ which, when distributed in equal portions among a number of bacterial suspensions, will provoke bac- teriophagy in a certain number of these suspensions, leaving others untouched. Inasmuch as the concept of the corpuscular nature of the bacterio- phage dominates entirely the study of this principle, I believe it wise to introduce the protocols of a few other experiments, taken at random from among more than fifty which I have performed, and which, uni- formly, have given the same results as regards the demonstration of the corpuscular state. * During my residence at the University of Leiden, in discussing this question with my colleague, Professor Einstein, he told me that, as a physicist, he would consider this experiment as demonstrating the discontinuity of the bacteriophage. I was very glad to see how this deservedly-famous mathematician evaluated my experimental demonstration, for I do not believe that there are a great many biological experiments whose nature satisfies a mathematician. 84 THE BACTERIOPHAGE AND ITS BEHAVIOR The following protocol deals also with B. dysenteriae, but with another race of the bacteriophage. It has already been published^^^ in reply to the communications of several authors, Bordet among others, who have affirmed that the bacteriophage principle exists in a soluble form, despite the proof of corpuscular nature which I had given from the time of my first publication''^'' and which has been re-stated at different times. Incidentally, none of these authors have discussed this experiment; they have all passed it over in silence. In an effort to make the matter clear, and to settle the point at issue I have presented the data several times, and have even offered, in case any doubt remained, to actually demonstrate the phenomenon. No one accepted the challenge, indeed, no one has since even mentioned the question. In this experiment, 10 cc. of a 10~^*' dilution of a bacteriophage prin- ciple active for B. dysenteriae were distributed, 1 cc. to each tube, into 10 supensions of dysentery bacilli. In 5 of these suspensions bacterio- phagy was complete; in the other 5 the phenomenon did not take place; they remained cloudy. Additional experiments of the same nature, carried out with a princi- ple extremely active for the Staphylococcus aureus follow. Ten cubic centimeters of a 10"^" dilution of this bacteriophage prin- ciple were divided, 1 cc. to each tube, among 10 tubes each containing a suspension of 100 million staphylococci per cc. After 72 hours of incubation at 32°C. all 10 suspensions were clear. Ten cc. of the 10~^^ dilution of the same principle were Ukewise distributed among 10 tubes of the same staphylococcus suspension. After 72 hours at 32°, 2 were clear, 8 were turbid. In the same way, 10 cc. of the 10~^- dilution were distributed among 10 tubes of the suspension. After 72 hours all were turbid. Each of the two dissolved suspensions, obtained by adding the material of the 10^^^ dilution, was in turn diluted to 10~^'. The 10 cc. of each of these two dilutions were distributed into 10 tubes of staphylo- coccus suspension. After 72 hours incubation at 32°C., of the 10 suspensions to each of which was added 1 cc. of one of the dilutions, 1 was clear, 9 were turbid; for the second dilution, 2 were clear, 8 were cloudy. All of the suspensions which remained turbid were subjected to numerous control examinations and all yielded normal cultures of the staphylococcus. These turbid suspensions have been filtered serially, and in no case did the filtrates cause the shghtest reaction which could be ascribed to bacteriophagy. THE BACTERIOPHAGE CORPUSCLE 85 Identical experiments have been performed with bacteriophage principles active for B. coli, for B. typhosus, and for B. pestis* With B. coli, the last active dilution, for the race under investigation was 10-^ This dilution divided among 10 tubes of suspension caused bacteriophagy in 8, the other 2 remaining unattacked. And, finally, the dilution 10"^ of the bacteriophage principle acting upon B. pestis, distributed among 10 suspensions of this organism caused bacteriophagy in 5, the other 5 remaining turbid. Control tests showed that the turbid tubes did not contain a trace of the bacteriophage principle, even after repeated filtrations. We have then, a definite proof that the bacteriophage exists in the form of corpuscles. If further proof of the corpuscular nature of the bacteriophage is desirable, it has certainly been provided by the very beautiful demon- stration presented by Eijkman at the meeting of the Society of Bacteriol- ogists of Holland. His experiment was based upon the well recognized fact that if a drop of a liquid, which contains a substance in solution, is allowed to evaporate slowly, when it is completely dry the substance present will be found evenly distributed over the entire surface previ- ously occupied by the drop; while, on the contrary, if the substance in the liquid is insoluble, in the form of corpuscles, as the drying proceeds the phenomenon of capillarity becomes operative and the corpuscles are attracted toward the periphery, so that when the evaporation is finished, the substance will be found in a circle indicating the circum- ference of the area previously occupied by the drop. Eijkman placed on an agar plate a very dilute suspension of bacterio- phage corpuscles, and allowed the fluid to evaporate slowly. When desiccation was finished he covered the surface of the plate with a sus- pension of the susceptible bacterium. After incubation, the plate revealed an appropriate number of plaques, and these were arranged in a circle, representing the contour of the drop of fluid which had been originally placed on the agar. Thus, making application of the phe- nomenon of capillarity, Eijkman has afforded further proof of the cor- puscular nature of the active principle in a bacteriophage filtrate. From the fact of the corpuscular nature, it becomes evident that the important thing permitting bacteriophagy to take place is not the con- centration of the bacteriophage principle in the suspension, but the * The bacteriophage filtrates utilized in these experiments were from suspen- sions of 200 million bacteria per cubic centimeter rendered limpid by bacteri- ophagy. 86 THE BACTERIOPHAGE AND ITS BEHAVIOR extent of the dilution of the bacteriophage principle which is inoculated into the suspension. In other words, with a Staphylo-bacteriophage still causing bacteriophagy in the tube diluted to 10"^^ (the dilutions being made by tens in volumes of 10 cc.) and not causing bacteriophagy in the tube with 10~^- dilution, it is not because this last tube has a dilu- tion of 10~^2 that bacteriophagy does not result, but simply because it has not received a single bacteriophage corpuscle. To further emphasize this point, let us take, as did Gratia and deKruif"" a liter of a staphylococcus suspension* and let us inoculate it with 1 cc. of a 10^^*' dilution of Staphylo-bacteriophage. The dilu- tion of the principle in this liter of suspension will be 10~^^, yet bacterio- phagy occurs. The reason is plain. We know that 1 cc, of the 10~^° dilution causes bacteriophagy when added to 9 cc. of bacterial suspen- sion, clearly showing that it contains at least one bacteriophage cor- puscle. And it makes no difference whether this corpuscle is inoculated into 10 cc. or into 10 liters of suspension. The result is the same, the principle multiphes and causes bacteriophagy. In brief, then, the concentration of the bacteriophage principle in a suspension where bacteriophagy is to take place is of little importance. For the phenomenon to occur the necessary and sufficient condition is that at least one bacteriophage corpuscle be introduced into the suspen- sion of organisms. In conclusion, let me repeat just once more, that to be conclusively demonstrable, these experiments must be made with a very active bacteriophage principle, that is, with one capable of causing a complete and permanent dissolution of the susceptible bacterium when 250 mil- Hon bacteria per cubic centimeter are present.! * These authors performed the experiment with a Coli-bacteriophage. I have repeated it with a Staphylo-bacteriophage, and the results have been identical in principle. They could not be otherwise. These authors stated that their experiment was open to several interpretations. This rather vague conclusion appears to have satisfied them, for the suggested interpretations have not yet appeared. As a matter of fact but a single interpretation is possible. The bacteriophage exists in the form of corpuscles. The experiment of Gratia agrees with experiments that I had published on several occasions, and this is all that Gratia could have said. t In studying the phenomena of the resistance of the bacteria to the bacterio- phage we will see the reasons why the experiment demonstrating the corpuscular form must be carried out with an extremely active bacteriophage principle. THE BACTERIOPHAGE CORPUSCLE 87 3. THE plaque: a colony of bacteriophage corpuscles Each plaque, scattered throughout the bacterial layer upon agar, represents, then, the point where, during the spreading of the suspension, a bacteriophage corpuscle was deposited. This being the case, the number of plaques must be strictly proportional to the number of cor- puscles inoculated into the bacterial suspension. This is what the experiments presented in the first section of this chapter have already shown. The number of plaques, on the other hand, should be entirely independent of the number of bacteria in the suspension. This is shown by the following experiment (d'Herelle^^^). Ten tubes, each containing 10 cc. of a suspension of Shiga dysentery bacilH in different concentrations— 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 million bacilli per cc, — are inoculated with a constant quantity of a bacteriophage filtrate, 5 X 10"". After shaking, 0.02 cc. is removed from each of the tubes and is carefully spread upon a corresponding agar slant. After incubation, each of the 10 slants shows a culture layer of bacteria, studded with plaques, and the number of plaques is practically the same in all of the tubes (the actual figures being— 19, 25, 20, 21, 19, 19, 22, 20, 16, and 18). Let us repeat this experiment, reversing the order of the factors. Let us use 10 suspensions of Shiga bacilli, all of the same concentration, 200 milKon bacilli per cubic centimeter, and let us inoculate these suspen- sions with an increasing quantity of a dilution of bacteriophage filtrate, in such a way that the first tube will receive a millionth of a cc, the second a 900 thousandth, the third an 800 thousandth, the fourth a 700 thousandth, and so on up to the tenth tube, which will get a 100 thousandth. Shake the tubes vigorously, and spread 0.02 cc from each upon an agar slant. After incubation each of the agar slants has a layer of bacterial growth studded with plaques, but the number of the plaques varies with the quantity of bacteriophage filtrate inoculated into the tubes. Practically the proportions are: 1:10, 1:9, 1:8, 1:7, etc., up to 0,5:1; the actual numbers observed being, 4, 4, 5, 6, 8, 10, 10, 14, 19, 42.* I have repeated this experiment with different races of the bacterio- * The differences between the numbers observed and the numbers theoretically calculated are of the same magnitude as those which we find in counting bacterial colonies plated on agar from serial dilutions of a suspension. The numbers ob- served approach more and more closely to the calculated numbers as the number of tubes counted increases and average figures are obtained. 88 THE BACTERIOPHAGE AND ITS BEHAVIOR phage, acting upon different bacterial species. It is needless to detail them here, for they are all comparable to the one described above. The experiments all agree in showing that each plaque originates in a bacteriophage corpuscle which dissolves the bacteria in its environ- ment. But this plaque occupies a certain area. With certain races particularly active for the dysentery bacillus each plaque may attain a diameter of 8 mm., all of the bacteria found within a radius of 4 mm. of the corpuscle being destroyed. Is this dissolution the result of a distant action of the corpuscle deposited on the agar at the time of spreading, or, does the original corpuscle multiply at the expense of the bacterial bodies as it proceeds with their dissolution? It is easy to sub- ject this question to experimental proof and determine which of the two possibilities accords with the facts. Touch the margin of a plaque with a platinum wire and then wash it off in a tube of sterile bouillon. Prepare also 10 tubes of a bacterial suspension susceptible to the action of the bacteriophage. Inoculate each of these tubes with a drop of the bouillon in which the needle has been washed off. After incubation, we will find that the 10 suspensions have undergone bacteriophagy. There must have been, then, at least one bacteriophage corpuscle in each drop of the bouillon with which these suspensions were inoculated. This proves, therefore, that the surface of the plaque is covered by bacteriophage corpuscles, and this, in turn, means that the initial corpuscle must have multiplied. The plaque represents, then, a colony of bacteriophage corpuscles, the issue of the original corpuscle which was the origin of the plaque. Upon a solid medium, as in a liquid medium, the dissolution of the bac- teria is accompanied by a multipHcation of the bacteriophage corpuscles. 4. CONDITIONS ESSENTIAL FOR PLAQUE FORMATION Before considering the characteristics of agar cultures of the bacterio- phage corpuscles, let us note the conditions of the medium most favor- able for their development. First, let us state, but it is not necessary to emphasize this, that all of the conditions, as discussed in the preceding Chapter which bear upon the nutritive quahties of liquid media for the bacteria subjected to the action of the bacteriophage, such as the reaction (pH), are entirely applicable to agar media. On solid media, as in liquid media, the state of the bacteria is important. Young bacteria are most readily attacked, and the critical period is the moment of division. With regard to the consistency of the medium, I have shown^^i ^^^t THE BACTERIOPHAGE CORPUSCLE 89 Martin's alkaline bouillon (pH 7.6 to 7.8) containing 2 per cent of agar serves perfectly well for the formation of plaques. More recently Nakamura^'^ has studied comparatively the formation of plaques upon media containing different concentrations of agar. He found that the plaques were the larger as the concentration of agar was reduced. The most favorable medium was made up of bouillon containing 0.4 per cent of agar. Practically, in view of the semi-fluid state of such a medium which makes it rather difficult to work with, a medium composed of bouillon with 0.8 to 1 per cent of agar is the most convenient. What is the cause of this reduction in the size of the plaque propor- tionate to the increase in the concentration of agar? Certainly it can not be, as Nakamura has suggested, a result of the inhibitory action of the agar colloid, for if this were the case it would hardly be clear why a medium containing 1 per cent would be very favorable, since the quan- tity of agar colloid here is sufficient to give the medium the character of a solid. Furthermore the fact that bacteriophagy takes place in a liquid medium in the presence of negative colloids, such as colloidal silver, and even in the presence of gelatin up to a certain concentration, shows indeed that this is not the real reason. The stronger and stronger inhibition of bacteriophagy in media con- taining increasing quantities of agar is certainly related to the con- sistency of the substratum. Products resulting from the dissolution of the bacteria exercise an inhibiting action upon the phenomenon, as is shown by the arrest of bacteriophagy in a liquid medium when a certain number of bacterial cells have been dissolved. Furthermore, it seems to be a general bio- logical law that the accumulation of the products formed during a reaction inhibits a biological process. Such products not only limit the growth of organisms, but also impair the action of ferments. What- ever may be the nature of the bacteriophage corpuscle, an inhibition of the action which it causes, brought about through the accumulation in the medium of the products resulting from this action, is, then, in con- formity with all that is known. It might even be said that if such an inhibition did not occur it would be unique, a new fact. From this it is clear that the higher the amount of agar in the sub- stratum the greater is its consistency, and consequently, the slower will be the diffusion into the substratum of the products formed during the dissolution of the bacterial cells. If the consistency is such that these products accumulate in the surface layer of the medium, there is, from the beginning, an arrest of the phenomenon, and, as a result, a reduction in the area of the plaque. Even its formation may be prevented. 90 THE BACTERIOPHAGE AND ITS BEHAVIOR The following experiments permit of no doubt on this question. Bail/^ Doerr and Berger/^^ and Nakamura''^^ have observed that plaques do not form on gelatin, and they have invoked the intervention of colloidal reactions to explain this fact. The deduction of these authors is absolutely correct, and the following experiments provide the true explanation for the inhibition which they observed. Prepare three series of Petri dishes, as follows : (a) containing an agar medium (bouillon with 2 per cent agar) ; (6) containing a gelatin medium (bouillon with 15 per cent gelatin; (c) containing agar (the 2 per cent agar of (a)) to a depth of 8 mm. Immediately after the introduction of the agar the dishes of this series are placed in a horizontal position (obtained by means of levelling screws) and the agar is allowed to solidify. They are then placed in an incubator at 45°C. to warm them somewhat, and over the surface of the agar is poured a quantity of gelatin (the 15 per cent gelatin mentioned above, in (6)), melted and cooled to about 60°C., in such a way that by tipping the dish in all directions the entire surface is covered by a thin layer. Adjust the dishes with the levelling device so that they are perfectly horizontal and allow the medium to harden in the ice-box. Prepared in this way these dishes will con- tain a layer of agar upon which is superimposed a thin layer of gela- tin. When planted the material is distributed solely upon the gelatin, and this medium will differ in no way from that present in the dishes containing gelatin alone (series b, above). The only difference will be that the c series will have a substratum of agar into which those pro- ducts resulting from reactions taking place on the surface may diffuse. When these Petri dishes are thus ready, take 4 tubes, each containing a suspension of Shiga bacilh, 250 million per cubic centimeter. Inocu- late the first with 0.1 cc. of a Shiga-bacteriophage; the second with 0.1 cc. of the first: the third with 0.1 cc. of suspension 2; and the fourth with 0.1 cc. of number 3. With these four suspensions the Petri dishes are implanted, 0.05 cc. of each of the individual suspensions being spread uniformly over the surface of a dish. When the results are read after 4 days, we find : I. Agar plates held at 37°C. Suspension 1 . . . Surface sterile Suspension 2. . . A few scattered traces of growth Suspension 3 . . . Fragments of growth Suspension 4. . .About 50 plaques, having a diameter of about 5 mm. THE BACTERIOPHAGE CORPUSCLE 91 //. Agar plates held at 30°C. Suspension 1 . . . Sterile Suspension 2. . .Traces of growth Suspension 3. . .Fragments of growth Suspension 4. . .About 50 plaques, having a diameter of about 7 mm. III. Agar plates held at 18°C. Suspension 1 . . . Sterile Suspension 2. . .Sterile Suspension 3. . .Scattered traces of growth Suspension 4. . .Confluent plaques (about 50), with a diameter of about 13 IV. Gelatin plates held at 18°C. Suspension 1 . . . Continuous bacterial layer, absolutely like a normal culture Suspension 2. . .Continuous bacterial layer Suspension 3. . .Continuous bacterial layer Suspension 4. . .Continuous bacterial layer V. Gelatin plates with a substratum of agar Suspension 1 . . . Sterile Suspension 2. . .Sterile Suspension 3. . .Culture debris Suspension 4. . .About 50 plaques, with diameters of 3 to 4 mm. This experiment, repeated upon three different occasions, has ahvays given similar results. By varying the depth of the gelatin layer I have observed that the size of the plaques varies inversely with the depth of the layer. When the layer was about 3 mm. in depth the bacterial culture appeared normal. Obviously it is not the gelatin as such which interferes with the process of bacteriophagy. It is simply because the gelatin is but slightly permeable to the products arising in the course of the phenom- enon that an inhibitory effect is manifested. When the layer is very thin and is superimposed upon a permeable substratum bacteri- ophagy occurs just as it does on agar. The experiment also shows some points with regard to the effect of temperature. The dimensions of the plaque resulting, one might say, from a ''race" between the rate of multiplication of the bacterium and that of the bacteriophage, it follows that at a given temperature the one or the other may be ''handicapped." On the other hand, experiment definitely 92 THE BACTERIOPHAGE AND ITS BEHAVIOR shows that the bacteriophage corpuscle is able, on a solid medium, to dissolve bacteria only if they are found in an extremely thin layer on the surface of the substratum. This is precisely the reason that the plaque, already formed when the bacterial growth has hardly become perceptible, no longer increases in size; the surrounding bacterial layer has become too thick. To summarize all of this, the formation of the plaque, and its extent if its formation is possible, depends upon a whole series of factors, of which two appear to be ; first, the greater or less facility with which the products resulting from the action diffuse into the substratum, and second, the respective powers of multiplication of the bacteriophage corpuscle and of the bacterium at the temperature at which the phe- nomenon takes place. 5. THE CHARACTERS OF PLAQUES It is of interest to note further some of the characters of the colonies of the bacteriophage corpuscle on agar. Let us take, as an example, a very active race of Shiga-bacteriophage, although any other race could be taken, acting upon any bacterial species whatever, for in all cases, the formation and the behavior of the plaques are identical. The only variant is the extent; the diameter of the plaque. Observation shows that in general, the size of the plaques formed with different bacterial species is the smaller the more rapidly the bacterium grows upon agar and the thicker the layer of growth becomes. Even with extremely active races of the Staphylo-bacteriophage the plaques are always small ; their diameter (for the races studied) does not exceed 1.5 mm. The plaque of the Shiga-bacteriophage, on the contrary, may reach a diameter of 8 mm. But even here uniformity does not obtain, for against a single bacterial species the size of the plaque varies. With all other conditions the same, the size of the plaque is related to the race of the bacteriophage which is acting. We will see the cause for this in the next chapter. When the surface of the agar remains bare because of the large num- ber of bacteriophagous corpuscles and maintains this appearance indefinitely it has become unsuited for the cultivation of the Shiga bacillus. When inoculated at such a time with a culture of this bacillus, even in a very abundant sowing, not the slightest development can be detected. The medium is, however, normal for another bacterium. If inoculated with the cholera vibrio, for example, the growth will be as luxuriant as if planted upon fresh medium. Hence, if B. dysenteriae THE BACTERIOPHAGE CORPUSCLE 93 does not grow it is only because the bacteriophagous corpuscles remain on the surface of the agar and exercise their dissolving action on the bacteria deposited thereon. This is readily confirmed. If we take a tube of agar which has remained apparently sterile after having been inoculated with a suspension of the bacteria containing a bacterio- phagous filtrate, and if the surface of the medium in such a tube is washed with a few drops of sterile bouillon and to this is added a fresh suspension of bacteria, this suspension will be dissolved within a few hours. It sometimes happens, especially when using agar somewhat dried out, that a few colonies of Shiga are obtained, always located at the extreme edge of the layer of agar. We will return to this extremely interesting particular in the discussion of secondary cultures. If, instead of spreading the bacteriophage filtrate over the entire surface the corpuscles are deposited in Kmited areas — and this is readily accomphshed by placing drops of filtrate on the sterile surface of a tube of agar, or again, by drawing lines over the surface with a platinum loop dipped in the suspension of bacteriophage, and after the tubes have remained inclined for a few hours in the incubator to secure drying — ■ we find that the areas impregnated with the bacteriophagous filtrate remain free of Shiga bacilh, but that these organisms grow, on the con- trary, perfectly well on the parts not covered by the bacteriophage. When in the suspension planted upon agar the number of bacilli is infinitely great and the number of the corpuscles is sufficiently small, the bacteriophage principle as individual units is distributed over the surface of the agar, and under such circumstances the bacterial layer will appear studded with apparently sterile areas. These areas, or plaques, have a circular form varying in size from those spoken of as "pin-point" up to those with a diameter of 8 mm. The plaques are in general of the greatest extent when the bacterial suspension is somewhat weak although sufficiently concentrated to give a continuous layer of growth rather than isolated colonies. On such a tube the areas are larger as the subjacent medium becomes thicker, that is, toward the bottom of the tube. Upon a Petri dish, where the agar layer is of essen- tially the same thickness throughout, all of the plaques of a given cul- ture are of approxunately the same diameter. As will be seen, the area of the plaque bears a relationship to the virulence of the bacteriophage which causes it. If a tube or plate presenting plaques is held in the incubator at 37°C., or at an entnely different temperature, no change occurs in the plaques; 94 THE BACTERIOPHAGE AND ITS BEHAVIOR their diameter remains indefinitely what it was at first. They are never covered or encroached upon by the bacterial culture. At no time does there exist within the extent of the plaque, whatever its size may be, microscopically visible bacterial cells. The plaque is always rigorously sterile. As soon as the culture is well developed, as after 18 to 24 hours of incubation, if the centre of such a plaque is touched with a platinum wire and this is immersed in a culture of Shiga bacilli the bacteriophage develops in this suspension and the latter is dissolved after a few hours. The plaque, although sterile, is not ultrasterile; it is in fact a colony of the bacteriophage corpuscles. Furthermore, if a trace of the bacillary growth at the periphery of a plaque is taken with a platinum wire and seeded on agar it remains sterile and inoculation into a bacterial culture shows that the bacterio- phage is present there also. But when the bacillary layer is taken, not at the immediate edge of the area, but at a distance of two milli- meters from it, for example, and planted, the tubes show the growth of a normal culture. The bacteriophage is not found. If the culture showing the plaques is returned to the incubator and the tests are repeated three or four days later, that is, culturing the bacillary growth at a distance of two millimeters from a plaque onto agar and into a suspension it will be found that the bacteriophage is there present at that time. The bacteriophage has, therefore, gradually invaded the bacillary layer. This invasion is always slow — ^proceeding more and more slowly as time progresses — so that the ring invaded, even after several months, amounts to a zone but a few milUmeters wide. Beyond the hmits of this zone the Shiga organisms remain cultivable just as long as they do in a normal control culture without the bacterio- phage. The question immediately arises as to why the bacteriophage does not invade the entire layer of bacterial growth. For this there are two reasons. The bacteriophage attacks the bacterial cell most readily when the bacterium is young. When placed upon agar the bacterio- phagous corpuscles find themselves located in the immediate vicinity of bacilli which reproduce actively as soon as they are deposited upon a nutrient medium. They find then, within their range, very young bacilh distributed in a very thin layer over the agar. Dissolution is thus possible and the apparent sterihty of the plaque results. But beyond this zone invaded by the bacteriophage during the first few hours the bacilh develop freely forming a layer of increasing thickness THE BACTERIOPHAGE CORPUSCLE 95 comprised of organisms of increasing age. In other words, a thicker and thicker layer of bacilh always becoming more and more resistant to dissolution develops. This can be readily demonstrated by direct experimental proof. If the agar surface in a Petri dish is heavily seeded with a Shiga culture and at some point on this a drop of the bacteriophage filtrate is placed, and after a three-hour incubation period another drop of the bacteriophage is placed on the surface and this same process repeated after six, twelve and twenty hours, with continuous incubation of the plate during the intervals, it wiU be found fifteen hours later that the areas upon which the first three drops were placed have remained ster- ile — no bacillary growth has taken place. At the point where the fourth drop was placed, that is, after the culture had been incubated for twelve hours, there is a thin layer of growth composed of dead bacilli. The area where the drop of bacteriophage was placed after twenty hours presents an appearance practically normal. These five spots, then, represent the diverse aspects of an isolated colony of the bacteriophage, as from the centre to the periphery. The second reason is of a more general nature, representing a pheno- menon common to the majority of cultivable organisms. The colonies of the bacteriophage act absolutely Hke colonies of those bacteria which, except for organisms such as B. proteus, never progressively invade the surface of sohd media. Thus, if the Shiga bacillus is planted upon agar in an amount suitable to yield isolated colonies, after 18 to 24 hours, each colony will be from two to four miUimeters in diameter, the largest colonies to be found at the points where the medium has the greatest depth, that is, toward the bottom of the tube. Such colonies increase in size but very slowly, always more and more slowly as time progresses, and even after two months, the zone of increase will not be greater than a few millimeters. From the bacteriological point of view it is not peculiar, as has been suggested, that the bacteriophage does not invade the entire bacterial layer. Far from being dissimilar to other cultivable organisms, an isolated colony of the bacteriophage behaves exactly like a colony of bacteria. Why does the bacterial colony fail to increase in size and invade the entire surface of the medium? Because the soluble substances result- ing from the vital activity of the bacteria diffuse into the agar and these substances constitute a true specific antiseptic which limits the growth. The medium is "vaccinated" around the colony. The deeper the agar layer, or the farther the colonies are separated, the greater the volume 96 THE BACTERIOPHAGE AND ITS BEHAVIOR of substratum capable of diluting this antiseptic substance, and the larger will be the colony. The situation is precisely the same with the bacteriophage ; the more scattered the colonies and the deeper the sub- stratum, the greater the diameter. This is also the reason why the plaques are larger, other conditions being equal, when the medium contains less agar. We know that dif- fusibility in an agar substratum is diminished as the percentage of agar in the medium is increased. 6. ENUMERATION OF BACTERIOPHAGE CORPUSCLES* Since we have now presented the evidence proving the corpuscular nature of the bacteriophage we will no longer make use of such vague expressions as bacteriophage "liquid," ''fluid," or "filtrate," but will employ the more precise term "suspension of bacteriophage corpuscles," or even more simply, "bacteriophage suspension." A bacterial sus- pension which has become limpid because the bacteria have disappeared through bacteriophagy, and in which, on the other hand, the bacterio- phage corpuscles have multiplied from the beginning, has, then, become a "bacteriophage corpuscle suspension." The evidence adduced above also shows that each plaque represents a colony of bacteriophage corpusclesf and experiment shows that this colony originates in a single corpuscle deposited on the agar in the midst of the bacteria. Obviously, this finding provides a method for the enumeration of the bacteriophage corpuscles to be found in a suspension (d'Herelle^^'^'^^^'^-^). The experiments described in a preceding section suggest, moreover, a second method. Since the hmiting dilution of a suspension of bac- teriophage distributed in equal fractions in bacterial suspensions, causes bacteriophagy in a certain number of them, the others remaining unat- tacked, it follows that each bacteriophaged suspension must have been * We are considering here only the question of the enumeration of the corpus- cles, not that which might be called the "titration" of the bacteriophage; a question much more complex, the study of which is reserved for a later chapter. t No concept of the nature of the corpuscle is here involved. This is a question which we will approach when, having accomplished the study of bacteriophagy, we will consider the characters of this corpuscle. The word "colony" is em- ployed because it signifies a "collection of individuals of the same type." As there is on agar a collection of corpuscles of the same type, having for their origin a corpuscle which has multiplied, it is evidently a colony, without predicating whether the corpuscles are living or not. THE BACTERIOPHAGE CORPUSCLE 97 inoculated with a single corpuscle (d'Herelle^^°'^^^'^2i)_ j^ simple calcu- lation gives then the number of corpuscles to each cubic centimeter of the suspension. And these two methods of counting should agree with each other, yielding comparable results. Applied to a specific case these two methods give the following values. A suspension of the Shiga-bacteriophage is titrated by successive dilu- tions, as in the experiments described previously. The last active dilution is found to be 10"^". Of this 10~^° dilution 10 cc. are distrib- uted, in amounts of 1 cc. each, into 10 suspensions of Shiga bacilli. After incubation, we find that 5 of these suspensions have undergone bacteriophagy, the other 5 have not. There were then, 5 bacteriophage corpuscles in the 10 cc. of the 10"^*' dilution. These 5 corpuscles must have been introduced into this 10~^° dilution with the cubic centimeter of the 10~^ dilution which was com- bined with 9 cc. of bouillon to yield the 10"^° dilution. Manifestly, the 10 cc. of the 10~^ dilution contained 50 corpuscles. Continuing the same reasoning for the successive decreasing dilutions to the point of the undiluted suspension, we find that the latter contained 5,000 mil- lion bacteriophage corpuscles per cubic centimeter.* Let us now take the 10"" dilution of the series of dilutions above. Inoculate a normal suspension of dysentery bacilH with 1 cc. of this dilution. The suspension will then contain the same quantitiy of bacteriophage as the 10~^ dilution of the original bacteriophage. Spread immediately, on 20 agar tubes, one drop (0.05 cc.) to each tube, 1 cc. of the inoculated suspension. After incubation we find that each agar * More simply, the 10~' dilution contained 5 corpuscles per cubic centimeter. The initial suspension undiluted contained then 5 X 10', or 5000 million per cubic centimeter. The method for counting is the same, indeed, as that which was devised by Miquel for counting bacteriji and this in turn was patterned after the procedure followed by Pasteur for the purification of bacterial cultures (the dilution method). Insofar as the bacteria are concerned, every tube of the medium which receives at least one bacterium gives a culture and every tube which does not receive one remains sterile. With the bacteriopha§,e every bacterial suspension which receives at least one corpuscle undergoes bacteriophagy while the sus- pensions which receive none fail to reveal the phenomenon. Obviously it is possible that the tubes which show a growth in the case of the bacterial counts or those which show bacteriophagy in the case of bacteriophage corpuscle counts may not have received a single element but, indeed, some of them may have received two or even more. The result obtained is, therefore, a mini- mum, a minimum which approaches more closely to the true number as the count is based upon a large number of tubes. 98 THE BACTERIOPHAGE AND ITS BEHAVIOR tube is covered by a growth of B. dysenteriae spotted with plaques. The total number of plaques is 478, distributed among the tubes in the following way : 1 slant shows 19 plaques = 19 3 slants show 20 plaques = 60 3 slants show 21 plaques = 63 4 slants show 23 plaques = 92 1 slant shows 24 plaques = 24 1 slant shows 25 plaques = 25 1 slant shows 26 plaques = 26 4 slants show 27 plaques = 108 1 slant shows 29 plaques = 29 1 slant shows 32 plaques = 32 Total = 478 The 478 ''formers of plaques" were found in 1 cc. of the lO"^ dilution. Calculation indicates that the initial suspension of the bacteriophage contained 478 X 10^ or 4.78 X 10^ The second procedure, therefore, shows that each cubic centimeter of the undiluted suspension contained 4780 milHons of "plaque formers," whereas the dilution method indicated that this suspension contained 5000 milUon corpuscles per cubic centimeter. To all intents and purposes these two figures agree, hence we can conclude that each plaque had its origin in a single bacteriophage corpuscle and that the method of count- ing the plaques forms a means of enumeration of the bacteriophage cor- puscles present in a suspension. I have repeated this experiment with two different races of Staphylo- bacteriophage. It is unnecessary to give the protocols of these experi- ments, since it would prolong this section needlessly, but both of them were comparable throughout with that which has just been described. I will only say that the undiluted suspension (that is, a suspension of staphylococci, 250 milHon per cubic centimeter, bacteriophaged and thus transformed into a suspension of bacteriophage corpuscles) of the more powerful race contained 200,000 million (2 X 10^^) corpuscles per cubic centimeter according to the dilution method and 121,000 million (1.21 X 10^0 according to the plaque method. For the second race the number of corpuscles was 6 X 10» by the first method and 8.1 X 10» by the second.* * It is quite incorrect to assume that all suspensions of the same race of bac- teriophage always contain the same number of corpuscles, indeed, the case is quite the contrary. The number of corpuscles present after bacteriophagy depends, as we will see in the following chapter, upon the number of bacteria bacterio- phaged. The above experiments are given simply to show the agreement between the two methods of enumerating the corpuscles. THE BACTEEIOPHAGE CORPUSCLE 99 In general, these two methods for the enumeration of bacteriophage corpuscles do not differ materially from those employed for counting the living bacteria present in a culture. The single difference resides in the fact that, for the bacteria the dilutions are planted in sterile bouillon or upon sterile agar, while for the bacteriophage corpuscles the counts can only be made in the presence of living bacterial cells. It could not be otherwise, for although the bacterium utihzes for its development the nutritive substances present in the medium itself, the bacteriophage corpuscle multipUes only at the expense of the living bacterium, which constitutes the medium within which it multipKes. RESUME When a suspension of susceptible bacteria, inoculated with a rela- tively large quantity of bacteriophage, is spread upon an agar medium no further growth results: the medium remains bare indefinitely. Furthermore, the surface of this agar remains permanently unsuited to the development of these susceptible organisms, but if it be seeded with a bacterial species insusceptible to the bacteriophage involved a normal culture is obtained, just as though it had been spread upon sterile agar (d'Herelle,3io. ^'i^. 321), A suspension, or a culture, of a susceptible bacterium, inoculated with a minute quantity of the bacteriophage principle, spread upon agar gives a layer of bacterial growth studded with bare spots, circular in form, where the agar is free of all traces of growth. These bare spots or "plaques" once formed are unchanging; they do not increase in size nor are they ever covered by the surrounding bacterial growth. The area occupied by the plaque has become unsuited to the growth of susceptible organisms (d'Herelle^^"- ^^-' ^^^). The number of plaques is in direct proportion to the quantity of bac- teriophage filtrate inoculated into the suspension spread upon the agar (d'Herelle^i"' ^^i). These facts suggest the hypothesis that the bacteriophage exists in the physical state of corpuscles (d'Herelle^^°). The corpuscular state is demonstrated by the fact that dilutions at the limit of activity distributed in equal amounts among different sus- pensions of the susceptible bacterium induce bacteriophagy in certain of these suspensions while the phenomenon fails to take place in others. Either bacteriophagy occurs, or it does not occur; there is no intermedi- ary stage. This proves, not only from the biological point of view, but from the point of view of physics as well, that the bacteriophage is 100 THE BACTERIOPHAGE AND ITS BEHAVIOR found in the liquid in a "discontinuous" state, that is to say, in the form of corpuscles (d'Herelle^^^). A bacteriophage filtrate is then, simply a suspension of bacteriophage corpuscles (d'Herelle^^"). Each plaque is a colony of bacteriophage corpuscles derived from a single corpuscle (d'Herelle^^"- ^^^). The phenomenon of bacteriophagy leads to the same results upon solid media as in fluid media. There is a dissolution of the bacterial cells and a multiplication of the bacteriophage corpuscles (d'Herelle^^"- ^^0- As a result of the mode of action of the bacteriophage corpuscles, the enumeration of the corpuscles present in a suspension may be accom- plished in two different ways, by the method of dilutions and by that of counting the plaques (d'Herelle,^^"' ^^2- ^^^ and following). CHAPTER III The Mechanism of Bacteriophagy 1. THE corpuscle: obligatory bacteriophage Whatever the nature of the medium, in the absence of a susceptible bacterium the bacteriophage corpuscles do not multiply. Nor is multiplication to be observed even though the medium be favorable for the phenomenon if the corpuscles are placed in contact with killed bacterial cells. The method of killing the bacteria is without signifi- cance: the reaction does not take place with bacteria killed by aging, heat, chloroform, essences of thyme, cinnamon or mustard, by alcohol, mercuric chloride, or by carbolic, sulfuric or hydrochloric acids.* The living bacterial cell is indispensible for the multiphcation of the bacteriophage corpuscle (d'Herelle*^"). Indeed, it is even essential that the living cell be "normal," that is, not exposed to the action of substances which may modify its characters appreciably even though they do not kill it. It is unnecessary to repeat the experiments bearing upon this point, since they have been considered in Chapter I in the section entitled "Influence of Chemical Conditions on the Phenom- enon of Bacteriophagy." It can readily be shown that the phenomenon fails to take place solely because of the disturbing influence of the medium upon the bacterium, for, if the unattacked bacteria are separated by centrifu- gation from the medium and suspended in a pure bouillon they undergo bacteriophagy. With such organisms the reaction is, however, more or less delayed; a fact entirely in keeping with the idea that the "ab- normal" bacteria, now multiplying in a pure medium, become again "normal" and when this happens, become subject to the attack of the bacteriophage. What is the nature of this anomalous condition which renders the bacteria unattackable? We will be able to interpret and reply to this * This applies only to the multiplication of the bacteriophage corpuscles. A dissolution of the bacterial cells may be effected even if the bacteria are dead. But this last reaction is not in reality bacteriophagy. We will return to this fact, since in several instances the dissolution of dead bacterial cells has led to the erroneous conclusion that bacteriophagy had taken place. ^-'-^^^ 101 102 THE BACTERIOPHAGE AND ITS BEHAVIOR question when we have studied the effect of an antibacterial serum upon the phenomenon of bacteriophagy. Here let us simply say that it is probable that this anomaly consists in a modification of the sur- face tension of the bacterial cell. We have seen above that the bacteriophage corpuscle multiplies only in the presence of living and normal bacteria. While this is in general true, it is likewise true that under very particular conditions, as observed by Wollman,^^- a certain amount of bacteriophage develop- ment appears to take place in the absence of intact bacterial cells. This investigator prepared a series of collodion sacs of different densities, thus regulating the permeability.* After sterihzation he filled the sac, having a capacity of 6 cc, with a bouillon implanted with Shiga- Kruse bacilli. In the outer tube, within which the sac was suspended, he placed 20 cc. of bouillon containing bacteriophage filtrate. The con- centration of bacteriophage in this external fluid was such that when 10 cc. of a suspension of B. dysenteriae was inoculated with 1 drop of the fluid and 1 drop of the resulting mixture was spread upon agar but two plaques would form. In this way he prepared a series of collodion sacs of increasing per- meabihty, all arranged in the same manner, with a suspension of B. dysenteriae within the sac and a bouillon suspension of the bacterio- phage in the tube into which the sac was immersed. After incubation he observed the following: Very -permeable sacs. It will be recalled that, as I had shown pre- viously,^^^ the bacteriophage corpuscle passes through collodion mem- branes which are sufficiently permeable to permit the passage of the molecule (perhaps it would be better to say micella) of serum albumin. In this experiment of Wollman the same fact appears, for he found that the corpuscles passed through the permeable membranes, penetrating into the sac. There coming into contact with the dysentery bacilli they caused bacteriophagy, as would be expected. Less permeable sacs. With these sacs the baclilary culture within the sac remained normal, but the number of corpuscles inoculated into the bouillon surrounding the sac increased. A preliminary test showed, as we have seen, that a drop of the bouillon outside gave two plaques on agar. After incubation, a test conducted in the same manner with the same amounts, yielded from 20 to 30 plaques. There were, then, from 10 to 15 times as many corpuscles after incubation as be- * For the methods of preparation and arrangement of collodion sacs see the section "Ultrafiltration" in the Introduction. THE MECHANISM OF BACTERIOPHAGY 103 fore. Nevertheless, no bacteria had been introduced into the bouillon and obviously none of the bacteria present in the sac had been able to reach the external medium, since the bacteriophage corpuscles, them- selves much smaller than the bacteria, did not pass through the sac. This last fact is the more certain since the culture in the sac did not undergo bacteriophagy. Sacs of least permeability. Here no change took place after incuba- tion. The number of corpuscles in the bouillon did not increase nor did the bacterial culture within the sac show any evidence of bacteri- ophagy.* WoUman concluded that even though bacteria were not present the bacteriophage commenced to develop because of the presence of certain diffusible bacillary products. These substances passed through the membranes impermeable to the bacteriophage corpuscles. He compared this behavior of the bacteriophage corpuscle to that of Dictyostelium mucoroides. With this, a Myxomycete, Pinoyf has shown, by means of a technic comparable to that of WoUman, that develop- ment starts because of the presence of diffiusible products of B. fluorescens, although normally, the IMyxomycete develops only in the presence of living bacteria. Asheshov has personally told me that he has repeated Wollman's experiment with the same results. It is necessary, then, to conclude that the multiplication of bacterio- phage corpuscles can take place, at least to a certain degree, in the absence of the bacterial cell, and that for this development the cor- puscles utilizes certain diffusible products present in the culture of susceptible bacteria. In any case, it is certain that these diffusible products can be utiUzed by the bacteriophage corpuscle only immediately after their derivation from the bacterium, for on several occasions, employing a variety of procedures, I have tried to cultivate the corpuscles in filtered bac- terial cultures or in autolysates, always unsuccessfully. This fully agrees with further observations made by Pinoy on the Myxomycete, which, although developing to some extent in bacterial products dif- fusing through a collodion membrane never multipUes in autolysates or in culture filtrates. * This last statement is based upon a verbal communication; it does not appear in the paper of WoUman. ^^- t Pinoy, E. — Role des bacteries dans le developpement de certaines Myxomy- cetes. Ann. Inst. Pasteur, 1907, £1, 622; 686. 104 THE BACTERIOPHAGE AND ITS BEHAVIOR 2. FIXATION OF THE BACTERIOPHAGE CORPUSCLE In view of the fact that it is possible to enumerate the bacteriophage corpuscles present in a liquid, we can now go somewhat further into the mechanism of the phenomenon of bacteriophagy. One of the first questions to consider bears upon the sphere of activ- ity of the bacteriophage. If, in an appropriate liquid, we combine bacteriophage corpuscles and susceptible bacteria, do the corpuscles act at a distance or must they first come into immediate contact with the bacterial cell? The following experiment (d'Herelle^^^) answers this question. The following suspensions are prepared: 1. One hundred cubic centimeters of a suspension of the Shiga bacil- lus containing 250 million bacilli per cubic centimeter. This is inocu- lated with 0.25 cc. of bacteriophage suspension. 2. One hundred cubic centimeters of a suspension of the cholera vibrio, containing 250 million bacilli per cubic centimeter. This also is inoculated with 0.25 cc. of the same suspension of Shiga-bacteriophage. 3. One hundred cubic centimeters of bouillon containing only 0.25 cc. of the same bacteriophage. The material of all three flasks is incubated at 37°C. Immediately after the inoculation, after 30 minutes, and again after 1 hour, 20 cc. are taken from each of the three flasks and centrifuged at 4000 revolu- tions per minute for 10 minutes. There are thus 9 tubes which have been centrifuged. From the supernatant fluid of each of these, 0.02 cc. is taken and introduced into other tubes containing suspensions of the Shiga bacillus, and counts of the corpuscles are made by plating 0.02 cc, of each of these 9 tubes on six plates of medium. In this way an average of the counts can be obtained, and the results of the counts indicate the number of corpuscles remaining in the medium, since those which have penetrated the bac- terial cells before the centrifugation have been thrown down with the cells during this procedure and, as a result are to be found in the sedi- ment. The results of the counts are as follows: Tube 1. Shiga suspension plus bacteriophage. a. Counts of the material made immediately after the preparation are 214, 193, 187, 221, 229, and 183 plaques. The average is 204, representing 5,000,000 corpuscles per cubic centimeter in the original suspension immediately after inoculation. THE MECHANISM OF BACTERIOPHAGY 105 b. Counts on the suspension after incubation for 30 minutes are 3, 7, 4, 6, 6, and 3 plaques. The average is 5. This indicates that there are 125,000 bacteriophagous corpuscles in the suspension 30 minutes after the inoculation. That is, of each 41 corpuscles inoculated, 40 have disappeared from the fluid. A count made directly upon the suspension, without centrifugation, gives 5,000,000 elements per cubic centimeter. It is therefore certain that the corpuscles which have disappeared from the fluid during the centrifugation have gone down with the bacteria. And, as we will see in the two control experiments, in the absence of Shiga bacilli this sedimentation of the bacteriophage does not occur (at least, when centrif uged at a speed of 4000 revolutions) . c. After 1 hour, the count, made as before upon the supernatant fluid gives an average of 8 plaques, or 200,000 corpuscles per cubic centimeter; a number essentially the same as that secured after 30 minutes. At this time a count of a suspension which has not been centrifuged gives 6,500,000; a number very close to that secured im- mediately after the inoculation. d. Counts made upon the suspension with and without centrifu- gation after one and one-quarter hours give the same number of cor- puscles — about 90 million. The inoculated corpuscles have therefore increased from 5 to 90 millions; the increase being in a proportion of about 1:18. And this increase has taken place in apparently a very abrupt manner, only to be explained as a result of the liberation of actual colonies containing an average of about 18 corpuscles. We will see by ultramicroscopic examination that the dissolution of a para- sitized bacterium takes place brusquely, by bursting. Tube 2. Control. Suspension of V. cholerae plus the Shiga-bacterio- phage. Counts made immediately after inoculation of the bacteriophage give; for the centrifuged material, 201; for the non-centrifuged, 211 plaques. After 30 minutes the counts are: for the centrifuged, 210; for the non-centrifuged, 216. After 1 hour the counts are: for the centrifuged, 203; for the non- centrifuged, 199. After one and one-haK hours the non-centrifuged suspension gives 207. Tube 3. Control. Sterile bouillon plus Shiga-bacteriophage. The counts immediately after the inoculation are: for the centri- fuged, 206; for the non-centrifuged, 210. 106 THE BACTERIOPHAGE AND ITS BEHAVIOR After 30 minutes the corresponding counts are; 201 and 211. After 1 hour the counts are: 203 and 206. After one and one-half hours the non-centrifuged medium contains 198. As is evident, in the absence of bacteria capable of being attacked nothing happens. The corpuscles remain inert in the medium. The nature of the multiplication taking place in the presence of the Shiga bacillus does not permit of any doubt on the following points: 1. After a contact of 30 minutes at 37°C. the corpuscles have almost entirely disappeared from the fluid; they are fixed by the bacteria. After 1 hour the situation is essentially the same. 2. The fixation is elective. It does not occur with V. cholerae, for example, for which the bacteriophage in question is without action (d'Herelle^^i)^ A complementary experiment, conducted in the same fashion, but centrifuging the suspension at 10-minute intervals during the first half-hour, has shown that very few of the bacteriophage corpuscles are fixed during the first 10 minutes, although they are almost all fixed after 20 minutes. The union, therefore, requires about a quarter of an hour. Additional data obtained with the staphylococcus may be introduced as bearing upon this subject of fixation. Ten cubic centimeters of a normal suspension of cocci are combined with 0.1 cc. of the bacterio- phage suspension. The temperature is held throughout at 30°C. With this particular race of bacteriophage, of relatively high but not of maximum potency, the fluid still contains 97 per cent of the intro- duced corpuscles after 45 minutes. After 1 hour 62 per cent are pres- ent; after 75 minutes, but 8.5 per cent remain. On the other hand, with two races of the bacteriophage of maxiinum potency the fixation was extremely rapid. When 0.1 cc. of a suspension containing 20,000 million corpuscles per cubic centimeter was intro- duced into 10 cc. of bacterial suspension (250 million cocci per cubic centimeter) and the material was filtered through a candle after 10 minutes there was not a single corpuscle in 0.05 cc. of the filtrate. The fixation was complete. This experiment shows particularly well how the activity of the bacteriophage race influences the speed of fixation. The fixation of the bacteriophage corpuscle to the susceptible bac- terium constitutes then, the first act of bacteriophagy (d'Herelle'^''' ^'O- This fact is accepted by all authors. The only difference to appear in THE MECHANISM OF BACTERIOPHAGY 107 different experiments is the time requisite for the fixation, and this varies with the bacterial species and with the race of the bacteriophage, as well as with the reaction of the medium, the temperature, and other environmental conditions. With a single strain of bacteria the fixation is the more rapid and complete the more active the bacteriophage. It is particularly perti- nent in this connection to remember that the mechanism of bacteri- ophagy can be studied only with races of the bacteriophage of maximum activity. With races of less potency the phenomenon of bacterial resistance masks to a greater or less degree the processes of attack. By a series of experiments performed with different bacterial species I' am convinced that with bacteriophage races of low potency it is impossible to demonstrate the process of fixation to the bacteria. At least, it is impossible to demonstrate that the corpuscles disappear from the liquid. The reason for this is obvious, for with such races fixation takes place slowly and instead of the process occur- ring almost simultaneously with practically the entire number of inocu- lated corpuscles, as is the case when the bacteriophage is very active, the time of fixation varies enormously among the different corpuscles. It thus happens that a great number have not been fixed when those which are first fixed have already commenced to reproduce. Conse- quently, the fixation remains undetected. Carrying out experiments analogous to those which I have described but employing filtration instead of centrifugation, Janzen and Wolff^" obtained the following results in two experiments with B. typhosus using different races of the Typhoid-bacteriophage. Number of free corpuscles per cubic centimeter, unfixed to the bacteria I. Immediately after inoculation 18,000,000 After 15 minutes 280,000 II. Immediately after inoculation 30,000,000 After ISminutes 4,000,000 It is probable that the number of bacteriophage corpuscles fixed would have been still greater if the counts had been made some 5 or 10 minutes later. The process of fixation is lacking when a bacteriophage is placed in contact with a bacterium insusceptible to bacteriophagy by the partic- ular bacteriophage involved.^^i ^i^h but one exception all of those who have studied this question agree on this point. Later we will 108 THE BACTERIOPHAGE AND ITS BEHAVIOR see that a Typho-bacteriophage, and this is also true for a CoH-bacterio- phage or a Staphylo-bacteriophage, is as a rule not active upon all strains of B. typhosus; certain strains being susceptible while others are naturally resistant. This is what I have meant when a bacterial species has been termed heterogeneous toward a bacteriophage. To return to the exception mentioned above, Janzen and Wolff""* have reported that a Typho-bacteriophage may be fixed to typhoid bacilli of strains against which this Typho-bacteriophage is inert. This must be regarded as an exceptional case, for Jaumain and Meulemans"^ have shown with different races of Coli-bacteriophage and Staphylo- bacteriophage that fixation does not occur with bacteria belonging to insusceptible strains. My own observations agree with those of Janzen and Wolff, for I have observed a fixation, although but partial (it is, however, the same when these races act upon susceptible bac- teria, it being simply a question of degree), with insusceptible staphy- lococci. We will see shortly that this is also what happened in some experiments reported by Flu. As a modification of the above experiments da Costa Cruz^'^^ has shown that fixation takes place upon heat-killed bacteria, provided they were of a susceptible strain. Working with a Flexner-bacteriophage he has seen that the corpuscles are fixed to dysentery bacilli which have been killed by heating at 60°C., but that they are not fixed to staphylococci, also killed at the same temperature. This has been confirmed by several authors. Prausnitz and Firle^^^ have seen that fixation took place with sus- ceptible bacteria after they had been heated at 60, 70, 80, 90 and 100°, but that it did not take place when the bacteria had been exposed to a temperature of 120°C. These observations warrant the conclusion that fixation proceeds in the same manner whether the susceptible bacteria are Uving or dead, although the bacteriophage corpuscles can develop only at the expense of the former. These facts make clear the reason for the delay in bacteriophagy caused by the viscosity of the medium, whether this viscosity is due to gelatin, to a gum, or to any other substance which is, of itself, without action upon the phenomenon. When we place bacteria and bacterio- phage corpuscles within a liquid it is evident that inasmuch as the first act of bacteriophagy consists in a fixation of the corpuscles to the bacterial cells, these corpuscles must first of all traverse the distance which separates them from the nearest bacteria. This necessitates THE MECHANISM OF BACTERIOPHAGY 109 the assumption that there be a positive chemotaxis of the bacteriophage corpuscle for the susceptible bacterium, or, if one prefers (it is of no consequence at the moment), of the bacteria for the corpuscle. What- ever may be the nature of the force which leads to the union, the attraction exists, and it is evident that anything which augments the viscosity of the liquid tends to interfere with the approach of the corpuscle to the bacterium, and, as a result, with its fixation. If the viscosity is sufficiently high the number of corpuscles attaining fixation will be so small that the phenomenon will be incapable of detection macroscopicallij because of the small number of bacteria dissolved. Only the proof provided by a demonstration of the multiplication of the corpuscles will show that the bacteriophagy of a few organisms has taken place. Doerr and his collaborators^^^- ^^' have attempted to draw a parallel- ism between the fixation of the bacteriophage corpuscle to the bacterium and the fixation of an antibody. Such a comparison is inadmissible, for the characteristics of the two phenomena are entirely different. We know that the agglutinin content of a serum can, to all intents and purposes, be completely exhausted by "saturation" with homolo- gous bacteria, and that the saturation required is in direct proportion to the agglutinating potency of the serum; that is to say, fewer bac- terial cells are required to exhaust a weakly agglutinating serum than to exhaust the same quantity of a strongly agglutinating serum. With the bacteriophage the situation is exactly the reverse. In a later chapter we will see that races of the bacteriophage may be isolated which differ widely in their activity for a single bacterium, some being weakly active, others possessing extreme activity. This difference does not involve a difference in the number of corpuscles, but is due rather to a difference in the activity of the corpuscles. But the partic- ular point as regards fixation is precisely this, that the greater the activity of the bacteriophage the fewer bacterial cells are requisite to give fixation. This fact has been disclosed consistently by several experiments made with both B. dysenteriae and the staphylococcus and their homologous bacteriophages. With a bacteriophage of maximum activity, using 0.1 cc. of a sus- pension containing more than 10,000 million corpuscles per cubic centimeter in conjunction with 10 cc. of a bacterial suspension (250 million per cubic centimeter), the fixation is complete with the staphylococcus, and almost complete with B. dysenteriae, within a period of 20 minutes. 110 THE BACTERIOPHAGE AND ITS BEHAVIOR Employing a less active bacteriophage, 0.1 cc. of a suspension with 1000 million corpuscles per cubic centimeter, added to 10 cc. of a bac- terial suspension, shows very little fixation after contact for 30 minutes. Even with the contacts repeated for 5 successive times, the material being centrifuged between each contact and fresh organisms added each time, a complete fixation is not attained. In brief, therefore, the course of fixation is exactly opposite to that observed in the fixation of agglutinins. We must regard the fixation of bacteriophage corpuscles to the bacteria as a phenomenon of col- loid nature — and nothing could be more legitimate since all of the reactions of living matter are colloidal reactions — and it would be strange indeed if bacteriophagy formed an exception. It is assuredly true that as yet we do not know the intimate mecha- nism of the process, yet we may unquestionably affirm, without appear- ing too radical, that it is a colloidal process. This, however, means but little since this is true for all of the phenomena of life. Beyond this statement all we can definitely say is that the first phase of bac- teriophagy consists in the approach of the bacteriophage corpuscle to the bacterium, and that this is followed by its fixation to the bac- terial cell. Unquestionably, the actual fixation is elective, as all available ex- perimental data indicate. But with regard to the process which leads to the contact between the bacteriophage corpuscle and the bacterium there is the question as to whether it is a passive phenomenon or whether it is a true chemotaxis. It might be assumed that the corpuscles, in violent motion because of the brownian motion which animates them, become fixed only when they come into contact with a susceptible bacterium. With these the fixation is then elective, but it takes place only after contact is effected. In this connection Kabelik^^* has recently published some experiments which enforce the conclusion that a real chemotaxis exists between bacteriophage corpuscles and susceptible bacteria. The statements of Kabelik, embodying his results, are here inserted. Bacterial migration may readily be studied in glass U-tubes which contain the nutritive fluid and in which the bottom of the tube is filled with sterile sand. For our tests these simple U-tubes are not quite adequate, and we have therefore devised an apparatus which is, in effect, a combination of several of these tubes. A horizontal tube, 9 mm. in diameter, and 21 cm. long, is provided with seven vertical arms, about 8 cm. in length, spaced about 3 cm. from each other. In the bottoms of all of these vertical tubes some very fine sand, thoroughly washed and THE MECHANISM OF BACTERIOPHAGY 111 sterilized, is placed, in such a way that migration through the material and the fluid is rendered very difficult. These tubes are then filled with bouillon to a height of 3 or 4 cm. above the sand. (They may be used for several purposes, particularly for separating B. typhosus from B. coli.) In an apparatus of this type (no. 1*) we inoculated a drop of a virulent bac- teriophage suspension (strain H of d'Herelle) in the first vertical arm. Tests were made after 2, 4, 6, 8, 12, and 24 hours to see if the bacteriophage had pene- trated to the other vertical arms. These tests consisted simply in seeding a loopful of the contents of each arm on to agar previously implanted with Shiga bacilli. These agar cultures were allowed to incubate for 24 hours, and then the number of plaques, that is, the number of bacteriophage corpuscles appearing were recorded (see the table). When the plaques were completely fused together, resulting in a large sterile area covering the entire surface of the medium the result was expressed as infinity (o°). In another apparatus (no. 2), under the same conditions, we inoculated the bacteriophage into the first upright arm after this had been filled with bouillon seeded with Shiga bacilli. In a third apparatus (no. 3) the bacteriophage was inoculated into pure bouillon in the first arm, and the last, or seventh, we im- planted with Shiga bacilli. In a fourth apparatus (no. 4) only Shiga bacilli were implanted; this set to test (as a control) the rapidity with which bacterial infil- tration into the neighboring arms occurred. This control showed that after 4 hours the bacilli were to be found in the second arm only. Obviously, the abso- lute rate of penetration of bacilli and bacteriophage depends, ceteris paribus, upon the size of the grains of sand. The results of these tests are summarized as follows: TIME RESULTS OF TESTS UPON THE FLUIDS WITHIN APPARA- TUS MATERIALS INTRODDCED ELAPSING BETWEEN PLANTING AND TESTING THE DIFFERENT VERTICAL ARMS NDMBER 1 2 3 4 5 6 7 hours 1 Bacteriophage only n CO 6^ CO 12 00 CO 1 24 00 OO 00 36 00 00 oo 00 CO 30 2 Bacteriophage -1- Shiga 6i 00 00 00 00 oo 50 bacilli in the first 12 CO 00 00 CO oo CO 00 arm 3 Bacteriophage in the n 00 first: Shiga in the 6* 00 so 2 seventh arm 12 00 00 00 CO 00 00 24 00 00 CO oo oo 00 ? 36 00 00 00 00 00 oo 00 See the table below. 112 THE BACTERIOPHAGE AND ITS BEHAVIOR TIME RESULTS OP TESTS UPON THE FLUIDS WITHIN APPARA- TUS MATERIALS INTRODUCED ELAPSING BETWEEN PLANTING AND TESTING THE DIFFERENT VERTICAL ARMS NUMBER 1 2 3 4 5 6 7 hours 4 Shiga bacilli only 6 gr. gr. gr. 24 gr. gr. gr. gr. gr. gr. 20 colo- nies 5 Bacteriophage only 61 CO 00 24 CO 00 2 6 Bacteriophage + Shiga 6i 00 00 00 oo CO bacilli in the first 24 00 00 CO 00 00 CO CO arm 7 Bacteriophage in the 61 00 oo 100 first: Shiga in the 24 00 00 00 2 seventh arm Tests 1 to 4 were made in bouillon; 5 to 7 were made in physiological saline, gr. = bacterial growth. In view of these results it would seem that we must assume that a true chemotactic influence is operative in the behavior of the bacterio- phage corpuscle.* 3. PENETRATION OF THE CORPUSCLE INTO THE BACTERIUM With the bacteriophage fixed to the bacterium, does it remain ad- herent to the surface or does it penetrate to the interior of the cell? "Macroscopic" experiments are inadequate to determine this point, but "microscopic" observation, chiefly by means of the dark-field method, provides some information. Inoculate 0.1 cc. of a very active Shiga-bacteriophage suspension into 10 cc. of a suspension of 250 milfion Shiga bacilU per cubic centimeter. During the period when bacterial dissolution is taking place most vigorously remove a drop of the suspension and examine it under the dark-field. Not a single bacterium will be seen which appears to be undergoing disintegrative changes. The only visible abnormality is that in the midst of the normally appearing bacteria some few will be seen presenting an "inflated" form. Those departing farthest from the typical cell are completely spherical, with a diameter of 3 to * In this preliminary note, adequate however to allow of a decision, Kabelik announces the publication of a more extended memoir on the question. THE MECHANISM OF BACTERIOPHAGY 113 5ju, and between the normal forms and the spherical forms are to be found all intermediates. But despite this variable morphology all of the cells have a sharply outlined contour. If the spherical cells are observed with care it is seen that after a variable length of time, sometimes amounting to only about 10 minutes, an actual bursting takes place; a process consuming only a fraction of a second. Immediately afterward, in the place of the spherical cell there remains a slightly cloudy floccule which slowly dissolves. These spherical cells are particularly abundant at the time when the dis- solving process is at its maximum rate. There can be no question concerning the nature of these cells; they are bacilli which, operated upon by a force exerting its effects from within, take at first a globoid form and later rupture. This is the more certain since at times one can witness the rupture of the swollen bacilh, even before they have assumed a completely spherical contour. This observation provides direct proof that the corpuscle develops and exerts its action within the bacterial cell. Destruction of the bacilh would be an entirely dif- ferent process if the dissolving action were exerted on the exterior. The spherical form and the bursting process prove beyond doubt that the operating force is internal (d'Herelle^^^* ^23^* Although, as stated above, it is best to make microscopic observa- tions with a suspension in the process of being dissolved under the action of a poiverful bacteriophage, this does not mean that the rupture of the bacterial cell takes place in this case only, for the phe- nomenon has occurred in the same manner with all of the races of the bacteriophage which I have isolated. It is, however, more readily observed when bacteriophagy is intense. I am convinced, because of various experiments with bacteria of varied species and with bac- teriophage races of different types, that the destruction and the dissolu- tion of the bacteria occurs always by bursting. But in the case of a slightly active bacteriophage the process may pass unobserved, for the number of ruptures occurring at a given moment is then extremely small, and it is pure "chance" if the rupture of a cell takes place at a given time within the extremely minute quantity of material under the objective of the microscope. With such materials it may at times * The bursting phenomenon was first observed by P. Jeantet, Chief of the Laboratory of Microphotography at the Pasteur Institute. There are few who have the capacity for microscopic observation as highly developed as he. More- over, he has the habit, as rare as it is original, of not publishing the things which he observes. Instead of taking to himself the credit for those things disclosed in the studies in which he takes an interest, there are many who have benefited from his powers of observation. 114 THE BACTERIOPHAGE AND ITS BEHAVIOR require an hour of continuous search and observation before the first rupture is seen. It is for this reason that those who desire to witness this curious phenomenon should use Shiga bacilh in contact with a highly potent bacteriophage. When they have once observed the reaction, recognizing the manner in which it takes place, they can then investigate other cases where the phenomenon is less conspicuous. Personally, I have observed the rupture of bacteria contaminated by bacteriophage corpuscles with B. dysenteriae Shiga and Flexner, with B. typhosus and the paratyphoid strains, with B. pestis, and with the staphylococcus. As a matter of fact, I have never failed to see it when I have sought for it. With the staphylococcus the individual coccus undergoing rupture will have a diameter 2 to 3 times as great as that of a normal coccus. The rupture of bacteriophaged bacteria has also been observed by da Costa Cruz,^^^ Pondman,^^^ as well as by Hauduroy^^^ and by Flu. Naturally, a quite logical question is. How can the bacteriophage corpuscle penetrate the bacterium? The following observations per- mit an hypothesis, although they do not give a clear and complete picture of the mechanism. At the moment when bacteriophagy is most intense we see by dark- field observation that the single unusual feature presented by the bacteria is a more or less outspoken swelling. None of the bacteria appear damaged, and with the exception of the ''floccules" which follow the bursting and which disappear after a few minutes there is no bac- terial debris. If a drop of the suspension in which the process of rupture is taking place is removed and spread upon a slide, dried, and stained, it will be seen that along with the normal bacteria there are some swollen organisms and some amorphous material which certainly represents stained floccular material. In such a stained smear the background is not colorless as in an ordinary preparation of a suspen- sion of normal young bacteria, but is tinted. All of the bacteria are sharply defined, even those which are distended. Another interesting situation develops if we take a drop of the suspension when bacteriophagy is at its maximum and place it between a shde and cover-glass (selecting a thick cover-glass to avoid breakage) exerting strong pressure upon the cover, as though a crushing of the bacteria was desired. Allow the preparation to dry in the incubator, then, after removal of the cover-glass, fix the smear and stain with any of the ordinary dyes (Loeffler's blue, carbol-f uchsin) . Examina- tion will reveal many of the bacteria presenting a curious appearance, for about some of the well-stained bacterial cells may be seen one, two, THE MECHANISM OF BACTERIOPHAGY 115 three, or sometimes several, stained "discharges." The material gives a definite impression that a portion of the contents of the bac- terium have escaped by means of one, or of several, apertures. This observation suggests that the wall of the bacterial cell (formed beyond doubt of condensed protoplasm) has been perforated by one, or several, bacteriophage corpuscles, and that the avenue of entrance has remained open. However this may be, the single fact that the phenomenon of rup- ture is "explosive" in nature shows that the bacteriophage corpuscle certainly penetrates to the interior of the bacterium, and that it is in this location that the multiphcation takes place. 4. MULTIPLICATION OF THE BACTERIOPHAGE CORPUSCLE At the very beginning of this section I believe it wise to state once more that when I cite an illustrative experiment it is not equivalent to stating that the rate of the reaction of all experiments which may be carried out upon the same subject must be identical. Such an erroneous deduction has been reached by a number of authors. For example, the experiment presented in the second section of the present chapter, and to which we will return, shows that the first increment in the bacteriophage was in a ratio of 18:1. This numerical ratio holds necessarily for this experiment only. In other cases it is possible to have ratios of increase anywhere between 6:1 and 60:1. Everything depends upon the conditions of the experiment, chiefly upon the "viru- lence" of the bacteriophage with which one is working, Bacteriophagy always takes place in the same manner; the sequence of events is always the same. The bacteriophage corpuscle must in- variably become fixed to the bacterium to exercise its action. Destruc- tion of the bacterium is always accomphshed by bursting. The bac- teriophage corpuscles always multiply within the bacterial cell and are always liberated with the rupture of this cell. But the time required for the fixation to take place, the time necessary for the bac- terium to undergo rupture, the number of young bacteriophage cor- puscles developing within the bacterium to be liberated with its rupture, all vary in each particular case, according to a multitude of conditions which vary from one experiment to another. Having again emphasized this, let us consider the manner in which multiplication of the bacteriophage corpuscle takes place, and the nature of the conditions which exercise an effect upon their develop- ment. 116 THE BACTERIOPHAGE AND ITS BEHAVIOR The course of tmdli'plication The experiment described in the second section of this chapter shows that: After 30 minutes of contact at 37°C. the bacteriophage corpuscles have almost entirely disappeared from the liquid. After 60 minutes the situation is the same. After 90 minutes the corpuscles have suddenly reappeared in the liquid, and their number is 18 times greater than was that of the inocu- lated corpuscles. In other words, each inoculated corpuscle has yielded 18. Another experiment may be inserted, likewise showing the sudden multiplication of the bacteriophage corpuscles. Six tubes of Shiga bacillus suspension are inoculated with a bacteriophage suspension (containing 3000 million per cubic centimeter) in such a way that each tube receives one six-millionth of a cubic centimeter. When incubated, four give normal cultures of B. dysenteriae and all subcul- tures on agar yield normal growths. These are, therefore, without interest for us. The other two, each of which received probably one, certainly not more than two corpuscles, show the following picture: The suspensions become more and more turbid. After 2 hours at 37°C. the opacity is about 2 times as great as at the beginning. After 3 hours it is about 2^ times as great, and after 4 hours, about 3 times. It then begins to diminish, so that after 5 hours the density is about twice as great as at the beginning of the incubation. This clearing continues gradually, so that after 14 hours the culture is almost en- tirely clear. If immediately after the inoculation with the bacterio- phage, and then every 30 minutes, 0.02 cc. of each of these two sus- pensions is transferred to agar slants, these tubes will show, after incubation, the following: Plantings after 30 minutes, 1, 1^, and 2 hours yield normal growths of B. dysenteriae. After 2^ hours the subcultures show 3 plaques in one tube and 5 in the other (average, 4). Therefore, after 2^ hours the inoculated suspension contains 2000 bacteriophage corpuscles. After 3 hours the subcultures show 5 and 4 respectively. There has been no material increase between 2^ and 3 hours. The 3 J hour plantings show 9 and 5 plaques (average, 7) . The num- ber of bacteriophagous elements has slightly increased. After 4 hours, the agar tubes show 101 and 111 plaques respectively (average, 106). After 4 hours, therefore, the number of corpuscles is between 50 and 60 thousand. THE MECHANISM OF BACTERIOPHAGY 117 After 4| hours, the counts are 145 and 160 (average, 152), indicating that the suspension contains 75,000; a number but shghtly higher than the count after 4 hours. After 5 hours the agar tubes remain sterile. "When diluted to 1 : 1000 in a suspension of Shiga bacilh and transferred immediately to agar in the same way, the tubes give 4 and 6 plaques. Thus, it appears that after 5 hours the suspension contains about 1,500,000 bacterio- phage corpuscles per cubic centimeter. Although all authors are virtually in agreement with me upon the question of the specific fixation of the bacteriophage to the bacteria, several have denied that the multiplication occurs through successive sudden increments. Thus, Doerr found that the increase in "lytic substance" was very rapid but took place gradually, the titre increas- ing by about 10 times every 15 minutes. After having carried out many tests upon a variety of bacteria with different races of the bac- teriophage I adhere definitely to my previous statement,^-^ namely, that the increase in the number of corpuscles does not take place in a continuous progressive fashion, but by successive liberations. It may be pertinent to observe that in order to clearly observe this phenom- enon it is essential that the experiments be performed in such a way that the course of the reaction is not obscured. To effectively demon- strate the phenomenon it is necessary to observe the following condi- tions: (1) To work with a bacterial species which undergoes a rapid bacteriophagy. Such a one is the Shiga bacillus. (2) To work with a bacteriophage of maximum activity for the bacterium in question. (3) To utilize a very small number of bacteriophage corpuscles, acting upon a large number of bacteria. The reasons which make these conditions essential if the phenomenon is to be observed distinctly can readily be understood. If one uses a bacteriophage of weak activity the corpuscles of the successive genera- tions become fixed very slowly and at a very unequal rate. Those fixed at first have already formed a colony and have caused the rupture of the bacterium before the other corpuscles are even fixed. Under such circumstances it can be understood that it is impossible to observe the true course of multiplication of each corpuscle, and Doerr is then apparently correct, for the total course of the multiplication is indeed progressively continuous. Even in the case of a bacteriophage of maximum activity the fixation of all of the corpuscles does not take place with mathematical precision within the same interval of time. Obviously, for this there are several 118 THE BACTERIOPHAGE AND ITS BEHAVIOR reasons. The first step in the process is the approach of the corpuscle to the bacterium (it can not be otherwise, since we know that the corpuscle can only act after it is fixed), and the more distant a corpuscle from the nearest bacterium the greater wiU be the time required for the fixation. Furthermore, we will see that even in the case of a bacteriophage of maximum potency all of the corpuscles do not have an equal virulence. The rapidity of fixation to a given bacterium, all of the other conditions being equal, is in direct propor- tion to its "virulence." This represents a further reason why the fixation of all of the corpuscles may be distributed over a certain period of time. Recognizing these facts, it is clear that the best condition for observing the true nature of the reaction consists in inoculating at the beginning only a very small number of corpuscles, so that complete fixation can be accomplished in a minimum of time. As a result of this all of the ruptures, and consequently the liberations of young corpus- cles, will occur after a like interval of time. This will permit one to observe that multiplication, the increase in the number of corpuscles, takes place suddenly. Another requisite condition, already mentioned, involves the presence of a large number of bacteria. The significance of this factor is evi- dent, for if there is only a small number each bacterium will be found at a considerable, and very variable, distance from the nearest corpuscle. This again means that fixation will occur in a very irregular manner. With due regard to the conditions mentioned anyone may demon- strate readily that the multipHcation of bacteriophage corpuscles takes place by means of successive jumps and not in a gradual pro- gressive fashion. But even here, this fact can be shown definitely only for the first hberation, as is quite natural, since as bacteriophagy progresses the greater will become the number of corpuscles, and the virulence of each of them, as individuals, being different, the time of fixation, and consequently, the speed of multipHcation, will proceed in an irregular manner.* The multipHcation by successive jumps, extremely clear-cut in the beginning of the process, becomes with time less and less sharply defined. The first series of Hberations of the young corpuscles is accompHshed within a short time, while for the * This fact is not astonishing. We have known since the days of Pasteur that in a bacterial culture each of the organisms presents individual characteristics, chiefly in those attributes dealing with its virulence. In the chapter devoted to "The Virulence of the Bacteriophage" we will see that the situation is exactly the same with the bacteriophage. THE MECHANISM OF BACTERIOPHAGY 119 following liberations this interval of time becomes progressively greater, and finally, the ruptures of the last cells of a series take place only when the first burstings of the following series have conunenced. That is, at this stage and under these circumstances the multipHcation of the culture as a whole is in effect a continuous process. The phenomenon of bacteriophagy is biological in nature. There- fore, it is impossible for its course to have the simplicity of a strictly chemical reaction. Since the publication of my experiments upon this question many students have studied the course of the multiplication of the bacterio- phage. It also appears from the work of Maitland^^^ that the sudden multiplication takes place only after an incubation period. I am not able to insert here his experiments for he has adopted a method of "titration" of the bacteriophage which does not allow of the exact enumeration of the corpuscles. I shall return to this later, when treating of the different methods proposed for measuring the activity of the bacteriophage. Let it suffice here to say that from his experi- ments it seems, as he has remarked, that at 37°C. there is no multipli- cation during the first hour; sometimes during an even longer period. Following this period of latency there is a period of rapid augmentation occurring between the second and the third hours after the mixture is made. During this increase the "titre" of the bacteriophage may attain 10,000 times the original titre. The rate of the increase then diminishes and the maximum concentration is reached at about the fifth hour. These results agree, in a general way, with those which I have pub- lished. But the great majority of those who have worked with the bacteriophage have wished to generalize from their results and in this tendency is to be found one of the causes of confusion in the study of the bacteriophage. All that one may correctly conclude from a given experiment is that under the conditions of this experiment such and such a result has been obtained. Beyond this nothing is permissible. Warranty for generalization is afforded only when a constant effect is produced, whatever may be the race of the bacteriophage and what- ever may be the bacterial species under investigation. Nevertheless, it may be stated that with a given bacterial species the rapidity of fixation and consequently the rate of multiplication is a function of the total 'Virulence," that is, an expression of the aver- age virulence of the different corpuscles which are acting upon the bacteria of the suspension. Each of the corpuscles present fixes itself 120 THE BACTERIOPHAGE AND ITS BEHAVIOR and multiplies the more rapidly as its "virulence" is the greater. But it is equally important to observe that against different bacterial species bacteriophage races of the same degree of virulence may be- come fixed and may multiply at different rates. From the experiments which I have carried out upon this subject the following may be cited as giving the maximum and minimum rates observed. With the most active race of Shiga-bacteriophage which I have isolated the fixation amounted to 94 per cent after -12 minutes (10,000 corpuscles per cubic centimeter in the presence of 250,000,000 bacteria per cubic centimeter) at a temperature of 30°C. Inoculating a single corpuscle into 10 cc. of a suspension containing 50,000,000 bacteria per cubic centimeter there were, after 11 hours, 12,000 million corpuscles per cubic centimeter. With a Shiga-bacteriophage of low activity, in inoculating 100,000 corpuscles into 10 cc. of a suspension containing 250,000,000 bacteria per cubic centimeter after 20 minutes only 10 per cent had been fixed. Here also the temperature was 30°C. After 60 minutes only 34 per cent had been fixed. Inoculating a single corpuscle into 10 cc. of a suspension containing 250,000,000 bacteria per cubic centimeter there were, after 24 hours, only 17,000,000 corpuscles present in the liquid. With an extremely potent race of the Staphylo-bacteriophage, work- ing under the same conditions as those described for the active Shiga- bacteriophage, the fixation was complete 20 minutes after the inocula- tion. Inoculating a single corpuscle into 10 cc. of a suspension con- taining 50,000,000 bacteria per cubic centimeter* the maximum value was reached only after 44 hours, and at this time there were 98,000 milHon corpuscles per cubic centimeter. Working under the same conditions as those of the preceding experi- ment, but using another race of Staphylo-bacteriophage, one much less potent, the fixation after 75 minutes amounted to 54 per cent. A single corpuscle inoculated into a suspension of staphylococci contain- ing 50,000,000 per cubic centimeter yielded, after 48 hours, only 780,000,000 corpuscles per cubic centimeter. In bacteriophagy the result of a single experiment always depends upon the conditions of the experiment, the most important of these * We have seen in Chapter I that in inoculating a very small amount of bac- teriophage the initial number of bacteria is of little consequence, for the latter develop abundantly up to the moment when the multiplication of the bacterio- phage is sufficient to effect bacteriophagy of all of the bacteria. THE MECHANIi^M OF BACTERIOPHAGY 121 conditions being resident in the ''characters" of the bacteriophage involved. Each bacteriophage presents, as we will discover from every page of this text, particular distinctive characteristics. Among the published experiments bearing upon the multiplication of the bacteriophage may be mentioned one presented by Doerr and Griininger^^^ carried out with a Coli-bacteriophage. These investigators have adopted a method of titration proposed l^y Applemans, concerning which we will offer certain criticisms later, where we will see that it gives an approximation of such a crude nature (and even absolutely fails in certain instances) that it can not serve for the enumeration of corpuscles. However this may be, Doerr and Griininger have concluded from this experiment that an intense "bacteriolysis" corresponds to a stabilization in the titre of the bacterio- TITRE OF BACTERIOPHAGE NUMBER OF BACTERIA TIME OF INCUBATION PER CUBIC CENTIMETER PER CUBIC CENTIMETER minutes 100 10,000,000 30 100 16,000,000 60 100 20,000,000 90 100 35,000,000 105 100 45,000,000 120 1,000 60,000,000 150 10,000 130,000,000 180 10,000,000 600,000,000 210 1,000,000,000 200,000,000 270 1,000,000,000 20,000,000 phage. This can hardly be the case, since their experiment indicates that the number of bacteria diminish from 600,000,000 to 200,000,000 within the interval between 180 and 210 minutes, while in this same period the bacteriophage increases from 10,000,000 to 1,000,000,000. It would appear that this demonstrates precisely that the maximum increase in the bacteriophage corresponds to the greatest destruction of the bacteria. As for the assumed stabilization in the titre of the bacteriophage, which remains at 1000 million while the number of bacteria diminish from 200,000,000 to 20,000,000, this results simply from the method adopted for the titration of the bacteriophage. In fact, the so-called "dilution method" does not allow one to say that there are 1000 milUon corpuscles per cubic centimeter; it simply per- mits the statement that there are more than 1000 million and less than 122 THE BACTERIOPHAGE AND ITS BEHAVIOR 10,000 million. Such being the case, the conclusion of Doerr is not supported by experimental proof, indeed, the contrary interpretation is more logical, for the experimental data indicate that the period of increase in the bacteriophage corresponds to the period of destruc- tion of a great many bacteria. In this same communication Doerr and Griininger'^*'* state that the dissolution of the bacteria contained in the suspension occurs when the concentration of ''lysin" equals e^^B, according to the notation of Werthemann.'^^' This is simply equivalent to saying that when the concentration of the bacteriophage is such that 1-10~^ cc. of a suspen- sion in process of being bacteriophaged is added to a fresh suspension of the same bacteria bacteriophagy of the latter ensues. In other words, and to state it somewhat more precisely, dissolution of the bacteria takes place when the number of corpuscles is between 100,000 and 1,000,000 per cubic centimeter. Again it is necessary to repeat that to attempt to establish precise rules as governing the reaction is an illusion. The statement of Doerr is the more remarkable in that I have never seen a macroscopically detectable dissolution of bacteria with such a small number of corpuscles per cubic centimeter. Ob- viously, if the number of bacteriophage corpuscles is very small, very few of the bacteria are attacked; they remain normal and multiply normally. On the other hand, the corpuscles multiply, proliferating at the expense of the bacteria, but the number of bacteria destroyed at the beginning of the process is infinitely smaller than the number which reproduce. If one bacterium is bacteriophaged while 100 reproduce, macroscopically it will be impossible to detect this destruc- tion. Only when the number of bacteria destroyed by bacteriophagy exceeds those bacteria which remain normal and which continue to multiply is it possible to perceive the change, and then macroscopic clearing of the medium begins. The facts that the bacteria undergo destruction through bursting and that the increase in the number of bacteriophage corpuscles is intermittent, as may be clearly observed at the beginning of the proc- ess, as well as the fact that the greatest multiplication of the bacterio- phage coincides with the moment when rupture of the bacterial cells occurs at the greatest rate can hardly leave a doubt concerning the mechanism of the liberation of the young corpuscles at the time of the bursting of each bacterium. THE MECHANISM OF BACTERIOPHAGY 123 The influence of temperature upon multiplication In the communication already mentioned Doerr and Griininger have also stated that the bacteriophage does not multiply at a tem- perature of 43°C.; a temperature at which, nevertheless, the bacteria concerned in the experiment (B. coli) reproduce perfectly. Prausnitz^^^ has carried out an experiment which shows clearly the error cormnitted by Doerr in his effort to generalize from the results of one experiment. I have reserved discussion of this experiment of Prausnitz until this time and it is here presented since it affords certain clues concerning the manner in which the bacteriophage multiplies at different tempera- tures. In the experiment here given a Flexner-bacteriophage was used. The culture medium was a bouillon with a pH of 7.6. The results obtained at different temperatures are given in table 11. TABLF 11 TEMPERAIURE 43° TEMPER ATURI 45° TEMPERATURE 47° TIME OP INCU- BATION Bacteria + bacteriophage Bacteria alone Bacteria + bacteriophage Bacteria alone Bacteria + bacteriophage Bacteria alone Bacteri- ophage Bacteria Bacteri- ophage Bacteria Bacteri- ophage Bacteria hours 100 380 140 0.1 116 265 12 180 120 1 100 180 0.7 110 260 17 185 88 2 600 172 195 1.5 186 228 15 150 80 3 500 193 1.8 148 238 23 220 74 4 700 226 236 1.4 140 214 12 160 62 6 900 160 206 1.1 97 230 13 115 58 8 1300 170 210 1.2 105 220 3 106 34 10 3400 185 185 2.0 90 111 2 22 20 24 6200 95 138 37 58 0.1 3 4 The number of bacteriophage corpuscles are determined upon the basis of 10~2 cc, that of the bacteria on 10~^ cc. Prausnitz calls atten- tion to the fact that in this experiment, at 43°C., the number of bac- teria increases two tunes and the number of bacteriophage corpuscles 62 times. At 45°C. the number of bacteria did not increase while at the 'tenth hour the corpuscles were 20 times as numerous as at the beginning. Even at 47°C. the corpuscles appear to begin to multiply during the early hours of the experiment. We have seen elsewhere (Chapter I) that in working with the Coli- bacteriophage a complete dissolution of a suspension containing 200 124 THE BACTERIOPHAGE AND ITS BEHAVIOR million B. coli per cubic centimeter was obtained within a very short time (3| hours) in an incubator at 46°C. In this experiment I did not measure the rate of increase of the corpuscles but it is certain that at the moment when bacteriophagy was complete the multiplication must have been considerable.* Multiplication as affected by the state of the bacteria Doerr and Griininger^^^ have suggested that when the bacteriophage is inoculated into an actively growing bacterial culture the bacteriophage develops immediately, without a latent period. It has been impossible for me to verify this, although I have made many experiments to this end with bacteria of different species and with races of the bacteriophage of diverse activities. Data on 21 such experiments are at hand, all carried out in the same manner, inoculating the bacteriophage corpus- cles into cultures containing about 50,000,000 bacteria per cubic centi- meter, (faintly turbid) and incubated at 36°C. for 5 hours before the introduction of the bacteriophage. Titrations of the bacteriophage made every 15 minutes have shown the minimum time before which the first increase was to be observed was 45 minutes, and indeed this was obtained in only one of the experiments. On this occasion al- though present, the increase was sHght (3-fold). In this same experi- ment the bacteriophage had increased 39-fold after 60 minutes; 41-fold after 75 minutes. In this single case the first rupture had taken place after 45 minutes. The first series of ruptures was complete after 1 hour. In 12 other experiments of this same type the first increase took place after 60 minutes (4 races of Coli-bacteriophage, 5 of Shiga-bacterio- phage, and 3 of Typhoid-bacteriophage; all very virulent races). In the other 8 experunents the first increase could be detected only after 90 minutes. And in all of these, races of the Coli-bacteriophage of relatively high activity were used. If we compare these experiments with the other results which have been mentioned it appears that although multiplication may not start immediately, the rate of reproduction may be accelerated when the bacteriophage is inoculated into an actively developing culture. While superficially this fact might appear to be significant, in reality the result is simply due to the fact that the temperature is favorable (37°C.) for the process at the moment of inoculation. If bacteria are suspended in a bouillon previously warmed to 37°C. and this is inocu- * But they are weakened, as we will see. THE MECHANISM OF BACTERIOPHAGY 125 lated at once with the bacteriophage multiphcation will take place just as promptly and as vigorously as in an actively developing culture. Multiplication in relation to the number of bacteria bacteriophaged Working with B. dijsenteriae Shiga and with B. coli, Meuli^^- reached the conclusion that the final ''lytic" titre is independent of the initial titre. This deduction is true or false according to the conditions of the experiment ; it all depends on the total number of bacteria available and suitable for serving for the multiplication of the bacteriophage corpuscles. If we combine in a medium a very few bacteria together with a very small number of bacteriophage corpuscles, the corpuscles which find a bacterium in their immediate vicinity readily available will be relatively few and thus the opportunity for multiplication will be restricted. Little by little, progressively, the number of corpuscles will augment, but before the number becomes sufficiently great for all of the bacteria, which meantime have had time to develop, to be "parasitized," a culture equivalent to several hundreds of millions of bacteria per cubic centimeter will have had time to mature. From this it is apparent that when but very few corpuscles are inoculated the initial titre of the bacteriophage is to a degree immaterial and has no great effect upon the final titre. Incidentally this view of Meuli is in some respects in accord with what I stated in the first edition of my collected papers,^-^ namely; "In a word, whatever may be the original titre of the suspension at the time when it is inoculated with a limited number of bacteriophagous organisms the latter must always operate on a suspension of about 650 million bacilli per cubic centimeter, since in all cases the bacilli reproduce until they attain this number." But MeuU has gone further, he has generalized, and his conclusions are entirely false when the conditions are changed. If the medium contains a small number of bacteria and a relatively large number of bacteriophage corpuscles, the final titre depends upon the initial titre in the sense that it varies but little, and solely in proportion to the number of bacteria implanted. This is to be interpreted in this way: each of the bacteria present at the moment of inoculation is in close proximity to one of the corpuscles, since the latter are very numerous, and all of the bacterial cells are parasitized and dissolved before they have had time to multiply to any appreciable extent. To state the situation correctly, it may be said that the final number of corpuscles depends upon the number of susceptible bacteria sub- 126 THE BACTERIOPHAGE AND ITS BEHAVIOR jected to bacteriophagy. This is true whether the process takes place solely with the bacteria implanted when the number of corpuscles inoculated is sufficiently great for all of the bacteria present to be bacteriophaged at the beginning, or whether, because of the small number of corpuscles present at first, the bacteria implanted have had time to multiply before they are subjected to bacteriophagy. Between these two extremes, — 1 corpuscle to 650 million bacteria, and 10,000 million corpuscles (and even more with the Staphylo- bacteriophage) to 1 bacterium,- — by varying the relative concentra- tions of the two factors there is an infinite number of combinations and of differing situations. But in every case the final number of corpuscles is determined by the number of bacteria susceptible to bacteriophagy, and, consequently, capable of serving for the multipli- cation of the corpuscles. Influence of the conditions of the medium And yet, the statements made in the preceding section are true only when the conditions of the medium are optimum for the process of bacteriophagy. For example, if we vary the reaction of the medium the final result of the multiplication of the corpuscles will vary, even though in all cases bacteriophagy may be complete. The following experiment clearly demonstrates this fact. A peptone water (peptone 25 grams, NaCl 5 grams, water 1000 cc.) is rendered neutral to phenolphthalein. The medium is definitely alkaline to litmus. After it has been distributed in 10 cc. amounts into tubes, HCl is added in appropriate amounts to provide a series of tubes having an increasing scale of acidity. All of the tubes are im- planted with a concentrated suspension of Shiga bacilli to give a nor- mal suspension, that is, 250 million per cubic centimeter. Then each tube is inoculated with 0.001 cc. of the bacteriophage. After an incubation period of 24 hours simple observation of the tubes indicates varying degrees of turbidity, and appropriate counts indicate the final number of corpuscles present in the individual tubes. In tabu- lated form the results of such an experiment are as shown in table 12. Here are, for example, two strictly comparable experiments which show that this is indeed the case. Inoculate 10 cc. of a suspension of staphylococci containing 50 million bacteria per cubic centimeter with but a single bacteriophage corpuscle. A count made after 3 days shows that although the medium is perfectly clear there are pres- ent 81,000 million corpuscles. Inoculate a like suspension (20 cc. of THE MECHANISM OF BACTERIOPHAGY 127 suspension was originally prepared and divided into two equal por- tions, one part being used in the test presented above) with 500 milHon corpuscles. After 3 days (bacteriophagy was complete in less than 24 hours) the number of corpuscles was 7000 million. Cause of the arrest of multiplication It may be asked why the bacteriophage ceases to multiply when the medium contains a certain number of them, even though bacteria are still present. Quite as logically it might be asked why bacteria stop multiplying even though food materials are left in the medium. The NUMBER OP TUBE REACTION TO MACROSCOPIC APPEARANCE OF THE BACTERIOPHAGE PHENOLPHTHALEIN SUSPENSION AFTER 24 HOURS CORPUSCLES PER CUBIC CENTIMETER 1 Very slight clouding 400,000,000 2 _2 Very slight clouding 500,000,000 3 -4 Clear 500,000,000 4 -6 Clear 1,250,000,000 5 -8 Clear 2,750,000,000 6 -10 Clear 1,000,000,000 7 -12 Clear 1,000,000,000 8 -14 Slight clouding 250,000,000 9 -10 Turbivl 500,000 10 -18 Turbid 1,000,000 11 -20 Turbid 500,000 12 -22 Turbid None answer is the same in both cases. Multiplication stops when the prod- ucts resulting from the "vital reaction" reach a certain concentration. Insofar as bacteriophagy is concerned, let us note first that with all of the conditions best suited to bacteriophagy the final number of corpuscles differs with the bacterium attacked. With the most active races of the Shiga-bacteriophage I have never obtained a final titre greater than about 10,000 million per cubic centimeter. With the Staphylo-bacteriophage the final titre often goes above 100,000 milhon. In the first chapter the statement was made that the substances resulting from the distinctive activity of the bacterium, that is, those substances which "vaccinate" the medium against the bacterium, do not exert an inhibitory effect upon bacteriophagy. The experi- ments carried out with Shiga baciUi leading to this conclusion-^ ^ have been confirmed by Maitland.^^^ 128 THE BACTERIOPHAGE AND ITS BEHAVIOR Another experiment, performed with the staphylococcus, may be cited since it substantiates further this conclusion. A flask containing 250 cc. of bouillon (pH 7.8) is seeded with a strain of Staphylococcus aureus. After incubation for 15 days at 27°C. the culture is filtered through a Chamberland candle. Two series of tubes are then prepared as follows: Series I Tube 1. 10 cc. of fresh bouillon Tube 2. 7.5 cc. of fresh bouillon + 2.5 cc. of the filtrate Tube 3. 5 cc. of fresh bouillon + 5 cc. of the filtrate Tube 4. 2.5 cc. of fresh bouillon + 7 .5 cc. of the filtrate Tube 5. 10 cc. of the filtrate, undiluted with bouillon To this series of tubes a suspension of the staphylococcus is added, the strain being the same as that used for the preparation of the filtrate. After 24 hours all of the tubes are turbid, but the turbidity in tube 5 is about half as great as that in tube 1. After 48 hours all tubes show the same degree of turbidity.* Series II The initial mixtures of fresh bouillon and of filtrate are the same as those in series I. To the 5 tubes a suspension of the staphylococcus is added to provide approximately 125 million organisms per cubic centimeter. All of the tubes are then inoculated with 0.001 cc. of Staphylo-bacteriophage. After 24 hours, the dissolution is complete in tubes 1 and 2, partial in the other three. After 48 hours it is com- plete in all. The number of corpuscles, per cubic centimeter at this time is: Tube 1. 52,000 million Tube 2. 46,000 milHon Tube 3. 38,000 miUion Tube 4. 44,000 million Tube 5. 50,000 mUlion I have not been able to determine the cause of these differences, but in spite of this variation it is possible to conclude that the products resulting from the distinctive activity of the bacterium itself have no effect upon the phenomenon of bacteriophagy, nor upon the multipli- cation of the bacteriophage corpuscles. * According to this experiment the staphylococcus, at least the strain under examination, has but little "vaccinatinj!;" activity. THE MECHANISM OF BACTERIOPHAGY 129 The situation is quite different as regards the effects of the products resulting from bacteriophagy. The following experiment is illustrative. A bouillon suspension containing 250,000,000 bacilli per cubic centimeter is inoculated with 0.001 cc. of bacteriophage suspension. The next morning, that is, after 14 hours, dissolution is complete. A count shows that there are 1600 milHon corpuscles per cubic centi- meter. At this time a concentrated bacterial suspension is added to the dissolved suspension to again yield 250 million bacteria per cubic centimeter. Seven hours later the medium is again clear, and a count shows that there are in each cubic centimeter 2100 million corpuscles. This second dissolution being completed the bacterial count is again restored. This time the dissolution is not quite complete after 48 hours; the medium still shows a slight clouding. The count is 2400 million. At this time, then, the medium contains in each cubic centi- meter the dissolved substance of 750,000,000 bacteria. For the fourth time the suspension is made up to a bacterial count of 250 million. After incubation for 8 hours the clearing is shght. The count now is 2600 million corpuscles. Inoculations upon agar or into broth remain sterile. From this it is clear that the more concentrated the medium becomes in dissolved substances the more marked becomes the inhibition and the less effective the process of bacteriophagy. As a matter of fact, such a result is not unexpected. Bacteriophagy and the resulting multiplication of corpuscles follow a general biological rule. Whether it be a bacterial culture, whether it be an enzyme reaction, whether it be bacteriophagy, the products resulting from all biological reactions first retard, then prevent, the reaction from con- tinuing indefinitely in the same medium. In concluding this section mention may be made of a statement by Bail and Matsumoto^"^ to the effect that there should be produced, in the course of bacteriophagy, as many bacteriophage corpuscles as bac- teria that have been destroyed. Nothing is less true. All experimental work demonstrates that the proportion of bacteriophage corpuscles which are formed, in proportion to the number of bacteria destroyed, may be 100 to 1, and even more. In order to demonstrate the error of these authors it is only necessary to count the corpuscles after bac- teriophagy of the staphylococcus. With potent races of the bacterio- phage one may readily find 100,000 million corpuscles per cubic centi- meter. Simply start with a staphylococcus containing 100,000 million cocci per cubic centimeter and see if the statement of Bail and Mat- sumoto is correct. 130 THE BACTERIOPHAGE AND ITS BEHAVIOR 5. BACTERIOPHAGY UNDER THE MICROSCOPE We have already seen how the destruction of the bacteria takes place through the action of the bacteriophage. Here are a few other observations made in studying the course of bacteriophagy with B. dysenteriaeP'^ We know that if the inoculation of the bacteriophage has been massive, all of the bacteria are attacked at the outset; the fixation of the corpuscles takes place immediately. If a very active race of the bacteriophage is used, within 2 or 3 hours the medium commences to clear little by little, and becomes completely limpid after a short time. If, on the contrary, the inoculation is minimal, the few corpuscles inoculated only affect an equal number of bacteria; the great majority remain unaffected and multiply as they would in a normal medium. But the corpuscles likewise multiply, following a progression more rapid than that pursued by the bacteria, so that within a few hours their number becomes equal to, or greater than, that of the bacteria. This is the time when macroscopic dissolution becomes evident. Let us consider the first case, that of the massive inoculation. If we take from time to time a drop of the suspension up to the point when dissolution is complete, spread these drops on slides and stain, either with the Gram stain, with carbol-thionin, or by the Romanowsky- Giemsa method (all staining methods give essentially the same picture), results such as the following are secured. A suspension of Shiga bacilli, 250,000,000 per cubic centimeter is inoculated with 0.1 cc. of a suspension of the bacteriophage and incu- bated at 37°C. After fifteen minutes it appears as a culture of normal bacilli. After thirty minutes it appears essentially the same, except that a few of the bacilli are poorly stained. After forty-five minutes about 10 per cent of the organisms stain poorly. Between one and two hours, the number of bacilh which stain badly continues to increase, and after 2 hours only a rare cell can be found which has taken the stain normally. At the same time, amorphous debris and granulations, derived most certainly from the bacteria al- ready dissolved are seen. Similar material is seen very abundantly in old normal cultures of the Shiga bacillus. These granulations dissolve more slowly than the remaining portions of the bacterial protoplasm. Finally, and this is a most important point, spherical forms, more or THE MECHANISM OF BACTERIOPHAGY 131 less ellipsoidal, of variable dimensions, always rare, measuring 4 to 7 by 3 to 5 M may be detected. We will see in a moment to what they are due. There are occasional bacillary forms, well-stained, having a length of from 8 to 12 yu. Between the second and third hours the amorphous debris consider- ably augments and the bacillary forms rapidly disappear. A few spher- ical forms are still to be seen. After four hours, solution becomes more and more complete. Only a single poorly stained bacillus will be found in two or three fields. Gradually the formless debris disappears, and, in turn, the granules. After thirty-six hours nothing whatever can be distinguished in stained preparations. With the ultramicroscope at no time can there be seen elements other than the bacilh (whose number gradually diminish, to disappear entirely in about two hours) and the extremely fine granules. It can hardly be said that the latter represent formed elements. At the beginning the bacilli present a normal appearance. After forty-five to sixty minutes fine granules are seen, ever becoming more and more abundant within the interior of the bacterial cells. The number of bacterial cells containing granules also rapidly increases with a cor- responding diminution in the number of normal bacilli. Not all of the amorphous material seen in the stained preparation is to be seen under the ultramicroscope. Apparently, strongly im- bibing water, it assumes the same refractile index as the medium. This amorphous debris is certainly composed of the "floccules" which remain after the rupture of the bacteria, floccules which hydrate gradu- ally and which thus become invisible under the microscope even though they still take the stain. Neither in the stained preparation nor under direct examination can corroded bacteria be observed. At the stage of the process when the number of refractile granules is the greatest the swelling of the bacteria, of which we have spoken, is particularly noticeable, and it is interesting to note that these dis- tended bacteria are the ones which contain the greatest number of refractile corpuscles. The number of corpuscles reaches its maximum within those bacteria which are spherical and ready to burst. What do the fine granules that can be seen under the ultramicroscope represent? While nothing can be affirmed with absolute assurance there is nothing to preclude the supposition that they represent the corpuscles of the bacteriophage, basing this upon the comparative examination of suspensions in which the number of corpuscles has 132 THE BACTERIOPHAGE AND ITS BEHAVIOR previously been counted. By such a procedure it is found that in taking two cultures presenting a great difference in count, a parallelism is always to be noted between the counts and the number of granules observed. It would likewise be well to recall what we have already seen with reference to the multiplication of the corpuscles, namely, that this multiplication appears to take place in successive jumps (which cor- respond to the rupture of a large number of parasitized bacilli) in which the number of corpuscles liberated after 1| to 1| hours cor- responds to about 18 to each single one inoculated. And we will see that the number of granules consequent upon the rupture of a cell amounts to between 15 and 25. There is, therefore, a great probability that the granules are actually the ultramicroscopic bacteriophagous corpuscles. We may consider a second case, that of a minimal inoculation. In this case the medium becomes more and more turbid before disso- lution actually commences. A suspension of Shiga bacilli, containing 250,000,000 per cubic centimeter is inoculated with 0.0001 cc. of a suspension of the bacterio- phage, a very active race being selected. After 30 minutes the medium has its original turbidity; essentially that of a normal culture of the Shiga bacillus. After one hour the original turbidity is still maintained. When smeared and stained all the baciUi are of normal shape, but an occa- sional form stains poorly. After two hours the culture is about twice as turbid as at first. There is amorphous debris in the bottom of the tube. All of the bacilli appear to stain normally. Many of the bacilli (about two in every three) are about four times the normal length, that is, of the bacilli used to seed the culture, and there are all intermediary forms. Oval and spherical forms are relatively numerous, but they are always fewer than would be expected from a comparative ultramicroscopic examination. These forms are indeed very fragile and are particularly liable to destruction during fixation upon the slide so that their demon- stration in stained preparations requires great care. After three hours the suspension is slightly cloudy. The bottom of the tube is covered with fine debris without definite form, with, from place to place, great amorphous masses and numerous granules re- sembling those encountered in very old cultures of normally grown Shiga bacilH. Only a single spherical fonn can be detected in a ten- THE MECHANISM OF BACTERIOPHAGY 133 minute search. Each field may contain a dozen large bacilli, well stained. After four hours the turbidity is very sHght. There is somewhat less material in the bottom of the tube, and this shows only a single poorly stained bacillus to a field. After sLx hours the medium is limpid. There is still less deposit in the bottom of the tube and it is with difficulty that a single poorly stained bacillus may be found in searching 25 fields. After eighteen hours nothing at all can be seen in the preparation. As is evident, the aspect of this preparation differs but little from that seen in the former case, the only departure being that the bacilli which have grown immediately after inoculation, before the action of the bacteriophage becomes operative, present abnormally large forms. A comparable ultramicroscopic examination in the two cases shows that in the last, where the inoculation was made with a bacteriophage which was extremely active, at the time when dissolution occurs with greatest intensity, that is, between two and three hours after the inocu- lation, the spherical forms were present in greatest numbers. There were as many as two to three to a field, and their rupture was readily observed. When the bacteriophagic process is once terminated the most careful search fails to reveal such forms. It is here fitting to recall an observation already made which should be noted by those wishing to investigate the subject. When a simple fermentative action is operative it proceeds with uniform rhythm when under identical conditions. This is not the case here. Up to the pres- ent time more than a hundred different races of the Shiga-bacterio- phage have been isolated and no two of them have been found to con- duct themselves in an exactly identical manner. The final result is always as has been indicated, the phases of the phenomenon always pro- gress in the same order, but the time of the reaction will vary. With one race of the bacteriophage complete dissolution is obtained in three hours, with another, only after twelve hours. The phases follow each other in one case four times more quickly than in the other. Another point which should be remembered is that all that which has been said up to the present time has been in reference to bacterio- phagous races which were extremely active; that is to say, races capa- ble of producing a complete dissolution of a normal suspension of bacteria. A summary of the foregoing shows that, in so far as the microscopic observations are concerned, there is no time when one can distinguish 134 THE BACTERIOPHAGE AND ITS BEHAVIOR in stained preparations, whatever the magnification, microorganisms other than B. dysenteriae. We have already stated that corroded or partially destroyed bac- teria, such as would necessarily occur if the dissolution was made from the outside inward, are never seen. Destruction always takes place by rupture. This is true not only for the dysentery bacillus but for all bacteria which undergo bacteriophagy. Furthermore, for all species the ''microscopic" picture of the phenomenon is the same. Examination under the ultramicroscope clearly indicates, then, that the bacteriophage corpuscles multiply within the interior of the bac- terial cell, and it is possible that the very fine refractile granules, so small as to approach the limits of visibility with the dark-field, ob- served within the interior of the bacteria in process of being bacterio- phaged represent these corpuscles. These granules are still visible in the floccules which float in the liquid for some time after the rupture of the bacteria. They cease to be visible when the floccules are com- pletely dissolved. This part of the discussion is, evidently, only an hypothesis, for as yet it is impossible to affirm that these fine corpuscles may not be due to changes in the bacteria. Nevertheless, the "coincidences" argue in favor of their bacteriophage nature. The fact that these granules are only visible when within the bacteria and that they cease to be so when they are free in the medium is not a basic objection to this view. That the bacteriophage is of corpuscular nature is undoubtedly true; indeed the fact is no longer questioned.* We will see that its dimensions are essentially the same as those of the protein micella. Its diameter has been determined by Prausnitz in one way and by von Angerer in another, and both methods agree in placing the size at between 20 and 30 millicrons. If it is not visible under the ultramicroscope it is most certainly because of its strong power of imbibition; the same thing that prevents the protein micella from being visible,! namely, because their refractile indices are essentially the same as that of the Hquid in which they are suspended. But the index of refraction of the bacterium is certainly different from that of the liquid medium, as is shown by the fact that they are perfectly visible without staining. It follows therefore that the index of refrac- * Doerr is the only author who is not quite convinced upon this point, although he does not deny it. f Metallic micella, even those whose diameter is much less, are visible because the index of refraction differs from that of the liquid. THE MECHANISM OF BACTERIOPHAGY 135 tion of the bacteriophage corpuscle must be different from that of the substance of the bacterium within which it multiples. And it violates no fundamental principle to assume that the corpuscle can be visible when found enclosed within the substance of the bacterium or even in the floccules before they dissolve, and that it may cease to be visible just as soon as these floccules are dissolved. However this may be, the visibility of the bacteriophage corpuscle within the bacterium is only an hypothesis, but it is a plausible and possibly a probable hy- pothesis. In concluding this section we may call attention to two facts which tend to show that, under the action of the bacteriophage, the electrical potential of the bacteria (which are, as we know, negatively charged) is diminished. In the first place it can readily be shown that their affinity for basic dyes is reduced, and in the second place, very frequently an agglutination takes place; the bacteria flocculate under the action of the bacteriophage. Flocculation results from an increase in the surface tension, associated with a reduction in charge. RESUME The bacteriophage corpuscle is unable to multiply in any medium in the absence of living and normal bacteria. The bacterial cell con- stitutes the sole culture medium for the bacteriophage (d'Herelle^^"). An experiment of WoUmann^^^ suggests that development, to some de- gree, may occur in the presence of diffusible bacterial products. The first act of bacteriophagy consists in the approach of the bac- teriophage corpuscle toward the bacteria, then in the fixation of the corpuscle to the latter (d'Herelle^^^). The rapidity with which fixation takes place depends upon various factors; principally upon the degree of activity of the bacteriophage. Fixation is the more rapid the higher the virulence of the bacteriophage corpuscle. The fixation is specific, that is to say, that it takes place only with susceptible bacteria (d'Herelle^-0> and it may occur even if the bacteria are dead (da Costa Cruz^^^). There is, however, an exception to this; the bacteriophage corpuscle fixes itself upon a bacterium naturally refractory to the action of this bacteriophage provided the latter at- tacks other strains of bacteria belonging to the same species (Janzen and Wolff^^^). This is not true for bacteria with an acquired resistance. The bacteriophage corpuscle penetrates into the interior of the bacterial cell. When, as a result of its faculty of multiplication, the bacteriophage corpuscle which has penetrated into the bacterium forms 136 THE BACTERIOPHAGE AND ITS BEHAVIOR a colony of a number of elements, the bacterium ruptures suddenly, liberating into the medium the young corpuscles which are then ready to continue the action (d'Herelle^''^'^^0- The extent to which the bacteriophage may multiply in the course of the process of bacteriophagy, that is, the final titre of the suspension, depends upon various factors, but the factor having by far the greatest importance is the total number of bacteria capable of being bacterio- phaged, and consequently available for serving as a ''culture medium" for the bacteriophage corpuscles (d'Herelle^-'). CHAPTER IV The Virulence of the Bacteriophage 1. variation in the activity of bacteriophage corpuscles Among the very first of the facts revealed by my early studies^^" '^^^ was the observation that bacteriophage principles, as isolated from natural sources, presented very considerable differences. Subsequent study has afforded abundant confirmation of this. With regard to their action upon a single bacterial strain different races of the bacteriophage possess differing degrees of activity. Just as there are races which provoke within a few hours a total dissolution of all of the bacteria to be found in a turbid suspension, so also there are other races of so low an activity that their presence can be detected only by the demonstra- tion of the rare and minute plaques which they form upon agar. Early in the first chapter the technic for disclosing the presence of the bacteriophage in different types of material was described. If, following this technic, a series of studies are undertaken for the purpose of isolating races of the Shiga-bacteriophage, for example, it will quickly become apparent that when bacterial suspensions, identical except for bacteriophage material, are inoculated with equal quantities of different filtrates a complete dissolution of the bacteria is not always obtained. The following experiment is ample to demonstrate this. Inasmuch as the intestinal contents of animals, — horses and fowl, in particular — almost always contain a bacteriophage active against B. dysenteriae Shiga,^^"' procure a dozen specimens of feces from animals of these species. Prepare filtrates according to the method described (Chapter I) for worldng with such materials. At the same time prepare 12 tubes from a young agar culture of B. dysenteriae Shiga, each tube containing 10 cc. of a broth suspension having 75 million bacteria per cubic centimeter.* The turbidity of such a suspension is slight, yet the broth is definitely clouded. Add to each of the tubes 5 drops of one of * I have shown that for such a study it is preferable to use suspensions con- taining but 75 to 100 million bacteria per cubic centimeter instead of bouillon cultures. 137 138 THE BACTERIOPHAGE AND ITS BEHAVIOR the 12 filtrates. Place them in the incubator at 30°C.* After incubation for 24 hours it will be found, as a usual thing, that some of the suspen- sions are limpid, indicating thus the presence of a very active bacterio- phage. Other tubes are but slightly less clouded than the control sus- pension without added filtrate. Others are as turbid as the control, sometimes even more so.f Filter these cloudy, or turbid, suspensions through candles. Add 1 cc. of each of the filtrates to a tube containing 10 cc. of a suspension (250 million per cubic centimeter) of Shiga bacilli, and immediately spread 0.05 cc. of the mixture upon a plate or an agar slant. After incubation, some of these agar cultures will appear sterile; others will show confluent or isolated plaques. In some tubes the plaques will be large; in others small, even pin-point in size. This experiment shows that different bacteriophage races, although active for a single strain of bacteria, present a whole range of potencies. Some of the races cause a prompt and complete dissolution of heavy sus- pensions; others can be detected only by the formation of minute plaques upon the agar. Between these extremes are all intermediate degrees of activity. Indeed, it is quite possible that there are still weaker races which escape detection because of an insufficiently dehcate technic. But, it may be said, we know that the bacteriophage principle is formed of corpuscles. May it not be that the differences in activity as manifested by different filtrates are due, not to a qualitative differ- ence among the corpuscles, but rather to a difference in the number of corpuscles present within a given volume of the different filtrates? Is it not possible that the very active filtrates contain a large number of corpuscles, while those which are weak contain but few? Two observations already recorded suffice to show that there is indeed a qualitative difference among the corpuscles. We have seen that as a matter of fact bacteriophagy may be complete in some instances if but a single very active corpuscle is introduced into the bacterial suspension. And yet in other cases, in the suspensions which remain turbid, there may be a great many corpuscles, as shown by the fact that a single drop * At first^i^ I stated that the temperature should be 37°C. More recently Hauduroy has suggested that it is preferable to allow the tubes to remain at room temperature. Taking into consideration the results obtained in all of the experi- ments performed it would seem that with weak races of bacteriophage the results are best when the temperature is held at 30°C. t In general, it appears that the feces of animals contain a more active bac- teriophage in summer than in winter, and that the fecal bacteriophage is more active in hot countries than in cold regions. We will return to this subject in Part III. VIRULENCE OF THE BACTERIOPHAGE 139 planted upon agar yields many plaques. This can only mean a qualita- tive difference. The many corpuscles present in the second case are not as powerful as the single very active corpuscle. The second observation bearing upon the idea of a quahtative dif- ference deals with plaque formation. Each plaque has its origin in a single corpuscle. The plaques vary in diameter with different races of the bacteriophage, and we know definitely that the formation of large plaques corresponds to races of the bacteriophage which cause a com- plete dissolution of the suspension, while those which do not dissolve the bacterial suspension completely yield little plaques. The more potent the corpuscle, the greater the area of the plaque (d'Herelle^'^). The idea of quahtative variation among races of the bacteriophage receives additional support from the fact that everyone who has studied the phenomenon is in agreement upon this point. But what is the real reason for this variation in activity among diff- erent races of the corpuscular bacteriophage? The following experi- ment contributes the answer.'^^ A. Ten cubic centimeters of a suspension of Shiga bacilh are inocu- lated with 1 cc. of a filtrate made directly from the feces of a patient with dysentery. The suspension is held at 37°C. Counts of the corpuscles, made at different times during the incubation, give the following results when 0.01 cc. is plated on agar. When plated immediately, IG plaques develop, representing 1600 corpuscles per cubic centimeter. The filtrate from the feces therefore contained 16,000 per cubic centimeter. After one and one-quarter hours, the count is 40 plaques, or 4000 per cubic centimeter. After two and one-half hours, a 1:10 dilution gives 42 plaques, or 42,000 per cubic centimeter. After three and three-quarter hours, a 1:100 dilution gives 18, or 180,000 per cubic centimeter. After five hours, a 1:1000 dilution gives 4, or 400,000 per cubic centimeter. After fourteen hours, the dissolution is not complete, the medium is cloudy and becomes more and more turbid, so that after forty-eight hours it is very turbid. Here there is an abundant culture, but the solution is never complete. The bacteria have, then, acquired a certain resistance which has allowed them to reproduce in spite of the presence of the bacteriophage. A result of this kind is usual when the filtrate is prepared from a stool taken shortly before the manifestations of convalescence appear. 140 THE BACTERIOPHAGE AND ITS BEHAVIOR B. Ten cubic centimeters of the Shiga suspension are inoculated with 1 cc. of the filtrate prepared from the feces from the same dysentery patient, but collected 24 hours later, the patient now being convalescent. Counts of this mixture give : When plated immediately, no plaques, or less than 100 corpuscles per cubic centimeter. Thus, the filtrate contained less than 1000 per cubic centimeter. After one and one-quarter hours the plating shows no plaques. After two and one-half hours there are 9 plaques, or 900 corpuscles per cubic centimeter. After three and three-quarters hours, in a 1 : 10 dilution, there are 27 plaques, or 27,000 per cubic centimeter. After five hours, a 1:1000 dilution shows 13 plaques, representing 1,300,000 per cubic centimeter. In this last experiment (B) the corpuscles were present in the filtrate in very small numbers, certainly less than 1000 per cubic centimeter, that is, there were less than one-sixteenth as many as in the filtrate of the first preparation (A). Nevertheless, the suspension was completely dissolved in ten hours and the fluid remained sterile indefinitely. It is unnecessary to insert here the many experiments made for the purpose of proving that the multipHcation of the bacteriophage cor- puscles is always proportionate to their activity. All have given results comparable to those presented above: The more active the bacterio- phage the greater the multipHcation of corpuscles. We have already seen in the preceding chapter that with a Shiga-bacteriophage of low activity a single corpuscle yielded only 17 millions after 24 hours, while under the same conditions, a single corpuscle of a very active race gave, in the same length of time, 12,000 milfion per cubic centimeter. With these two races the increase with the second is 700 times that of the first. With the Staphylo-bacteriophage I have observed an increase from 1 corpuscle to 780 millions with a race of average activity, and from 1 to 98,000 milHons with a race that is very active. Here, with two races acting under the same conditions one is 125 times more active than the other. For but sUghtly active races figures still lower have been observed. From these results it may be concluded that activity in the bacterio- phage corresponds to the vigor with which it multipHes at the expense of susceptible bacteria (d'Herelle^^^). This conclusion leads to an interesting deduction: the facts disclose a curious coincidence, not without significance. What do we mean by the VIRULENCE OF THE BACTERIOPHAGE 141 virulence of a pathogenic bacterium? Simply the power to develop within and at the expense of the host, and we consider the degree of virulence to be the higher as this development is the more rapid. Logi- cally, then, if these definitions are correct that which we have termed "activity" in a bacteriophage corpuscle represents a "virulence" in the strictest sense of the word (d'Herelle^^^- ^^^), It is evident, and not without interest, that the term "virulence" as apphed to the bacterio- phage is employed in this same sense by those authors (Otto, for ex- ample) ^^* who still consider the bacteriophage to be a ferment. 2. EVALUATION OF THE VIRULENCE OF A BACTERIOPHAGE The virulence of the bacteriophage being variable from one race to another, it is desirable to be able to express numerically the virulence of a given race. Let us consider the methods which have been proposed, and evaluate them with regard to their precision and their utility. From the very first of my studies I have advocated and employed solely the method involving an enumeration of the corpuscles. To this end I have carefully spread upon an agar slant in a 22 mm. tube, 0.02 cc. of a suspension of the susceptible bacterium containing 250 million bacteria per cubic centimeter inoculated with a dilution of the bac- teriophage of such a titre that, after incubation, the plaques are iso- lated.^-^ In some instances, as an alternative procedure, I have used Petri dishes, and in this case I have spread 0.05 cc, or 0.02 cc. of the suspension, according to the size of the plate.^^^ This method is the only one which should be employed for the study of the phenomenon of bacteriophagy if false interpretations, associated with poor methods of evaluating the bacteriophage, are to be avoided. When it is not essential to obtain results of the greatest accuracy, for example, when it is desired simply to observe the variations in virulence shown by the bacteriophage as isolated from the body at different stages of a disease and during convalescence, a more simple and rapid method may be employed, based upon the development of bacteriophagy in a liquid medium and on the general appearance of agar sub-cultures.^-^ Since it will be necessary, in many cases throughout this discussion to indicate the relative degree of virulence possessed by a given race of the bacteriophage, it may be well to indicate here a method to express this virulence. This system is somewhat arbitrary, but it meets aU practical needs, and will facilitate expression. = no virulence toward a given bacterium. Normal cul- tures of the bacterium develop in bouillon or on agar. 142 THE BACTERIOPHAGE AND ITS BEHAVIOR whatever the quantity of the filtrate from the feces which had been added. + = weak virulence. The growth in bouillon of the bac- terium to which the filtrate has been added is appar- ently normal. Transfer of this culture to agar gives, after incubation, a culture layer showing a few minute plaques. Some of the bacteriophagous corpuscles have therefore attacked the bacteria and have formed colonies. + + = medium virulence. The culture of the bacterium to which the filtrate has been added is almost normal in bouillon. Transfers of this culture to agar give, after incubation^ either a culture layer of the bacterium studded with very numerous colonies of the bacterio- phage, presenting an appreciable surface area, or of fragments of bacterial culture because of the very great number of bacteriophage colonies. + + + = high virulence. Dissolution of a bacterial suspension is obtained but secondary cultures constantly develop. The reinoculations on to agar remain sterile or give only rare colonies of the bacterium. + + + + = extreme virulence. The bouillon suspension shows complete, and, in general, permanent dissolution. Inoculations on to agar always remain sterile. Obviously, it would be possible to establish a more detailed scale of virulence. In fact, this has been done in the curves which will be given in Part III of this text, where the interval between no virulence and extreme virulence has been subdivided into ten steps, in accordance with the aspect of the cultures, the number of colonies of the bacterio- phage, and the size of the plaques, which bear a relation to its virulence. Practically, the appreciation is adequate with four steps, particularly in view of the fact of the extreme variability of virulence in the bacterio- phage in the body of a single individual from one time to another. Perhaps the first method for determining virulence which we should consider is that of Appelmans.^^ Objecting to the method of plaque counting on the grounds that colonies of the bacteriophage upon agar — ■ the plaques — are sometimes difficult to see (an ill-founded objection) and that plaques may be confused with bare spots on the agar due to the method of distribution of the material during the spreading (a difficulty encountered only when the technic is poor) he has proposed a method VIRULENCE OF THE BACTERIOPHAGE 143 analogous to that devised by Miquel for counting bacteria, that is, the procedure known as the ''method of successive dilutions." In accordance with this procedure 1 cc. of the bacteriophage suspen- sion to be "titrated" is added to 9 cc. of bouillon. This gives an initial dilution of 1:10, each cubic centimeter containing 1-10~^ cc. of the original suspension.* One cubic centimeter of this first dilution is removed and introduced into 9 cc. of bouillon. This second dilution is 1:100, containing per cubic centimeter 1-10"^ cc. of the original sus- pension. One cubic centimeter of this dilution is then carried on into 9 cc. of bouillon, giving a third dilution, 1 : 1000, each cubic centimeter containing 1-10^^ cc. of the original suspension. Continuing thus, by- tens, the dilution up to the twelfth tube, a series of dilutions is obtained in which in each cubic centimeter of the individual tubes there is 1-10~^, 1-10-2, 1.10-3, 1.10-4^ 1.10-5, M0-», MO-7, MO-S MO-9, MO-i", 1-10~", and 1-10-^2 ^c. of the original suspension. Having prepared these dilutions Appelmans next seeds each of them with a culture of the susceptible bacterium and allows bacteriophagy to proceed. The greater the dilution with which bacteriophagy takes place the more active was the original bacteriophage suspension. This method has proved very attractive, doubtless because of its appearance of "mathematical" precision. In reahty it has exerted an unfortunate influence upon the study of bacteriophagy, for this pro- cedure is, in great part, responsible for the errors which have been com- mitted by those who have employed it. Several circumstances render results obtained by this method invalid, among which we may mention the following. 1. The method can not be employed for measuring the virulence of a race of bacteriophage of weak virulence. The difficulty here rests in the fact that such a bacteriophage in no way inhibits the development of bacteria. The presence of such races in a fluid medium can be dis- closed only by the development of plaques when this fluid is spread upon agar.^i^ I am aware that many authors (Otto,^^^ and later Doerr'^^^ and their collaborators) have supported the reverse opinion, namely, that a bacteriophage of weak virulence may be detected by "lysis" in bouillon when plaques are lacldng. But these authors have fallen into a double error, as wiU be pointed out shortly. * Obviously, one might take 4.5 cc. of bouUion and add to it 0.5 cc. of the bacteri- ophage suspension. Then proceding by removing 0.5 cc. of this first dilution and introducing it into 4.5 cc. of bouillon, a second dilution would be obtained. Continuing thus a series could be prepared in all respects comparable to the series described. 144 THE BACTERIOPHAGE AND ITS BEHAVIOR 2. With bacteriophage races of average activity ''secondary cultures" (of which we will speak in the next chapter) develop, and where this is the case the method of dilutions gives results which are entirely false. 3. For very virulent bacteriophages the precision of the method is in inverse proportion to the degree of dilution. Yet, with such races, it is in the high dilutions that precision is most essential. As illustrating this, here is a titration of a suspension of Staphylo-bacteriophage made by the dilution method. Duphcate titrations are made. First titration. The last dilution in which bacteriophagy takes place is l-lO^i". From this one might conclude that in 1-10"^° cc. there is but one corpuscle, and that the original suspension contained, therefore, 10,000 million per cubic centimeter. Second titration. In this the last dilution to show bacteriophagy is 1-10~". Applying the same reasoning, this time the suspension should contain 100,000 million per cubic centimeter. Obviously these two determinations present a very considerable discrepancy. By means of counts of the colonies of the bacteriophage developing on agar we find an explanation of the difference. As a matter of fact these counts show that the original suspension contained 81,000 million corpuscles per cubic centimeter. There were in reality 8 corpuscles in the 10 cc. of the 1 •10"^'' dilution. Consequently, the l-10~^i dilu- tion shows, or does not show, bacteriophagy, depending upon whether the particular fraction removed from the 10~^° tube contained, or did not contain, a corpuscle. In the first dilutions, the error resulting from this fact is negligible. It would be a maximum of 90 corpuscles between 10~^ and 10"^, of 900 corpuscles between 10~2 and 10"^. But it becomes enormous in the higher dilutions. The error may amount to 900 million between 10~^ and 10-9; to 9000 million between 10-^ and IQ-i"; and to 90,000 milhon between lO-^" and lO-^i. This error of technic makes it readily apparent that errors of inter- pretation of considerable significance may result from the use of such a method, a fact all the more unfortunate since the procedure has an atmosphere of precision. We have mentioned in the preceding chapter one of the mistakes made by Doerr in relation to the multipHcation of bacteriophage corpuscles. With reference to this method it is interesting to cite an observation of Gratia and de Kruif^^ which, to them,, seemed "curious," although in reahty it is not so strange. These authors prepared a series of in- creasing dilutions of a suspension of Coli-bacteriophage, and found the VIRULENCE OF THE BACTERIOPHAGE 145 last active dilution to be 10~^. Working with these dilutions in a volume of 5 cc, they removed 4 cc. from the last active dilution and distributed it as follows: a. One cubic centimeter into a sterile tube. b. One cubic centimeter into a tube with 10 cc. of bouillon. c. One cubic centimeter into 100 cc. of bouillon. d. One cubic centimeter into 1000 cc. of bouillon. The four specimens were then seeded with a culture of B. coli. Bac- teriophagy took place in all of them. It seemed strange to Gratia and de Kruif that bacteriophagy should occur in the flask containing 1000 cubic centimeters, where the ''concentration of the lytic principle," ac- cording to their expression, was only 10~^^ when it did not take place in the 10"^ dilution when working with 5 cc. quantities. Inasmuch as a long time before this I had shown that bacteriophagy takes place if but a single corpuscle is added to a bacterial suspension,and that bacterial dissolution does not occur if this corpuscle is lacking (a condition re- calhng the "all-or-none law"), the results of Gratia and de Kruif should not have appeared so unusual. Incidentally, the term "concentration" as apphed by these authors to the bacteriophage principle is an expression without significance, and it can only lead to confusion when used in this connection. Werthemann-'^" has proposed a scheme of notation designed to express the "concentration" of the bacteriophage principle. According to this scheme "concentration" is indicated by the exponent of the last active dilution. For example, in the experiment of Gratia and de Kruif already mentioned, the CoU-bacteriophage would be termed active up to the concentration 1-10~^, or eL8 according to the scale of Werthe- mann. Since concentration is not the dominating factor in the phe- nomenon of bacteriophagy one might well question the significance of such a notation. Not only does it aggravate the defects inherent in the method of titration by dilution, but it adds a false conception. To ascertain how invalid such a method is I would recommend that those who employ the procedure repeat the experiment of Gratia. Beckerich and Hauduroy" have proposed the following method. Each of a series of tubes receives 1 cc. of a young culture of the sus- ceptible bacterium. The bacteriophage suspension whose activity is to be measured is then added in decreasing quantities — ^perhaps 1:3 in the first tube, 1:5 in the next, up to a dilution equal to 3 parts in 10,000,000,000. A portion of the contents of each tube is spread over an agar plate, while the remainder is retained in the tube to indi- cate what takes place in a fluid medium. 146 THE BACTERIOPHAGE AND ITS BEHAVIOR After incubation at 37°C. they note the presence or absence of plaques on the plates, and designate the result by Pi, P2, P3, etc., according to the number of plaques. If plaques are abundant they use the terms many, or very numerous. As for the results in the fluid media; Lo indicates no dissolution, Li means a slow and partial dissolution, L2 is a partial dissolution, and L3 impHes a complete solution. Here is an example cited by them, showing that a weak bacterio- phage can not be measured by the dilution method. Indeed, it may not even be detected. Expressed according to this scheme, using a bac- teriophage very weakly active for B. paratyphosus B the results were : Dilution, 1:3; Pmany, Lo Dilution, l:30;Prare, Lo Dilution, 1:700; Po, Lo This method of Beckerich and Hauduroy, based upon the principle which I have advocated ^^^ determines simultaneously both the dissolv- ing power in a fluid medium and the formation of plaques upon a sohd medium, and it permits an approximately accurate titration, disclosing bacteriophage races which would remain undetected by the dilution method alone. Nevertheless, there is one criticism which may be made to the work of Beckerich and Hauduroy. In certain of their titrations with potent Shiga-bacteriophages it appears that the formation of plaques does not always follow when a quantity of suspension, most certainly containing corpuscles, is spread over the agar. I have carried out some thousands of titrations, and I have never observed this sort of a result. It must be due to some error in their experiments which remains undetected. Janzen and Wolff^'^^ effect a titration employing the following technic. 1. One drop of the suspension of bacteriophage to be titrated is in- troduced into a culture of the susceptible bacterium. 2. A tube of bouillon hghtly seeded with culture is also inoculated with 1 drop of bacteriophage suspension. 3. Immediately after the mixture is made 1 di'op of culture no. 1 is spread over an agar plate. After incubation the results with culture no. 1 are recorded as + + + + (complete dissolution), + ++, ++, or + (partial dissolu- tion), or finally as ± if the dissolution is hardly perceptible. With culture no. 2, (which is a seeded bouillon, not a turbid suspension) no growth is expressed by + + + + , very weak, and weak growth by + + + and ++, and growth but slightly less than the control by -{-. Culture no. 3, designed to show plaque formation on agar, gives results VIRULENCE OF THE BACTERIOPHAGE 147 recorded as + + + +, meaning sterility, + ++, meaning confluent plaques, ++, many plaques, or +, indicating that plaques are few. This is an excellent method for approximating quickly the degree of virulence of a bacteriophage, but it is not sufficiently precise for a study of the detailed features of the process of bacteriophagy. The authors who devised the method have used it for studies to which we will give considerable attention, and for which the method yields fully adequate results. Pfreimbter, Sell and Pistorius^°" follow a procedure which consists in adding a drop of the bacteriophage suspension to be titrated to a sus- pension of the susceptible bacterium and spreading a definite quantity of this mixture upon agar plates, immediately after preparing the mix- ture, and again after intervals of 3, 6, and 24 hours. After the plates are incubated the presence of plaques and the number found provide the significant data. This is a very good method, especially for comparing the virulences of races that are weak. With highly active races it is necessary to work with dilutions. According to the technic of Otto and Munter,^**" a culture of the sus- ceptible bacterium is spread over the surface of an agar plate and upon this surface drops of the suspension to be titrated, diluted to different degrees, are deposited. After incubation the areas where the drops of bacteriophage were deposited are observed and the results are recorded as 0, meaning a normal bacterial culture, that is, no bacteriophage, ±, indicating a few plaques,* +i and +2 implying that plaques are numerous or confluent, +3 means that only isolated bacterial colonies develop, and +4 denotes that the surface of the ac,ar is sterile. A record of such a titration, employing this notation, is : Dilution, 1 : 1000, + 4 Dilution, 1:1,000,000, +2 Dilution, 1:100,000,000 ± In effect, this procedure is simply a modification of the method of counting colonies of the bacteriophage. But it lacks accuracy, and it is precisely this defect which has caused those who devised the method to arrive at certain erroneous deductions.^^* For example, they observe that when greater and greater dilutions of a fluid containing the bacterio- phage are made, a degree of dilution is reached where spreadings no longer show plaques, although in this dilution active principle is un- * A rather curious notation, for the sign ± usually indicates a doubtful result. If there are plaques, there is no chance for doubt, the bacteriophage is present. 148 THE BACTERIOPHAGE AND ITS BEHAVIOR doubtedly still present, since it may be revealed by passages. The cause for this difficulty is readily explained. Let us suppose that the hmiting dilution is one where there is but a single corpuscle in the whole volume of the dilution. This is, as we know, sufficient to cause bac- teriophagy. Let us remove a drop of this dilution and spread it upon agar. Either the corpuscle will be in the drop taken or it will remain in the rest of the fluid in the tube, and naturally, the second possibility is the greater. What will happen? In the first case we will have a plaque when the spreading is made on the agar, and as no bacteriophage corpuscles will be left in the dilution bacteriophagy will not take place there. The deduction here would be that bacteriophagy in the fluid medium does not take place when but few corpuscles are present, even though they are very active. In the second case, it may be concluded, as Otto and Munter decided, that bacteriophagy in a fluid medium takes place, even when plaques do not appear after the material is spread upon agar. Both conclusions are equally false, and a defective method of titration is responsible for the errors. Maitland^^- has proposed a method of titration also based upon the principle of counting the plaques found on agar. He prepares 6 dilu- tions, from 10~^ to 10~*', in suspensions of the susceptible bacterium. A fixed and uniform quantity of each of these dilutions is spread upon agar, and after incubation he denotes the appearance of the agar cul- tures by numbers, as follows : 1 = 1 to 10 plaques 2 = 10 to 20 plaques 3 = numerous plaques 4 = confluent plaques 5 = culture broken up into irregular masses 6 = traces of culture growth 7 = culture debris 8 = isolated colonies 9 = but a single colony, or medium sterile Here is an example of such a titration, showing how, by repeated platings he observes the multiplication of the bacteriophage in the course of bacteriophagy. The titration is performed with a culture of B. dysenteriae 24 hours old, to which is added an appropriate quantity of the filtrate prepared from the feces of a patient convalescent from bacil- lary dysentery. From time to time (as indicated) specimens of the mixture are removed, dilutions are made, and agar plates are spread. The results obtained are given in table 13. VIRULENCE OF THE BACTERIOPHAGE 149 This method is certainly a good one, but it would be still more ac- curate and much more simple if the number of plaques, that is to say the number of bacteriophage corpuscles, was plainly stated. Such are the different methods proposed. Can it be assumed that any of them permit a precise evaluation of the virulence of a bacterio- phage? A logical method of titration must take the facts into con- sideration; it must recognize the physical state of the bacteriophage as well as its behavior. We know several facts which should afford a basis for a proper method of titration. Thus, we know (1) that the bacteriophage exists in the form of corpuscles, and (2) that it is possible to enumerate these corpuscles. We also know (3) that the corpuscle possesses a "virulence" in the true sense of the word, that is, it possesses SPECIMEN REMOVED Immediately .... After 10 minutes After 1 hour. . . After 2 hours.., After 3f hours. . , After 5^ hours. . After 8| hours. . After llf hours. . After 25| hours. . After 7 days . . . DILUTIONS PLATED the faculty of multiplying at the expense of bacteria; and (4) that the virulence of bacteriophage corpuscles is variable, the degree of virulence of different corpuscles being strictly proportional to the rapidity with which each race multiphes. From these facts, based upon the experiments detailed in the preced- ing chapters, it follows that if we inoculate a series of tubes containing identical suspensions of a susceptible bacterium with equal quantities of bacteriophage suspensions derived from different sources, we will find, after a given time, the conditions throughout being the same, that the resulting bacteriophage suspensions will each contain a different number of corpuscles. These numbers will be strictly proportional to the virulence of the different bacteriophages.* * It may be noted that if it were possible to effect the same operation with a pathogenic bacterium we could likewise obtain a numerical expression of viru- 150 THE BACTERIOPHAGE AND ITS BEHAVIOR In view of the nature of the bacteriophage and considering the prin- ciples governing its reproductive activity it would seem that the fol- lowing technic should serve best for determining the rate of multi- plication of a race of the bacteriophage. Take a series of 12 tubes, each containing 4.5 cc. of the bacterial suspension with respect to which the virulence of the bacteriophage is to be measured. This suspension should be prepared from a fresh 24- hour agar culture of a known susceptible strain, and should contain 100 million bacteria per cubic centimeter. Introduce 0.5 cc. of the bacterio- phage suspension (whether it be fecal filtrate, bacteriophage culture, or what not) into the first tube of the series. Shake thoroughly. This will give an initial dilution in which each cubic centimeter will contain 1-10~^ cc. of the original suspension. Remove* 0.55 cc. of this 10~^ dilution and introduce 0.5 cc. into a second suspension, giving thus a dilution of 10"^. Spread the 0.05 cc. remaining in the pipette over an agar plate. t After carefully shaking the second dilution remove 0.55 cc. and spread lence. Thus, with 2 strains of B. pestis we could inoculate 2 guinea pigs with a like quantity of the 2 cultures. After a given time, for example, 48 hours, if it were possible to determine the exact number of plague bacilli present in each of the 2 pigs, we would have 2 figures which would represent the relative virulences of the strains. Such a determination would be infinitely more exact than the usual method, based upon the time factor or upon the lethal dose. * Various authors have observed that it is necessary to use a fresh sterile pipette for each successive dilution. This is self-evident, and I have always followed this procedure, for it is obvious, since the bacteriophage is corpuscular, that if but one single corpuscle remains adherent to the wall of the pipette it will suffice to completely falsify the result. This would be much less significant if the bacteriophage were soluble, like an antibody, for example. t It is essential that neither the surface of the agar nor the cover of the Petri dish contain excess moisture which will mix with the drop of suspension and thus form in effect a fluid medium in which the bacteriophage can develop. This would give an entirely erroneous result. To avoid such an excess of water of condensa- tion the agar should be cooled to 50 to 55° before it is poured into the plates, and as additional precautions, when the agar has solidified the covers of the plates should be replaced by fresh sterile covers, and the plates then placed in the incubator until the next day. In this way the agar becomes well dried out and the drop of fluid placed upon the surface evaporates rapidly, thus preventing growth of the corpuscles in the drop of liquid which otherwise would persist for a considerable time before evaporation. It is well to bear in mind the fact that a liberal amount of agar should be used; the layer in the plates should have a depth of at least 3 mm. If the agar is too thin the plaques will be too small, and will form poorly. The reason for this has been discussed. VIRULENCE OF THE BACTERIOPHAGE 151 0.05 cc. on another plate, inoculating the 0.5 cc. into a third suspension. This will give a 10~^ dilution. If there is reason to beheve that the race is very active continue in this same way up to the twelfth tube (dilu- tion 10~^2)_ If i\^Q pace is of but moderate potency it is unnecessary to go beyond the tenth dilution. It is also unnecessary to prepare plates corresponding to the last two dilutions, 10~^^ and 10"^^^ qj.^ jf ^j^g pg^^jg is of less virulence, those corresponding to the 10"^ and lO-^" dilutions. In these extreme dilutions the number of corpuscles is too small for them to appear, except by accident, in the 0,05 cc. spread over the agar. I would recommend placing the suspension dilutions in the incubator at 32°C. To obtain plaques of the largest possible diameter, and for this reason more readily counted, even in the case of weakly active bacteriophage races* it is preferable to hold the plates at a temperature of 20° to 22°C., in an incubator such as is used for gelatin media. After incubation the series of dilutions will permit an evaluation of the intensity of bacteriophagy in relation to the dilution of the active principle. But this is but a tentative measure, the less trustworthy in that in many cases "secondary cultures" associated with the phenom- enon of bacterial resistance, develop, rendering such a titration com- pletely false and obscuring the true result. The exact measure of virulence can be obtained only by counting the colonies of the bacterio- phage. In the series of plates there are always two, sometunes three, plates upon which the plaques are separated and easy to count. Since the quantity of suspension spread upon the surface and also the degree of dilution are known, it is easy to calculate the number of corpuscles contained in the original suspension. This method, so well suited to virulence determinations, is also the most practical procedure for studying the various phases of the phe- nomenon of bacteriophagy. If one wishes to follow, for example, the course of the phenomenon under definite determined conditions, it is only necessary to remove at suitable intervals 0.5 cc. of the medium in which bacteriophagy is taking place and to carry out the technic which has been outHned in order to know the number of corpuscles present at the moment when the specimen was removed. Can the method be used to measure the relative degrees of virulence of different bacteriophages, when by serial passages we know that the materials in question contain the active principle? Here is a typical * We have seen that the diameter of the plaque is proportionate to the viru- lence, at least when the bacteriophage is acting upon strains of the same bacterial species. 152 THE BACTERIOPHAGE AND ITS BEHAVIOR example, involving virulence determinations of three Shiga-bacterio- phages derived from different sources; the first isolated from the feces of a fowl, the second from the feces of a horse, both having undergone but a single culture passage (+ in potency), the third isolated in 1916 from the stools of a convalescent from dysentery, and since that time subjected to about 2000 culture passages. Take 3 tubes, each containing 10 cc. of a suspension of Shiga bacilli with 250 million organisms per cubic centimeter. Inoculate 0.01 cc. of the suspension of bacteriophage No. 1 into the first, 0.01 cc. of bac- teriophage No. 2 into the second, and the same quantity of bacterio- phage No. 3 into the third. After 24 hours at 32°C. examination shows that suspension No. 1 is cloudy, suspension No. 2 is slightly cloudy, and suspension No. 3 is clear. Filter the contents of the three tubes through Chamberland L3 candles, yielding thus 3 bacteriophage sus- pensions. Remove 0.55 cc. of suspension No. 1 and make a series of 10 dilutions, with 8 platings. This should be adequate to measure the activity for this race is of but low potency. For races Nos. 2 and 3, make the larger series of 12 dilutions and 10 spreadings. After incuba- tion the results are as follows : /. Dilutions. After incubation at 32°C. for 24 hours the tubes con- taining fluid media with successive dilutions of the filtrates show : Bacteriophage No. 1; the first 2 dilutions are cloudy, the others turbid. Bacteriophage No. 2; the first 4 dilutions are clear, the others cloudy. Bacteriophage No. 3; the first 5 dilutions are clear, the remainder cloudy. After incubation for 48 hours : Bacteriophage No. 1; all tubes are turbid. Bacteriophage No. 2; the first 5 are strongly clouded, the others turbid. Bacteriophage No. 3; the first 9 are clear, the last 3 turbid. It is obvious that these findings do not express the virulences of the 3 races in a precise manner. //. Platings. These are incubated for 3 days at 20°C., at the end of which time the readings can readily be made, and are as follows : Bacteriophage No. 1. Plating of dilution 10"^; bacterial layer abnormal, roughened. Plating of dilution 10"^; bacterial layer abnormal, roughened. Plating of dilution lO^^; bacterial layer abnormal, roughened. VIRULENCE OF THE BACTERIOPHAGE 153 Plating of dilution 10~^; bacterial layer smooth, with 141 small plaques. Plating of dilution 10~^; layer with 13 plaques (diameter about 0.25 mm.). Plating of dilution 10~^; normal culture without plaques. Platings of dilutions 10~^ and 10~^; normal culture without plaques. Computation upon the basis of the above would indicate that the original suspension contained 26,000,000 corpuscles per cubic centi- meter.* Bacteriophage No. 2. Plating of dilution 10~^; isolated colonies. Plating of dilution 10"^; scattered traces of growth with isolated colonies in the open spaces. Plating of dilution 10~^; the same, except that the fragments of culture are somewhat more extensive. Plating of dilution 10~^; the same, still more culture evident. Plating of dilution 10^^; culture layer with numerous plaques. Plating of dilution 10""; 27 plaques (diameter, 3 mm.). Plating of dilution lO"'^; 3 plaques. Plating of dilution 10~^; 1 plaque. Plating of dilution 10~^; normal culture without plaques. Plating of dilution 10"^°; normal culture without plaques. The original suspension of bacteriophage No. 2 contained therefore, 540,000,000 corpuscles per cubic centimeter. Bacteriophage No. 3. Platings of dilutions 10~^ to 10~^; plates sterile. Platings of dilutions lO""* and 10~^; traces of culture. Plating of dilution lO"**; confluent plaques. Plating of dilution 10~^; 38 plaques (7 mm. in diameter). Plating of dilution 10~^; 5 plaques. Platings of dilutions 10~^ and lO^^"; normal culture without plaques. The original suspension of this race contained, then, 7,600,000,000 corpuscles per cubic centimeter. The number of corpuscles in the three suspensions being respectively 26, 540, and 7600 million per cubic centimeter after bacteriophagy has continued in identical suspensions for the same length of time obviously means that these figures represent the power of multiplication of the * 0.05 cc. of dilution 10~^ containing 13 corpuscles, 1 cc. would contain 20 times as many, or 260. The original suspension would then contain 260 X 10^ = 26,000,000. 154 THE BACTERIOPHAGE AND ITS BEHAVIOR corpuscles of each of the three races. And since virulence, by common consent, is considered as an expression of the power of multiphcation in a foreign host, the respective virulences of the three bacteriophage races above could be represented by the ratios : 26 540 7600 . ^ , , „« .„^ 26 * "26" ■ "26~ " approximately 1 : 20 : 300 If the increase in the number of bacteriophage corpuscles taking place during bacteriophagy is determined for several bacteriophages of dif- ferent virulences it becomes obvious that the numbers derived will be proportionate to the rate of bacteriophagy with each. Such a deduction is, of course, obligatory. The contrary could not be comprehended, since insofar as the study of bacteriophagy is concerned the single meth- od permitting the recognition of the behavior of the bacteriophage con- sists in determining the virulence by means of the increase in the number of corpuscles. From the practical point of view it is convenient to designate the intensity of the virulence of a bacteriophage by the rate of the phenomenon which it causes. Such a procedure is, indeed, quite logical since there is always a parallelism between the rate at which the corpuscles increase and the rate of bacteriophagy. Different degrees of virulence may be designated by the following terms: the determinations being made always in bouillon having a pH of 7.6 to 8.0, and the temperature being between 30 and 32°C. Maximum virulence. Single corpuscles inoculated into 10 cc. of a normal suspension (each cubic centimeter containing 250 million bac- teria derived from an 18 to 24 hour culture on agar) cause complete bacteriophagy. The suspension of the bacteriophage resulting remains clear indefinitely provided the suspension is held at a temperature lower than 32°C. This virulence corresponds to a multiplication of such an order that for each corpuscle inoculated the final number is greater than 10,000 million per cubic centimeter. It may be mentioned that this number is apparently fLxed, that is to say, it does not apply to bacteriophagy of any particular bacterial species. Whatever the race of bacteriophage this titre of multiplication is that of a race of maximum virulence. Very high virulence. Here the phenomenon is the same as in the above case, except for the fact that the suspension of corpuscles resulting from bacteriophagy when a normal suspension is inoculated with a single corpuscle becomes clouded again through the development of a sec- ondary culture. On the other hand, secondary cultures do not usually VIRULENCE OF THE BACTERIOPHAGE 155 form in suspensions where bacteriophagy results from the dissolution of a normal suspension by a large number of corpuscles (a million or more) . Here the virulence corresponds to a multiplication such that for each corpuscle inoculated into the 10 cc, of normal suspension, there is an end result between 5000 and 10,000 million per cubic centimeter. High virulence. Here again the phenomenon is the same, with the exception that with races of this type secondary cultures are the rule, quite without regard to the number of corpuscles inoculated. The viru- lence corresponds to an increase whereby a single corpuscle in the course of the action becomes between 1000 and 5000 milHon per cubic centimeter. Moderate virulence. Bacteriophagy is never complete when a single corpuscle is inoculated. When the inoculation involves a million or more corpuscles complete bacteriophagy of a normal suspension results. The development of secondary cultures is constant. The virulence here represents an increase in the number of corpuscles to such an extent that for each one originally present there are at the end of the process between 100 and 1000 million per cubic centimeter. Weak virulence. Bacteriophagy is never complete whatever may be the number of corpuscles inoculated, even though the suspension con- tains very few bacteria. The opacity of the suspension may be reduced to a greater or less degree. If complete bacteriophagy by any chance takes place the number of corpuscles is always below 100 million per cubic centimeter. Very weak virulence. With such strains no clearing of the medium can be observed. The presence of the bacteriophage can be disclosed only by the development of plaques on an agar medium. The rate of in- crease of corpuscles is very low ; after 24 hours only a few hundred per cubic centimeter are to be found. 3. PURE RACES OF THE BACTERIOPHAGE Different races of the bacteriophage may be isolated which possess virulences for bacteria belonging to different species. On the other hand, the degree of virulence for each of the bacteriophages possessing an activity for a given bacterium is in its very nature variable. As a mat- ter of fact, among the hundreds of bacteriophage races which I have isolated I have yet to find two which are exactly alike. ^^^ It is, there- fore, evident that there are under natural conditions an infinite number of different races of bacteriophage corpuscles.* * This, of course, has no direct bearing upon the question of the unicity of the bacteriophage; a question which will be discussed in a later chapter. 156 THE BACTERIOPHAGE AND ITS BEHAVIOR When we filter a fecal suspension, a specimen of river water, or even tap-water, we obtain a filtrate which is a suspension of bacteriophage corpuscles, but there is no reason for affirming that all of the corpuscles present belong to the same race. Indeed, it is certain that quite the contrary is true. Therefore, it is essential, especially for a study of the processes of bacteriophagy, to isolate the corpuscles so that one may work with strictly pure races, the issue of a single corpuscle. In order to accomplish such an isolation we have only to imitate bac- teriological methods. For example, assume that we have a material containing, aside from a variety of banal bacteria, a very few organisms pathogenic for a given animal, such as the guinea pig. How would we obtain a pure culture of this pathogen? We would inoculate a guinea pig with some of the material, the pathogenic bacterium would multiply within the body of the animal and at the death of the animal we would find a pure culture of the bacterium in the blood or in some lesion. But it is evident that if the initial material contained two bacteria pathogenic for the particular animal used the two organisms might be recovered in a mixed culture. If we wish to separate all of the bacteriophages which may be found in a stool filtrate virulent for a given bacterial species, B. typhosus for example, eUminating all of the races which do not possess a virulence for this bacterium, we have only to adopt an analogous method. Inoculate 0.1 cc. of this filtrate into 10 cc. of a B. typhosus suspension. After 24 hours, bacteriophagy having taken place and the Typho- bacteriophage corpuscles having developed, filter through a candle. Inoculate 0.01 cc. of this filtrate into a fresh suspension of B. typhosus. Incubate, filter, and continue such passages. After a sufficient number of these have been carried out we may be sure that, because of the extreme dilution brought about through the passages, no trace of the original filtrate from the stool will remain. At this time all of the corpuscles present will obviously have reproduced at the expense of the typhoid bacillus; that is to say, there will be present only those resulting from the development of corpuscles present in the initial filtrate virulent for B. typhosus. None of the corpuscles of the original filtrate which were virulent for other bacterial species and non- virulent for B. typhosus will be recoverable after a certain number of transfers. At whose ex- pense could they have multiphed? And if they have not multiplied they have necessarily disappeared when the degree of dilution has reached a certain limit, readily calculable.* * Determination of the limiting dilution in which there no longer remains a VIRULENCE OF THE BACTERIOPHAGE 157 This method of purification by successive passages is the first one that I employed. ^^^ But this method permits, however, the reasonable objections that although it may be true that all of the bacteriophage corpuscles avirulent for the bacterium at the expense of which the pas- sages are made become ehminated it is none the less true that not all of those which are virulent and which have multiplied necessarily belong to the same race. As a matter of fact it is quite possible to assume that in a filtrate of feces or of water not all of the corpuscles virulent for a given bacterium are derived from a single corpuscle. According to this, then, in order to be sure of working with a pure race it is necessary, as is the case in bacteriology, to start with a single corpuscle. There are two methods which will permit this; the method of dilution in liquid media of Pasteur, and the method of colony isolation of Koch. We have seen that when we make serial dilutions, by tens, of a sus- pension of the bacteriophage and add susceptible bacteria to each of these dilutions bacteriophagy occurs up to a certain dilution. In the higher dilutions it does not take place. If we prepare a second series of comparable dilutions in sterile bouillon and distribute the 10 cubic centi- meters of the last active dilution among 10 suspensions of the susceptible bacterium, adding 1 cc. to each suspension, bacteriophagy will take place in some of these 10 suspensions, wherever a single bacteriophage corpuscle had been present. To be more certain of attaining the de- sired end we may filter one of these suspensions and repeat the same operation. This can be repeated even a third time. Such is the method of "successive dilutions" which I have most frequently employed. I prefer it to that of plaque isolation, for reasons which will be explained later. Let us observe that with very virulent bacteriophages determina- tion of the presence or absence of bacteriophagy is simple, inasmuch as when it is present the suspension becomes completely cleared. With less virulent bacteriophage races the formation of "secondary cultures" frequently masks the clearing of the medium. Here, in order to as- certain whether bacteriophagy has occurred it is necessary to spread the suspected suspension on agar and the culture developing there will provide the answer.* finite amount of the original filtrate involves the calculation of that degree of dilution where the volume of the original material present is less than the volume of one electron. The electron being the smallest possible mass of matter, to say that as the result of the successive dilutions there remains in the liquid a quantity of material less than one electron, is to say that there remains a hypothetical but not an actual amount. * The characters of secondary cultures will be discussed in a following chapter. 158 THE BACTERIOPHAGE AND ITS BEHAVIOR The dilution method can be appKed even in the case of races of low virulence, provided bacteriophagy in the hmiting suspensions is con- trolled by spreading them on agar. However, with races of low viru- lence a purification by means of isolation through plaques is certainly less compHcated. We have seen that it is only necessary to touch the centre of a plaque with a sterile platinum wire and then immerse this wire in a suspension of the susceptible bacterium to cause bacteriophagy of this suspension. Each plaque on the agar being a colony of bacteriophage corpuscles resulting from the multipHcation of a single corpuscle, we have here a method of isolation absolutely analogous to that discovered by Koch for the isolation of bacteria. But we may mention the fact that as is often the case with bacteria, the colony developing at a given point on the agar may be derived, not from a single corpuscle, but from a group of two or more. This being the case, it is unwise to rely upon a single isolation. Certainty demands that the process of isolation be repeated two or three times. The method of isolation by plaques has been employed especially by Bail and his collaborators^^ who were the first to call attention to the possibiUty that a filtrate possessing activity for a single bacterial species could contain a mixture of bacteriophage races. Wolif and Janzen''^*' have likewise made this observation in studying different races of Typho-bacteriophage. It may be well to emphasize the fact that if we would effect isolation by the plaque method it is necessary to proceed in the following manner. Inoculate a heavy suspension, 250,000,000 bacteria per cubic centimeter, with a very small quantity of the raw suspension of bacteriophage. When this mixture is spread immediately upon agar the surface should show, after incubation, but very few plaques. If too many corpuscles are inoculated into the bacterial suspension it is possible that several will become attached to a single bacterium and the plaque will be formed of elements not the issue of a single corpuscle. Furthermore, for this purpose Petri dishes should not be used. Agar slants are much better, since they may be placed in a vertical position immediately after plant- ing in such a way that it will insure that none of the liquid remains on the surface. The presence of fluid facilitates the development of the corpuscles, as in a Hquid medium, and if this occurs the isolation will be fictitious. VIRULENCE OF THE BACTERIOPHAGE 159 4. VARIABILITY IN THE VIRULENCE OF THE BACTERIOPHAGE From the time of Pasteur we have known that in a single bacterial culture each of the bacteria possesses certain characters which are pe- culiar to itseK and differentiate it from the associated organisms, even though all of the organisms present the general characters belonging to the species. Such qualities as vitaHty, resistance to heat and to anti- septics differ for every organism within a culture. This fact is partic- ularly striking as regards virulence. For example, let us take a culture of B. pestis and implant it on an agar slant in such a way as to secure isolated colonies. By the inoculation of susceptible animals we can show that cultures of some of these colonies kill the animal in an in- finitely small amount while others are inoffensive in very large quantities. The bacteriophage, just hke the bacterium, is of corpuscular nature and different races of the bacteriophage, just like different strains of bacteria, possess different virulences. One might, indeed, ask if the analogy does not go still further and if in a given suspension of bacterio- phage each corpuscle does not possess a particular virulence. It is very easy to determine that this is indeed the case. Isolate, by means of dilutions, an extremely active Staphylo-bacterio- phage, such a race as I have described in the preceding section. Dis- tribute 10 cc. of the Umiting dilution among 10 suspensions of the staphylococcus, 1 cc. to each. The results of such an experiment will be as follows: After 36 hours : 1 suspension will be clear 1 suspension will be clouded 8 suspensions will be turbid After 48 hours : 2 suspensions will be clear 2 suspensions will be clouded 6 suspensions will be turbid After 72 hours : 5 suspensions will be clear 1 suspension will be cloudy 1 suspension will be very cloudy 3 suspensions will be turbid After 7 days: 5 suspensions will be clear 2 suspensions will be very cloudy 3 suspensions will be turbid Spreadings made upon agar, and repeated passages will show that the 3 turbid suspensions are free of bacteriophage; that is, bacteriophage 160 THE BACTERIOPHAGE AND ITS BEHAVIOR corpuscles were not present in the 1 cc. quantities which each received. As for the 2 cloudy suspensions, they contain a bacteriophage whose virulence is much less than that of the suspensions which had undergone complete bacteriophagy.* It is unnecessary to state that the race of Staphylo-bacteriophage utihzed was derived from a single corpuscle ; as a matter of fact it had passed through multiple isolations carried out at different times either by the dilution method or by the plaque method. This experiment shows that, considering the end result as well as the rate of the reaction, among 7 corpuscles each possessed a different degree of virulence. We must, therefore, conclude that in a suspension of a race of the bacteriophage every corpuscle possesses a special degree of virulence. The same experiment carried out with Coli-, Typho-, and Shiga-bacteriophages has given in every case comparable results. This finding indicates the reason for the differences in virulence of different races of the bacteriophage as they are found under natural conditions. 5. INCREASES IN VIRULENCE We have seen that a bacteriophage suspension, originating from a single very virulent corpuscle, contains corpuscles possessing a virulence equal to that of the original corpuscle which served as the source of the race, together with others whose virulence is much lower. This simply means that virulence may be attenuated, as is the case with bacteria. But Pasteur showed that the virulence of a bacterium is capable of being increased by successive passages within the body of a susceptible animal. Is this likewise true for the bacteriophage? Again the analogy is complete. The virulence of a bacteriophage may be exalted by successive passages in suspensions of a susceptible bacterium (d'Herelle^^"' ^^^). There are several methods by means of which a bacteriophage but slightly active at the time of its isolation may be increased in virulence.^^^ For example, the following procedure will result in such a change in virulence. When an agar inoculation has shown that a bouillon suspension con- tains an active bacteriophage this suspension is filtered through infuso- rial earth and then through a bougie. A slightly turbid suspension is prepared, using the bacterial strain against which the bacteriophage has shown some activity, and into this suspension are introduced some four or five drops of the filtrate. After incubation, if dissolution has not been produced, this second bacterial suspension is filtered as before and * They contain secondary cultures also. VIRULENCE OF THE BACTERIOPHAGE 161 a third suspension is inoculated with four or five drops of the filtrate. Such transfers are continued until evident dissolution occurs. During the process it is easy to verify the presence of the bacteriophage in each passage, and to detect any increase in virulence, simply by spreading the successive cultures on agar slants. Comparison of the cultures secured with each passage reflects the degree of virulence. For ex- ample, the agar growth obtained from the first passage shows a culture growth with ten plaques, the second passage shows 100, with the third the layer of bacillary growth is broken up with an abundance of the areas, while with the fourth passage only a few isolated colonies of bacteria are seen. It can be readily seen that the virulence of the bacteriophage, that is, its ability to develop at the expense of the bacteria, increases with each transfer until a point is reached where complete dissolution of the suspension is obtained.* Usually it is relatively easy to increase the virulence of a weak race of the bacteriophage, but at times it may become very difficult, partic- ularly when working with races active against the Gram-positive cocci. In such cases it is necessary to effect a great number of passages, and there is considerable risk of losing the bacteriophage altogether, partic- ularly during the first few passages. I might cite as an example an anti-staphylococcic race with which Eliava was forced to make passages during four months in order to obtain sufficient virulence to induce complete dissolution of a suspension containing 500 million staphylo- cocci per cubic centimeter. It may be well to emphasize here a point of some importance. We will see that the virulence of the bacteriophage becomes considerably weakened when it is held in contact with bacteria which resist its action. For this reason it is wise to avoid, in each passage, contact with those organisms which have had time to acquire a resistance. To accomphsh this it is best at first to conduct the passages at a relatively low tem- perature; 30°C. is adequate to permit the active development of the corpuscles and it retards somewhat the acquisition of a resistance to the process of bacteriophagy by the bacterium. Bacterial resistance de- * The bacteriophage is not destroyed until a temperature of about 76°C. is reached. Bordet and Ciuca^e have proposed to utilize this fact to avoid filtration. They hold the bacteriophage suspension at 58°C. for an hour during which time the bacteria are killed while the bacteriophage resists. This method of isolating a virulent bacteriophage should never be employed during the course of a series of passages designed to increase virulence. For although heating may not kill, it attenuates the virulence of the bacteriophage with the result that the advantage gained by each passage is lost through the heating. 162 THE BACTERIOPHAGE AND ITS BEHAVIOR velops somewhat more rapidly at a temperature of 37°C., but even at the lower temperature of 30°C. the bacterium reacts vigorously. For this reason it is essential to restrict the period of contact to that just sufficient to afford the corpuscles opportunity to multiply. And at the same time a limited period of contact will prevent the bacteria from acquiring a resistance through processes of adaptation. As a matter of fact practical experience shows that this end is obtained by effecting two passages every day; one in the morning upon arrival at the labora- tory, the other in the afternoon just before leaving. An excellent procedure which provides through serial passages for the increase in the virulence of the bacteriophage by adaptation and at the same time for the selection of those corpuscles most apt at acquiring such a virulence consists in carrying out a series of isolations by the dilution method. The following scheme indicates how this can be effected. Among 10 suspensions, each inoculated with 1 cc. of the last active dilution of a bacteriophage, a certain number undergo bacteriophagy. Select the tube in which the phenomenon has been the most marked.* This suspension is filtered through a candle and the isolation procedure is begun again. Once more that suspension in which bacteriophagy is the most intense is filtered. This procedure is repeated up to the point where the maximum virulence has been obtained. The only difficulty with this resides in the determination of the limit- ing dilution, especially with very weak races of the bacteriophage, or where bacteriophagy can only be disclosed by agar cultures. More- over, in carrying out the passages one can not take time to make the preliminary studies necessary to ascertaining the limiting dilution. In order to obviate this difficulty the increase in virulence may be started by a few simple passages and later, when a degree of virulence has been obtained sufficient to cause bacteriophagy such as can be detected by microscopic examination, the limiting dilution may be determined. When this is once determined the filtrate is utifized and an isolation by dilution is made. The choice from among the bacteriophage sus- pensions falls on the one where the phenomenon occurred most intensely. This one is filtered, and the filtrate serves for a new isolation, arbitrarily choosing as the Hmiting dilution the same as that disclosed by the pre- * Needless to say, if for any reason it is desired to make a selection from among a very great many corpuscles, in the place of utilizing 10 cc. of the limiting dilu- tion in 10 suspensions, one might use 20 cc. of the limiting dilution, or even more, distributing it among 20 or more suspensions. VIRULENCE OF THE BACTERIOPHAGE 163 ceding titration. If this time it is found that the 10 suspensions inocu- lated are bacteriophaged, that one where the process occurred most vigorously is selected and is filtered. For the next passage, as is obvious, the next higher dilution is selected. Simple observation of the 10 sus- pensions here will show the dilution to be used for the following passage. If there are only one, two, or three of the 10 suspensions bacteriophaged, it may be well to use the same limiting dilution as that previously used. If there are 8, 9, or 10 suspensions bacteriophaged it is desirable to take the next higher dilution for the next passage. At first sight this method appears rather complicated but it gives excellent results. In this way I have been able to obtain bacteriophage races causing regularly a complete and invariably permanent dissolution of normal bacterial suspensions. Those who have worked with the bacteriophage realize how difficult it is to attain a virulence sufficiently high to preclude the development of secondary cultures. A number of authors, among whom may be mentioned Izar,^" and Janzen and Wolff"^ have observed that all races of the bacteriophage do not acquire virulence with equal facihty. This is quite true. And in this same connection I have found^^^ that certain races of very low virulence may lose their activity with the first passage, and for this we will see the reason later. But it is likewise true that the mode of pro- cedure has a very great influence upon the result. Just to indicate the importance of the matter of the bacterial strain selected I may state that upon several occasions, when working with a single race of bacterio- phage, I have made two parallel series of passages for the purpose of increasing the virulence. One series was made at the expense of one strain of the bacterium, the second at the expense of another strain of the same species. It was found frequently that virulence increased rapidly with one strain and but very slowly with the other. Further study showed that this difference in result was due to the fact that one of the two bacterial strains developed a resistance far more readily than the other. With certain strains which possess the faculty of developing resist- ance in a high degree, the result of serial passages is not an increase in virulence but the bacteriophage is overcome and sometimes it disappears even in the first passage. We will study the phenomenon of bacterial resistance in the next chapter so that we will not enter into further detail here, resting content with the statement that the choice of the bacterial strain at the expense of which the passages are to be made is of extreme importance. It might be well to add, however, that once the increase in 164 THE BACTERIOPHAGE AND ITS BEHAVIOR virulence for one strain is obtained (when working with organisms be- longing to homogeneous races — we will see later the significance of this) virulence is likewise enhanced for strains which were unsuited to the development of this virulence. The reason for this fact is that when the virulence is once increased, the resistance of the bacterium, which previously would have been able to withstand a low virulence, is over- come by a higher virulence. Upon this point again the story of patho- genic bacteria provides us with ample examples of facts of the same nature. Before ending this section I would like to call attention to an error of fact which has been made by Seiffert."^ This author has assumed that the increase in the virulence of the bacteriophage effected by suc- cessive passages at the expense of bacteria is due to an "adaptation" on the part of the bacteria and not to an adaptation of the bacterio- phage. With regard to "theories" of this type Pasteur stated, "they are under no particular obligations as regards the facts." Just how valid such a theory is may be shown by the following, which indicates exactly the sequence of events leading to the adaptation. In- oculate a drop of filtrate containing bacteriophage corpuscles of low virulence into a suspension of bacteria. After an appropriate time filter through a candle. This effectively discards all of the bacteria which have been subjected to contact with the bacteriophage cor- puscles. Introduce this filtrate, containing no bacteria whatever, into a second suspension of young bacteria — bacteria which have never yet been in contact with the bacteriophage. When bacteriophagy has occurred filter again and discard once more all of the bacteria which have been in contact with the bacteriophage corpuscles. Carry out in this manner a series of passages, introducing a filtration to ehminate the bacteria between each passage. None of the bacteria of the sus- pension in which one passage is made can be carried over into the suspension of the following passage. Under such conditions, since the bacteria are not involved in the passages, how can they undergo adapta- tion? Such an adaptation as a being may acquire can be transmitted only when there are descendents. It is easy to understand that the fact that the virulence of the bacterio- phage is subject to increase is disturbing to the partisans of certain theories which we must discuss later. Nevertheless, if objections are raised to the concept which I have evolved, the opposing arguments should, at least, take the facts into consideration. VIRULENCE OF THE BACTERIOPHAGE 165 6. ATTENUATION OF VIRULENCE Just as it is possible to produce experimentally an increase in the virulence of the bacteriophage, so also is it possible to cause an attenua- tion of virulence. Exposure to high temperatures is one of the methods of causing such an attenuation (d'Herelle and Pozerski^'^^) . The effect of heat is clearly indicated by the data which follow. In the following experiments the suspension of bacteriophage under test, previously filtered through a bougie, is taken up in capillary pipettes, sealed at both ends, and completely submerged in a water- bath maintained at the temperatures indicated in each experiment. In each series of experiments 8 tubes with the suspension are maintained for thirty minutes at temperatures of 60, 62, 64, 66, 68, 70, 72, and 75°C. Anti-Shiga bacteriophage Two drops of the suspension from tubes maintained at 60, 62, 64, and 66°C., when introduced into suspensions of Shiga bacilH, cause complete dissolution in less than fourteen hours. The tests repeated with a second strain of Shiga bacilli give identical results. The bacterio- phage heated to 68 and 70°C. causes dissolution with one strain of Shiga baciUi but not with the other. When heated to 72 and 75°C. the bac- teriophage fails to dissolve the organisms. One drop of each of these suspensions, which had received the bac- teriophage previously maintained at 68, 70, 72 and 75°C., and which had not been submitted to bacteriophagy is planted on slant agar. After incubation, all of the cultures, except the last, which is normal, show plaques characteristic of the presence of the bacteriophage. Serial passages may be effected, thus permitting the enhancement in virulence of the bacteriophage attenuated by the action of temperature. After two such passages, with the corpuscles heated to 68 and 70°C., and after three passages with those heated to 72°C., dissolution in liquid media is obtained. Comparable experiments have demonstrated that the bacteriophagous corpuscles active for B. dysenteriae Flexner, B. dysenteriae Hiss, B. coli, and B. paratyphosiis B, act in a quite similar manner. With the bac- teriophage active for B. paratyphosus A attenuation begins at about 64°C. (at least with the strain tested). With that virulent for B. typhosus attenuation is already apparent at about 62°C. In all cases, when heated to 75°C. the bacteriophage is completely inactive, either 166 THE BACTERIOPHAGE AND ITS BEHAVIOR actually destroyed or attenuated to such an extent that its presence can no longer be detected. In all these instances the bacteriophage shows a recuperative power, the virulence being restored when the tem- perature to which the corpuscles have been subjected is not higher than 72°C. Anti-staphylococcus hacteriophage Attenuation of this bacteriophage is already manifest after heating to 60°G. Sul^cultures of suspensions which have not been dissolved show that it is a simple attenuation, for, even with suspensions inocu- lated with a bacteriophage previously held at 72°C. for thirty minutes, plaques are obtained characteristic of the presence of an active bac- teriophage. Moreover, two passages suffice to restore the original viru- lence to corpuscles heated to 62, 64, 66, and 68°C. After heating at 70 and 72°C. the attenuation of virulence does not disappear until after six passages. When heated to 75°C. the bacteriophage is deprived of all activity. It may be concluded from these experiments that all races of the bacteriophage react to temperature in the same manner. When heated above 60°C. they are attenuated more or less rapidly, the speed de- pending to some extent upon the bacterial species for which they are active. But all are completely killed, or at least paralyzed, at a tem- perature of about 75°C. The results observed in these experiments can not be ascribed to a reduction in the number of corpuscles, for the Shiga-bacteriophage used, like the Staphylo-bacteriophage, had caused complete bacteriophagy in a unit volume before exposure to the high temperature, and it will do the same after the vu'ulence is again increased. My experiments have also shown that bacteriophage races of low virulence are attenuated at lower temperatures than are races possessing a more outspoken virulence for the same bacterial strain. Time also exerts an effect upon virulence, but here there is a very great difference in the effects, depending upon the race of bacteriophage, and especially upon its virulence. Bacteriophage races possessing a high virulence are in general but shghtly modified while those of low virulence are much more sensitive. Asheshov^* was the first to observe that certain races of the bacterio- phage may lose their virulence very rapidly. A Flexner-bacteriophage which I isolated, and which had a relatively high virulence, manifested a very considerable attenuation even within a period of two months, VIRULENCE OF THE BACTERIOPHAGE 167 in spite of the fact that it was preserved in sealed ampoules. Various observations made in the course of my studies contribute additional data upon this question of spontaneous attenuation. One race of the Shiga-bacteriophage, originally very virulent, has been held for a period of 9 years in a sealed tube, and throughout this period it has remained almost as active as it was at the beginning. The sole change consisted in the number of corpuscles, which diminished from 2400 million to 110 million, but despite this reduction in numbers those which survived retained their virulence without change and this viru- lence was equal to that of corpuscles having the same origin but which had undergone, throughout this time, between 1500 and 2000 passages. Other races of Shiga-bacteriophage from various sources, but all very virulent, hkewise maintained their activity unchanged during periods of 3 to 5 years. A very active Staphylo-bacteriophage, held in a sealed ampoule, retained its potency completely for a period of 3 years. A second race, somewhat less virulent, was attenuated after 4 years to such an extent that it was lost in the second passage when an attempt was made to restore its virulence. Two very active races of Coli- bacteriophage maintained their virulence for 6 years. Three races which originally showed a strong virulence, although somewhat less than that of the two races just mentioned, were dead or totally avirulent after the same interval of time.* A Barbone-bacteriophage of high virulence when sealed up in an ampoule had lost a large part of its virulence after 11 months, but with 3 passages the virulence was restored. After 26 months this particular race was dead or completely avirulent in some of the ampoules whereas in others it was still present, although attenuated to such a degree that it was lost during the first passage. A Cholera-bacteriophage tested after it had been preserved for 8 months was completely avirulent. During the attempt at rejuvenation a few minute plaques could be seen in the first passage, but with the second passage it disappeared. A Plague-bacteriophage (of rodent origin) retained almost all of its activity throughout a period of 26 months. Another race (of human origin), somewhat less virulent at the beginning, was very markedly attenuated in the same length of time. After 40 months the first race was still very virulent; the second was dead or avirulent. * The bacteriophage can be recognized only through its virulence for a given bacterium. Consequently it is not possible to tell whether the bacteriophage as such has disappeared or whether it has become avirulent, for in either case it would not be possible to disclose its presence. 168 THE BACTERIOPHAGE AND ITS BEHAVIOR It is obvious that it is impossible to state any specific rule as applying to the phenomenon in all cases. Each bacteriophage behaves in a par- ticular manner. The most that can be said is that in general races with a high virulence retain virulence for a long time, while a low virulence disappears relatively quickly. Antiseptics hkewise exert an effect upon virulence, but we will reserve a study of this subject until we consider the properties of the bacterio- phage corpuscle. Virulence is weakened through contact with resistant bacteria. This aspect of the subject will be considered in detail in the next chapter. 7. HOMOGENEOUS AND HETEROGENEOUS BACTERIAL SPECIES Different bacterial species behave in a different manner as regards susceptibility to bacteriophagy. With certain species it is found that when one strain is susceptible to a given race of the bacteriophage all other strains are also susceptible.* The Shiga dysentery bacillus is typical of those species which I have termed homogeneous insofar as the bacteriophage is concerned. During the past 10 years I have isolated several hundred races of bac- teriophage active for this bacterium and I have never yet found a strain of the Shiga bacillus which was not attacked by any of these races. The degree of virulence is the only variable. A given bacteriophage of max- imum activity for a single strain may possess only a moderate virulence for others, but a few passages are always sufficient to intensify the virulence for those strains which were at first but weakly attacked. B. pestis is another bacterial species which I have found to be homo- geneous as regards bacteriophagy. I have shown^i^ that certain races of Typhoid-bacteriophage have an extremely specific action, attacking but a single strain of B. typhosus to the exclusion of all other strains. Bordet and Ciuca^*^ have confirmed this finding, and they have shown that with B. coli a comparable speci- ficity may be demonstrated with some races of bacteriophage. My experiments have disclosed the fact that this non-susceptibility of certain strains of a bacterium when others are attacked, is limited to certain bacterial species, and these I have termed heterogeneous. * Naturally, this is true only insofar as it deals with typical strains, that is to say, with those presenting all of the characters of the species. In general, atypi- cal strains (those which are arbitrarily^ classified in a species to which they do not conform in all characters) are found to be resistant to the action of the bacterio- phage. We will return to this subject later. VIRULENCE OF THE BACTERIOPHAGE 169 The strains which are not attacked possess a true natural immunity, but this immunity is Hmited, not absolute. As a matter of fact, Janzen and Wolff,"^ working with B. typhosus, have shown that those strains of this bacterium not attacked by a certain bacteriophage are attacked by others. It is, however, quite essential that we do not confuse this natural immunity, possessed by certain bacterial strains toward some races of the bacteriophage, with the immunity acquired by a susceptible bac- terium in reacting to the action of a bacteriophage. Each race of the bacteriophage which has a virulence for a single bac- terial strain belonging to a heterogeneous species possesses an individual range of virulences. This range varies from one race to another. Cer- tain bacteriophage races will attack only a few strains of the heterogene- ous bacterium; other races will attack a large number, and there are some which will attack all. With respect to the last, the bacterial species will be homogeneous. Furthermore, each susceptible strain will be attacked with greater or less intensity, that is to say, the violence of the reaction will vary. Nevertheless, in these cases it is always possible to increase the virulence by means of passages with the strain that was at first but slightly attacked. Since all combinations of these variables are encountered and since there are all degrees of virulence for diverse strains, and since also there are differences in the intensity with which each strain is attacked it is obviously practically impossible to isolate two races of the bacteriophage possessing identical characters (d'Herelle^^^). A very typical case is that of the Staphylo-bacteriophage, where races may be isolated which are virulent for but a single strain of the staphylococcus. It is of some significance that the races of Staphylo- bacteriophage isolated from vaccinal lymph* are of this type. On the other hand I have isolated races of Staphylo-bacteriophage from the pus of abscesses, undergoing resolution, which were virulent for a number of staphylococcus strains. Gratia-" has isolated a race (race H) which is virulent for all strains of the staphylococcus, including strains of the * As we will see, in some specimens of vaccinal lymph a bacteriophage-bac- terium symbiosis between a staphylococcus and a bacteriophage may be found. The symbiotic organisms are continuously reinoculated at the same time as the ultravirus of vaccinia from calf to calf. This represents a true final adaptation of the bacteriophage to parasitism of the staphylococcus with which it has been associated in a parasitic relationship for a great many generations. 170 THE BACTERIOPHAGE AND ITS BEHAVIOR several species, albus, aureus, and citreus. As a matter of fact, with this particular race I have not yet found a typical staphylococcus which is not attacked, even Micrococcus tetragenus is dissolved. We will return to this question of homogeneous and heterogeneous bacterial species when we study the peculiarities of the phenomenon of bacteriophagy with the different bacterial species. We will then review the studies of Janzen and Wolff, particularly those dealing with the bac- teriophagy of the heterogenous B. typhosus. 8. MULTIPLE VIRULENCES OF THE BACTERIOPHAGE We have seen that certain races of the bacteriophage are virulent for only a single bacterial strain, while others are virulent for all strains of a given species. Furthermore, we have stated that still other races are virulent not only for all strains of a single species but also for strains of bacteria belonging to different species, sometimes to species rather remotely related (d'Herelle^^^). As a matter of common observation it has been found that a given race of the bacteriophage, when derived from the organism, is rarely active for but a single bacterial species. Usually at this time it attacks a number of species and possesses for each of them a varying degree of virulence (d'Herelle^^^). Such a race of the bacteriophage might have, for example, a very high virulence for the Hiss strain of B. dysenteriae, a moderate virulence for some strains of B. coli, and a low virulence for other strains of B. coli as well as for the Shiga bacillus. For B. paratyphosus B the action may be very weak, and for other species no virulence whatever can be demonstrated. Another race may be very active for certain strains of B. coli and of B. typhosus, less virulent for other strains of these or- ganisms, and at the same time it may show but little activity for B. dysenteriae Flexner with no virulence for all other bacterial species. But provided an activity, even though weak, is manifested by such a race it is always easy, by passages, to enhance the virulence for one of these organisms. We have seen further that because of variations in virulence a given race of the bacteriophage may vary materially at different times. All of the combinations of virulence, in quality as well as in quantity, being possible, that is to say, in the range of the action against various bacterial species and in the intensity of the action for each of the strains of these different species, one can readily understand, in view of the infinite number of possible combinations, that there can be no two races of the bacteriophage which can be absolutely identical (d'Herelle^^^). VIRULENCE OF THE BACTERIOPHAGE 171 All of those investigators who have studied the bacteriophage, with the exception of Bail and of Wagemans/^^ have confirmed the fact that a given race of the bacteriophage may show multiple virulences, and this conclusion is reached whatever may be the individual opinion as regards the nature of the bacteriophage itself. The two authors mentioned above as exceptions have advanced the suggestion that when a bacterio- phage filtrate causes bacteriophagy with different bacterial species the phenomenon is due to the fact that the material contains a mixture of several races of the bacteriophage. The experiments which will be detailed in the following paragraphs show that such a conclusion is in- admissible, simply because it is contrary to demonstrated experimental facts. Foreseeing this objection I had refuted it before it was ad- vanced*^^ but the authors named above have not even mentioned the experiments which I had reported as bearing upon this point. 9. PERSISTENCE OF VIRULENCE A race of the bacteriophage possesses the faculty of regaining its virulence for a given bacterium, and this capacity persists throughout a great many passages effected in vitro with a bacterium belonging to another species. In 1916 I isolated a bacteriophage extremely active for B. dysenteriae Shiga. At that time, as it was derived from the body it also showed a moderate virulence for one strain of B. coli and a low virulence for different strains of B. typhosus and the paratyphoids A and B. This bacteriophage was used in many experiments through- out the years 1916, 1917, 1918, and 1919, and during this time was sub- jected to a very great many passages, always at the expense of B. dysen- teriae Shiga. As a matter of fact the number of passages somewhat exceeded 1200. Early in 1920 I showed that, without a preliminary adaptation, it still possessed a moderate virulence for the strain of B. coli and a slight virulence for the strain of B. typhosus toward which it was active 4 years earHer. By means of passages with these bacteria the virulence was increased, up to the point where it caused complete dissolution. ^^^ Having preserved this race of Shiga-bacteriophage I carried out, in 1923, three successive purifications by the method of selection described above, without causing a loss in the virulences for B. coli and B. typhosus. This experiment has been repeated at different times with several races of the bacteriophage, the result being that the multiple virulences per- sisted after three successive isolations. Inasmuch as the suspension 172 THE BACTERIOPHAGE AND ITS BEHAVIOR of the bacteriophage which manifests these multiple virulences is most certainly derived from a single corpuscle, the effects can hardly be said to be due to a mixture of races. The fact that multiple virulences persist despite a great many pas- sages made at the expense of a single bacterial strain, is, however, the most vahd evidence that these multiple virulences are attributes of a single bacteriophage corpuscle. Let us assume that a stool filtrate causing bacteriophagy of several bacteria of different species contains several races of the bacteriophage. If, with this filtrate, we carry out passages at the expense of a single bacterium we must admit that only those corpuscles should multiply which possess a virulence for this bac- terium. We must hkewise admit that all corpuscles avirulent for this bacterium should disappear gradually in the course of the passages. And the number of passages necessary to eliminate all of the avirulent corpuscles within the initial filtrate is readily calculable. Physicists have determined that the smallest possible quantity of matter is the electron, whose mass is 1-10~" grams. When, as the result of succes- sive dilutions such as take place from passage to passage, we introduce into a suspension a quantity of the initial filtrate less than 1-10~" grams we may be very sure that there no longer remains any of the original filtrate, and, a fortiori, none of the bacteriophage corpuscles which were in this filtrate. All of these corpuscles present at this time result from a multiplication of virulent corpuscles. In carrying out the series of passages by introducing into the first suspension 0.001 cc. of filtrate, into the second 0.001 cc. of the first suspension after it has undergone bacteriophagy, and continuing thus, introducing each time 0.001 cc. of the last suspension dissolved into 10 cc. of fresh bacterial suspension, it can be readily calculated that in the seventh passage there can be but 1-10~2^ grams of the original filtrate, that is to say, a virtually non- existent quantity, since it is smaller than an electron. This seventh suspension can not contain any of the bacteriophage corpuscles aviru- lent for the bacterium at the expense of which the passages were made, even though such corpuscles were found in the original filtrate. If the corpuscles found in the last filtrate manifest a virulence for a bac- terium of a species other than that with which the passages were made the only inference is that these corpuscles possess the property of causing a bacteriophagy of different bacterial species at one and the same time. But experiment shows that, not only after the seventh, but after more than 1000 passages, a race of the bacteriophage still possesses the prop- erty of causing bacteriophagy with bacteria belonging to different VIRULENCE OF THE BACTERIOPHAGE 173 species. This affords, then, mathematical proof that multiple viru- lences are really attributes of bacteriophage corpuscles.* 10. THE MECHANISM OF THE PERSISTENCE OF VIRULENCE A bacteriophage which has received more than a thousand passages with B. dysenteriae has a relatively weak action upon the typhoid bacil- lus. This can be demonstrated by spreadings made on agar, with sub- sequent observation of the formation of characteristic plaques. If we introduce into a tube of bouillon about 10 drops of an anti-dysentery bacteriophage and then a small amount of typhoid culture we secure, after incubation for 18 to 24 hours, an apparently normal culture of B. typhosus, but if it is spread upon agar a few plaques are obtained. This finding provides us with additional information as to the mechanism of the virulence of the bacteriophage. For here we have introduced into the suspension of B. typhosus several thousand million bacteriophage corpuscles, all virulent for B. dysenteriae. Each plaque to develop on the agar, upon which the suspension of B. typhosus in- oculated with the Shiga-bacteriophage is spread, represents a colony derived from a corpuscle virulent for B. typhosus. And this shows that among several billions of corpuscles virulent for B. dysenteriae there are only a few which are also virulent for B. typhosus. Furthermore, by such a procedure it can be shown that the number of corpuscles having a virulence for B. typhosus hardly varies throughout the series of passages with B. dysenteriae, for the number of plaques ob- tained by spreading a suspension of typhoid bacilli inoculated with the suspension of bacteriophaged dysentery bacilli is approximately the same, whether the bacteriophage has undergone 50, 100, 500, or 1000 passages, always with B. dysenteriae. Nevertheless, the number of corpuscles active for B. typhosus diminishes, although the aptitude for the bacteriophagy of B. typhosus is lost but very slowly. After 1500 passages the virulence was so weak that spreadings upon agar no longer showed plaques, yet plaques appeared after only two passages. After 17 passages with B. typhosus the dissolution of a normal suspension of typhoid bacilli was complete. * At least, if it is understood that the bacteriophage is not derived from the bacterium itself. This is a point which we will examine in another chapter, but I believe it wise to state here that there is very conclusive evidence which has not been discussed or contradicted by anyone, which demonstrates that the bacteriophage corpuscle is autonomous, independent of the bacterium which is subject to its action. The evidence supporting this will be presented when we discuss the question of the nature of the bacteriophage. 174 THE BACTERIOPHAGE AND ITS BEHAVIOR It is probable that by continuing the passages the aptitude for reacting with B. typhosus would have gradually diminished and finally would have been completely lost. This would have represented, then, a strict adaptation to the bacteriophagy of B. dysenteriae. As I have said above, I have repeated at different times, with diverse races of the bacteriophage, experiments which show that after a series of passages followed by three successive isolations the virulence of a bacteriophage for several bacterial species persists. Here is such an experiment. A race of Typhoid-bacteriophage, isolated in 1918 from the stools of a convalescent from typhoid fever, manifested at its origin a strong viru- lence (complete bacteriophagy of a normal suspension but with almost always the development of secondary cultures) for many strains of B. typhosus and B. coli, for the paratyphoids A and B, and for all strains of B. dysenteriae, Shiga, Flexner, and Hiss. In 1924 I made two or three hundred passages with B. coli, B. typhosus, and B. dysenteriae, alternat- ing the bacterium with which the bacteriophage was placed in contact. A series of a dozen passages were then made with B. dysenteriae to elimi- nate avirulent corpuscles if any were present (in each passage 0.001 cc. of the suspension previously dissolved was introduced into 10 cc. of a fresh suspension) . And finally, the race was carried through three successive isolations by selection. This procedure assured two things; first, that all of the corpuscles avirulent for B. dysenteriae must have been ehminated by dilution, and second, that the derived suspension represented the progeny of a single corpuscle. With the final suspension the virulences were the same as those shown immediately after the iso- lation of the race in 1918. This can only mean, therefore, that a single bacteriophage possesses multiple virulences. In all of the experiments performed, where consideration was given to this point, it has been possible to show that among the corpuscles comprising a race of the bacteriophage, there are some — many or few, as the case may be — which show a virulence for a bacterium of a species other than that at the expense of which the passages have been made. The persistence of a latent virulence is a function of certain particularly apt corpuscles. In view of the very great number of corpuscles present after bacteriophagy, with the law of large numbers applying to each passage, it follows that the number of corpuscles presenting a latent virulence for a bacterium of another species continues practically the same. It is only after a very great many passages, always at the ex- pense of a single bacterium, that the aptitude for bacteriophagy with VIRULENCE OF THE BACTERIOPHAGE 175 other organisms becomes lost. In this connection we will see later many examples showing that the characters pertaining to each race of the bacteriophage are very stable. When, after a number of passages at the expense of a given bacterium, the bacteriophage corpuscle is placed in contact with a bacterium of another species for which some of the corpuscles possess a latent viru- lence, a selection occurs. Only the corpuscles having this virulence multiply; the virulence for the new bacterium is thus increased so that after a few passages the virulence reaches such a degree that complete bacteriophagy is effected. It is necessary, however, to state that this tendency toward an increase in virulence differs very markedly for different races of the bacteriophage. Given two races of the bacterio- phage, both presenting a latent virulence for a single bacterial strain, one may obtain, after a few passages, a high virulence with one of the races while it may require a considerable number of passages before the second race attains a comparable degree of virulence. Indeed, with certain races this may never be accomplished. We have seen in the experiments recorded above that even in the case of a bacteriophage having a maximum activity for a given bacterium each corpuscle possesses its own individual virulence. Certain of them are high in virulence, others are less active. We will see that this is also true for the latent virulences which these corpuscles may manifest. This behavior of bacteriophage corpuscles is identical to that of patho- genic bacteria. Since the time of Pasteur we have known that in a pure bacterial culture each individual cell, although possessing the general characteristics of the species, presents "variations" which are peculiar to it. A bacterium may be considered, according to the expression of Maurice Nicolle, as a "mosaic" of properties, each of these properties — vitality, resistance to such and such an agent, virulence for such and such an animal — -being subject to continual variation. And the varia- tions in each of these factors follow different lines with each cell division. Everything would indicate that the situation is precisely the same for bacteriophage corpuscles. Each race of the bacteriophage possesses characters which distinguish it from other races, and each of the cor- puscles of a given race possesses characters which are subject to con- tinual variation. 11. THE ACQUISITION OF VIRULENCE From the very first of my communications upon the subject I have insisted upon the fact that a bacteriophage can acquire, experimentally, 176 THE BACTERIOPHAGE AND ITS BEHAVIOR by passages, a virulence for a bacterium toward which it previously manifested no activity. In the first edition of my collected papers^^^ experiments were presented supporting this idea. Since that time a great many authors have confirmed this fact. The contributions of Otto and his collaborators in particular present many experiments dem- onstrating the truth of this idea. But I will mention here only the following, for these experiments are extremely interesting from the theoretical point of view since they show how the virulence of the bac- teriophage is acquired under natural conditions, and from the practical point of view they are significant in that they indicate a procedure par- ticularly well adapted to bring about, experimentally, such an acquisi- tion of virulence. STRAINS OF VIRULENCE DETERMINATIONS IMMEDIATELY AFTER ISOLATION VIRULENCE DETERMINATIONS AFTER A SERIES OF PASSAGES WITH STRAIN Sm Dissolution Inhibition Plaques Dissolution Inhibition Plaques Wi 1 +++ +++ ++++ ++++ 24 ± ++++ 27 29 ++ +++ ++++* * The method employed by Janzen and Wolff for measuring virulence has been described. In the tables given here, dissolution refers to bacteriophagy in a fluid medium; inhibition to a retardation of growth when the bacteriophage is added to a seeded medium; and plaques, of course, refers to the formation of these areas upon agar. Janzen and Wolff were the first to show that a race of Typhoid-bac- teriophage, manifesting at its isolation a virulence for certain strains of B. typhosus and at the same time completely avirulent for others, was able to acquire a definite virulence for some of the latter strains, and this simply by means of passages at the expense of certain susceptible strains. I am including here two of their experiments.*^^ The virulences of the bacteriophage involved were determined for 5 different strains of B. typhosus immediately after its isolation from the intestinal tract and again after a series of passages with a single strain of the typhoid bacillus. The results obtained with bacteriophage race Re, are shown in table 14. Additional data are given for bacteriophage, race Wi (table ] 5) . The results are essentially these: bacteriophage Re, by virtue of passages at the expense of typhoid strain Sm has acquired a virulence VIRULENCE OF THE BACTERIOPHAGE 177 6 m 1 < \ a, El 1 + + + + + + + + + + + + + + + + + + + + 1 c + + , + + + t ^ + + + "^ + 1 1 s + + + + 1 i \ I 1 + + + + + + + + + ° + + + + + + + 1 2 + + + + ^ + + + 1 .2 Q + j[ o o o o + o H 2 i \ s « 3 1 S + + + + + + + + + + + + + + + + 1 + + + + 4^ + + + 1 5 + + o o o o + i q •^ -" ^ ^ s 178 THE BACTERIOPHAGE AND ITS BEHAVIOR for strains 24 and 29, while bacteriophage Wi, through passages with this same strain Sm, has acquired a virulence for strain 1. Da Costa Cruz^^^ has applied the same procedure to develop a race which would give bacteriophagy of a strain which was originally not attacked. When isolated this bacteriophage had but a slight virulence for strain 1 of B. typhosus, but it was very active for strain 18. With these two strains as extremes, the virulence for a number of other strains was more or less marked. The first passages were made with strain 18, then with strains which originally were less susceptible. Contacts were thus made involving successive passages with strains which were less and less susceptible. When the series of contacts was completed the race showed a very marked virulence for strain 1. It would appear, therefore, that contacts with species more and more remote from the normal host may, in reality, be the device whereby a given race of the bacteriophage acquires a virulence for a bacterium previously insensitive to its action. Eliava and myself^^^ have adapted a Staphylo-bacteriophage to bacteriophagy of B. dysenteriae Shiga, but thus far it has been impossible to accomplish the reverse change. McKinley,*^^ working with a race of Shiga-bacteriophage* quickly obtained a high virulence for the meningococcus by means of four pas- sages in a bouillon containing glucose and blood. This bacteriophage, after its adaptation to the meningococcus, was found to be virulent for Staphylococcus albus, aureus, and citreus, for M. tetragenus, for Strep- tococcus hemolyticus, and for pneumococci of Groups I, II and III. This same Shiga-bacteriophage, without the passages with the menin- gococcus was avinilent for all of these different organisms. The virulence of a race of bacteriophage is not fixed. No race is immutable; all vary continually. Some of them lose virulence, al- though very slowly, and others acquire virulences by adaptation. We will see that this behavior of the bacteriophage, here demonstrated experimentally, is but a reproduction of processes occurring in the same way in nature. 12. BACTERIOPHAGY IN MIXED CULTURES If one introduces a bacteriophage possessing multiple virulences into a bacterial suspension prepared by mixing cultures of several different * A race which I sent him; indeed, one of the races which is mentioned here in a number of the experiments. It had undergone, at the time when it left my laboratory, about 1200 passages with B. dysenteriae. VIRULENCE OF THE BACTERIOPHAGE 179 susceptible bacterial species, bacteriophagy occurs, but not all of the organisms are attacked with the same intensity. Bacteriophagy is complete for the species toward which the virulence is maximal or very high, it is partial for others, and is, indeed, the less marked as the viru- lence for the organisms is the less pronounced. As a matter of fact, whether the bacteriophage acts upon different susceptible organisms separately or whether it acts upon mixtures of these bacteria, the attack occurs in just the same manner. It is rather interesting that it appears that the virulence for bacteria which are attacked but weakly may be increased more rapidly in com- bined cultures, as is indicated by the following experiment. Three tubes of bouillon receive respectively 0.01, 0.1, and 1 cc. of a known Shiga-bacteriophage. The thi'ee tubes are then Hghtly planted with B. coli. Normal cultures develop in the three tubes. Platings on agar give few plaques. Each of the three cultures is transferred to fresh bouillon. Normal B. coli cultures develop. Transfers to agar give two plaques for the first tube, none for the other two. The culture yielding the two plaques is again re-inoculated. A normal culture develops. The bacteriophage has been eliminated. This strain of Shiga-bacteriophage possesses, therefore, an extremely feeble virulence for the strain of B. coli under test. To 10 cc. of bouillon is added 1 drop of a concentrated suspension of Shiga bacilli (this should give a slight turbidity equal to about 50,000,000 bacilli per cubic centimeter) and 1 drop of an equally concentrated suspension of B. coli. This double suspension is then inoculated with 0.01 cc. of the Shiga bacteriophage used in the above experiment. After twenty-four hours there is a slight turbidity. A new passage into a double Shiga-Colon suspension is made. Perfect dissolution takes place after eleven hours. The dissolved suspension is then introduced, in a quantity of 0.04 cc, into a simple suspension of B. coli. Dissolution is complete in seven hours. The corpuscles have developed at the expense of the Shiga bacilli, and thus being maintained in the medium they have gradually acquired a virulence for B. coli (d'Herelle^-^). RJESUME The multiplication of bacteriophage corpuscles belonging to differ- ent races does not take place with the same intensity, even though the conditions for the process are identical for all. The activitv — the 180 THE BACTERIOPHAGE AND ITS BEHAVIOR ''force" — of a bacteriophage is always strictly proportional to the in- tensity of its power of multiplication. The activity of a bacteriophage corresponds, then, to a 'Virulence" in the strict sense of the word (d'Herelle^'i^). A method of "titrating" the activity of a race must take the attributes of the bacteriophage into consideration, in particular, the fact that it is corpuscular in nature and that the corpuscle possesses a virulence. Inasmuch as the activity of a bacteriophage for a particular bacterium is causally related to the intensity of its power of multiplication at the expense of this bacterium the only correct method of numerically expressing virulence consists in a determination of the number of corpuscles per cubic centimeter to be found in a suspension after bacte- riophagy is complete. Results are comparable only if the figures repre- senting the intensities of multiplication are derived through experi- ments carried out under comparable conditions. The larger the number of corpuscles found the more intense has been the development, and the higher is the virulence of the race (d'Herelle). Particular emphasis is placed upon this point because of its very great importance; because of its lack of precision the so-called "dilution method" of counting bacterio- phage corpuscles can not be used in the study of bacteriophagy. It represents the greatest single cause of error appearing in the results of those who have, unfortunately, employed it. Inasmuch as different races of the bacteriophage show varying de- grees of virulence it is necessary, in experimental procedures, to work with pure races, that is to say, with a race derived entirely from a single corpuscle (BaiP^). There are two methods of purification which permit one to obtain a pure race. The first consists in the removal of corpuscles from a colony or plaque arising from the multiplication of a single cor- puscle (d'Herelle^^"-^^^); the second is the method where dilution is car- ried to the point where but a single corpuscle per unit volume is present (d'Herelle^is-^^'*^). In a given suspension of the bacteriophage not all of the corpuscles are endowed with exactly the same properties. Virulence, in particu- lar, is variable for each one of them (d'Herelle^-^- The method of isola- tion by the limiting dilution permits the selection of the most virulent corpuscles as the origin of pure races. This procedure is at one and the same time a method of isolation and of selection (d'Herelle). The virulence of a bacteriophage is variable. It can be increased ex- perimentally. Increase in virulence may be obtained by serial passages with a bacterium toward which one desires the virulence to be increased VIRULENCE OF THE BACTERIOPHAGE 181 (d'Herelle,^'"). It is also possible to produce, experimentally, an atten- uation in the virulence of a bacteriophage. Attenuation occurs when it is subjected to an appropriate degree of heat (d'Herelle and Pozerski^''"). It may occur spontaneously with the passage of time (Asheshov^^). The action of certain races of the bacteriophage is strictly specific; they act upon but a single strain of a given bacterium. Examples of this are afforded by certain races of the Typhoid-bacteriophage (d'Her- elle^^^). Certain bacterial species are homogeneous with regard to the bacteriophage, that is to say, when a race of the bacteriophage is virulent for one strain it is also virulent for all other strains. B. dysenteriae Shiga and B. pestis are examples of homogeneous species. Other bac- terial species are heterogeneous, that is to say, certain strains may be attacked while others are insusceptible (d'Herelle^-'). This natural resistance of certain strains is not absolute inasmuch as a strain may be unattacked by one race of the bacteriophage although it is attacked by others (Janzen and Wolff^^"*). Ordinarily a bacteriophage is virulent, not only for bacteria of a given species, but also for bacteria belonging to different species, sometimes to organisms very distantly related (d'Herelle''^^). The range of viru- lence and the intensity of virulence may differ for each race of the bacteriophage and as the result of this it is impossible to conceive of two races as having absolutely identical characteristics (d'Herelle^^^). The manner in which virulence for different bacterial species persists in a bacteriophage indicates that virulence is a fairly stable property. Virulence will persist throughout a very great number of passages, all carried out at the expense of a single strain (d'Herelle'^^^). Multiple virulences persist in spite of the fact that the race is subjected to purifi- cation procedures, indicating that they are, therefore, an inherent property of a race derived from a single corpuscle (d'Herelle). The persistence of virulence for a bacterium other than that with which the passages are made is not an attribute of all corpuscles but of certain ones only, and the number of these varies but slightly during a series of passages (d'Herelle^-'-). It is possible, by experimental adaptation procedures, to cause a race of the bacteriophage to acquire a virulence for an organism against which it was originally completely inactive (d'Herelle^^^'^-^. CHAPTER V Resistance of the Bacteria 1. secondary cultures Throughout the first three chapters we have studied the typical phenomenon of bacteriophagy as it is brought about through the action of powerful races of the bacteriophage. Here the bacteriophage cor- puscles develop in proportion as the bacteria become dissolved and when the process is once completed that which was a few hours previously a suspension of bacteria has become a suspension of bacteriophage cor- puscles, free of all bacteria. We have also seen that the power of pro- voking bacteriophagy varies among races of the bacteriophage. For some of them the power to reproduce at the expense of the bacteria is considerable, for others it is less, and this reproductive capacity cor- responds exactly to a virulence more or less great. As a rule each race of the bacteriophage will attack bacteria belonging to different species and will have for each of them a different degree of virulence, capable of being increased by adaptation. But there is another aspect of the phenomenon. In biology there is a general law to the effect that all living beings react against an agent which attacks them. If we consider the bacteria, endowed with virulence for some animal species, we do not know of any whose viru- lence can be such that an opposing immunity may not possibly be acquired.* The natural law of resistance comes into play in the same way when a bacterium is attacked by a bacteriophage. Bacteria have the capacity to resist, and may thus acquire an immunity to the bacter- iophage corpuscles which attack them (d'Herelle^^^). There are races of the bacteriophage of such virulence that under cer- tain determined conditions of temperature and of medium the suscep- tible bacterium is always overcome. Resistance is impossible. Bacteri- ophagy is then complete and the medium becoming clear remains so indefinitely. I have obtained, by repeated selection of the most apt corpuscles, * This has occurred and will occur in the future. The inescapable result of the possession by a bacterium of an "absolute virulence" for an animal species means the disappearance of this animal species within a short period of time. 182 RESISTANCE OF BACTERIA 183 races of the Shiga-bacteriophage capable of effecting a complete dissolu- tion of normal suspensions and with such a race it is not necessary to filter the dissolved suspension through a candle to insure that the medium remain clear and sterile indefinitely. With such races bacteri- ophagy can be continued throughout an unlimited series without filtra- tion between the successive passages. After a process of selection it is the same for the Staphylo-bacteriophage described by Gratia as race H, but in order to obtain this result it is necessary that bacteriophagy take place at a temperature of 32°C. and that the bouillon have a pH greater than 7.5. If these conditions are not satisfied a number of the suspen- sions, after a complete clearing, again become cloudy after an interval of time, and it is of interest that the number of suspensions to become turbid increases as the conditions become more divergent from the optimum. The following experiments were carried out with Staphylo-bacterio- phage, race H. Normal suspensions, containing 250 million cocci per cubic centimeter, prepared from a 24-hour agar culture were used. Ten cubic centimeters of the suspension were inoculated with 0.001 cc. of the bacteriophage. Each of the experiments comprised 24 tubes, each containing 10 cc, subjected to the same treatment at the same time. Experiments 1 to 5 illustrate the effect of temperature. In all of these the reaction of the bouillon was 7.8. 1. Temperature, 25°C. After 32 hours bacteriophagy was complete in all 24 tubes. After 7 days, at 25°C., the 24 suspensions were limpid. After 2 months at laboratory temperature the 24 suspensions remained unchanged, — perfectly clear. 2. Temperature, 31°C. After 19 hours bacteriophagy was complete in the 24 tubes. After 7 days, at 31°C. the 24 suspensions were clear. After 2 months at laboratory temperature they still remained clear. 3. Temperature, 36°C. After 18 hours bacteriophagy was complete in the 24 tubes. After 72 hours at 36°C. the 24 suspensions were limpid. After 4 days at 36°C., 23 were limpid; one was cloudy. After 5 days at 36°C., 22 were limpid and 2 were cloudy. After 7 days at 36°C. the result was the same. After 2 months at room temperature the results still remained the same; 22 tubes being clear, 2 being cloudy. 4. Temperature, 40°C. After 18 hours bacteriophagy was complete in all 24 tubes. After 28 hours, 11 tubes were clear, 13 were cloudy. After 72 hours all 24 tubes were cloudy. 5. Temperature, 31°C. After 24 hours all 24 of the suspensions were clear. After 6 days they still remained clear. At this time 20 of the 184 THE BACTERIOPHAGE AND ITS BEHAVIOR tubes were placed in the incubator at 41.5°C., the other 4 remained at laboratory temperature. After 24 hours at 41,5°C., 7 of the 20 suspen- sions were clear, 13 were cloudy. After 48 hours at this temperature all 20 of the suspensions were cloudy showing an abundant secondary cul- ture. The four suspensions held at laboratory temperature were clear and remained so for 2 months. A second part of this experiment, designed to show the effect of the reaction of the medium was carried out at a temperature of 31°C. 6. The bouillon used had a pH of 7.0. Bacteriophagy of all 24 tubes was complete after 24 hours. After 48 hours the 24 tubes were turbid. This, compared with part 5 above shows clearly the importance of the hydrogen ion concentration. Experiments carried out with a potent Shiga-bacteriophage have given results of the same nature ; the conditions of temperature and of reaction exerting a comparable influence. It may be well to state, and later we will consider this point further, that the optimum temperature may vary through some degrees. Fur- thermore, we have seen that the optimum temperature is not the same for all races; it is simply a question of adaptation as may be readily demonstrated by experiment. But it is none the less true that insofar as the bacteriophagy of B. dysenteriae and of the staphylococcus is concerned, once an adaptation has been accompHshed, a temperature of 32°C. is optimum — the critical temperature. Above this, whatever may be the virulence of a bacteriophage and although it may be adapted to induce bacteriophagy at a higher temperature, permanent sterility of unfiltered suspensions of the bacteriophage is not uniformlij obtained. Experiment 5 (above) is of significance, for it shows that although media in which bacteriophagy has been complete will remain sterile indefinitely if conditions favorable to the bacteriophage are maintained, they will yield secondary cultures if placed under conditions favorable for the bacterium, although the media were perfectly clear and no bacteria could be found even by examination of the sediment secured by centrifugation. From where then, in these cases, are the resistant bacteria derived? This is a question which we will consider in a later section entitled Ultrabacteria. Macroscopic examination and biological reactions show that the tur- bidity which occurs in bacteriophaged suspensions is due to the develop- ment of bacteria of the same kind as those which formed the suspension prior to bacteriophagy. These cultures which develop under these circumstances, i.e., after bacteriophagy, I have termed secondary cultures. ^^'''^^^ RESISTANCE OF BACTERIA 185 Once bacteriophagy is completed with the suspensions perfectly clear; those tubes which are to give a secondary culture can not be distin- guished macroscopically or microscopically in any way from those which are to remain clear indefinitely. Transfers to bouillon and on to agar of l)acteriophaged suspensions in which a secondary culture is to develop later remain sterile up to the time that the secondary culture appears. This does not often occur until 5 or 6 days after the dissolution, some- times even later. A suspension of Shiga bacilh, containing 250 milHon bacilh per cubic centimeter, is inoculated with 0.001 cc. of a culture of the bacteriophage. Normal bacteriophagy takes place in 5 hours, with the medium perfectly limpid. The dissolved suspension is planted on agar and in bouillon 1, 2, 3, 4, 5, 6, and 7 days after the dissolution is complete. All of the plantings remain sterile. On the eighth day the dissolved suspension is slightly clouded. On the ninth day a drop is introduced into broth and drops are spread over 3 tubes of agar. Two of the agar tubes remain sterile, the third shows 4 small colonies. The broth tube gives an agglutinated, sedimented culture.^^^ The following experiments show that the number of secondary cul- tures to develop diminishes as the virulence of the bacteriophage is increased. The most potent race of the bacteriophage which I have yet isolated (these experiments were carried out in 1918) was combined with 2 strains of the Shiga bacillus. One of these bacterial strains has been for a long time under artificial cultivation, being used by the Pasteur Insti- tute for the inoculation of horses in the production of anti-dysentery serum (type strain) . The other was recently isolated from the stool of a patient with dysentery (strain Jerv.). (A) Twelve tubes of the suspension of the type strain of the Shiga bacillus are each inoculated with 0.001 cc. of a culture of the bacterio- phage. This latter has been carried on for a great number of genera- tions always at the expense of a single bacihary strain. In all twelve tubes dissolution is perfect, with complete clearing in four hours. After three days at 37°C. one of the tubes is slightly cloudy, the others are clear. (Five other experiments, each consisting of 12 tubes, with the same race of the bacteriophage and the same bacihus give the follow- ing results: — tubes showing secondary cultures in each set, 0, 2, 0, 3 and 1. There develop, then, 7 secondary cultures in the 60 tubes, or 12 per cent.) (B) Twelve tubes of suspension were prepared with the strain Jerv., a strain with which the bacteriophage in question had never been in 186 THE BACTERIOPHAGE AND ITS BEHAVIOR contact. Each of these tubes is inoculated with 0.001 cc. of the same culture of bacteriophage as that used in the preceding experiment (A). Seven of the 12 tubes give secondary cultures. The results from five other experiments with the same strains are, 9, 5, 10, 5, and 6 secondary- cultures, or 70 per cent. A week later 12 cultures of the Jerv. bacillus are inoculated from one of the previous tubes that had remained clear. From these, 5 secondary cultures are secured. A further passage made after another week, gives 4 secondary cul- tures in the 12 suspensions. After another week, a fourth passage, still taking the bacteriophage from a perfectly hmpid culture, yields but one secondary culture among the twelve inoculated. (C) At the beginning of convalescence in the dysentery case (Jerv.) a bacteriophage was isolated which was tested in the same manner both on the type Shiga strain and on the Jerv. strain. This last was derived from the patient early in the infection at a time when the intestinal bacteriophage had manifested no activity for this organism. With the bacteriophage Jerv. on the type bacillus 4 secondary cul- tures develop among the 12 suspensions dissolved. With the bacteriophage Jerv. on the bacillus Jerv., there are no secondary cultures among the 12 tubes dissolved. When repeated upon an additional 12 suspensions a single secondary culture develops. To state the situation briefly, the frequency of secondary cultures is strictly related to the virulence of the bacteriophage. With the bac- teriophage of maximum virulence they do not occur when the conditions are optimum for the development of the bacteriophage corpuscles. If the conditions are not best suited to the development of the bacterio- phage, secondary cultures may develop and the number to appear becomes increasingly large as the conditions are more remote from the optimum. With bacteriophage races but slightly below a maximum virulence a certain number of secondary cultures can always be obtained even though the conditions are optimum. A number of the bacterio- phage suspensions, many or few as the case may be, will remain limpid indefinitely while other tubes will give secondary cultures. With races still less virulent, but yet capable of causing a total dissolution of the bacteria of the suspension, secondary cultures always develop. And finally, for races where the virulence is only moderate or weak, the bac- teria acquire, within the first few hours, a resistance sufficient to enable them to develop in spite of the presence of bacteriophage. Under such conditions clearing of the medium can not take place. Coincident with the dissolution of those bacteria which are least capable of developing a RESISTANCE OF BACTERIA 187 resistance there occurs a multiplication of the bacteria which are the more apt to acquire a resistance (d'Herelle^^^). This makes it clear why it is absolutely necessary, during a series of passages made to enhance the virulence of a bacteriophage, to separate the bacteriophage corpuscles in the process of increasing their virulence from the bacteria which have acquired a resistance. As we know this can be done either by filtration or by heating. By causing in each new passage the corpuscles whose virulence is gradually being increased to react upon fresh, normal bacteria which have never yet been in contact with the bacteriophage, the phenomenon of gradually increasing resist- ance does not intervene to counterbalance the progressive acquisition of virulence. We have already refuted the rather peculiar theory of Seiffert^^^ according to which it is not the bacteriophage which increases its viru- lence but rather the bacterium which adapts itself to the secretion of a "lysin." An adaptation can not be transmitted to non-existent descendants. As a matter of fact the result of the adaptation of the bacterium to the bacteriophage is exactly the opposite to the production of a "lysin," for the bacterium adapts itself to resist the agent which provokes its dissolution. Bacteriophagy is in reality a very complex phenomenon. The two cardinal factors which come into play are the bacteriophage corpuscles on the one hand with their virulence and on the other, the bacterium with its capacity to resist. Each of these factors is by its nature a variable, subject to the conditions of the moment. The virulence and the resistance fluctuate continually; they are increased or they are diminished. The macroscopic result of bacteriophagy, that is to say, the dissolution of the bacterial cells, is the resultant of the two factors which operate in opposition to each other (d'Herelle^'^). An experiment of Gratia-"^^ exteriorizes, one might say, this struggle between virulence and resistance. Working with B. coli he has shown that in an acid (pH. 6.8), neutral (7.0), or even shghtly alkaline (7.2) medium one may observe a succession of waves of growth and of dissolu- tion of the bacteria exposed to the action of a bacteriophage. With each wave the growth is a little more accentuated and the dissolution follow- ing is less complete. With the same bacteriophage but under condi- tions which were more favorable, that is, in an alkaline medium (pH 7.5), the phenomenon of bacteriophagy occurred normally. If the conditions are still more unfavorable to the bacteriophage, for example, if the medium is definitely acid, the bacteriophage no longer 188 THE BACTERIOPHAGE AND ITS BEHAVIOR attacks the bacterium. Under these circumstances the latter does not acquire a resistance since it is not attacked. Thus Scheidegger^^'^ work- ing with B. coli, has shown that in a bouillon having a pH of 4.5, in which this organism is still able to develop, the bacteriophage remains inert, and the bacterium acquires no resistance. But if such a medium is neutralized bacteriophagy takes place and the colon bacillus develops a resistance. The optimum conditions for development are not the same for bacteriophage corpuscles and for bacteria. Conse- quently the conditions favoring the one or the other of the two antago- nists determines whether the first or the second will finally win out. 2. THE ORIGIN OF SECONDARY CULTURES What is the intimate mechanism of the process that results in the formation of secondary cultures? A 'priori two hypotheses can be for- mulated. Two factors are present, a bacteriophage whose virulence may be attenuated, and a bacterium whose resistance may be aug- mented. Thus, are secondary cultures due to a weakening of the activ- ity of the bacteriophage, or, do there exist in the bacterial suspension certain individual cells which acquire an immunity to the bacteriophage, thus leading to the development of a resistant race? The following experiments clearly settle the question in favor of the last hypothesis. In the section treating of the isolation of the bacteriophage we have seen that in the large majority of cases the races which are freshly iso- lated are of too low activity to effect a complete dissolution of a bacterial suspension; cases where the presence of the corpuscles could only be detected by the presence of plaques upon the agar slants. These same races were able to acquire, by successive passages, a very high activity, a potency which enabled them to bring about dissolution of very heavy suspensions. This method of serial passages of the bacteriophage, in which it is forced to develop in vitro at the expense of a given bacterium, corresponds exactly with the method of Pasteur for effecting an enhance- ment in virulence of a bacterial strain by repeated passage through a given animal species. This single experiment, repeated a considerable number of times, — ■ in fact, each time that a bacteriophage of low virulence is isolated from the body^ — shows that secondary cultures are not produced by a simple diminution in the virulence of the bacteriophage. Indeed, there is, on the contrary, an enhancement with each passage, even if macroscopic dissolution is not to be seen. For this the following experiment offers direct proof: RESISTANCE OF BACTERIA 189 The contents of a tube that gave a secondary culture is filtered through infusorial earth and a bougie. Twelve tubes of a Shiga suspension are inoculated, each receiving 0.001 cc. of the filtrate. Per- fect dissolution is seen in all tubes, and in all but one the dissolution is permanent. This single tube again becomes turbid after 4 days. From this it is clear that the bacteriophage has not lost in virulence, and that secondary cultures can not be ascribed to a change in that direction. The bacteriophage remains virulent, coexisting with bacteria which have become resistant. The secondary cultures, then, are the result of an adaptation undergone by the bacterium which acquires an immunity to its parasite. It has already been shown that the number of corpuscles inoculated is without influence on the appearance of secondary cultures. The TUBE AMOUNT OF BACTERIOPHAGE FILTRATE INOCULATED RESULTS 1 2 3 4 5 6 CC. 0.1 0.02 0.004 0.002 0.0002 0.00002 Normal dissolution, secondary cultures Normal dissolution, no secondary cultures Normal dissolution, no secondary cultures Normal dissolution, secondary cultures Normal dissolution, no secondary cultures Normal dissolution, no secondary cultures conflict is not one of numbers; it is rather a struggle in which the signifi- cant factors are virulence on one side and ability to resist on the other. A suspension of B. dysenteriae, 250,000,000 per cubic centimeter, is distributed into 6 tubes and these are inoculated with variable quanti- ties of the same bacteriophage filtrate. The results obtained are given in table 16. The tubes yielding secondary cultures are distributed at random throughout the series, showing no fixed relationship to those tubes in which the dissolution was permanent. Another experiment may be presented, indicating as it does, the ran- dom manner in which secondary cultures may develop. This experiment shows the serial activity of the bacteriophage together with the appearance of secondary cultures. Each tube of the series is prepared with a suspension of B. dysenteriae, 250,000,000 per cubic centimeter, and into each is introduced 0.001 cc. of the dis- 190 THE BACTERIOPHAGE AND ITS BEHAVIOR solved suspension of the preceding tube. Transfers are made after twenty-four hours, that is, at a time when dissolution is complete. (See table 17.) Certain salts, when added to the suspension in very minute quantities, 0.1 mgm. to 10 cc. of culture, favor the development of secondary cul- tures. The salts of lead (nitrate and acetate) and of silver (nitrate and sulfate) act in this way. The soluble phosphates and magnesium sulfate appear to be without action. With a single race of bacteriophage and a given strain of bacillus the development of secondary cultures is, in A FRESH SUSPENSION RECEIVED THE DATE MATERIAL INDICATED RESULT July 8 0.001 cc. of bacteriophage suspension Permanent dissolution July 9 0.001 cc. of suspension bacteriophaged Julys on Permanent dissolution July 10 0.001 cc. of suspension bacteriophaged on Secondary cultures in 3 July 9 days July 11 0.001 cc. of suspension bacteriophaged July 10 on Permanent dissolution July 12 0.001 cc. of suspension bacteriophaged July 11 on Permanent dissolution July 13 0.001 cc. of suspension bacteriophaged July 12 on Permanent dissolution July 14 0.001 cc. of suspension bacteriophaged on Secondary cultures in 4 July 13 days July 15 0.001 cc. of suspension bacteriophaged July 14 on Permanent dissolution July 16 0.001 cc. of suspension bacteriophaged July 15 on Permanent dissolution July 17 0.001 cc. of suspension bacteriophaged July 16 on Permanent dissolution general, more frequent when the suspension is prepared from agar cul- tures several days old than when made from fresh cultures. At first thought it appears strange that when secondary cultures develop with a race of bacteriophage of high potency, they appear in some tubes and not in others. The following experiment offers an explanation for this. Two flasks, each containing 200 cc. of a B. dysenteriae suspension (250,000,000 per cubic centimeter) are inoculated with 0.04 cc. of a culture of the bacteriophage (the same race as that used in the preced- ing experiments). Immediately after inoculation the contents of the RESISTANCE OF BACTERIA 191 first flask is distributed into 20 tubes, 10 cc. to each. In all of these dissolution takes place normally, being permanent in 19, showing a secondary culture in 1 . The second flask is portioned out the next day, that is, after dissolution is completed, 10 cc. being placed in each of 20 tubes. None of these become turbid. When this second part of the experiment is repeated, 18 remain clear, and 2 tubes yield secondary cultures. Each flask of suspension contained 50,000 milhon bacilh, and the above experiments show that of this number but one or two were capable of acquiring an immunity to the very active bacteriophage. It is these "immune" bacilli which give rise to organisms that enjoy the same degree of resistance. Secondary cultures, then, have their origin in the operation of the phenomenon of natural selection, whereby some bacilli show a greater aptitude than others to the acquisition of a resistance to the bac- teriophage. The phenomenon of secondary culture formation is governed by the individual properties of the bacterium and bacteriophage. Against a single strain of bacterium the less virulent the bacteriophage the greater will be the proportion of secondary cultures, or, in other words, the greater is the number of bacilli in the suspension capable of acquiring a resistance. Gratia^^" has suggested that resistance to the action of the bacterio- phage may not consist in the acquisition of resistance by the bacterium but in a selection of those bacteria naturally endowed with this property prior to the action of the bacteriophage. That a selection takes place is precisely what I had shown previously^^^ by means of an experiment which has been presented in this present section. But it operates not through a selection of the bacteria naturally endowed with a resistance, but through a selection of those susceptible bacteria which are the more apt at acquiring resistance. This experiment shows definitely that what takes place is really an acquisition, in the strictest sense of the word, of resistance to the action of the bacteriophage. This is empha- sized by the fact that a resistant bacterium gradually loses resistance in the absence of virulent bacteriophage corpuscles. If resistance can be lost, obviously it is only because it has been acquired. We will consider further this phenomenon in a later section. 3. VARIABILITY IN ACQUIRED RESISTANCE Appelmans and Wagemans^*' have published some experiments designed to show that bacteria may become resistant to one race of the 192 THE BACTERIOPHAGE AND ITS BEHAVIOR bacteriophage and remain susceptible to another and this quite without regard to the virulence of the bacteriophage against which the resistance is acquired. Unquestionably this observation is correct, but it is not legitimate to base too broad generahzations upon it. Everything depends upon the respective characters of the race of bacteriophage and the strain of bacteria. In my experience, it has seemed that the matter of virulence usually exerts the greater influence, but to this there are many excep- tions, particularly when the bacterium involved belongs to a hetero- geneous species. A bacterium which has acquired a resistance to a bacteriophage of low virulence may remain susceptible to another bac- teriophage of high virulence. But a bacterium which has acquired a resistance to a bacteriophage of very high virulence is usually resistant to bacteriophagy by other races which are of lower virulence. In cases where two races of the bacteriophage have approximately the same degree of virulence a bacterium may become resistant to one of them and remain susceptible to the attack of the other. But even here, the matter of virulence often plays a role as is proved by the fact that if a bacterium acquires a refractory state toward a bacteriophage of maxunum virulence this bacterium usually resists the action of all other races of the bacteriophage even if they are likewise endowed with a maximum virulence. An experiment illustrative of these facts may be given. The Staphylo-bacteriophage v has a maximum virulence for but a single strain of the staphylococcus, Staphylococcus albus V. Staphylo- bacteriophage h also possesses a maximum virulence, but its action is exercised indiscriminately upon all strains of the staphylococcus. By carrying out the process of bacteriophagy in a bouillon having a pH of 6.8, leading thus to secondary cultures, I have obtained a strain of staphylococcus resistant to bacteriophage h. With this bacterium a series of cultures were made in the presence of increasing amounts of bacteriophage h, first incubating them at 37°C., and then at 30°C. Finally a strain of Staphylococcus V was derived which was refractory to bacteriophage h, that is to say, at this time the staphylococcus developed in a normal manner, macroscopically, in a pure suspension of bacteriophage h corpuscles in a medium with a pH of 7.8 and at a tem- perature of 30°C. At this time this staphylococcus was found to be refractory to the action of race v also, developing normally insofar as macroscopic obser- vation could reveal in a pure suspension of corpuscles of bacteriophage V with the mediiun of a pH of 6.8 and the temperature at 30°C. RESISTANCE OF BACTERIA 193 These two races of the bacteriophage are, however, as different from each other as it is possible for them to be. As I have already stated, one of them, V, is virulent only for the V strain of the Staphylococcus albus. The other race, h, is virulent for all staphylococci, whether they are classed as albus, aureus, or citreus.* I have obtained the same results with three races of the Shiga-bacterio- phage. A Shiga bacillus, having become resistant to one of the races was also refractory to the action of the other two. These experiments warrant the conclusion that the resistance of a bacterium is by no means limited to the action of a single race of the bacteriophage, ])ut that a bacterium having acquired a resistance against one race of the bacteriophage may manifest a resistance to the action of any other race whatever. It is only in the cases where the resistance acquired is relative that this resistance may be lacking with respect to the action of a bacteriophage of another race. It is, indeed, quite impossible to state any fixed rules governing the phenomenon of resistance to the bacteriophage. All that can be said is that usually such and such is true, but that there are many exceptions. For example, in an experiment reported by BaiP" deahng with a strain of B. coll which was naturally resistant to one race of the bacteriophage but susceptible to another, by suitable treatment this strain was induced to acquire a resistance to the last race of bacteriophage, and when this had developed the strain was found to be susceptible to the race for which it previously was resistant. It would be just as unwise to generalize from the outcome of this experiment as to draw sweeping conclusions from the fact that under certain circumstances resistance appears to be specific. Let us bear clearly in mind, in order to avoid confusion such as has occurred with certain authors, that the natural resistance to a bacterio- phage presented by strains belonging to a heterogeneous species bears no relation to the acquired resistance developing in a susceptible bacterial strain. These two distinct phenomena are comparable to those proc- esses which are designated in immunology by the terms "natural immunity" and "acquired immunity." * As we know, an acquired resistance is gradually lost during successive trans- fers. In this experiment, the resistance of the V strain of the staphylococcus to bacteriophage v is lost after 7 transfers; resistance to bacteriophage h is lost only after 19. 194 THE BACTERIOPHAGE AND ITS BEHAVIOR 4. THE ACQUISITION OF RESISTANCE How can this acquisition of immunity by a bacterium be explained? Numerous experiments have shown that if a certain quantity of a slightly active suspension of a bacteriophage is introduced into a rela- tively heavy (1000 to 2000 milHon per cubic centimeter) suspension of bacilli, the corpuscles, readily demonstrated at first by the presence of plaques on plantings on agar, disappear from the medium after an inter- val of time varying from one hour to two or three days, and that they can not later be demonstrated. Subcultures give normal cultures of bacteria. On the other hand, we have seen that with a very virulent bacteriophage the corpuscles disappear from the fluid between ten and twenty minutes after introduction into a suspension, but that they reap- pear in about twenty times as great a number in from one to one and a half hours later — they have multipHed within the interior of the bac- teria. In the case of a bacteriophage of low virulence it seems, there- fore, that penetration of the bacteria takes place but that multipHca- tion can not be effected. The bacterium resists and the corpuscle is actually destroyed in vivo. These parasitized bacteria which "recover" acquire by this an immunity. Another fact has been sometimes observed which shows that certain bacteria are able to become "carriers." As has been said, heavy sus- pensions which are inoculated with a filtrate containing a relatively avirulent bacteriophage give after a few hours absolutely normal cul- tures on agar, free of plaques. If serial transplants are made of these cultures, the plantings being made in such a manner as to yield an even layer of growth, it sometimes happens that after a certain number of transplants, two to four, a very definite plaque appears, which is indeed a colony of the bacteriophage corpuscles. This is evidenced by the fact that successive passages from this plaque yield a very active bac- teriophage. From where could this corpuscle have so suddenly come? The corpuscle had remained alive within a bacillus, and at a given moment, it overcame the resistance of the latter and multiplied. Its virulence being increased, the young corpuscles were able to parasitize the neighboring bacilli and form a colony. Any other explanation seems impossible, since, immediately after the inoculation of the bacterio- phage, seeding upon agar shows plaques characteristic of the presence of virulent bacteriophagous corpuscles, then these corpuscles completely disappear, the bacteria ,however, remaining sensitive to the action of a more active bacteriophage, for perfect dissolution is secured if the suspen- RESISTANCE OF BACTERIA 195 sion is inoculated with a trace of a very active race of the bacteriophage, and finally, the active bacteriophage reappears after a series of subcul- tures on agar in the course of which all the bacillary cultures have been normal. This corpuscle can only be one of those which had disappeared. The fact, demonstrated by experiment, of the penetration of virulent bacteriophage corpuscles into the bacteria, warrants us in thinking that this corpuscle (but slightly virulent) has been preserved in a latent living state within the interior of the bacterium. At a given moment the resistance of the bacterium is broken down and infection results (d'Herelle'^^i), The fact that a bacteriophage corpuscle may penetrate a bacterium and be destroyed there has been confirmed by Flu-^^ w^ho has carried out the following admirable experiment. Flu found, among the cultures of the Institute of Tropical Medicine at the University of Leiden, a bacterium (isolated from the stools of an individual affected with sprue) which presented all of the cultural, biochemical, and serological characteristics of B. dysenteriae Flexner, except that it was inagglutinable. This bacillus was refractory to all races of bacteriophage virulent for B. dysenteriae Flexner. Neverthe- less, suspensions of this bacillus, whether living or killed by heat, fixed the bacteriophage corpuscles of these races. By a method of grinding with anhydrous sodium sulphate, a method which will be described in a later section, Flu was able to recover the corpuscles which had been fixed to the killed bacteria. When they had been fixed to living bacilli he was unable to recover them. As he has observed, this fact can only be explained by a destruction of the bacteriophage corpuscles by the protoplasm of the bacillus. I have since proved by this same method that a bacterium rendered experimentally refractory behaves in the same manner. As a matter of fact, this experiment was carried out with the refractory strain of staphylococcus V, mentioned in a preceding paragraph. Destruction of the bacteriophage occurred not only with the corpuscles of race v, but also with those of race h. The destruction was, as a ruk;, complete when the number of corpuscles in proportion to the number of cocci was not too great, for example, when there was not more than 1 corpuscle to 100 cocci. When the ratio was higher very frequently unfixed corpus- cles remained. A bacterium possessing a moderate degree of resistance may destroy bacteriophage corpuscles of low virulence. A refractory bacterium may destroy corpuscles of maximum virulence. 196 THE BACTERIOPHAGE AND ITS BEHAVIOR 5. THE BEHAVIOR OF THE BACTERIOPHAGE IN SECONDARY CULTURES The way in which the bacteriophage behaves in secondary cultures involves a number of very interesting pecuharities. We have seen that a single bacteriophage corpuscle, provided it is endowed with a high virulence, may cause complete bacteriophagy in a normal bacterial suspension and dissolution is still more certain if the suspension is somewhat less dense. Dissolution of the bacterial cells is complete. If one simply seeds the bacterium into bouillon inoculated with a single very virulent corpuscle there is a simultaneous develop- ment of a culture of the bacteria and of the corpuscles. When the latter have become sufficiently abundant to parasitize each of the young bac- teria, dissolution occurs rapidly and the medium becomes clear. Under all circumstances, then, the introduction of a single corpuscle of maxunum virulence into a medium containing from 1 to 250,000,000 susceptible bacteria per cubic centimeter leads to a complete bacteri- ophagy, and secondary cultures never appear, provided the conditions are optimum for bacteriophagy, that is to say, provided the medium has a pH greater than 7.6 and the temperature is lower than 32°C. Furthermore, in all such cases the corpuscles derived from the single corpuscle also have a maximum virulence. With a bacteriophage of high, but not maximum, virulence the inocu- lation of a single corpuscle causes a bacteriophagy manifesting itself in the same manner as the preceding, but with further incubation a second- ary culture always develops in the medium which had previously become completely cleared.* Let us, then, observe the course of bacteriophagy as it takes place when a single corpuscle of high virulence is inoculated into a series of tubes each containing 10 cc. of a normal suspension of susceptible bac- teria.f When bacteriophagy is complete and secondary cultures appear separate these suspensions in which the phenomenon has taken place into two groups. Filter the first group immediately while the medium is still clear. With the second group allow secondary cultures to form and filter them after a few days. Comparing the two filtrates it will be found that the number of bac- teriophage corpuscles is practically the same in both, but while those of the first filtrate have a very high virulence, equal to that of the original corpuscles, the virulence of the second filtrate is attenuated. If pas- * See the section "Evaluation of Virulence." t Following the technic which has been described, distributing 10 cc. of the last active dilution into 10 suspensions, 1 cc. per tube. RESISTANCE OF BACTERIA 197 sages are continued in this same manner, allowing the filtrate which has been in contact with the resistant bacteria to remain unfiltered in each passage until secondary cultures form, it will be found that a gradual attenuation of virulence takes place. Hadley-^^ has reported that a Shiga-bacteriophage (one which I sent him) produced, at the beginning, plaques with a diameter of about G mm. During a series of passages continued throughout a period of 2 years the diameter of the plaques became progressively less, up to the point where they ceased to be larger than 1 mm. At this time, on going back to the original suspension which had been preserved for 2 years in a sealed tube, he found that the plaques formed were just as at the begin- ning, i.e., about 6 mm. in diameter. And since the diameter of the plaques, all conditions being equal, is directly proportional to the viru- lence of the bacteriophage (d'Herelle^^^-^^^), Hadley has concluded that by successive cultures the bacteriophage has degenerated.' But I have maintained this same race of the bacteriophage* and it has undergone in my laboratory a great many passages and the plaques still actually measure between 5 and 6 mm. in diameter. This would seem to prove that the degeneration observed by Hadley is a result of the conditions under which bacteriophagy took place throughout his experiments. Whatever may have been the unfavorable conditions, there was most certainly a formation of secondary cultures and these must always be avoided if it is desired to maintain intact the virulence of a bacteriophage. To accomplish this it is only necessary to filter the material just as soon as bacteriophagy is complete. This is the best means of avoiding, even from the beginning, the development of second- ary cultures which macroscopically might pass undetected. A bacteriophage, then, becomes attenuated during the same process which leads to an acquisition of resistance by the bacterium. Even a bacteriophage of maximum virulence may be ''overcome." To bring this about it is only necessary to inoculate it into a very concentrated suspension of bacteria. f It can be shown that under these conditions the virulence of the bacteriophage l^ecomes weakened. After a series of passages is made in very heavy suspensions (8000 to 10,000 million * Intentionally every time that I have given out a Shiga-bacteriophage I have always selected this same race. t We have seen that if a suspension contains more than 700 to 800,000,000 bacteria per cubic centimeter the dissolution of the bacteria is incomplete, even under the action of a bacteriophage of maximum virulence. With a bacteriophage of less virulence the number of bacteria capable of being dissolved is less. 198 THE BACTERIOPHAGE AND ITS BEHAVIOR per cubic centimeter) the virulence is attenuated to such a degree that after a few passages the bacteriophage is lost. In a word, each time that a secondary culture develops the virulence of the bacteriophage concerned becomes attenuated and this attenua- tion is the more pronounced when, on the one hand, the initial virulence was low and when, on the other, the number of resistant bacteria in proportion to the number of bacteriophage corpuscles present was high. This is precisely the experiment performed by Bordet and Ciuca^'^ who inoculated a relatively large amount (a twenty-millionth of a cubic centimeter) of a Coli-bacteriophage suspension of average virulence* into a tube of bouillon which they then seeded with a culture of B. coli. After a few days in the incubator at 37°C., that is to say, after a long contact of the corpuscles with a heavy culture of bacilli which had acquired a resistance, they heated the mixture at 57°C. in order to kill the bacilli (a further cause of the attenuation of the virulence). They demonstrated that the fluid contained a bacteriophage attenuated both quantitatively and qualitatively. These authors have attempted to explain this attenuation by assum- ing that a degeneration of the "lytic principle" occurred. As a result of its low concentration each bacterium in the suspension could fix but a very small amount and being thus but very slightly stimulated it could regenerate only a weak "lytic principle." Such an explanation is not admissible for it does not take into account the fact that the bacteriophage exists in the form of corpuscles. Fol- lowing this publication of Bordet and Ciuca, I offered to give a demon- stration of the experiment proving the corpuscular nature of the principle,^''^ but the offer was not accepted. As a matter of fact, shortly after Bordet had published his experiment one of his collaborators. Gratia, published an experiment confirming the fact that the "concen- tration" of the bacteriophage principle plays but a secondary role.^^-f It is very evident that the explanation of Bordet can not be correct since if one inoculates the smallest active dilution of a suspension of bacterio- phage into 1, 10, 100, or 1000 cc. of bacterial suspension bacteriophagy takes place in the 4 suspensions in the same manner. As a final proof let us prepare from a young agar culture of the staphy- lococcus 2 cc. of suspension each cubic centimeter containing 10,000 million cocci. Remove 1 cc. and introduce it into 99 cc. of sterile bou- * As indicated by their experiments. t The experiment of Gratia and DeKruif has been referred to in the section dealing with "Evaluation of Virulence." RESISTANCE OF BACTERIA 199 illon. We will thus have on the one hand 1 cc. of bouillon containing 10,000 million cocci and on the other hand a flask containing the same number of cocci suspended in 100 cc. of bouillon. Inoculate the two suspensions with the same quantity (1- 10~^) of a bacteriophage of maxi- mum virulence. After incubation it will be found that the hquid in the flask is clear and remains so indefinitely, and the corpuscles present show a maximum virulence. At this same time the cubic centimeter quantity is extremely cloudy and after filtration it will be found that only attenuated corpuscles are present, their virulence being not even moderate. It is possible to demonstrate this same fact in a still more conclusive manner. The cubic centimeter of suspension is inoculated with 0.1 cc. of a bacteriophage suspension; the hundred cubic centimeters with a hundred-millionth of this amount of the same suspension. After incubation the results are absolutely comparable to those of the preced- ing experiment. Corpuscles of a maximum virulence are found in the 100 cc. quantity and corpuscles of a low virulence in the cubic centi- meter. In view of the fact that the number of bacteria was the same in both cases,* the attenuation of virulence can not, then, be explained by assuming that the bacteriophage disseminates its action through too large a number of bacteria. This is, however, a priori certain since we know that the bacteriophage acts as a unit; a single bacteriophage inoculated into 10 cc. of a normal suspension, that is, placed in contact with 2500 million bacteria, effects a complete bacteriophagy. What, then, can be the cause of the attenuation in virulence? The single point which differentiates the process of bacteriophagy as it occurs in one suspension from that taking place in the other is that in the 100 cc. of suspension a secondary culture does not develop. None of the bacteria there acquire a resistance. In the 1 cc. the bacteria become resistant. All of the other conditions such as the number of corpuscles and the number of bacteria, the state of these bacteria and of the cor- puscles combined with them, the nature of the medium and the tem- perature are alike in the two suspensions. This being the case, it seems necessary to conclude that the single difference observed, that is, the acquisition of a resistance by the bacteria, is responsible for the attenuation of the bacteriophage corpuscles. Of interest in this same connection are the observations of Bordet,^'' as well as those of Gratia and de Kruif."^ They have found that if * One might even accomplish the experiment by placing three or four times as many bacteria in the 100 cc. as in the cubic centimeter. 200 THE BACTERIOPHAGE AND ITS BEHAVIOR bacteriophage corpuscles are removed from the center of a plaque on agar, the corpuscles are virulent while in the periphery of the plaque, at the margin of the bacterial growth an attenuated bacteriophage is found. The reason for this attenuation is still the same. Let us recall the manner in which the plaque is formed. A corpuscle is deposited upon the surface of the agar in the midst of many bacteria. This corpuscle parasitizes the bacterium in its immediate vicinity and multiples, and young corpuscles are liberated by the destruction of the parasitized organism. These freed corpuscles in their turn parasitize the bacterial cells with which they come in contact and the process thus continues in this manner. But during this time those bacteria which are found beyond the reach of a corpuscle multiply. The bacterial layer becomes thicker and thicker and consequently more and more difficult to attack. If the corpuscles are very virulent, that is to say, if they are reproducing actively the plaque has reached a diameter of several millimeters by the time the critical period is reached, when the bacterial layer becomes sufficiently dense to "suffocate" the bacteriophage corpuscles.* If the bacteriophage is of low virulence this period is reached when the plaque is small, simply because of the slowness with which the corpuscles have multiphed. When the layer of growth has reached a certain thickness the products resulting from the activity of the bacteriophage corpuscles can rio longer diffuse into the agar.f We have seen already that it is precisely because of the non-diffusion of the products resulting from bacteriophagy that the process on agar is limited. The activity of the corpuscles becomes paralyzed and the bacteria become resistant and acquire an immunity. This is precisely what takes place at the periph- ery of the plaque. The same conditions are to be found there as when corpuscles are inoculated into extremely dense bacterial suspensions, * We have seen that a number of factors limit the multiplication of bacterio- phage corpuscles on agar. The first of these is the thickness of the layer of me- dium which to some extent regulates the rapidity with which the products which result from the activities of the bacteriophage and which impede its action, diffuse. Furthermore, the critical moment for the bacterium is that time when it is di- viding. Division is intense when the layer on the agar is very thin, but it is much less active when the layer becomes somewhat thicker for it then contains a num- ber of old bacteria but slightly susceptible to attack. t The fact that bacteriophagy takes place perfectly well on gelatin provided that the layer is very thin on a substratum of agar shows beyond possible con- tradiction the effect of diffusible products upon the activity of the bacteriophage corpuscles. RESISTANCE OF BACTERIA 201 and the result on the corpuscles is the same in both cases,^ — an attenua- tion of virulence. Bordet^" has further shown that if a Petri dish is seeded with a cul- ture of B. coli (and all other bacteria behave in the same manner) and if, then, a drop of bacteriophage of moderate or weak* potency is deposited upon the surface the bacteria do not develop in this region, but after some time colonies of resistant bacteria appear. If these mixed colonies, which, as we will see in a later section, contain both bacteria and bac- teriophage corpuscles the latter possess an attenuated virulence. The reason is always the same; virulence becomes attenuated through con- tact with bacteria which have acquired a resistance, Bordet and Ciuca^^ have stated that a bacteriophage attenuated in one or the other of these experiments which have been cited, is no longer able to increase in virulence by passages with susceptible organisms. Brutsaert^"' has shown that this is not the case. After 12 to 20 passages he obtained an increase in virulence to such a degree that it became equal to the virulence of the bacteriophage prior to its attenuation. I have con- firmed this fact entirely. The erroneous conclusion reached by Bordet may be ascribed to the fact that the virulence of a bacteriophage attenuated through contact with resistant bacteria becomes increased only very slowly during the first passages with susceptible organisms. It is only after 7 or 8 pas- sages that the virulence begins to be increased to an appreciable degree. Once it has started to augment the increase is rapid. One might conclude from these facts that the bacteriophage corpuscles become increased in virulence by passages with susceptible bacteria, and that virulence is attenuated by passages with bacteria which have acquired a resistance, that is, an immunity. Bacteria which have acquired a completely refractory state may even destroy the corpuscles. Here again the manner in which the bacteriophage corpuscles and the susceptible bacterium which has acquired an immunity behave is exactly like that of the pathogenic microorganism and the susceptible animal which has acquired an immunity.! It is, on the other hand, * If the bacteriophage is of maximum virulence or is very active no resistant bacterial colonies develop. Bordet did not state, in his paper, the virulence of the bacteriophage with which he worked, but it is obvious that the virulence was weak because of the fact that resistant colonies developed. t Among all of the experiments which might be mentioned and which have been of interest to all biologists there is one which is of particular interest because t he conditions, experimentally, are almost identical with those of corpuscles at- tenuated through contact with resistant bacteria. WoUmann inoculated a few drops of an attenuated culture of B. anthracis into 202 THE BACTERIOPHAGE AND ITS BEHAVIOR evident that the attenuation of the virulence of a bacteriophage takes place only when the resistance of the bacteria dominates the virulence. In the opposite case, when the resistant bacteria are overcome there results, naturally, an increase in virulence. A bacteriophage, when overcome, is attenuated. A bacteriophage when overcoming is enhanced and the increase in virulence is in direct proportion to the resistance of the bacterium overcome. Whether these events take place in the scale of beings that involve the susceptible animal and the virulent bacterium or the susceptible bacterium and the virulent bac- teriophage is of no fundamental significance. The result is exactly the same. Some experiments of Gratia and of Wollstein illustrate particularly well the increase in virulence resulting from the contact of a victorious bacteriophage with bacteria which have acquired a resistance.^^® A bacteriophage at its time of origin presented a specific activity, limited to a single strain of B. coli. Successive passages were made at the expense of resistant bacilli of this same strain. The results are sum- marized in table 18 (+ + + + = complete bacteriophagy, no resistant colonies forming when the material is spread upon agar; 4- + + = almost complete dissolution, less than 12 colonies developing when spread upon agar; +-F = partial dissolution, many resistant colonies appearing; + = no dissolution, a few plaques forming when spread upon agar; — = no bacteriophagy; S = the susceptible strain, R = the resist- ant strain). the peritoneum of a guinea-pig. After a few hours he removed the peritoneal exudate and centrifuged it at moderate speed, the leukocytes, together with the phagocytized bacteria, collecting in the sediment. The free bacteria, i.e., those which had resisted phagocytosis, remained suspended in the supernatant fluid. With these materials he inoculated two guinea-pigs intraperitoneally. To one he gave a few drops of the supernatant fluid, containing, of course, "vic- torious" bacteria, and to another he gave a portion of the sediment containing phagocytized bacteria, that is to say, "conquered" organisms. He continued these passages in a double series and demonstrated that the virulence of the strain resulting from the selection of the "victorious" bacteria increased with each passage and ended by being very high. On the contrary the strain resulting from the selection of "conquered" bacteria became more and more attenuated. It is only necessary to substitute in the experiment of WoUmann the word "guinea-pig" or better yet "leukocytes of the guinea-pig" by "bacterium" and the word "bacterium" by "bacteriophage corpuscle" in order to realize how completely the facts observed by him conform to the behavior of the bacteriophage. RESISTANCE OF BACTERIA 203 These experiments are of great interest for they indicate, as Gratia himself has remarked, a method of increasing the virulence of a bacterio- phage and they show how virulence toward diverse bacterial species may be acquired. 6. THE LOSS OF RESISTANCE Bordet and Ciuca^^ were th