Gornell University Library Ithaca, New York BOUGHT WITH THE INCOME OF THE SAGE ENDOWMENT FUND THE GIFT OF HENRY W. SAGE 1891 RETURN TO ALBERT R. MANN LIBRARY ITHACA, N. Y. Cornell University Library The transmutation of bacteria, Cornell University The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003199316 THE TRANSMUTATION OF BACTERIA CAMBRIDGE UNIVERSITY PRESS C. F. CLAY, MANAGER LONDON : FETTER LANE, E.C. 4 LONDON : H. K. LEWIS AND CO., Lrtp., 136 Gower Street, W.C, 1 LONDON : WILLIAM WESLEY AND SON, 28 Essex Street, Strand, W.C. 2 NEW YORK : G. P. PUTNAM’S SONS BOMBAY CALCUTTA} MACMILLAN AND CO., Lrpv. MADRAS TORONTO : J. M. DENT AND SONS, Lrp. TOKYO : MARUZEN-KABUSHIKI-KAISHA ALL RIGHTS RESERVED THE TRANSMUTATION OF BACTERIA BY S. GURNEY-DIXON, M.A., M.D. (Cantas.) M.R.CS. (Ene.), L.R.C.P. (Lowp.) “Ogni primaio aspetto ivi era casso: due e nessun I’ imagine perversa parea,... Cosi vid’ io la settima zavorra mutare e trasmutare; e qui mi scusi la novita, se fior la penna abborra.” Dante: Inf. XXV. CAMBRIDGE AT THE UNIVERSITY PRESS 1919 PREFACE HIS essay is based upon notes and observations which I collected previous to the year 1913. It was only partly written when, in August 1914, I proceeded on active service. I was able, however, to complete it in the following summer while serving with a Field Ambulance in France, and in the autumn of the same year (1915) I submitted it in the form of a Dissertation for the degree of M.D. at the University of Cambridge. The difficulties in carrying out work of this character while serving at the Front—remote from libraries and amidst “alarms and excursions” which break up one’s scanty leisure —are sufficiently obvious and I trust may excuse some of its defects. Some valuable materials which I had hoped to utilise, including chapters on Viability and Agglutination Reactions, were buried by a shell explosion and could not be replaced. The claims of Army work have also precluded any attempt on my part to bring the work up to date by reference to papers published since the beginning of the war. I particu- larly regret having learnt too late to include any mention of it in the following pages of the valuable research carried out by Dr Thiele and Dr Embleton on the part played by the body ferments in the pathogenicity of bacteria. Though I have endeavoured to suppress all irrelevant matter, I am only too conscious of the discursiveness of this essay. The topic is one of absorbing interest and at every step one is tempted to digress. In the words of Dante, which I have quoted on the title page, “The novelty must be my excuse if my pen has wandered at all.’ vi PREFACE I have only touched the fringe of the subject. An inex- perienced sailor in my “ piccioletta barca,” I have been tossed about in the breakers of this uncrossed sea. To others, better equipped by knowledge and training than I am to explore it, | I would say— “Metter potete ben per I’ alto sale vostro navigio,... Quei gloriosi che passaro a Colco non s’ ammiraron, come voi farete, quando Jason vider fatto bifolco.” (Dante : Par. IT.) 8. G.-D. CONTENTS PREFACE SYNOPSIS. . INTRODUCTION CHAP, I. II. XI. XII. THE SCOPE OF THE ENQUIRY CONDITIONS MODIFYING THE CHARACTERS OF BACTERIA A CONSIDERATION OF THE EVIDENCE VARIATIONS IN MORPHOLOGY VARIATIONS IN FERMENTING POWER VARIATIONS IN VIRULENCE . VARIATIONS IN PATHOGENICITY THE POSSIBLE OCCURRENCE OF TRANS- MUTATION IN THE LIVING BODY SUPPOSED INSTANCES OF TRANSMUTATION BROUGHT ABOUT EXPERIMENTALLY . SUMMARY THE ENZYME THEORY OF DISEASE CONCLUSIONS APPENDIX. REFERENCES . PAGE 107 116 140 153 170 171 SYNOPSIS INTRODUCTION CHAPTER I THE SCOPE OF THE ENQUIRY Derinrrion or Turms. Transmutation not evolution—evolution in bac- teria—its stages. Natural variation—“Spontaneous” and “impressed.” Varia- tion easily studied in bacteria—unicellular organisms—method of generation —rapidity of generation—environment easily modified. Natural selection. Artificial selection. “Transmutation of Species” apparently contradictory— meaning of “species”—based on characters. Arbitrary nature of distinction between species—illustrated by streptococci—classified according to food- stuffs and haemolytic power, adhesiveness, staining, cultural characters, virulence and pathogenicity, agglutination, fermenting power. “Species” not a rigid term. A CONSIDERATION OF THE POSSIBILITIES. 1. Simple variation. 2. Varia- tions in different directions associated. 3. Development of intermediate forms. 4. Slight changes in closely allied organisms. 5. Complete change in characters. (Pages 3—12) CHAPTER I CONDITIONS MODIFYING THE CHARACTERS OF BACTERIA 1. Spontaneous variations. Pleomorphism. Unexplained variations, 2. Geographical distribution. 3. Prolonged cultivation—extends survey— permits natural selection—influence of saprophytism. 4. Conditions of culti- vation, (a) lowered vitality, (b) crowding of colonies, (c) temperature, (d) at- mospheric pressure, (¢) oxygen, (/) sunlight. 5. Ultra violet rays. 6. Elec- trolysis. 7. Age of culture—pleomorphism—other variations. 8. Culture medium—(a) age of medium, (5) reaction of medium, (c) nature of medium —natural secretions—pathological exudations—water, (d) chemical sub- stances—carbolic acid, antiseptics, boric acid, potassium bichromate, sodium benzoate, glycerine, iodine trichloride, lactic acid. 9. Prolonged contact with particular foodstuff. 10. Artificial selection—method sometimes in- effective. 11. Symbiosis—lichens—nitrifying organisms—parasitism—anaer- obes. Symbiosis may confer new powers—may have no effect. Methods of studying symbiosis—mixed growth, adjacent colonies, criss-cross planting, surface and deep growths, double celluloid sac, successive growth. 12. Para- sitism, (#) transmission through alimentary canal, (6) passage, (c) celloidin sac in body cavity, (d) residence in living tissues, (¢) during disease, (°) in (13—27) “carriers.” x SYNOPSIS CHAPTER III A CONSIDERATION OF THE EVIDENCE 1. Contamination. Growth from single bacterium. 2. Mixed infection— error due to (a) unequal growth of two strains, (b) incomplete recognition. Proof of continuity necessary. 3. Secondary invasion. Bacteria in healthy organs. Post mortem invasion. 4. Repetition of experiment. 5. Constancy of new feature—meaning of “permanent.” 6. Perseverance necessary. 7. Faultless technique (e.g. agglutination) and accurate observation (e.g. staining) required. 8. Methods may require to be improved, (a) irregular results due to media—eg. sugars, may be impure, contaminated by glass, affected by sterilisation, deteriorate—age of medium—composition—re- action, (6) age of culture, (c) time allowance. 9. Clinical observation im- portant, e.g. Widal’s test in jaundice—effect of drugs—pre-existing disease. (28—36) CHAPTER IV VARIATIONS IN MORPHOLOGY A. ZoogiEtc ForMs—not fortuitous—B. radicicola—Beggiatoa versatilis. Zoogleae not in strict sense individuals—analogy of regiment of soldiers and crowd of pitmen—typical formations assumed—not separate individuals like a tree—formations not invariable—may simulate each other—this does not imply transmutation. I. Zoogleic forms occurring spontanecously—stages in life history—or variations. B. rubescens—other examples. II. Zoogleic JSorms artificially produced, 1. Due to chemical substances—salt, sewage, urea, saliva, bile, acid, caustic soda, 8 naphthol, alcohol, potassium bi- chromate, boric acid, nitrates, antiseptics, tartaric acid. 2. Temperature. 3. Absence of oxygen. 4. Ultra violet rays. 5. Growth in animal body. B, VaRIaTIONS IN INDIVIDUAL oRGANISMS. I. Pleomorphism—B. ru- bescens—other examples. II. Variations due to environment. 1.Geographical distribution. 2, Prolonged cultivation. 3. Crowding of colonies. 4, Changes in medium—reaction. 5. Chemical substances—urea, urine, carbolic acid, creosote, nitrogenous substances. 6. Ultra violet rays. 7. Electrolysis. 8. Symbiosis. 9. Growth in living tissues, C. Variations IN coLonies. 1. Colonies of the same organism vary. 2. Different organisms produce similar colonies. 3. Addition of various substances to medium affects colonies. 4. Influence of heat. 5. Effect of “passage.” Variation in other morphological characters. (37—49) SYNOPSIS xi CHAPTER V VARIATIONS IN FERMENTING POWER THE FERMENTATION OF CARBOHYDRATES—its stages. Different types of variation. I. Different strains may possess different fermenting properties. II. The same strain may vary spontaneously. III. Fermenting properties modified by conditions of growth. 1. Temperature. 2. Oxygen. 3. Atmo- spheric pressure. 4. Age of culture. 5. Age of medium. 6. Composition of medium—effect of carbolic acid, sodium benzoate, monochloracetic acid. 7. Influence of source—milk, urine, ascitic fluid. IV. Symbiosis. V. In “carriers.” VI. After “passage.” VII. In disease. VIII. Prolonged con- tact with a particular sugar. IX. Artificial selection—method often in- effective. THE SIGNIFICANCE OF VARIATIONS IN SUGAR REACTIONS. 1. Fermentation due to enzymes which are destroyed by antiseptics. 2. Distinct enzyme for each different sugar. 3. Different enzyme for each different stage in fer- mentation. 4. Distinct enzyme for forming each acid. 5. Distinct enzyme for producing gas from each acid. 6. New fermenting power an adaptation to environment. 7. Such adaptation advantageous to organism. 8. En- couraged by natural selection. 9. Explanation of incubation period—oc- cupied by preparatory changes ?-—this disproved,—interval before variation appears!—this disproved,--time required for variants to predominate ?— does not explain definite length of period. 10. Reason for shortening of period by subculture—subculture hastens reproduction. 11. Artificial selection. 12. Reversion. 13, Variations apparently spontaneous—possibly due to contamination of medium—or to impure sugar. No explanation of spontaneous variation. ; THE VALUE OF THE SUGAR KEACTIONS—unsatisfactory as tests. 1. Time allowance not fixed. 2. Reactions vary with temperature and other con- ditions. 3. Media often unreliable—sugars impure—altered by sterilisation —contaminated by glass vessel—deteriorate on keeping—acid reaction masked. 4. Tests inconstant. 5. Positive or negative reaction a matter of degree only. 6. Different carbohydrate groups yield different classification —if designed to correspond with other tests useful for identification only. Comparison between fermentation and agglutination tests—fermentation tests may vary while agglutination constant—fermentation and agglutination properties may both be altered—they yield a different classification—two tests not related but may supplement each other. THE VALUE OF VARIATIONS IN THE SUGAR REACTIONS IN THE IDENTIFICA- TION OF BACTERIA—Variations themselves constitute a test—may be specific (cf. morphology of B. diph.)—may identify source of strain. (50—70) xii SYNOPSIS CHAPTER VI VARIATIONS IN VIRULENCE Bacteria pathogenic and non-pathogenic. Pathogenic character due to two factors—parasitism—nature of activity in tissues. Most bacteria cannot invade tissues—activities of some invaders harmless—actual invasion not essential, Effects of bacterial invasion due to (a) their metabolism, (0) their disintegration, (c) their mechanical action, (d) response of living tissues. Viability—pathogenesis—virulence. VARIATIONS IN VIRULENCE. 1. At different stages of epidemic—possibly explained by unequal resistance met with. 2. Sporadic cases of infectious disease imply weakened virulence. 3. Endemic diseases become less viru- lent—possibly explained by acquired immunity. 4. Epidemics vary in severity with date and locality. 5. Intensity of infection by same specific organism varies. 6. Virulence altered by abnormal conditions of cultiva- tion, (a) temperature—possibly protective influence of fever—disproved, (6) presence of antiseptics—carbolic acid, potassium bichromate, iodine tri- chloride, saliva, (c) oxygen, (2) sunlight, (e) reaction of medium. 7. Virulence altered by prolonged cultivation outside the body. Results due to several factors, (a) chemical composition of media—blood media—pathological exu- dations—urine, (b) physical character of artificial media, (c) response of tissues, (¢) purity of culture. 8. Virulence increased by growth in patho- logical secretions. 9. Symbiosis—affects viability—also affects virulence. 10. Virulence altered by “passage”—passage alternating with culture more effective. 11. Simultaneous inoculation with another organism intensifies results—even when symbiosis of same organism outside the body ineffective. Simultaneous subcutaneous and sub-peritoneal inoculations with different organisms also effective. Exalted virulence is towards species used for passage—not necessarily towards others. THE SIGNIFICANCE OF VARIATION IN VIRULENCE. Evolution of bacteria —virulence is latest property acquired and first to be lost. Its re-acquire- ment an example of the survival of the fittest —“fittest” not necessarily most robust, but most capable of defence. Virulence results from adaptation and is not due to increased robustness, (a) increased virulence to one species of animal does not apply to another, (6) most virulent not always most robust —contrary true of pneumococeus, (¢) analogy suggests adaptation, eg. increased resistance to antiseptics, (d) increased virulence accompanied by other changes obviously adaptive, eg. growth at body temperature. Difi- culties in accepting natural selection as developing virulence. (a) Intra- cellular toxins only set free after death of organism—may nevertheless be of advantage to strain—their effect perhaps purely physiological and not the result of adaptation. (6) Why are common infective diseases not of deadly virulence?—death of host involves death of organism. (c) Virulence established by single “passage”—virulence possibly results from sudden change in metabolism. (d) Toxic saprophyte assists non-toxic as well as SYNOPSIS xiii itself—nevertheless may benefit strain. Invasion of tissues by virulent saprophyte involves change in foodstuffs—relationship between altered metabolism and acquirement of toxicity—possibly a change in excretion following change in assimilation—experimental evidence, («) B. colt does not attack proteid if carbohydrate present, (b) B. diph. does not yield toxin if much carbohydrate present—suggest toxins may result from alteration in food material. Altered metabolism of saprophyte facilitates invasion of tissues—this supposed alteration in metabolism does not always confer toxicity—toxins may be regarded as an excretion or as a secretion—or as product of enzyme—activity of enzyme may be due to adaptation, encouraged by natural selection. THE VALUE OF VIRULENCE IN CLASSIFICATION. Classification according to virulence inconsistent. Non-virulent B. diph. in “carriers” regarded as lineal descendant of virulent Klebs-Loeffler bacillus—other non-virulent B. diph. provoke antitoxin, therefore same species as Klebs-Loeffler bacillus. Non-virulent and virulent pneumococcus regarded as varieties of same species. Non-virulent and virulent B. colt communis thought by some to be different species—cf. amoeba colt. Non-virulent “B.anthracoides” described as different species from virulent B. anthracis. S. erysipelatis and S. pyogenes formerly regarded as distinct species, Virulence not a specific character. (71—93) CHAPTER VII VARIATIONS IN PATHOGEN ICITY Pathogenicity is power to produce in certain animals certain symptoms and certain lesions—quite distinct from virulence and other characters, Generally regarded as more fixed than other characters—constitutes final appeal in doubtful cases, e.g. Hofmann’s bacillus and Klebs-Loeffler bacillus —gonococcus and meningococcus—gonococcus does not cause meningitis nor meningococcus urethritis. Pathogenicity a variable character in all three aspects. J. VARIATION IN KIND OF ANIMAL AFFECTED. IJ. VaRiaTIon IN SYMPTOMS CAUSED. (1) Same organism causes different symptoms in different cases. Symptoms may depend upon organs affected—cf. lead. poisoning—this determined by route of infection and vitality of organs—also by patho- genicity of organism—eg. tubercle bacillus causes phthisis, osteitis, arthritis, lupus—unlike lead poisoning types remain distinct—skin rarely infected by tuberculous sputum—contrast between gonococcus and meningococcus no greater than between different strains of tubercle bacilli. Fallacy due to pre-existing disease, ¢.g. nephritis in cerebrospinal fever. (2) Pathogenicity can be artificially modified, e.g. that of B. anthracis by ultra violet rays. (3) During epidemic different cases exhibit different symptoms. Xiv SYNOPSIS S. scarlatinae causes scarlet fever in some cases and puerperal fever in others. J. catarrhalis produces symptoms of many diseases—common cold, influenza, scarlet fever, diphtheria, typhoid fever, cerebro-spinal fever. ' (4) In different epidemics different types of disease presented— B. influenzae causes epidemics simulating coryza, rheumatic fever, typhoid fever, cerebrospinal fever. (5) Same train of symptoms follows infection by different organisms —typical rabies due to 2B. diph,—typical scarlet fever, cerebrospinal fever and influenza due to I. catarrhalis—typical cerebrospinal fever due to B. typhosus and to Klebs-Loefiler bacillus—symptoms resembling diphtheria due to pneumococcus—typical typhoid fever due to B. coli. III. Variation IN LESIONS PRODUCED—studied in two ways—lesions pro- duced during disease and by artificial inoculation in animals. 1. Variations in lesions produced during disease. In many cases characteristic—not invariably so—lesions typical of one infection may be produced by a different one—lesions influenced by other factors than species of organism—e.g. age of patient, route of invasion, secondary infection, treatment, etc.—possibility of excluding such factors by inoculation. 2. Variation in lesion caused by artificial inoculation. Method “standardises” lesion—lesions said to be invariable under these conditions. B. pseudo-diphtheriae distinguished from #&. coli—avian tubercle bacillus distinguished from human type— S. mastitidis distinguished from S. anginosa and S. pyogenes—pneumo- coccus of lobar pneumonia distinguished from that of lobular pneumonia. Are the lesions caused by artificial means invariable ?—certainly very constant—eg. tubercle bacillus—but not absolutely fixed—eg. a strain of B. diph, causes lesions of rabies—a strain of S. mastitidis loses its power to cause typical lesion—various types of tubercle bacilli fail to cause their typical lesions. Two strains causing different lesions arise from single strain during cultivation—D. lanceolatus capsulatus isolated from different organs causes different lesions—type of lesion altered if organism first grown anaerobically. Every aspect of pathogenicity subject to variation. Other characters of bacteria equally variable. (94—106) CHAPTER VIII THE POSSIBLE OCCURRENCE OF TRANSMUTATION IN THE LIVING BODY Organisms closely resembling each other except in pathogenicity often found associated—can one of these be a derivative of the other ?—¢g. B. anthracis and B. anthracoides, in hides of cattle. Other instances in the human body. B. coli and B. typhosus. Klebs-Loefier bacilius and Hofmann’s bacillus —pathogenesis—fermenting properties—seasonal pre- valence—during convalescence from diphtheria—recent work. Staph. epidermidis and staph. pyogenes—pathogenesis—fermenting properties— SYNOPSIS XV pigment formation. Zhe meningococcus and M. catarrhalis—morpho- logy—fermenting properties—pathogenesis—mixed infection—habitat. The meningococcus and the pneumococcus—symptoms produced—common com- plications—seasonal prevalence—age incidence and mortality —distribution. Such transition less credible than it appears—but not less credible than instances known to occur—saprophytic and parasitic types of pneumococcus. Conclusions, (107—115) ‘CHAPTER IX SUPPOSED INSTANCES OF TRANSMUTATION BROUGHT ABOUT EXPERIMENTALLY I. Mason Horrocks’s experiments (Journal of R.A.M.C. Vol. xvi). Importance of the claims made by him. Criticism. Possibilities to be considered—purity of original strain, peritoneum possibly not sterile, possible contamination from skin, possible invasion from gut before or after death, continuity of strain not confirmed by reversion or presence of intermediate forms, different results obtained on repeating experiments, results possibly explained by variation. Criticism. Conclusions. II. ReLatioNsHIP BETWEEN PARATYPHOID ORGANISMS. A. Schmitt's experiments. Experiment I. “Fligge” type given to calf in food, injected beneath skin—second strain isolated from blood. Experi- ment II. Second strain injected into nasopharynx of second calf—third strain isolated from blood and injected into vein, fourth strain recovered after death—later strains resembled B. Gaertner in agglutination. Possible fallacies, (a) contamination in original strain? (6) contamination in bodies of calves ?—not absolutely excluded by agglutination tests—B. Gaertner in intestines of healthy calves, possible increase in numbers and virulence due to local inflammation, might lead to systemic invasion, (cf. saprophytes. in inflamed uterus) and fresh agglutination reactions of blood serum. . Experiments of Mihlens, Dahm and Fiirst. Mice fed on infected meat—faeces of some contained B. Aertryck, of others B. Gaertner—due to transmutation ? Possible fallacies, (a) contamination of original source ? (0) contamination in bodies of mice ?—control—B. Aertryck in healthy mice, presence possibly overlooked ?—their appearance favoured by in- flammation and disturbed function of bowel. C. The Author's experiments. Experiment I. Guineapigs given B. Gaertner in food—B. Gaertner and B. Aertryck isolated from faeces at different times—control—two organisms transmutable ’— intestinal bacteria undetected if few in number—disturbed function of bowel reveals their presence—multiply in inflamed intestine (cf. B. colt in cholera)—such factors may explain result of experiment—qualification—proof that in disordered intestine unsuspected organisms make their appearance. Experiment II. Faeces of six guineapigs examined—ZB. proteus found in one case—guinea- xvi SYNOPSIS pigs given unwholesome food—faeces again examined—B. proteus found in four cases. Conclusions—suggest presence of secondary invaders in experi- ments quoted—possible error from identifying organism by agglutination— variable agglutination of paratyphoid organisms. Application to experiments quoted. Results no evidence of transmutation. (116—139) CHAPTER X SUMMARY All characters of bacteria show variation—“spontaneous” or “impressed” ~—modifying influences already discussed (Chap. 11). Variation may be ap- parent only. Apparently spontaneous variation may be due to unrecognised influences. Variation itself may be specific (morphology of B. diphtheriae and S. scarlatinae). No one character specific—variation need not imply loss of specific character (morphology of B. coli). Analogy of regiment of soldiers and crowd of pitmen. Variation may be specific because it indicates racial character. Many variations represent past stages in evolution (Mor- phology, Chap. rv)—others represent new steps in evolution (Fermenting Power and Virulence, Chaps. v and v1). TRANSMUTATION DIFFERS FROM VARIATION IN DEGREE ONLY—different species derived from a common stock—differentiation more advanced in some than others—reversion therefore differently interpreted—necessity of regarding characters as a whole and their stability—danger of relying upon one character alone already shown (Chap. rv—vit). Analogy of human race groups. Variation may indicate recent environment—and so reveal source of particular strain (streptococcus from milk—general coli infection from biliary passages). -STABILITY OF VARIATIONS. “Spontaneous” variations. (1) Imperfect development—tend to disappear. (2) Senility or lowered vitality—tend to persist. (3) Atavistic tendencies—tend to recur. (4) Fresh stage in evolu- tion—therefore unstable. Two variations constantly associated—both due to lowered vitality—both due to higher evolution—both due to imperfect development or degeneracy. “Jmpressed” variations—may lapse when in- fluence withdrawn—may persist for a time—may appear permanent—danger of assuming variation is permanent—examples—ability to ferment sugar or produce pigment—inability to ferment sugar or display virulence. Duration of impressed variation. (1) If only part of strain varies it may appear to revert—danger of assuming reversion has occurred. (2) If readily acquired is long retained—if slowly acquired quickly lost (ability of B. typhosus to ferment)—not true of spontaneous variations. (3) The longer the training the more lasting the effect (streptococcus at different stages in disease— ability of B. typhosus to ferment). Same principle governs development of races. Absence of reversion does not imply inability to revert (pigment SYNOPSIS xvii production by B. ruber—mycelial development of B. diph.). Tendency to revert does not imply loss of specific character. Variation differs from transmutation in degree alone. TRANSMUTATION DIFFERS FROM EVOLUTION IN DEGREE ALONE. Analogy of different branches of family. Possibility of transmutation. Saprophytic and parasitic pneumococcus. Other examples already discussed (Chap. vit). Experiments suggesting transmutation already discussed (Chap. 1x). Ist series, strains not guaranteed pure, results explained by variation. 2nd series, results explained by variation, secondary invasion not excluded. Transmutation improbable. Enzyme theory of disease. (140—152) CHAPTER XI THE ENZYME THEORY OF DISEASE Predicates disease uot due to bacteria but to their ferments. (1) Ac- quisition and loss of pathogenic powers. (2) Different organisms may cause same type of disease—rabies due to. B. diph. (3) Same organism causes different types of disease—in different epidemics (8. influenzae)—cases differ in same epidemic—scarlet fever and puerperal fever—M. catarrhalis infection simulating other diseases, coryza, influenza, scarlet fever, diphtheria, typhoid fever, cerebrospinal fever. (4) Same conditions influence virulence and fermenting power, (a) antiseptics, (b) oxygen—virulence of cholera, toxicity of B. diph. fermenting power of B. dysent., of streptococcus, (c) temperature—optimum temperature—digestive enzyme in cold blooded animals—germ barley—marine enzymes—fermenting power of B. coli— virulence of B. diph., B. tetani, B. anthracis, etc.—enzymes killed at 60° C. and virulence destroyed, (@) sunlight, (¢) symbiosis—tetanus and pyogenic cocci, B. colt and B. dentrificans. (5) Virulence due to “passage” through. an ‘animal and fermenting power due to growth in a sugar, (a) specific, (6) repeated inoculations or subcultures more effective, (c) power readily acquired is easily maintained, (d) if recently lost is quickly regained. (6) Intra- and extra-cellular toxins—intra- and extra-cellular enzymes, yeast, digestive enzymes—emulsion of gland or bacteria more potent. (7) Virulence associated with fermenting properties—M. catarrhalis, gonococcus and meningococcus, Hofmann’s bacillus and Klebs-Loeffler bacillus, B. cofi—both due to adaptation? (8) Living tissues defended by enzymes. (9) Other functions of bacteria due to enzymes—influenced by same conditions as virulence, ¢g. pigment formation. (10) These ferments separable from bacteria—enzyme which liquefies gelatin survives bacteria—passes filter— soluble. (11) M. ureae—enzyme separable. (12) Isolation of pathogenic enzymes (pneumococcus), (13) Bacteria deprived of a pathogenic function by environment—same conditions influence ferment activity, (a) ultra violet rays—pathogenesis of B. anthracis, (b) oxygen—power of pneumococcus to Xvili SYNOPSIS produce skin lesion, (c) growth in milk—fermentation by ZB. coli, rash in scarlet fever, (d) effect of substances added to media and of drugs in disease —sod. benzoate and JB. coli—sod. salicylate and acute rheumatism. (14) Different symptoms due to different enzymes?—analogy with sugar ferments of bacteria—complexity of action—association with particular vegetable and bacterial cells—possibility of complete dissociation of patho- genic enzymes? (15) Two results obtainable—organisms deprived of patho- genic functions (B. typhosus)—functions maintained in absence of organism, eg. filter passers. (16) No enzyme isolated which forms toxins outside the body—true also of other recognised ferments—artificial media differ from vital fluids. (17) The enzyme theory and transmutation—suggests transfer of function possible—certain conditions essential. Analogy of ships at sea. Conclusion. (153—169) CHAPTER XII CONCLUSIONS (170) APPENDIX REFERENCES (171—179) INTRODUCTION THE mediaeval alchemists conceived the idea of the trans- mutation of metals and dreamt of changing the baser metals into gold. The task which baffled them the scientists of our own generation seem destined to achieve. The transmutation of bacteria is a problem of more recent date but it bears a certain resemblance. If silver and gold are the currency of wealth by means of which it changes hands, bacteria represent the currency of disease by means of which this also is passed from one person to another. The resemblance, however, goes much deeper than this, for just as the metals have hitherto been regarded as “elements” of matter so the functions of the unicellular organism have been thought to represent the “elements” of life. The physicist has learnt that the so-called “elements” of matter are themselves composed of infinitely small particles or “ions”; the pathologist is learning that the functions of bacteria in many cases result from the activity of ultra-microscopic bodies, of the nature of “enzymes.” The occurrence of transmutation in the case of bacteria would prove as revolutionary in our conception of disease as its occurrence in the case of certain rare metals is already proving in our conception of matter. The idea of the permanence of characters in the animal world is at least as old as the question “Can the Ethiopian change his skin or the leopard his spots?” but. it is only in recent times that the fixity of animal species has’ been scientifically demonstrated. Amongst the less highly organised structures of plant life variation is of more frequent occurrence and, though it is not possible to “gather figs from thistles,” it is generally acknowledged that “species” in the case of plants are less rigidly defined than in the animal world. In the realm of bacteriology still simpler forms are met with in which are recognised the beginnings of both animal D. 1 2 INTRODUCTION and vegetable life, and amongst these variation is of still greater frequency. This fact, confirmed by personal observa- tion and by a perusal of the literature of the subject, suggested to the writer the question whether actual transmutation of species might not occur amongst bacteria, and it was in the hope of answering this question that the investigation here recorded was undertaken. An endeavour was made in the first instance to collect the published records of all experiments in which transmuta- tion was alleged to have occurred. These were found to be few in number. In the second place, a series of experiments was carried out by the writer with the object of disproving the contention put forward in one of these cases. Thirdly, with a view to criticising the claim made in the remaining cases, and in the hope that it might throw some light on the problem of transmutation as a whole, a study of the subject of variation amongst bacteria was undertaken. The material on which it is based has been collected from the scattered literature of the subject. With a few exceptions, only papers written in English have been consulted. In Chapter I the scope of the enquiry is outlined. In Chapter II the conditions which modify the characters of bacteria are enumerated and in Chapter III the value of the evidence adduced in proof of such modification having occurred is considered. Examples of variation are then studied in detail and their significance is discussed, reference being made more particularly to morphological characters, fer- menting properties, virulence, and pathogenesis (Chapters IV—VII). In Chapter VIII the possibility of transmutation occurring in the animal body is considered. In Chapter IX instances of supposed transmutation are examined. In Chapter X the subject is reviewed at length and the results of the investigation summarised. In Chapter XI the Enzyme theory of disease is discussed, together with its bearing upon the subject of transmutation. In Chapter XII the author’s conclu- sions are briefly stated. References are given in the Appendix. CHAPTER I THE SCOPE OF THE ENQUIRY DEFINITION OF TERMS THE phrase “transmutation of bacteria” is not synonymous with “evolution of bacteria.” “Evolution” is the gradual de- velopment of new species and tends towards further differentia- tion. “Transmutation” is the changing of members of one recognised species into those of another and, if proved, would tend towards unification by undermining existing barriers. There is no reason to doubt and abundant evidence to support the opinion that in this field of life, as in others, the forces of natural selection and the survival of the fittest have been at work and have resulted, in the course of ages, in the evolution and differentiation of the various types of bacteria which we recognise and distinguish today. Andrewes, in the Horace Dobell Lecture for 1906, “traced the evolution of streptococci from the condition of harmless mineral-feeders, through that of saprophytism in the alimen- tary canal, to the development of weak powers of parasitism which have culminated, in certain instances, in the fully developed property of aggressive parasitism seen in the streptococcus pyogenes.” He showed how, at different stages, natural selection and the survival of those best adapted to the environment in which they found themselves, resulted in the permanent acquisition of new characters, such as the ability, when they had once entered upon a saprophytic career in the alimentary canal, to flourish most vigorously at the body temperature of their host and to utilise the foodstuffs available in their new habitat; to resist desiccation during the intervals between their discharge from one host and their reception by another; and later still to support themselves in the actual living tissues of the host : 1—2 4 THE SCOPE OF THE ENQUIRY [cH. I and to defend their position there by the manufacture of haemolysins and toxins. This process of evolution is, no doubt, going on continually in bacteria as in higher forms of life. It is rendered possible in both cases by the occurrence of natural variation. This variation in bacteria is of two kinds, namely, spontaneous or intrinsic variation between the individuals of a pure culture— that is to say, bacteria derived from a single organism,—and impressed variation, the effect of special environmental con- ditions upon a succession of bacterial generations, due either to the direct reaction of the bacterial protoplasm to the environment, or to selection acting upon slight spontaneous variations and producing a cumulative effect. Itis reasonable to expect that amongst the bacteria natural variation would occur with greater frequency than amongst higher forms of life for, being unicellular organisms, changes in their environment can operate directly upon the germ plasm. Moreover the common method by which bacteria multiply, namely the division of the parent cell into two daughter cells, ensures the ready transmission of any acquired character from parent to offspring. The variation in character may be said to be retained by the daughter cells rather than transmitted to them. Such retention of parental characters by the daughter cells is not, however, invariable. For example, McDonald (1908) has published photographs of a young culture of the meningococcus showing diplococci in which one member is stained while the other is not. Thirdly, such variations would be more readily noted in their case since as many as 30 or 40 successive generations may be observed in the course of 24 hours. In the case of some bacteria division may occur as frequently as once every 17 minutes (Barber, 1908). Yet a fourth factor might be mentioned, namely the ease with which the environment of any strain of organisms can be modified in any direction and to any extent. As a matter of fact examples of such variation, as we shall show, are innumerable, no matter what particular property or character of bacteria we investigate. Differences occur in size and shape, in staining properties, in power of growth on various CH. I] THE SCOPE OF THE ENQUIRY 5 media, in viability, in virulence, in the power to ferment sugars, and so on. z The environment in which bacteria grow and multiply tends, in the course of time, to “fix” some of these variations by offering to the possessors of them a better chance of survival or perpetuation, so that they ultimately become characteristic of a new species. This is evolution through natural selection. By asimilar process of artificial selection, as will be shown, we can encourage variation in almost any direction we choose. “Within certain limits the simple forms of life are able to adapt themselves to their surroundings and the adaptation cannot be ascribed to chance for, with a given environment, the one particular alteration in properties surely results.” (Adami, 1910.) If weso vary the characters of a member of one species that it comes ultimately to possess all the characters of a member of another species, that is “transmutation.” The question is, how far can we go in this direction, and to what extent are the recognised species of bacteria really Exod. in their characters? THE MEANING OF SPECIES. The objection may be raised at this point, that the phrase “transmutation of species” involves a contradiction in terms, since the very definition of “species” excludes the possibility of transmutation. This leads us to a further question, namely, what do we mean by the word “species” as applied to bacteria?—in other words, what determines our present classification ? The distinction between different species of bacteria and their recognition depends upon the observation of their charac- ters—morphological, biological, chemical, physiological and pathological. Briefly enumerated these are as follows:—the naked eye appearance of colonies and of a stab culture: microscopic appearances, size, shape, motility, adhesiveness: method of generation and life history, involution forms: power to produce pigment: staining properties: cultural characters, power of growing on different media, in the presence or absence of oxygen, and under different conditions of temperature and 6 THE SCOPE OF THE ENQUIRY [cH. I moisture, with or without production of gas or odour; power to liquefy gelatin, to reduce neutral red, to clot milk, to ferment various carbohydrate substances: power to form agglutinins and susceptibility to agglutination: viability under different conditions: virulence or the nature of the toxins they produce : pathogenicity or the nature of the lesions they cause and the kind of animal susceptible to their invasion. It is seen from this list that the characteristic qualities of bacteria are very numerous and it would be thought that their classification would on this account be very thorough and complete. But, as will be shown in the course of this enquiry, every one of these characters is liable to variation and the occurrence of these variations renders the task of classification very difficult and in many cases uncertain. If certain characteristics were invariable, even though others varied, a definite criterion would be afforded, but where all alike are subject to modification the division into species is necessarily an arbitrary one. Amongst the higher animals, where sexual production prevails, mutual fertility or sterility offers a guide in determining the limits of species. Here no hard and fast line can be drawn. Nevertheless we see exhibited amongst bacteria, in the words of De Bary, “the same periodi- cally repeated course of development within certain empirically determined limits of variation,’ which is considered to justify the recognition of a species. Many species of bacteria do show characters apparently quite fixed and rigid. The anthrax bacillus and the tetanus bacillus are quite as good species in the natural history sense as any that can be found amongst flowering plants. But the classification of others is still a matter of dispute. This can be illustrated by reference to the streptococci. THE CLASSIFICATION OF THE STREPTOCOCCI. Marmorek held the opinion that the human streptococci constituted one species. “He based his chief argument on the observation that bouillon in which one sort of streptococcus had grown would not serve afterwards as a culture medium for any other streptococcus, and that the same haemolytic CH. I] THE SCOPE OF THE ENQUIRY 7 power was possessed by them all.” (Andrewes and Horder, 1906.) In 1891 von Lingelsheim (2bd.) proposed a division into two groups according to the length of chains formed: “strepto- coccus brevis” and “streptococcus longus.” Andrewes and Horder (1906) with a view to further classification on the same lines suggested the adoption of the terms brevissimus, brevis, medius, longus, longissimus, conglomeratus. The quality of cohesiveness by itself was, however, considered too trivial a character to base a fundamental classification upon. The power of retaining stains was found to offer no means of differentiation, since all stained well. Minute differences in their mode of growth on different media were found to be too inconstant to be of any value, though Schottmiiller (quoted by Muir and Ritchie)attempted to classify the streptococci according to theappearance of colonies. Classification according to pathogenicity and virulence appeared to have the advantage of being practical and signifi- cant from a clinical standpoint. Virulence, however, was likewise found to be an inconstant character, being lost and regained with great readiness by these organisms. It was lost after a few days on certain culture media. On the other hand, after a few “passages” through a susceptible animal, a strepto- coccus of feeble virulence might become intensely pathogenic. Clinical experience confirms this variability invirulence. “One and the same strain of streptococcus may at different stages in its career produce now a rapidly fatal septicaemia, now a spreading erysipelas, now a localised suppuration and now no effect at all” (Andrewes and Horder, 1906), so that the degree of virulence was an uncertain aid to classification. The streptococcus erysipelatis, for instance, is no longer considered on account of its virulence to be a distinct species from the streptococcus pyogenes. Agglutination tests have not been found to be sufficiently specific. Marmorek’s contention, therefore, for the unity of species of the human streptococci continued to hold the field successfully until the introduction of Gordon’s tests. 8 THE SCOPE OF THE ENQUIRY [cH. I Gordon (1903-4) isolated from human saliva 300 strains of streptococci. He tested separately the power of these different strains to ferment various substances, consisting of 14 carbo- hydrates, 13 glucosides, and 6 polyatomic alcohols. Many of these test substances proved of no differential value as regards streptococci, either because they were uniformly attacked by all or because no streptococci could ferment them, but he was led to select a series of seven substances—namely saccharose, raffinose, inulin, salicin, coniferin and mannite—as being of special value as tests for streptococci, and to these he added two further tests—the clotting of milk and the reduction of neutral red under anaerobic conditions. By such means he was enabled to distinguish 48 chemical varieties. Houston (1903-4) applied the same tests (with the omission of one—the action on coniferin) to 300 strains of streptococci derived from human faeces and was able to distinguish 40 chemical varieties amongst them. Gordon demonstrated that these chemically different strains were remarkably constant in their reactions and this was confirmed later by the work of Andrewes and Horder (1906), who tested his strains and, in addition, some 200 new strains derived from foci of disease in human beings. “Gordon himself was careful to abstain from claiming specific value for his different chemical types and he did not venture to propose any reasoned scheme of scientific classification based upon his tests.” Andrewes and Horder attempted to do this. They collected from various sources particulars of the behaviour of over 1200 different strains of streptococci when subjected to Gordon’s tests. Asa result they found that these 1200 strains fell into some half a dozen main groups. By adding one further test—the power of growth in gelatin at 20° C.—and by taking into consideration also the morphological characters and pathogenesis, they were able to define five varieties of streptococci which they regarded as of “approximately specific value” though connected by a multiplicity of intermediate varieties. They named these S. anginosa, S. salivarius, S. faecalis, S. pyogenes and S. pneumococcus. Though confirming, on the whole, the stability of the CH. I] THE SCOPE OF THE ENQUIRY 9 reactions constituting the tests, these observers noticed that variation in virulence was sometimes accompanied by changes in chemical behaviour. They also acknowledged that slight differences in the composition of the media might possibly affect the series of reactions to some slight extent. Subsequently Ainley Walker (1911) offered evidence to show that greater differences in the media do actually affect the reactions to a remarkable extent, so much so as, in his opinion, to invalidate any claim that they should be re- garded as specific, and he fell back on the position held by Marmorek. Still later Jensen and Holth, after a prolonged investiga- tion, came to the opposite conclusion, that is to say in favour of the stability of the differences brought out by Gordon's tests, but they also showed that these differences were in no way closely related to virulence or pathogenic action so that the method of classification founded on them was imperfect. We have thus demonstrated that the term “species” when applied to bacteria must be interpreted much more loosely than in the case of plants or the higher animals and the phrase “transmutation of species” is thereby absolved of the accusa- tion of being self-contradictory. The aim of this paper is to show how far transmutation does occur, and what is its significance. It will be obvious, from what has already been said, that in considering the evidence of transmutation it will be neces- sary to consider also the evidence of variation. The difference between the two is one of degree only. A member of one “species” of bacteria is distinguished from a member of another “species” by its morphological and other characters. If these characters become altered, within certain limits, the process may be regarded simply as variation; if outside these limits it must be regarded as transmutation. We have first of all to consider then, what are the possi- bilities in the direction of such alteration in character. 10 THE SCOPE OF THE ENQUIRY [CH. I A CONSIDERATION OF THE POSSIBILITIES. The various possibilities to be considered are five in number. It will be seen that the first three are instances of variation and the remaining two, instances of transmutation. 1. Simple variation. Modifications may occur in the characters of an organism, involving either the loss of some feature previously regarded as characteristic or the acquisition of some other feature not hitherto considered to be so,—such modifications not being so numerous, however, or so funda- mental, as to lead to any doubt as to the proper identification of the organism. For example, Twort (1907) succeeded, in the course of two years, in training a strain of B. typhosus to ferment lactose, and both Twort (1907) and Penfold (1910 A) produced pure strains capable of fermenting dulcite, which the usual variety of typhoid bacillus is practically unable to do. These new strains retained qualitatively all the other properties of the B. typhosus unchanged. Similarly Miss Peckham (1897) induced indol formation in numerous strains of B. typhosus. 2. Variations in different directions associated. The acquirement of some fresh character may be associated with the loss simultaneously of some other character previously possessed or, with the acquisition of a second new character, and in some cases this association may prove to be invariable under the same modifying conditions (vide p. 146). For example, Eyre and Washbourn (1899) observed that a non- virulent strain of the pneumococcus growing readily at 20° C. could by “passage” be converted into a highly virulent strain which was then unable to grow at a temperature below 37° C. The reverse change showed the same relation between the virulence of the organism and the temperature at which it would grow. Jenner (1898) was able to revert B. colt capsulatus to an unencapsulated form by cultural methods and found that the new variety had lost the power to coagulate milk, and instead of being highly pathogenic to white mice had become much less so or even non-pathogenic. CH. I] THE SCOPE OF THE ENQUIRY 11 The acquirement of power to ferment a certain carbo- hydrate may coincide with the loss of fermenting power in other directions. Penfold (1910-11) has shown that the development of new fermenting powers on the part of B. typhosus towards lactose and dulcite is frequently associated with the formation of papillae on its colonies. Diminished gas-production in glucose media on the part of certain coliform organisms (B. Griinthal, etc.) was likewise associated with papillae formation. The same observer found that colonies of B. typhosus which had lost the property of fermenting glycerine showed impaired agglutinability also, though typical fermenting colonies on the same plate were normal as regards agglutination. Adami, Abbott and Nicholson (1899) found that the as- sumption of coccic and diplococcic forms by B. colt in the organs of healthy animals was associated with a loss of power to ferment carbohydrates and to produce indol. Gordon (1900-1) observed that the tendency of the strep- tococeus of scarlet fever to assume a bacillary form was abolished by “passage” and at the same time its virulence was increased. Rosenow (1914) obtained a strain of streptococci from the throat in a case of scarlet fever, which yielded on blood agar two distinct kinds of colonies. These displayed marked differ- ences in their fermenting power and also in their pathogenicity. Many other instances might be given. ; 3. The development of intermediate forms, i.e. the possible derivation from one or other of two known species of forms intermediate between them in their characters. For example, W. J. Wilson obtained from the urine of a supposed typhoid carrier (1910), and also from the urine in certain cases of cystitis and pyelitis (1908), coliform organisms intermediate in their characters between B. typhosus and B. colt communis and derived presumably from B. typhosus in one case and from B. colt in the others. Many other observers have described organisms resembling both B. typhosus and B. coli communis in their characters. Klotz (1906) has described such an organism, isolated from water, and called by him Bacillus perturbans. Mcnaught (1905) described two varieties, 12 THE SCOPE OF THE ENQUIRY [CH. 1 also derived from water, under the term Bacillus typhosus simulans. One organism, for example, described by Wilson (1910), resembled B. typhosus in forming acid without gas in glucose and in failing to ferment lactose at 37° C.; but it failed to agglutinate with typhoid serum, and it resembled B. col in producing acid and much gas from mannite and in pEneene lactose at 22° C. The Bacillus perturbans of Klotz was ean by high dilutions of typhoid serum (1-1550 in 15 minutes) and it produced slight acidity in milk without coagulation ; but it differed from B. typhosus in fermenting both lactose and saccharose, in giving the neutral red reaction and forming indol, and in other ways. Major Horrocks obtained from the urine of a patient convalescent from typhoid fever a typical strain of B. typhosus which however on subculture gave rise to an organism inter- mediate in its characters between B. typhosus and B. coli (vide p. 118). 4, Slight changes in closely allied organisms, i.e. the possibility in the case of closely allied organisms of a modifica- tion in the few distinguishing features they possess, so that they may appear to change, the one into the other. For example Schmitt (1911) concluded from his experiments that paratyphoid bacilli of the ‘Fliigge’ type and of the ‘Gaertner’ type, generally regarded as distinct species, could be trans- formed from one into the other in the animal body. 5. A complete change in characters, i.e. the possibility of the occurrence, more or less suddenly, of a complete change simultaneously of all the characters of an organism, or at least of all the fundamental ones by which it was distinguished. For example, Major Horrocks (1911) concluded that he had been able gradually to modify a strain of B. typhosus, by changes in its environment, to such an extent that it assumed eventually the characteristics of a Gram-positive coccus having the cultural characters of Streptococcus faecalis. Before these several possibilities are studied more fully, the conditions which modify the characters of bacteria will be mentioned and examples given under each head. CHAPTER II CONDITIONS MODIFYING THE CHARACTERS OF BACTERIA THE factors which appear to influence the growth and de- velopment of bacteria and to produce modification in their characters are many in number and diverse in nature, and it is often impossible to state with certainty which of these various factors is the one primarily responsible for the modification observed in a particular case. 1. Many variations appear to be spontaneous, not due, that is to say, to any external agency, but the result of developmental or atavistic tendencies inherent in the organism itself. We know as little of the nature of these tendencies to variation as we know of the nature of those which control normal development and, in the vast majority of cases, prevent variation occurring. Some spontaneous variations are examples of “pleomorph- ism” and represent stages in the life history of the individual organism or of the race. Others cannot be explained in this way. For example, one component of a diplococcus may retain a stain while its fellow fails to do so. In such a case faulty technique cannot be held responsible, nor can the variation be attributed to differences in environment. Moreover in the case of the meningococcus it has been found to persist after animal passage (McDonald, 1908). The variation would appear to date from the cell division which constitutes the “birth” of the organism. Denny (1903) observed the same inequality in staining properties in different segments of a segmented form of B. Xerosis. Many other examples of spontaneous variation will be found in later pages. 2. Differences in characters are sometimes associated with differences in geographical distribution. Thus Schultz (1909) 14 CONDITIONS MODIFYING [CH. II found that in Cleveland U.S.A., during the 12 months covered by the investigation, “barred” forms of the diphtheria bacillus had almost disappeared; during the same period, in Boston and Providence, another observer noted that “barred” forms were unusually common while “granular” forms were very rarely met with. 3. Many organisms after prolonged cultivation on artificial media display variation in character. In some of these cases the length of the period of cultiva- tion is not the cause of the modification, it merely extends the survey over a large number of generations and so enables the observer to detect variations spontaneously occurring. In other cases the length of time permits “natural selection” to play its part and produce modifications which, in a shorter interval, would not have advanced far enough to be apparent. In other cases, again, the prolonged exclusion from animal tissues does lead directly to a modification in character which is proportionate, as regards its extent and its permanence, to the duration of such exile, but disappears when the organism is again “passed” through the body of an animal. This is true more particularly of the property of virulence (gq. v.). As an example of the influence exerted by prolonged cultivation in modifying the character of an organism may be cited the statement of Mohler and Washburn (1906) that astrain of bovine tubercle bacilli after cultivation for 11 years was found to have become modified in morphological and cultural characters to the human type. Lentz (quoted by Bahr, 1912) found that a “Flexner” type of B. dysenteriae after 9 years’ laboratory cultivation completely lost the power to ferment maltose. Arkwright (1909) mentions a strain of the meningococcus which when first isolated did not ferment glucose but after ten months’ artificial cultivation developed power to do so. Rettger and Sherrick (1911) describe the gradual loss of power to produce pigment on the part of an old stock culture of B. pyocyaneus after 5 years’ artificial growth. Prolonged cultivation in a medium containing a particular carbohydrate may develop in a strain of bacteria the ability CH. IT] THE CHARACTERS OF BACTERIA 15 to ferment that carbohydrate. B. typhosus for example after two years’ growth in a medium containing lactose acquires the power to ferment this sugar (vide p. 58). 4. In other cases again the length of the period of cultiva- tion is of less importance than the conditions under which such cultivation takes place. (a) Conditions which lower the vitality of a strain may modify its characters. Such conditions include starvation (for example, growth in pure water), acidity of the medium, want of oxygen, the presence of antiseptics, exposure to sunlight, high or low temperatures, symbiosis, ete. One example will suffice. A strain of B. ruber of Kiel if heated to a temperature just below that known to kill the organism loses its power to produce pigment (Adami, 1892). (b) The crowding together of the organisms on the surface of the medium may lead to a diminution in pigment produc- tion in the staphylococcus aureus (Andrewes and Gordon, 1905-6) and an earlier appearance of granular staining forms of the diphtheria bacillus (Denny, 1903). (ce) The temperature at which organisms grow is respon- sible for certain variations. Laurent (1890) found that a selected strain of B. ruber which had grown for 12 months at a temperature of 25°—35° C. without exhibiting a trace of colouration, yielded its characteristic pigment when the tem- perature was lowered to 18°C. An apparent staphylococcus “albus” growing at 37°C. may become a vivid “aureus” at 22° C. (Andrewes and Gordon, 1905-6). The virulence of B. anthracis is greatly modified by growth at 43°C. and that of B. diphtheriae may be destroyed by subjection to a similar temperature (Hewlett and Knight, 1897). Wilson (1910) describes an atypical B. typhosus which fermented lactose at a temperature of 22°C. but failed to do so at 37°C. Coplans (1909) found that dulcite was more quickly fermented by certain colon bacilli at 20°C. than at 37°C. Rodet (quoted and confirmed by Adami, Abbott and Nicholson, 1899) found that at a temperature of 45°C. B. colt developed in a few hours into long filaments. The same 16 CONDITIONS MODIFYING [CH. II agency will abolish the power of some bacteria—such as B. anthracis—to form spores, and may modify the character of the colonies it forms (Bainbridge, 1903). Bacteria of the paratyphoid group agglutinate much less readily after being heated (Sobernheim and Seligmann, 1910). (d) Differences in atmospheric pressure may modify the activity of certain organisms. B. coli yields formic acid and gas from glucose at ordinary atmospheric pressure. If the pressure is raised the yield of gas diminishes but the yield of formic acid increases (Harden, 1901). A great pressure of carbon dioxide is said to deprive B. anthracis of its power to form spores though it has no effect on the vitality of the organism (Muir and Ritchie). Certain organisms which do not readily lose their virulence on artificial media do so rapidly if grown in an atmosphere of compressed air (zbid.). (e) The presence or absence of oxygen is another factor of importance. Strains of B. typhoid and B. coli growing in water maintain their viability better if plentifully supplied with oxygen (Whipple and Mayer, 1906). In the absence of free oxygen B. pyocyaneus ceases to produce pigment (Adami, 1892) though the spirilum rubrum produces it more plentifully (Muir and Ritchie). Torrey (1905) observed that by alternate aerobic and anaerobic culture a certain type of dysentery bacillus had its power to ferment maltose greatly augmented. Andrewes and Horder (1906) found that a certain strepto- coccus which refused to ferment lactose, under ordinary con- ditions of cultivation, did so readily when deprived of oxygen. Kruse (quoted by Glenn, 1911) found that a staphylococcus which, similarly, refused to liquefy gelatin did so at once under the same altered conditions. Anthrax bacilli in the absence of oxygen may develop torula zoogleic forms (Wood, 1889). Noguchi (1910) discovered that B. bifidus communis only exhibited the bifurcating phase under anaerobic conditions and in the absence of oxygen became less pathogenic. CH. It] THE CHARACTERS OF BACTERIA 17 It is well known that B. diphtheriae produces toxins more plentifully in a free supply of air (Clark, 1910). The bacillus of malignant oedema is said to lose virulence when grown aerobically (Harass, 1906) and that of cholera to gain virulence when grown anaerobically (Hueppe, quoted by Adami, 1892). Foa (1890) describes how a strain of the pneumococcus could by anaerobic growth be deprived of its property of causing a characteristic inflammatory oedema of the skin when injected into an animal. Wood (1889) attributed the diminished infectivity of virulent organisms discharged from the bowel in many diseases to the fact that in the bowel they are practically deprived of oxygen. (f) Bright sunlight destroys the virulence of some patho- genic organisms (Marshall Ward and Blackman, 1910) and leads to the loss of pigmentation in others (Laurent, 1890). The bacterium mycoides, on the other hand, will only produce its red pigment in the dark (Scholl, quoted by Wood, 1889). 5. Exposure to the ultra violet rays has recently been shown by Madame Henri (1914) to effect a startling change in both the morphology and the pathogenicity of B. anthracis, Cocci and filamentous forms were produced which differed from the original bacilli in their power of retaining stains, of forming spores, of liquefying gelatine and coagulating milk, and which gave rise, on injection into an animal, to symptoms quite unlike those produced by normal anthrax bacilli. The new forms did not revert after daily subculture for over two months. 6. Electrolysis. Electrolysis may produce changes in the morphology and staining properties of bacteria. Russ has observed the production of elongated forms of B. coli, with altered reaction to Gram’s stain, in urine (within the human bladder and outside the body) as a result of the passage of a galvanic current of jth m.a. strength for one hour. The modification persisted for many months. 7. The age of the culture is of importance in the case of many pleomorphic organisms. For example, B. megatherium D. 2 18 ' CONDITIONS MODIFYING [CH. II and B. subtilis pass in a few hours from a bacillary motile stage with cilia, to one of filamentous growth preceded by the casting off of cilia (Marshall Ward and Blackman, 1910). This factor influences the characters. not only of bacteria known to be “pleomorphic” but of others also. Young tubercle bacilli are said not to be “acid-fast” (Hamer, 1900). Young cultures of B. diphtheriae more often show branched and clubbed forms (Kanthack and Andrewes, 1905) and solid staining bacilli (Denny, 1903) than do older cultures; at the ‘same time they are unable to ferment glycerine and lactose ‘ though older cultures usually ferment both (Muir and Ritchie). A young culture of B. coli does not yield indol (MacConkey, 1909). Wood (1889) found that an old culture of cholera failed to liquefy gelatin but did so readily after subculturing, and that a young culture of the same organism was much more susceptible to the action of antiseptics than a younger one. Old cultures of pigment forming bacteria are often colour- less (Adami, 1892). Arkwright (1909) found bacillary forms of the meningo- coccus in old cultures. The tubercle bacillus also in old cultures displays elongated and even branched forms. 8. The character of the culture medium employed may influence bacteria in many ways. (a) The age of the medium. S. pyogenes normally does not ferment saccharose, raffinose or salicin, but if old media be used this organism will ferment all three substances (8S. Martin, 1908-9). On the other hand B. diphtheriae, which in fresh beef serum gives its characteristic “sugar” reactions, fails to do so if this medium is old (Fisher, 1909). (6) Changes in the reaction of the medium employed is in many cases accompanied by changes in the morphological characters of organisms growing in it, bacilli giving place to cocci and diplococci, and vice versa, and pigment formation being modified or lost (Adami, 1892). The reaction also affects the vitality of many bacteria (Wood, 1889), and their virulence (Peckham, 1897). ' (e) The nature of the medium is important. Individual morphology, the appearance of colonies, fermenting power, cH.1] THE CHARACTERS OF BACTERIA 19 indol formation, pigment production, and virulence, all vary with the kind of medium used. Gordon (1900-1) states that the streptococcus of scarlatina may form, on serum, rods which closely resemble B. diph- theriae. but in a liquid medium it grows in a typical strepto- coccal form. B. diphtheriae does not form toxins readily if there is much carbohydrate present in the medium (Fisher, 1909). Many media contain substances derived from the living body such as serum, or blood, and to this extent are “natural” rather than “artificial” media and the alteration in the character of organisms growing in them, particularly as regards virulence, is possibly to be attributed to this factor. Penfold (1914) mentions the fact that vaccination with a plague strain grown on agar will protect rats against itself but not against the same strain grown on serum. ‘If the ordinary artificial media are replaced by the natural secretions of the body the modifications in character on the part of the organisms growing in them may be even more marked. Rosenow (1912-13) found that a streptococcus which presented certain morphological and cultural characters on ordinary media, underwent a profound modification in respect to both as a result of growth in unheated milk. Horrocks (1911) found that a strain of B.typhosus, obtained from the urine of a “carrier,” lost virulence on ordinary media (broth, and agar) in a few days but maintained it for over a year in urine. Both in milk and in urine B. coli may form a dense network of branching filaments (Revis, 1908, Wilson, 1908) and give atypical fermenting reactions ( District case (E) Puerperal fever is well Puerperal fever Hospital nurse (C) Scarlet fever The original case of scarlet fever infected two other people, one with scarlet fever and the other with puerperal fever ; this case of puerperal fever also infected two other people, one with puerperal fever and the other with scarlet fever. An even more remarkable instance is recorded by Dunn CH. vi] VARIATIONS IN PATHOGENICITY 99 and Gordon (1905). They describe an epidemic in Hertford- shire characterised by an extraordinary diversity of symptoms in different patients. In some cases there were sneezing, coryza, and the ordinary symptoms of a common cold. In other cases patients had “aches and pains all over,” stiff neck and suffered subsequently from great debility ; such cases had all the appearances of influenza. In others, again, the illness closely simulated scarlet fever; it began with sore throat, rigors, vomiting, headache, fever and rapid pulse, and was accompanied by a punctate rash at the end of the first 24 hours (followed later by desquamation), the “strawberry ” tongue, circumoral pallor, enlarged cervical glands which in some cases suppurated, and, in some patients, complications such as nephritis, arthritis and otorrhoea. A fourth type resembled diphtheria and exhibited a suspicious membrane on the tonsil. A fifth type was notified in some cases as typhoid fever and was characterised by epistaxis, melaena, prostra- tion and, in some cases it is stated, a positive Widal reaction. Finally, a number of cases, particularly amongst children, resembled cerebrospinal fever and were so diagnosed ; these were characterised by profuse nasal discharge, pain in the back of the neck, headache, photophobia and irritability, dilatation of one or both pupils, persistent vomiting, drowsi- ness, head retraction, paralysis, coma and, sometimes, con- vulsions and death. Sometimes these widely divergent types were exhibited by the different members of a single family or household struck down by the disease, either simultaneously or con- secutively. After a thorough investigation these observers were convinced that the outbreak of these various types of illness was due to the prevalence and spread of only one disease and not a number of different diseases, and a bacterio- logical examination of a large number of cases by Gordon showed that the disease was due to infection by an organism closely resembling, if not identical with, M. catarrhalis. 4. In the fourth place, the same species of organism may give rise in different epidemics to widely different types of disease. For example, strains of B. influenzae may give rise 7—2 100 VARIATIONS IN PATHOGENICITY [cn. vit to epidemics of “influenza” characterised by symptoms resembling in one epidemic a simple coryza, in another rheumatic fever, in a third typhoid fever, and in a fourth cerebrospinal meningitis. 5. In the fifth place, the train of symptoms characteristic of infection by one organism may develop as a result of infection by a totally different organism. A striking instance of this is recorded by Head and Wilson (1899) who proved that a supposed case of rabies was actually due to infection by the diphtheria bacillus. The diagnosis of rabies was founded on the history and clinical symptoms. “The well authenticated history of a bite on the cheek by an unknown animal, the two months’ incubation period, the onset with extreme pain and numbness in the region of the scar, the development of the characteristic laryngeal and respiratory spasms on attempting to take liquids, the spasm at first being slight but later more pro- nounced and towards the close again feeble or absent, the insomnia, the absence in the beginning of fever which later in the illness became pronounced, the rapid pulse at all stages, the attacks of violent delirium interspersed with periods of calm and complete rationality, the absence of all symptoms pointing towards any other simulating disease and the fatal termination—all serve to make an almost complete picture of rabies.” The Klebs-Loeffler bacillus was isolated from the ventricular fluid and detected in the nerve cells of the medulla. The recognition of this organism was complete and beyond doubt. “Not less suggestive of rabies than the clinical history were the results of subdural inoculations of rabbits with emulsions prepared from the medulla of the patient. There occurred the long period of incubation (20 and 21 days) followed by phenomena similar to those in experimental rabies of rabbits, and other rabbits inoculated subdurally with the medulla of the first rabbits behaved in a similar manner.” B. diphtheriae was demonstrated after death in the medulla of the rabbits. By a thorough investiga- tion, full details of which are given, infection by the virus of rabies was definitely excluded. CH. viI] VARIATIONS IN PATHOGENICITY 101 Dunn and Gordon (1905, vide supra p. 99) have described almost typical cases of scarlet fever, of cerebrospinal fever and of influenza, which proved to be due to infec- tion by the micrococcus catarrhalis. Gordon has described elsewhere typical cases of cerebrospinal fever due to B. typhosus. Nash has recorded a remarkable case of malignant en- docarditis characterised by fever, constipation, headache, drowsiness and delirium, photophobia, strabismus, head re- traction and the appearance of a petechial rash. The illness, in fact, presented all the clinical features of cerebrospinal fever. A copious growth of a pure culture of the Klebs- Loeffler bacillus was obtained post mortem from the spinal fluid and a similar growth from the heart’s blood. There was a history of a discharge from the ear at the beginning of the illness but no history of sore throat. Thomson (1911) has recorded his own experience of an acute inflammation of the throat simulating diphtheria in producing, in the fourth week of the illness, temporary para- lysis of the tongue, arms and legs, but proved to be due to pneumococcal infection. Colman and Hastings (1909) state their conviction that some strains of B. coli are capable of causing a disease clini- cally identical with typhoid fever. III. The pathogenicity of bacteria presents yet another aspect, namely the character of the lesions produced by them in the living tissues. This can be studied in two ways. Firstly, by observing the lesions produced in the body at various stages in the course of an infective disease ; and secondly, by observing the lesions produced by the artificial inoculation of organisms into animals, both at the site of inoculation and elsewhere. 1. The lesions produced in the course of disease and ob- served post mortem not infrequently enable one to identify the infecting organism. For example, tuberculous ulceration of the intestine, tuberculous consolidation of the lungs, and tuberculous invasion of the skin, present altogether different features from typhoid ulceration of the intestine, pneumococcal 102 VARIATIONS IN PATHOGENICITY [cx. vii consolidation of the lung and streptococcal invasion of the skin, respectively. It is however common experience that even in the post mortem room a certain diagnosis of the nature of the infec- tion cannot always be made. Sydney Martin, in speaking of tuberculosis, says “There is, with the exception of the presence of the tubercle bacillus, no element in the structure of the tu- berculous lesion which is diagnostic of the disease.” In other words the lesions regarded as characteristic of infection by one species of organism may be produced by infection by a totally different species. Such departures from what experience has taught us to regard as the normal or characteristic lesion in the case of a given organism may be accounted for by the influence of other factors beside the nature of the organism itself—such factors, for example, as the age of the patient, the route of invasion, the presence of a secondary infection, the effect of treatment, and many others. The question arises, how far, if it were possible to exclude such disturbing influences, would the lesions retain their specific character ? 2. This leads us to a consideration of the second method of studying the question—by observing the lesions produced by artificial inoculation of animals, both at the site of inocu- lation and elsewhere. Such a method enables one to, so to speak, “standardise” the lesion. A healthy animal of the same species, age and weight can be utilised at each experi- ment, the inoculation made in the same manner, at the same site, with the same number of organisms and these of the same degree of virulence, and the animal can be killed after the same interval of time. Many investigators maintain that under such conditions the lesions produced by a certain species of organism are constant in their appearance—that, however much the other characters of an organism may vary, this character at any rate is invariable and will establish beyond dispute to which of two species a doubtful organism actually belongs. Thus, Klein as long ago as 1899 in describing the “ bacillus of pseudo-tuberculosis” stated that in cultural and morpho- CH. vi] VARIATIONS IN PATHOGENICITY 103 logical characters this organism showed certain resemblances to B. coli. The two organisms could be distinguished from each other most certainly by animal inoculation. Subcutaneous inoculation of the first named into the guineapig gave rise to typical nodular, necrotic, purulent changes in the lymphatic glands, omentum, pancreas, liver, spleen, and lung, an effect which B. coli and its varieties did not produce. Again, Shattock (and others, 1907) regards the avian tu- bercle bacillus and the human tubercle bacillus as two distinct species on the ground that, whereas the former when inoculated into guineapigs produces merely a local or a local and glandu- lar disease, the latter produces visceral disease as well. Savage (1908-9) has recorded some interesting experiments illustrating the value of animal inoculation in revealing differ- ences in pathogenicity. He found that streptococcus mastitidis, which causes mastitis in the cow, was non-virulent to mice and other rodents but possessed to a marked degree the power to produce mastitis in goats when inoculated into the mammary ducts, and was thereby differentiated from streptococcus an- ginosa (isolated from human sore throat) which, though virulent to mice, did not possess the power to produce mastitis in goats. Continuing his experiments with pyogenic streptococci derived from many sources, he found that, although in their cultural properties and their virulence to mice they displayed wide differences, they all resembled each other in their inability to produce mastitis in goats. One streptococcus, for example, isolated from a fatal lymphadenitis in a boy, after it was in- oculated into the teat of a goat survived for seven months as a harmless saprophyte in the milk passages. One other example will suffice. We recognise clinically two types of pneumonia, lobar or croupous pneumonia and lobular, catarrhal or broncho-pneumonia. Both types may result from infection of the lung by the pneumococcus. The invading organism is apparently identical in the two cases, judged by the ordinary cultural and morphological tests, and the difference in the results produced are therefore attributed to differences in the age and vitality of the patient and the route of infection. 104 VARIATIONS IN PATHOGENICITY [cu. vit Eyre, Leatham and Washbourne (1906) endeavoured by the method of animal inoculation to ascertain whether the difference in the lesions caused depended upon specific differ- ences in the pathogenicity of the infecting strains. They found that strains of the pneumococcus isolated from cases of lobar pneumonia when inoculated subcutaneously into the guineapig almost invariably gave rise to a local inflammatory exudation of a fibrinous type, whereas strains isolated from cases of broncho-pneumonia, when similarly inoculated, almost invariably gave rise to a local inflammatory exudation of a cellular type, easily distinguished from the other. A number of strains of pneumococci obtained from a “neutral” source, such as the mouth, likewise showed differences in the nature of the inflammatory reaction they provoked at the site of in- oculation, some belonging to the “fibrinous” type and others to the “cellular” type. They further showed that this feature was not associated with any other differences between the strains as regards morphology or cultural characters or fer- menting properties and was quite independent of their degree of virulence. They therefore regarded it as a specific character. If the lesions produced in the body during the course of an infective disease are subject to variation, are those which result from the artificial inoculation of animals any more constant? The materials from which to form an opinion on this point are somewhat scanty. That the feature in some cases is very constant was shown by Shattock (and others, 1907) by means of the following experiment. They grew a strain of human tubercle bacilli for eight weeks in the spleen of a pigeon. The subsequent inoculation of the organisms into a guineapig gave rise, not as might have been expected to the lesions charac- teristic of avian tubercle, but to those characteristic of the human type. Baldwin (1910) likewise grew the human type of tubercle bacillus for 19 months continuously in the bovine tissues without in any way affecting its pathogenic powers to- wards rabbits and guineapigs. On the other hand, we have quoted in an earlier paragraph an instance of a certain strain of the diphtheria bacillus which CH. vil] VARIATIONS IN PATHOGENICITY 105 not only gave rise to atypical symptoms and lesions (namely those of rabies) in the human body in the course of disease but produced no less atypical lesions when inoculated into a rabbit (vide p. 100). Again, Savage (1908-9) found in further experiments that a virulent strain of the streptococcus mastitidis from the udder secretion in a case of bovine mastitis, under certain conditions (namely 3 days’.residence in the human pharynx), was almost deprived of its characteristic power to produce mastitis in goats. Again, Mohler and Washburn (1906) claim that the various types of tubercle bacilli—human, bovine, avian—can be readily converted one into another, by prolongedresidence in a suitable animal host, so as to be indistinguishable by the ordinary in- oculation tests. Rosenow (1914) obtained a strain of haemolysing strepto- cocci from the throat in a case of scarlet fever. A culture on blood agar yielded two distinct kinds of colonies, (a) non-ad- herent colonies of a haemolysing organism which fermented mannite but failed to ferment maltose and saccharose, (b) adherent, green-producing colonies of a non-haemolysing organism which would not ferment mannite but fermented maltose and saccharose. When injected into a rabbit, the former attacked primarily the joints while the latter showed a predilection for the heart valves. In other words, the original strain on artificial cultivation gave rise to two strains which differed in their pathogenicity. Finally, may be mentioned Foa’s experiments (1890). He inoculated a rabbit with the diplococcus lanceolatus capsulatus with a fatal result. From this dead rabbit he inoculated two others, the first by injecting organisms derived from some of the fresh fibrinous pneumonic exudate in the lung, and the second by injecting organisms derived from the cerebrospinal fluid. He found that the disease set up in these two rabbits differed. The first rabbit showed, for example, an inflammatory oedema of the skin; the second did not show this. He found, however, that if the strain isolated from the lung were grown anaerobically and then injected into a rabbit the effects it 106 VARIATIONS IN PATHOGENICITY [CH. vit produced were indistinguishable from those produced by the strain isolated from the spinal fluid. Whatever aspect of pathogenicity, therefore, we study, the same feature becomes apparent—namely, that this property of bacteria is, like others, subject to variation. VARIATION IN OTHER CHARACTERS OF BACTERIA. In the foregoing pages variations in morphology, ferment- ing power, virulence and pathogenesis have been discussed in detail. There remain many more characters of bacteria to be considered—such as their viability, their staining properties, their power to produce indol and to liquefy gelatin, their ag- glutination reactions and many others. It would be easy to illustrate the variations these characters also undergo under different conditions. Many examples will be found in Chapter IT. CHAPTER VIII THE POSSIBLE OCCURRENCE OF TRANSMUTATION IN THE LIVING BODY THE significance of the variations recorded in the foregoing sections, with reference to the question whether actual trans- mutation of bacteria can be brought about artificially or not, will be dealt with later. It is proposed, at this point, to consider another aspect of the problem, namely the possibility of transmutation occurring in the tissues of the living body. In-certain regions of the body one finds growing side by side two strains of organisms closely resembling each other in every respect save one—namely their pathogenicity. One strain is capable of causing a definite train of lesions and symptoms; the other, as a rule, does not give rise to any signs of disease. The suggestion that one strain may be in some way a derivative of the other offers a tempting hypothesis to explain both their resemblance and their proximity to each other. An illustration will, perhaps, make this clearer. In the hides of cattle may sometimes be found non-virulent bacilli closely resembling B. anthracis. Such an organism was discovered by Andrewes and described by Bainbridge (1903) under the name B. anthracoides (vide p. 92). The organism was stated to differ from B. anthracis in the appearance of its colonies, in its rate of growth, in possessing slight motility and in being non-virulent. By slightly modifying the conditions of growth, colonies on agar could be made to assume the typical appearance of anthrax colonies, while its virulence proved capable of increase by “passage.” The differences in character between this organism and B. anthracis were deemed sufficient by these observers to justify them in classifying it as a distinct species, but it is difficult to resist the conclusion either that 108 THE POSSIBLE OCCURRENCE OF [cH. vill the non-virulent organism was a direct derivative of the true anthrax bacillus or that it would be capable of giving rise to the latter under suitable conditions. Such a supposition is favoured, firstly, by the admission that the bundle of horse hair from which the B. anthracoides was isolated contained also the spores of true anthrax, and, secondly, by the discovery of Hueppe and Wood some years before (1889) of a similar non- virulent saprophytic anthrax-like organism in earth, which however on injection into a mouse rendered the animal immune to anthrax. Similar examples of association between non-virulent and virulent organisms, otherwise closely resembling each other, may be found in the human body—in the intestine B. colt and B. typhosus, in the throat Hofmann’s bacillus and the Klebs- Loeffler bacillus, in the skin the Staphylococcus epidermidis albus and the Staphylococcus pyogenes aureus, in the naso- pharynx the micrococcus catarrhalis and the meningococcus. The exact relationship in each case has never been satis- factorily determined. Over twenty years ago Adami (quoted by Arloing, 1891) put forward the suggestion that B. coli might give rise in the presence of fermenting faecal matter to B. typhosus, a theory which has been recently revived by Tarchette (1904) and others (quoted by Hamer, 1909). The precise relationship between the virulent Klebs-Loeffter bacillus and Hofmann’s bacillus is still a matter of controversy. The latter is a harmless saprophyte not infrequently found in the pharynx of healthy persons. It is distinguished from the true diphtheria bacillus by the somewhat different appearance of its colonies on artificial media, by slight and, according to some observers, inconstant differences in its morphology and staining, by its inability to ferment glucose and other sugars, and by being non-pathogenic to man and to the guineapig. It has not been found possible to produce immunity against true diphtheria by inoculation with Hofmann’s bacillus, and the injection of a filtered broth culture of the latter does not give rise to antitoxin formation in the horse (Petrie, 1905) though the filtrate in the case of even avirulent Klebs-Loeffler bacilli will do so (Arkwright, 1909). Nevertheless many in- CH. vil] TRANSMUTATION IN THE LIVING BODY 109 vestigators claim to have converted the Klebs-Loeffler type of organism into the Hofmann type—by prolonged cultivation (Lesieur, 1901), by growth at a high temperature (Hewlett and Knight, 1897), by growth in the subcutaneous tissues of an immune rat (Ohlmacher, 1902) and other methods—and main- tain that the reverse change can be brought about by “passage” (Lesieur, 1901, Hewlett and Knight, 1897, OhIlmacher, 1902, etc.). Salter (1899) has stated that, by five successive passages through goldfinches, he was able to convert four strains of typical Hofmann’s bacilli into no less typical Klebs-Loeffler bacilli, the transformation being complete as regards virulence, morphology and acid production, and in the power to form a toxin neutralised by diphtheria antitoxin. Thiele and Embleton claim to have converted Hofmann’s bacillus into a bacillus morphologically indistinguishable from the diphtheria bacillus and capable of secreting an exotoxin which can be neutralised by diphtheria antitoxin. This was accomplished by inoculating a succession of guineapigs with an emulsion of Hofmann’s bacillus containing a certain pro- portion of gelatin, the organism being recovered from the peritoneal cavity after each passage. As regards the fermenting properties of the two organisms, Clark (1910) has shown that Hofmann’s bacillus does produce slight acidity in dextrose broth; while Goodman (1908), by a process of selection, obtained strains of the true diphtheria bacillus which exhibited differences in fermenting power as wide as those naturally existing between this organism and Hofmann’s; and he concluded that the fermenting power was a poor guide in determining whether an organism was a pathogenic one or a harmless saprophyte. Finally, Boycott’s statistics demonstrate (Muir and Ritchie) that the period of maximal seasonal prevalence of Hofmann’s bacillus immediately precedes that of true diphtheria, and Hewlett and Knight (1897) have offered evidence in support of the opinion that Hofmann’s bacillus is present in increasing numbers in the throats of diphtheria patients during recovery from the disease. Recent work by Graham Smith and others, and the inability 110 THE POSSIBLE OCCURRENCE OF [cu. vu of these observers, on repeating the experiments of earlier investigators, to obtain the same results, somewhat invalidates the conclusions of the latter, so that the question of the possi- bility of a mutation between the two species remains sub Jjudice. Several species of staphylococci are recognised,—S. epidermidis albus, S. pyogenes albus, S. pyogenes aureus. The distinction between these three rests on their inequality in virulence, on their different powers of fermenting carbo- hydrates, and, as their names imply, on their dissimilarity in the production of pigment. _As regards virulence, the first-named organism is normally present in the skin of healthy persons and is non-pathogenic; the second possesses slight virulence, producing mild local inflammatory conditions; while the third is a virulent organism found in pathological conditions such as suppurative cutaneous and subcutaneouslesions, acute bone infection and septicaemia. Staphylococcus epidermidis albus may however assume a certain degree of virulence and give rise to a stitch abscess or mild inflammation (Dudgeon and Sargent, 1907) and plays an important role in peritonitis (¢bid.). Andrewes and Gordon (1905-6) isolated it in pure culture in one case of otitis media and also from a boil. The Staphylococcus pyogenes albus can be made much more virulent by artificial passage. It has been known to become parasitic, invading the human body and circulating in the blood stream (Panichi, 1906, Southard, 1910). Tn the second place, as regards their fermenting properties, Gordon (1904-5) has shown that strains of Staphylococcus albus isolated from the skin of healthy persons show very great diversity in their fermenting power. In an earlier paper (1903-4) he describes two strains, one a Staph. albus derived from the skin and the other a Staph. pyogenes aureus derived from pus—which, when “put through” no less than 20 carbo- hydrate substances, revealed different fermenting power in one only, namely mannite. In the third place, as regards pigment formation, it has been proved by many investigators (Neumann, Dudgeon, 1908, Andrewes and Gordon, 1905-6) that non-pigmented cocci can CH. vi] TRANSMUTATION IN THE LIVING BODY 111 be obtained on culture from pigmented ones, and that cocci which fail to produce pigment under certain conditions will do so readily if the conditions are altered (vide p. 15). Dudgeon (1908) cites one experiment in which a Staphylococcus aureus was injected into an animal and a Staphylococcus albus was recovered from the spleen at its death. In the last case Gordon’s tests were identical in both instances, showing that the character of pigment production was the only one to undergo modification, but it does not require a great stretch of imagination to suppose that just as the virulent parasitic pnheumococcus and the avirulent saprophytic variety may undergo mutation (vide p. 82) so the highly virulent “aureus” and the less virulent “albus” might under certain circum- stances be converted the one into the other. Many more hypotheses of the same nature, and based on similar evidence, might be put forward with varying degrees of plausibility. One other example will suffice, namely the question of the relationship of the meningococcus to two other diplococci—Wie. catarrhalis on the one hand and the pneumococcus on the other. The Micrococcus catarrhalis which is frequently present in the mouths of healthy persons, especially children, is an organism resembling in many respects the meningococcus, but of low virulence. The two organisms are, as a rule, easily distinguished by important differences existing between them. Thus the meningococcus is much smaller than Wic. catarrh- alis: its colonies are also smaller and their outlines more regular ; it liquefies blood serum and forms acid in dextrose, maltose and galactose which Mic. catarrhalis fails to do; it is more virulent also and gives rise to a different train of symptoms. The difference in size, however, is not invariable, an organism no greater than the meningococcus occasionally proving on examination to be Mic. catarrhalis (Hachtel and Hayward, 1911), while the appearance of a colony is a character of bacteria liable, as we have shown, to undergo great modification (vide p. 47). The power of the meningococcus to produce fermentation in sugars is subject to variation. Arkwright (1909) found that 112 THE POSSIBLE OCCURRENCE OF [c#. vit 9 per cent. (out of 36 cultures) failed to produce acid in dextrose. Summers and Wilson (1909) state that out of 80 strains “nearly all” fermented the usual sugars but a few gave the fermentation reactions of Mic. catarrhalis. The organism isolated from sporadic cases of meningococcal menin- gitis shows such marked differences in its sugar reactions when compared with a typical meningococcus that some writers regard it as a distinct species (Batten). Arkwright (1909), though he refutes this, acknowledges that the sporadic type is less uniform in its fermenting powers. Some of his strains of meningococcus permanently failed to ferment any sugars; others, which failed to do so when first examined, gradually acquired the power in the course of many months ; others, again, which did ferment sugars, completely lost this property after cultivation for a certain time. Another interest- ing fact, in this connection, is mentioned by Andrew Connal (1910), namely that in the late, chronic stages of cerebrospinal fever the meningococcus isolated from the cerebrospinal fluid is found to have lost its power to break up sugar. Mic. catarrhalis on the other hand may acquire power to ferment sugars. Gordon (quoted Martin, 1911) found that, out of 25 strains examined by him, three fermented dextrose, saccharose, galactose and maltose. The meningococcus and Mie. catarrhalis differ in virulence but this property in the latter can be artificially raised by “passage.” As regards pathogenesis, this distinction, again, between the two organisms sometimes breaks down, symptoms typical of infection by one organism being in reality due to infection by the other. The symptoms attributable to Mic. catarrhalis infection differ widely. Thus it may cause an acute pharyn- gitis (Gordon, 1906) or a tonsilitis; it may cause a “common cold” or give rise to an infective cold and sore throat spreading from person to person (Allen, 1908); it may set up otitis media and a secondary meningitis (Barker, 1908), or, again, a primary meningitis (Arkwright and Wilson) or, finally, an epidemic so closely resembling cerebrospinal fever in its symptoms that this disease has actually been diagnosed until CH. vi] TRANSMUTATION IN THE LIVING BODY 113 a bacteriological examination demonstrated the absence of the meningococcus and the presence of Mic. catarrhalis (Dunn and Gordon, 1905). Conversely, the meningococcus may cause a simple coryza (20th century Dict. of Med.). Sometimes a “mixed infection” occurs; thus, Arkwright (1909) describes a case in which Mic. catarrhalis was isolated from the heart’s blood after death although the typical meningococcus was proved to be present before death in the cerebrospinal fluid. Prof. McDonald (1908) has commented upon the frequency with which, in cerebrospinal fever, leptothrix forms are found in the spinal fluid and compares this with the similar frequency of leptothrix forms in the pharynx. He considers these leptothrices to be merely secondary invaders but regards their presence as confirmatory of the opinion, now generally held, that the route of invasion of the meningococcus in cases of cerebrospinal fever is from the nasopharynx. If the appearance of these “camp followers” tends to support the opinion as to the locality from which the regiment was drawn, still further light is thrown on the question by the presence of “disbanded soldiers” in the form of non-virulent meningococci in the nasopharynx of healthy persons. Out of a total of 810 healthy persons, examined by different observers all over the world, the meningococcus was isolated from the nose in 164 cases (Hachtel and Hayward, 1911). We have already referred to the fact that organisms normally non- pathogenic may become pathogenic when growing and multi- plying in inflammatory exudations (wide p. 79). Cerebro- spinal fever is a disease more particularly of young children and it is in children that Mic. catarrhalis is most often discovered as an inhabitant of the pharynx in health. It has been observed that an attack of cerebrospinal fever very often commences with a purulent nasal discharge. Thequestion arises, does this area of suppurative inflammation in the vicinity of its natural habitation afford a training ground, so to speak, for the peaceful Micrococcus catarrhalis preparatory to its entry upon a military career in the uniform of the meningococcus? D. 8 114 THE POSSIBLE OCCURRENCE OF [cu. vi The oft mooted question of the relationship of the meningo- coccus to the pneumococcus is prompted by clinical rather than by bacteriological evidence. Pneumococcal meningitis, like all pneumococcal infections, is characterised by certain features which are also observed in meningococcal meningitis (Preble), namely an acute onset, a polymorphonuclear leucocytosis, a diminution in the chlorides in the urine, and herpes. In the second place, certain complications are common to both, namely endocarditis, pericarditis, arthritis and otitis media. In the third place, Preble observes that there is an extraordinary similarity in the seasonal distribution of the two diseases. On these grounds he suggests that the meningococcus is a variant of the pneumococcus. Certain differences between the two diseases exist. The petechial eruptions which formerly gave a name to one disease are rare in the other, but this haem- orrhagic tendency is altogether absent in some epidemics of “meningococcal” meningitis. Again, “meningococcal” menin- gitis is a disease more especially of childhood and frequently ends in recovery; “pneumococcal” meningitis is a disease more commonly of adult life and is invariably fatal. The differences in age incidence and mortality are however com- patible with the view that the causal organism is the same but of different virulence. Finally, the sporadic nature of meningococcal meningitis, which is difficult to“explain if one admits the meningococcus to be an independent organism, ceases to be so if one assumes it to be a modified form of the ubiquitous pneumococcus. It may be argued that, although each of the several differ- ences in character which distinguish the organisms we have been comparing, when considered by itself, may appear trivial and may prove to be variable, nevertheless all these differences, if taken together and viewed as a whole, represent a degree of divergence in type which cannot be so lightly dismissed. A series of surmises, no matter how credible these may be made to appear, does not constitute a proof. It is in our power to prove, however, that in other cases differences no less diverse in character and no less marked in degree, differ- ences moreover which, taken together and viewed as a whole, CH. vi] TRANSMUTATION IN THE LIVING BODY 115 might be thought to represent no less wide a divergence in type, may disappear entirely under certain conditions—con- ditions, be it noted, precisely analogous to those which we have surmised might bring about a similar result in the cases we have been considering—namely, invasion of the living body. The experiments of Eyre, Leatham and Washbourn (1906) with strains of the pneumococcus furnish an example. These observers describe the virulent, parasitic pneumococcus as re- quiring for its growth a certain reaction and temperature, and particular media (blood agar) ; it would not grow if the reaction were even faintly acid or at a temperature much below 37° C. and rapidly died out on agar or in broth. It would not liquefy gelatin and in broth formed a dust-like deposit. The avirulent saprophytic variety, on the other hand, grew luxuriantly at temperatures ranging from 37° to 20°C.—on agar, gelatin, potato or in broth, whether acid or alkaline, slowly liquefying gelatin and producing a uniform turbidity in broth. It retained its vitality for many months. It also exhibited differences in ‘its morphology,—“ instead of isolated diplococci and strepto- cocci large masses of cocci and diplococci were found and forms dividing into tetrads were common.” Nevertheless this avirulent saprophytic pneumococcus could, by a single “ pas- sage” through a rabbit, be converted into a typical parasitic pneumococcus of high virulence. Such a remarkable transition, if it did not actually happen, would seem to us quite as improbable as a transition from, let us say, the micrococcus catarrhalis to the meningococcus. The purpose of this section is to suggest that a change in character, comparable to that brought about in the case of the saprophytic pneumococcus by a single animal passage, might be brought about in the case of other saprophytic organisms by an analogous process, namely by their invasion of the living body when the lowered vitality or the inflamed condition of the tissues enable them to gain a foothold therein. CHAPTER [x SUPPOSED INSTANCES OF TRANSMUTATION BROUGHT ABOUT EXPERIMENTALLY I. Magsor Horrocks’s EXPERIMENTS. (Journal of R.A.M.C. Vol. XVI.) In March, 1911, Major Horrocks published the records of a series of experiments of great interest. The results of these experiments may be briefly summarised as follows : (a) From a strain of B. typhosus (derived from the urine of a carrier) he obtained, by subculture, an organism intermediate in its characters between B. typhosus and B. colt. ‘ (6) From a second (laboratory) strain of B. typhosus, by symbiosis with B. coli, he obtained an organism which produced slight acidity in mannite but fermented no other sugars, and which later reverted to B. typhosus. (c) From a third (laboratory) strain of B. typhosus, by symbiosis with the same strain of B. coli, he obtained an or- ganism closely resembling B. faecalis alcaligenes. (d) From a fourth strain of B. typhosus (derived from the urine of another carrier), after injection into the peritoneal cavity of a guineapig, he obtained a Gram-positive coccus resembling streptococcus faecalis. (e) From a fifth strain of B. typhosus (derived from the urine of a third carrier), after injection into the peritoneal cavity of a guineapig, he obtained in three different ex- periments a coliform organism which differed widely in its fermentation and agglutination properties from B. typhosus. (f) From a sixth strain of B. typhosus (derived from the stool of a fourth carrier), after growth in the diluted and filtered urine of another carrier “S,” he obtained B. faecalis CH. Ix] SUPPOSED TRANSMUTATIONS 117 alcaligenes. The latter organism in one experiment, after three passages through the guineapig, gave rise to B. coli which however reverted subsequently to B. faecalis alcali- genes. (g) From the second (laboratory) strain of B. typhosus referred to above (6), after exposure to the same conditions, he obtained in two different experiments B. faecalis alcali- genes. The latter organism in two later experiments (in the first after 5 months’ further growth and in the second after 8 passages through the guineapig) gave rise to streptococcus Jaecalis; while in a third experiment (after 18 successive passages through the guinea-pig) it gave rise to B. colt which, after the 19th passage, reverted to B. faecalis alcaligenes and this, after further “passages,” in two different experiments yielded the streptococcus faecalis. (h) From the same (laboratory) strain of B. typhosus, after growth in the diluted and filtered urine a different carrier “T,” he obtained again B. faecalis alcaligenes. To summarise these results even more concisely, it appears that Major Horrocks was forced to the conclusion that not only had an organism arisen from a strain of B. typhosus in- termediate in character between B. typhosus and B. coli, but that other strains of B. typhosus, derived from three distinct sources, had in no less than five of his experiments undergone mutation into B. faecalis alcaligenes as a result of changes in their environment; and, further, that the B. faecalis alcali- genes so obtained had later, in two instances, become changed into B. col (reversion taking place in both cases, however, subsequently) and, in four instances, become changed into streptococcus faecalis, once after prolonged cultivation and three times as the result of passage; and, finally, that one of the original strains of B. typhosus had undergone a similar change into streptococcus faecalis after passage. Major Horrocks’s statements are so startling and, if sub- stantiated, would prove so revolutionary in character that they demand careful examination. It may not be possible to disprove either his facts or his inferences, but it is not necessary to do so. The onus of proof 118 SUPPOSED INSTANCES [ cH. Ix rests with the claimant. If it is possible to show that he has failed to exclude a single possible source of error, a verdict of not proven must be returned. When considering the value of evidence adduced in sup- port of supposed instances of variation or transmutation (vide Chap. IIT) we mentioned varioussourcesof error. Bearing these in mind, and also the wide limits within which we have found variation may occur (vide Chaps. IV-VII) we will now consider in detail the processes by which Major Horrocks obtained the results he claimed and the value of the evidence he brings forward to support his contentions. (a) The alteration of B. typhosus to an organism inter- mediate between B. typhosus and B. coli. (Page 246.) A strain of B. typhosus was isolated from the urine of a typhoid carrier “TS ”—from whose blood a pure culture of B. typhosus had previously been obtained. After 3 days’ incubation on bile salt glucose litmus agar the strain gave the typical reactions of B. typhosus. At the end of a week, however, the following characters were displayed: lac- tose, salicin and dulcite were rendered slightly acid, broth gave a marked indol reaction, the neutral red reaction yielded a slight yellow colouration, the organism appeared only slightly motile and was not agglutinated by anti-typhoid serum. The organism, however, gave rise to typhoid agglutinins when injected into a rabbit, and this rabbit’s serum deviated com- plement in the same manner as a known antityphoid serum, and the organism further had the power of absorbing the specific agglutinins from a known typhoid serum. After 4 passages through the guineapig the organism lost its lactose fermenting property and only differed from the original B. typhosus by forming a trace of acid in salicin. After 4 further passages it reverted to the unusual fermenting type described. The urine of carrier “TS” from which the strain was origin- ally derived was again carefully tested but only typical typhoid organisms were obtained. Criticism. We have already quoted (vide p. 11) instances of the occurrence of organisms, derived in some cases from CH. Ix] OF TRANSMUTATION 119 the urine of typhoid carriers, intermediate in character between B. typhosus and B. coli. Wilson (1910) described such an organism as fermenting glucose and mannite but, unlike B. ty- phosus, fermenting lactose also at 22°C. (but not at 37°C.) and failing to agglutinate with typhoid serum. The Bacillus perturbans of Klotz (1906), though agglutinated by high dilu- tions of typhoid serum, fermented lactose and saccharose, gave the neutral red reaction and produced indol. Examples have also been given (vide Chapter V) to show the variability of organisms with respect to their power to ferment sugars and their ability to acquire fresh fermenting properties. B. typhosus, for example, may acquire the power in a few days to ferment dulcite. The organism described here by Major Horrocks is another example of temporary variation in character with respect to the power to ferment sugars and to produce indol, associated with some modification also in agglutination properties. (b,c) The change from B. typhosus to B. faecalis alcaligenes due to symbiosis with B. coli. (Page 233, exp. 1.) The strain of B. typhosus used was a stock laboratory strain “R,” from which stock vaccines were prepared—a strain, that is to say, of unimpeachable character. The strain of B. coli was derived from the urine of a typhoid carrier (“BombS ”). The two organisms were added to 1 c.c. of sterilised tap water and the suspension plated. 10 days later, examination showed typical typhoid colonies and others white and opaque. The latter were planted on the usual media and in 48 hours yielded slight acidity in mannite only; no other sugars were fermented in 7 days. The original strain of B. typhosus used was replanted on agar and the resulting growth gave the typical reactions of this organism. (Page 234, exp. 3.) The experiment was repeated, a dif- ferent typhoid strain (“Bombay”) being used. After an interval of two months, 1 cc. of the inoculated water was added to MacConkey’s bile salt broth and this plated on lactose bile salt litmus agar. A few blue colonies were seen consisting of bacilli which resembled B. faecalis alcaligenes in not ferment- ing any sugars and producing an alkaline reaction in milk. 120 SUPPOSED INSTANCES [CH. Ix No B. typhosus could be isolated and at later examinations only B. coli was recovered. Criticism. Conditions of growth inimical to the life of an organism might be expected to deprive it gradually of its functions. A strain of typhoid bacilli whose vitality is at its lowest ebb would hardly be likely to ferment sugars vigorously, if at all. In both these experiments two factors were at work inimical to the life of B. typhosus, namely the presence of B. coli, and growth in water—a non-nutrient medium. After 10 days, in the first experiment, slight fermenting power persisted. After two months, in the second experiment, all fermenting power was lost. No attempt was made to resusci- tate the strain of organisms on ordinary media to ascertain whether with returning vitality fermenting power would be restored. That this explanation is the true one and that no new race of organisms was produced is suggested further by the obser- vation that no organisms giving the ordinary reactions of B. typhosus survived side by side with the non-fermenters and that, at a later stage, the strain was found to have died out altogether. (d) The change from B. typhosus to Streptococcus faecalis in the peritoneal cavity of the guineapig. (Page 230, exp. 4.) The urine of a typhoid carrier “S” was plated and found to contain typhoid bacilli. One colony was subcultured on agar and a standard loopful of a 24 hours’ growth was injected into the peritoneal cavity of a guineapig. The animal was found dead in the morning. No typhoid ba- cilli were found in the peritoneal fluid which contained a pure culture of a Gram-positive streptococcus giving the reactions of S. faecalis. (e) The change in the peritoneal cavity of a guineapig Jrom B. typhosus to a coliform organism giving atypical re- actions. The urine of a typhoid carrier “I” was plated after being kept 12 months in a flask. Two colonies of B. typhosus were planted on agar and labelled JB, and JB, respectively. (ei) (Page 230, exp. 6.) One standard loopful of a 24 hours’ CH. 1x] OF TRANSMUTATION 121 growth from the culture 7B, was injected into the peritoneal cavity of a guineapig. The animal was found dead the next morning and a pure culture of B. typhosus was obtained from the heart’s blood. From the peritoneal fluid and spleen was obtained, in addition to B. typhosus, a coliform organism possessing the following characters: a gram-negative motile bacillus, forming acid and gas in glucose, mannite, lactose and dulcite, but producing no change in salicin or cane sugar but giving rise in the neutral red medium to gas and fluorescence, not liquefying gelatin or forming indol in broth and giving an acid reaction in litmus milk without any clotting. A broth culture from the original agar slope was carefully tested but typical B. typhosus alone found. The broth culture was planted on agar and the experiment repeated with a loopful of this growth. The animal did not die and a pure culture of B. typhosus was recovered from the peritoneal cavity. (eli) (Page 230, exp. 6.) One standard loopful of a 24 hours’ growth from the culture 7B, was then injected into the peri- toneal cavity of a guineapig in the same manner. The animal was found dead next morning and a pure culture of B. typhosus was obtained from the heart’s blood and spleen. From the peritoneal fluid was obtained, in addition to B. typhosus, a coliform bacillus. (eiii) (Page 231, exp. 7.) The urine of the typhoid carrier “T” was again plated after having been kept over 14 months in a flask. A colony was again planted on agar and a standard loopful of the growth again injected into the peritoneal fluid of a guineapig. The animal was found to be dying the next morning and was killed with chloroform and a pure culture of B. typhosus was obtained from the heart’s blood. From the peritoneal fluid and spleen a pure culture of a coliform or- ganism was obtained. The latter organism failed to produce any typhoid agglutinins when injected into a rabbit, or to ab- sorb agglutinins from a known typhoid serum. The last experiment was repeated, the same strain being used (“1”) after 14 days further growth on agar. The injection did not prove fatal to the guineapig and from the peritoneal fluid a pure culture of B. typhosus was obtained. 122 SUPPOSED INSTANCES [CH. Ix Criticism. The last four experiments (d, ei, eii, and e¢iii) may be discussed together. In one experiment (d) B. typhosus derived from a carrier apparently gave rise, in the peritoneal cavity, to aGram-positive coccus. In three experiments (¢i, eii, and eiii) B. typhosus, derived from another carrier, apparently gave rise, in the peri- toneal cavity, to atypical coliform organisms. The questions to be discussed are two—whether the strain of organisms isolated from the peritoneal cavity were derived from the original strain of B. typhosus injected in each case, and whether, if such continuity is established, the alteration in character is to be regarded as a temporary variation or a transmutation. The possibilities to be considered are (1) whether the original strain of B. typhosus was pure ; (2) whether the peri- toneal cavity in each case was sterile before the injection was made; (3) whether it was contaminated from the skin at the time the injection was made; (4) whether it was invaded from the gut after the injection was made or after the death of the animal; (5) whether the later strain was linked up with the original one by the occurrence of reversion or the discovery of intermediate forms; (6) whether a repetition of the ex- periments confirmed the results; (7) whether the alteration in character falls within the recognised limits of variation discussed in the earlier part of this work. (1) There are grounds for viewing the original cultures with suspicion. They were not made in any instance from a single organism. The urine of carrier “S” and of carrier “I,” from which they were isolated, admittedly contained streptococci, B. coli, bacilli closely resembling B. faecalis alcaligenes and other coliform organisms. These other organisms were present in comparatively small numbers. In one instance it is stated that B. coli and B. typhosus were present in the proportion of 1 to 30,000. Such disparity in numbers might easily account for the less common organisms being overlooked. It is men- tioned that a change in the character of the medium, brought about by simple dilution with water, enabled the associated microbes to multiply so much more rapidly than the B. ty- CH. Ix] OF TRANSMUTATION 123 phosus that the latter organism was soon “swamped,” as it were, and disappeared altogether. If the strain of B. typhosus injected contained one or two specimens of a streptococcus or coliform organism, might not growth in the peritoneal cavity yield a similar result?—not before, however, some of the ty- phoid bacilli had succeeded in escaping from the peritoneum into the blood vessels and setting up a systemic infection. In two instances, in which the experiments were repeated, the injection of the original culture of B. typhosus into the peri- toneal cavity did not kill the animal although a pure culture of B. typhosus was recovered from it. The original culture, therefore, apparently contained strains differing from each other in virulence. They may conceivably have possessed other differences. (2) No control experiments were carried out to prove that the peritoneal cavity before the experiment was sterile. Dudgeon (1908) states that in healthy animals the omentum may normally contain the staphylococcus albus. There is evidence to show that even in healthy animals the internal organs may contain both pathogenic and non-pathogenic bacteria. Ford (1900) showed by experiments, in which rigid precautions against contamination were adopted, that the kidneys, liver and spleen of healthy animals, in a large majority of cases, contained organisms such as the staphylococcus, mesentericus, colon and paracolon bacilli, B. subtilis and proteus. In rabbits 66 per cent. of the organs examined contained bacteria, in cats over 77 per cent., in dogs over 88 per cent. In guineapigs the percentage was 77 per cent. of the organs examined and the organisms that predominated were B. subtilis, staphylococci and the colon bacillus. Adami, Abbott and Nicholson (1899) found in the livers of healthy animals (cows, sheep, rabbits, guineapigs) diplococci and chains of 3 or 4 cocci, which on culture yielded B. coli in many cases. (3) No control experiments were conducted to exclude the possibility of skin contamination at the site of the inoculation, but such a supposition is inerdog uate to explain all the results obtained. 124 SUPPOSED INSTANCES [CH. 1x (4) The injection of organisms into the peritoneal cavity would have a threefold effect, it would make the animal ill, produce a more or less marked peritonitis, and finally kill the animal. All three events would favour the invasion of the peritoneal cavity by organisms. If the vitality of the body and consequently of the peritoneum is lowered, organisms can penetrate it from the gut even in the absence of any definite lesion or inflammation. Ford (1900) noted that in animals whose vitality was lowered, by fasting or unhealthy conditions, bacteria were more abundant in the internal organs, and that this applied particularly to bacteria of the colon type. Dudgeon and Sargent (1907) have shown that at the earliest stage of peritonitis the staphylococcus albus (either normally present on the surface of the gut or pene- trating from within it) increases with enormous rapidity. Miiller (1910) remarks that when organisms (eg. typhoid bacilli) are injected into the peritoneal cavity they at first decrease in number owing to the bactericidal effect of the body fluids but later on increase again. It is during this early stage when the injected organisms are decreasing rapidly that the staphylococcus albus (and possibly, in animals, the bacteria present in the internal organs) are increasing rapidly. A culture removed during this period might well convey the impression that a mutation had occurred. Dudgeon and Sargent (1907) mention that the staphylo- coccus albus is often quite non-pathogenic in the peritoneal cavity of the guineapig. After death there is a rapid invasion of the peritoneal cavity by organisms, particularly by B. coli from the gut, so that the true nature of the infection becomes obscured. In illustration of this point, Dudgeon and Sargent (1907) record a case of pneumococcal peritonitis in which the peritoneal exudate one hour after death gave a pure culture of pneumo- cocci, whereas 26 hours later B. coli alone could be recognised in the same exudate. It is not possible, therefore, to exclude in Major Horrocks’s experiments the possibility of an invasion of the peritoneal cavity from the gut, following either the inoculation or the death of the animal. CH. Ix] OF TRANSMUTATION 125 (5) In none of these four experiments was any attempt made to test the new strain by subculture or passage to ascertain whether it would revert. In two experiments the original strain of B. typhosus had, within a few hours of its injection, completely disappeared and in none of the experi- ments were any intermediate forms observed which might be regarded as linking up the new strain with the original one and suggesting a transmutation. All the organisms were apparently of the same type. Moreover the agglutination reactions betrayed no sign, in the only instance in which they were tested, of any connection between the new strain and the original B. typhosus. The continuity, therefore, of the two forms cannot be regarded as proved. Moreover if the variants were really derived from the original strain of B. typhosus one would have expected them to be present, like the typhoid bacilli, in the blood stream as well as in the peritoneal cavity. One possible explanation is that the change was dependent upon the influence of some agent existing in the peritoneum but absent elsewhere. This will be referred to again (vide p. 126). (6) A repetition of the experiments was made in only two cases (ei and eiii) and without yielding similar results— indeed the results differed from those first obtained in such a way as to suggest that the original cultures, in both cases, contained strains of bacteria differing widely, at any rate in their virulence. (7) If the continuity of the two different strains were established in each case, what would be the significance of these changes? The transition from B. typhosus to an atypical coliform organism may be regarded as a variation on the part of the typhoid strain, probably temporary in character and of the same nature as those discussed in an earlier part of this work (vide p. 11). The transition from B. typhosus to a Gram-positive coccus is more difficult to explain but the observations of Adami and others would suggest that, in this case also, a temporary variation and not a true transmutation might have been brought about. Adami (1892) observed that the addition to a medium of substances inimical to the life 126 SUPPOSED INSTANCES [CH. Ix of B. typhosus—for example, carbolic acid or creosote— made this bacillus in such a medium assume temporarily the form of non-motile cocci or diplococci. Again, Adami, Abbott and Nicholson (1899) obtained from the bile in guineapigs and also from the peritoneal fluid in man, under certain conditions, coccic forms of B. colt. These were present as diplococci or short chains of 3 or 4 cocci; they were non-motile, non-fermenting and did not produce indol; their growth on the surface of agar at first closely resembled that of a strepto- coccus, the colonies were white and opaque. Intraperitoneal inoculation into a guineapig increased their fermenting power and, after 3 passages, yielded normal B. colt. They found evidence that B. typhosus yielded similar modified coccic forms when acted on by peritoneal and other fluids. They describe coccic forms of B. typhosus in the mesenteric and retroperitoneal glands. They mention that in some cases the action of ascitic and peritoneal fluids in this respect is so marked that it was difficult to obtain complete reversion to type. They found, also, that B. colt injected into the blood stream in a rabbit appeared in these coccic forms within half an hour in the liver and the bile, though similar forms were not found in the systemic circulation. If the modification in a strain of B. typhosus which Major Horrocks describes was due to the same agency, one can understand why the variant was only found in the peritoneal cavity and not in the heart's blood. In his later experiments, however, cocci possessing the characters of S. faecalis were obtained not only in the peritoneal cavity but during culture on artificial media. In the account of these experiments, however, there is much to suggest that the original strain was not a pure one. (f) The change from B. typhosus fo B. faecalis alcaligenes after growth in the diluted and filtered urine of a typhoid carrier and the further changes from B. faecalis alcaligenes to B. coli on passage. (Page 237, exp. I.) The urine of a typhoid carrier “S” was diluted 1 in 10 with tap water and allowed to stand 11 days. It was then filtered through a Pasteur candle (F) with- out pressure and shown to be sterile by “prolonged incubation” CH. 1x] OF TRANSMUTATION 127 at 37°C. after plating. The filtrate was then inoculated with a 48 hours’ growth of B. typhosus isolated from the stool of a carrier “C” and a week later plated out on bile salt neutral red lactose agar. Two or three colonies were examined. One gave the typical reactions of B. typhosus but two other colonies failed to agglutinate with typhoid serum and cor- responded in their reactions to B. faecalis alcaligenes. One week later the latter organism alone was found and _ it persisted unchanged for several months afterwards. Criticism. The same criticism applies to this experiment. The original strain was not grown from a single organism. “A particle,’ we are told, of the growth was added to the solution, sufficient to yield 480 million bacteria to each cubic centimetre. The purity of such a strain cannot be guaranteed. The original culture was derived from a stool and it is said to have yielded an organism closely resembling B. faecalis alealigenes. The experiment was however repeated twice over with a laboratory strain with the same result—a fact which considerably discounts this objection. The new strain might, again, be regarded as a variant of the original B. typhosus which, as a result of growth under conditions inimical to its vitality, had suffered a loss of power with respect to its fermenting properties and also its property of agglutinating with typhoid serum. Its other agglutinative characters were not examined, but a similar organism obtained in the same manner from a laboratory strain was found to possess slight power of absorbing agglutinins from antityphoid serum. The new strain was, however, further tested (“culture 33” page 242) by being alternately passed through the peri- toneal cavity of a guineapig and subcultured on agar. In one experiment, after the 3rd passage, the peritoneal fluid removed at the end of 6 hours contained B. colt, but the fluid removed at the end of 12 hours contained not B. coli but the new strain of B. faecalis alcaligenes which had been injected. Only three passages were made—the investigation, that is to say, was not persisted in long enough to decide whether the new strain was capable of reverting or not. The recovery of B. coli after the 3rd passage Major 128 SUPPOSED INSTANCES [CH. 1x Horrocks considers may have been due to an invasion by this organism from the gut but was more probably due, in his opinion, to the still further modification of the strain injected, inasmuch as “reversion” apparently took place a few hours later. The disappearance of the B. coli might however be explained on other grounds. The strain of B. faecalis alcaligenes undergoing passage may have been contaminated with B. coli between the second and third passages; the former organism after its two passages might be expected to be more resistant to the body fluids which destroyed the latter. (9g) The change from B. typhosus fo B. faecalis alcaligenes after growth in the diluted and filtered urine of a typhoid carrier and the further change from B. faecalis alcaligenes to Streptococcus faecalis. (Page 238, exp. II.) The sterile filtrate from the urine of a typhoid carrier “S” was again inoculated with B. typhosus—the strain used, on this occasion, being a stock laboratory strain “R” of unimpeachable character. The result was similar to that in the previous experiment. Side by side with colonies of typical B. typhosus were found colonies of an organism (“35 A, Col. 2”) which corresponded with B. faecalis alcaligenes but possessed a slight power of absorbing agglutinins from antityphoid serum (p. 242). Later the latter organism alone was found and it persisted unchanged for many months. The experiment was repeated in exactly the same way and with the same result, B. faecalis al- caligenes emerging (p. 240, exp. IV). These two experiments are simply a repetition of the experiments already discussed, a laboratory strain of B. typhosus being used instead of a carrier strain. When the inoculated filtrate used in the first experiment (p. 238, exp. II) was 6 months old, a loopful of it was added to a broth tube and a 48 hours’ growth plated on MacConkey’s medium (p. 239). Colonies of B. faecalis alcaligenes were again found but with them smaller colonies of a streptococcus closely resembling S. faecalis. The strain of B. faecalis alcaligenes obtained in the CH. Ix] OF TRANSMUTATION 129 first experiment (p. 238, exp. II)—known after this as “35 A ”— was further tested (p. 243) by successive passages through the peritoneal cavity of the guineapig. The fluid removed after the 8th passage when subcultured into broth showed short-chained cocci and diplococci but when subcultured on to agar gave, in addition to these cocci, the original bacillus “35A.” The latter in broth again yielded the short-chained cocci and these on agar gave a bacillus once more—but this time not “35 A” but a fermenting coliform organism. The original strain of B. faecalis alcaligenes (obtained in Exp. II, p. 238) was a second time tested in the same way (p. 243). It showed no change in character until the 18th passage when it gave rise to a fermenting bacillus of the B. colt type which on the 19th passage reverted to B. faecalis alcaligenes. This last-named organism, after 7 more passages in one experiment and 8 more passages in another, gave rise to a fermenting B. colt type of organism “in pure culture” and this, on planting in broth, yielded B. faecalis alcaligenes once more but, with it, cocci corresponding to S. faecalis. The latter after 3 passages remained unchanged. Criticism. The transition from the non-fermenting B. Jaecalis alcaligenes to the fermenting coli type and back again, may be regarded as no more than another example of variation similar to those quoted in an earlier section of this work, Again, in the “passage” experiments, one or other type may have been an invader from the gut, the apparent reversion at a later passage merely representing the de- struction of the invader which had not been “hardened,” so to speak, by previous passages. Only one guineapig was used for each “passage” experiment in a series of passages. If more than one had been used some check would have existed on possible errors due to this cause. The repeated transitions from B. faecalis alcaligenes to S. faecalis and vice versa are more difficult to explain, for S. faecalis appeared not only after passage but during cultivation also on artificial media. One is ‘almost forced to the conclusion that Major Horrocks was dealing with a mixed strain of the two organisms and that changes in the conditions of growth at one time D. 9 130 SUPPOSED INSTANCES [CH. 1x fostered the growth of the first organism almost to the exclusion of the second, and at another time fostered the growth of the second almost to the exclusion of the first. If, after each appearance of S. faecalis, the strain had been guaranteed pure by the method of successive plating and growth from a single organism, the results obtained would have had more weight. It is worth noting that the strains of B. faecalis al- caligenes used in all these experiments, and supposed to have been derived from B. typhosus in the first place, showed no tendency to revert to B. typhosus. It is also interesting to compare these experiments with those of Adami, Abbott and Nicholson (1899) who obtained from B. coli grown in peri- toneal fluid cocci which did not ferment sugars or form indol, and yielded colonies which were white and opaque and resembled those of a streptococcus, After 3 passages through the guineapig these cocci yield short bacilli which however were still unable to ferment sugars. They state that B. typhosus was modified in much the same way under similar conditions. (h) The change from B. typhosus éo B. faecalis alcaligenes after growth in the diluted and filtered urine of a typhoid carrier. (Page 239, exp. III.) This experiment was practically a repetition of the last, the same strain of B. typhosus being used (laboratory stock “R”) but the urine was that of a different typhoid carrier “I”. The result was the same, colonies of typical B. typhosus being found at first but after a month’s interval colonies of a non-fermenting and non- agglutinating coliform organism being alone found. To this experiment the same criticism applies. 19 other experiments were made but with negative results. Summary. The evidence that Major Horrocks brings forward in support of the claim that in the course of his experiments transmutation occurred is inconclusive. He is unable in any case to guarantee as pure the culture with which he was dealing. He is unable to exclude definitely, in some of his experiments, the occurrence of a secondary CH. Ix] OF TRANSMUTATION 131 invasion in the living body. Lastly, even if the evidence established the continuity between his original strains and the new ones he obtained, the changes may possibly have been merely examples of variation no greater in degree than many that have been recorded by other observers. In other words, the strains of atypical B. coli and those closely resembling B. faecalis alcaligenes or S. faecalis, may con- ceivably have been variants of his original strains of B. typhosus whose true identity would have been disclosed by more prolonged efforts to obtain reversion or more thorough investigation with regard to their agglutination reactions. II. THERELATIONSHIP BETWEEN MEMBERS OF THE ENTERITIS GROUP OF BACILLI—B. ENTERITIDIS “GAERTNER” AND THE PARATYPHOID BACILLUS OF THE “ AERTRYCK ” OR “ FLUGGE ” TYPE. The question of the specific character of these organisms has been much discussed. They are generally recognised as distinct species but evidence has been brought forward from time to time suggesting that they may be transmuted one into the other. A. Schinitt’s experiments. Schmitt (1911), for example, claims that in certain experi- ments conducted by him strains of the paratyphoid bacillus of the Fliigge type became changed within the animal body into the Gaertner type of bacillus. The details of the experi- ments are briefly as follows. Experiment I. On July 17th, 21st and 28th he fed a young calf on milk to which had been added (in amounts varying from 1 to 50 cc.) a broth culture of a Fliigge type of organism—without apparent effect, except that the blood serum which previously did not agglutinate the organism now did so in dilutions of 1 in 35. On August 3rd the same strain of organisms was injected subcutaneously into the calf, with the result that the calf became ill. An organism (“Pgst I”) was isolated from the 9—2 132 SUPPOSED INSTANCES [OH. Ix calfs blood on the same day. It was found to be agglutinated by the animal’s blood serum in dilutions of 1 in 50—60 although, again, serum which had been taken from the calf before the experiments began failed to agglutinate it. (A calf serum, immunised against a Gaertner strain from cattle, was on the same day injected intravenously but only made the calf more ill.) A broth culture of this organism, Pgst I, was injected into the same animal on August 7th and again on August 10th, giving rise only to a slight febrile reaction on each occasion. ‘Eaperiment II, On August 25th, the strain already mentioned (Pgst I) as having been isolated from the first calf’s blood was suspended in normal saline and sprayed into the nose of another calf—without apparent effect. On August 28th a similar suspension was injected into the animal’s mouth with the result that the calf became ill. An organism (Pgst II) was isolated from the calf’s blood. On September Ath a saline suspension of this organism was injected intra- venously into the same calf, which died a few hours later. From the blood, intestines, muscles and bone marrow was obtained an organism Pgst III. The “Schluss-serum” was found to agglutinate the original Fliigge type and also the strains Pgst I, II, and I1J—in dilutions of 1 in 3000—4500. The great interest of the experiments lies in the obser- vation that these later strains isolated from the blood of the calf were found to correspond in their agglutination reactions, not with the original Fliigge type but with the Gaertner type of bacillus—a conclusion confirmed by absorption tests. Schmitt maintained, therefore, that passage through the calf modified the agglutination properties of the human para- typhoid bacillus (“Fliigge”) so that it came to resemble the calf paratyphoid bacillus (“ Gaertner ”’). Two possible fallacies at once suggest themselves. The type of organism which made its appearance later in the experiment might have been present as a contamination either (a) in the original strain or (6) in the bodies of the animals. inoculated. (a) As regards the first alternative, it is conceivable that CH. Ix] OF TRANSMUTATION 133 if bacilli of the Gaertner type were present in the original strain, but in such scanty numbers as to escape detection, these few bacilli might in the living tissues multiply with such rapidity as to become ultimately the predominant organism. The pre- cautions taken to secure the purity of the original culture would presumably exclude such a source of error. (b) In the second place, bacilli of the later or Gaertner type might conceivably have been present, before the inocu- lations were made, in the bodies of the calves themselves. This possibility appears at first sight to be excluded by the fact that the serum of the calf before inoculation failed to agglutinate the organism recovered afterwards, although the serum after inoculation was able to do so. This observation is not, however, final. Savage (1907-8) and other observers have recorded the presence of B. Gaertner in the intestines of healthy young calves. Its presence in the intestines of the calves inoculated in these experiments was not definitely excluded. The repeated administration in the food of as much as 50 c.c. of a young broth culture of a pathogenic organism would be likely to cause an inflammatory reaction in the bowel and such inflammation might lead, not only to an enormous increase in numbers on the part of any other pathogenic organism present, but also to an exaltation in their virulence. We have referred elsewhere (vide p. 80) to such a sequence in the case of B. colt in the intestine during an attack of typhoid fever and in inflammatory conditions resulting from improper food. Such an increase both in numbers and in virulence on the part of the organism we are discussing might well pave the way for its invasion of the system as a whole and lead to its appearance in the blood and internal organs, in a manner analogous to the invasion of the body by saprophytic organ- isms of heightened virulence in the cavity of an inflamed uterus. At the same time the blood serum would acquire agglutination properties which it did not possess when the bacilli were few in number, of low virulence and restricted to the lumen of the intestine. 134 SUPPOSED INSTANCES (CH. Ix B. The experiments of Miihlens, Dahm and First. These writers (1909) have recorded experiments which suggest a similar transmutation. They fed a large number of mice on meat which was thought to have been infected although a preliminary bacteriological examination proved negative. Over 50 per cent. of the mice died. A bacteriological examination of the faeces of 56 mice was made. In 20 cases none of the paratyphoid group of bacilli were detected. Aertryck’s bacillus was found to be present in 24 and Gaertner’s bacillus in 13 cases. These results suggest that one type may have arisen from the other within the animal body. As in the experiments already discussed, two possibilities have first to be excluded—namely, the possibility of con- tamination (a) in the original strain and (6) in the bodies of the mice used for the experiment. (a) All that can be said by way of excluding the first of these alternatives is that the simultaneous presence of both the Aertryck and the Gaertner type of organism in infected meat is contrary to experience and was thought by other writers (Zwich and Weichel, 1910) to be highly improbable in this instance. (6) With regard to the second alternative, no preliminary bacteriological examination of the mice used in the experi- ment was made. The faeces of 40 control mice were examined and B. Gaertner was discovered in one case only. Zwich and Weichel (1910), on the other hand, found that out of 177 healthy mice 28 gave B. Aertryck in the faeces. The bacteriological examination of the 40 control mice with a negative result in 39 cases is, again, open to the criticism that paratyphoid organisms might have been actually present at the time but in such small numbers that they escaped detection. Any such organisms present, in equally small numbers, in the other mice would find a nidus for their growth in the unhealthy and inflamed condition of the intestine which would result from feeding the mice on infected meat, while the disturbed functions of the bowel, by CH. 1x] OF TRANSMUTATION 135 hastening its evacuation, would bring about the subsequent appearance of these organisms in the faeces. C. The author's experiments. The following experiments were suggested to the writer by Professor F. A. Bainbridge as likely to throw some further light on this aspect of the question and were carried out at the Lister Institute under his kind supervision. Experiment I. 24 healthy guineapigs were chosen and 12 of these were confined in 3 cages. On August 13th one of the four guineapigs in each cage was removed and given in its food 1cc. of a 48 hours’ broth culture of B. enteritidis Gaertner. Every precaution was taken to prevent, as far as possible, any external contamination of the guineapigs with the food and they were then returned to their respective cages. This culture of B. Gaertner was made from a labo- ratory strain the agglutination reactions of which had been repeatedly tested and had been found to be constant. A bacteriological examination of the faeces of the 12 guineapigs was made subsequently on three occasions, namely on August 23rd, September 19th and October 9th—bacilli of the paratyphoid group being identified by agglutination tests. During this period each cage was kept separate from the others and none of the guineapigs were removed from their cages except for the necessary examination on the three dates mentioned. Up to the time of the first examination on August 23rd all the guineapigs remained well, but subse- quently three of them died and the remainder exhibited varying degrees of malaise and intestinal disturbance. The following scheme shows the type of organism found in the faeces at each examination. N.B. Guineapig No. 1 in each cage was given 1 cc. of a broth culture of B. enteritidis Gaertner on August 13th. “0” indicates that neither the Gaertner nor the Aertryck type of organism was isolated. The faeces of the remaining 12 guineapigs, which had been kept quite apart from the others, were carefully investi- 136 SUPPOSED INSTANCES [CH. Ix gated at the date of the first examination (August 23rd). Gaertner’s bacillus was not found in any case and Aertryck’s bacillus in one case only. “ “ 1st examination | 2nd examination | 3rd examination Cage | Guineapig | “august 23rd Sept. 19th Oct. 9th No. 1 Aertryck Dead — No. 2 0 Aertryck Gaertner A | No.3 Gaertner Dead — P. M: Gaetner No. 4 Gaertner Aertryck Gaertner No. 1 Aertryck 0 0 B No. 2 Aertryck 0 0 No. 3 Gaertner Dead — No. 4 ‘ 0 0 (no faeces) No. 1 Gaertner Aertryck 0 No. 2 Aertryck Gaertner 0 C | No.3 Aertryck and (no faeces) _ Gaertner : No. 4 0. (no faeces) _— These experiments appeared to lend further support to the theory that the two types of organisms were capable of transmutation. It is necessary, however, again to emphasise the fact that a negative result in the case of all but one of the animals examined as a control, does not prove the absence of organisms—it only proves their scarcity. A subsequent in- crease in their numbers might at once have revealed their presence. Such an increase might be apparent only, due to a simple disturbance of the functions of the bowel, such as diarrhoea, which would dislodge the organism from its usual habitat, carry it to a lower part of the bowel and hasten its evacuation. The increase in numbers might, on the other hand, be a real one, brought about by a lowered vitality of the body as a whole and local inflammatory changes. It is to such factors that we attribute the enormous number of B. coli found in the stools of patients suffering from cholera. All these factors were, no doubt, operative in the case of the three guineapigs which were actually fed with the culture of B. Gaertner and may explain the subsequent discovery of CH. Ix] OF TRANSMUTATION 137 B. Aertryck in the faeces of all of them. The remaining guineapigs may have been infected by B. Aertryck from the faeces of these, at one stage or another, owing to their food becoming contaminated. The same factors would explain, in their case, the later appearance of B. Gaertner. It is, however, to be noted that at the 1st examination (August 23rd) of the guineapigs in cages A and B, while both those which had been fed with the Gaertner culture were passing B. Aertryck in their faeces, three of the six animals which had noé been fed with the Gaertner culture were passing B. Gaertner. To explain this on the grounds that the food of these three had been contaminated by the faeces of the Gaertner fed guineapigs, we must assume that the latter had, at some time previous to the examination, been also passing B. Gaertner in their faeces and that this organism had only later given place to B. Aertryck. That the factors we have been discussing do actually lead to the detection in the faeces of organisms which previously did not appear to be present, the writer endeavoured to prove by further experiment. Experiment II. On August 25th six apparently healthy guineapigs were chosen and labelled Nos. 1 to 6. A frag- ment of the faeces from each guineapig was shaken up in malachite green broth and after incubation the latter was plated out, two sterile plates being used for each guineapig. Both plates from No. 3 showed white colonies. A culture from these colonies was grown in broth for 48 hours and then passed through the sugars by which means it was identified as B. proteus. In the case of the remainder the five pairs of plates all proved to be sterile. The guineapigs were then for several days fed on green vegetables and bran soaked in castor oil—a diet designedly unwholesome and calculated to make the animals ill and also to set up some intestinal catarrh. On August 30th a fragment of faeces from each guineapig was again shaken up in malachite green broth and this, after incubation, plated out as before on two sterile plates. In the case of No. 1 and No. 5 no growth was apparent in 138 SUPPOSED INSTANCES (CH. Ix the tubes and both pairs of plates remained sterile. In the other four tubes growth took place with gas formation and the four pairs of plates all showed colonies. Broth cultures were made from these colonies and yielded strains which gave the sugar reactions of B. proteus. The writer failed to demonstrate the presence in any case of organisms of the paratyphoid group. The experiment however was successful in demonstrating that bacteria which failed to give evidence of their presence in the faeces of a healthy guineapig might make their appearance in the faeces of the same animal after it had been given for a few days unwhole- ‘some and irritating food. This conclusion lends weight to the suggestion already made that the results obtained by Schmitt, and also by Miihlens, Dahm and First, might possibly be explained by the presence of a secondary invader. Their experiments are, in both instances, open to one further criticism. The identification of the paratyphoid or- ganisms was made to depend solely upon their agglutination reactions. If it is admitted that the power to form and to absorb specific agglutinins on the part of an organism is subject to variation it must be recognised that such tests alone are insufficient to establish the identity of the organism. In other words, it is within the bounds of possibility that only one type of organism was actually present, but that its agglu- tination properties varied. Such a contingency would be likely to arise in the case of two organisms so closely allied as B. Aertryckand B. Gaertner. An elaborate investigation into the agglutination properties of these two organisms was conducted by Sobernheim and Seligmann (1910). Pure colonies of numerous strains were secured by the Indian ink method. They found that colonies derived from the same strain and growing side by side differed in their agglutination reactions. The same strain differed at different times. The agglutination reactions, in some instances, became altered after passage through the mouse and after a culture had been heated. In some instances the injection of living bacilli yielded a serum which was much more variable CH. Ix] OF TRANSMUTATION 139 in its agglutinative powers than a serum obtained by means of a dead culture. Some strains gave doubtful reactions. The power of the same strain to form agglutinins and to bind agglutinins appeared in some cases to differ. They therefore concluded that the agglutination reactions did not constitute a specific test. We may interpret these results in one of two ways. We may decline to recognise the two types as representing dis- tinct species ; or we may continue to regard them as distinct species and acknowledge that their agglutination properties are liable to variation. In either case the experiments quoted are deprived of all significance as examples of transmutation. CHAPTER X SUMMARY Ir will be evident from the foregoing pages that practically every character of bacteria is liable to vary at different times and under different conditions. These variations are of two kinds, spontaneous or “intrinsic”—that is to say due to tendencies inherent in the organism itself—and impressed as a result of external influences. These modifying influences have been enumerated (Chapter II) and examples given of the variations they produce. In many cases an organism may appear to vary although no variation actually takes place, and in other cases what appears to be a “spontaneous” variation is actually an “im- pressed” variation due to external influences which have not been recognised by the observer. These various sources of error have been enumerated and discussed in Chapter III. A tendency to vary in a particular way—either spontane- ously or in response to external stimuli—may be so charac- teristic of a certain organism as to be in itself almost specific in character, and so far from confusing its identity may actually make this more apparent. The pleomorphism of B. diphtheriae, the tendency of S. scarlatinae to assume a bacillary shape, the tendency of B. paratyphoid B to form papillae on raffinose agar, will serve as examples. No single property of bacteria can be regarded as specific nor does the occurrence of variation in respect to any one quality or function, or to several of them simultaneously, necessarily imply a loss of specific character on the part of the organism concerned. This is well illustrated by the mor- phology of bacteria. A certain appearance may be spoken of as “characteristic.” This does not mean that it is in- variable but merely that the organism shows a tendency to CH. X] SUMMARY 141 present such an appearance rather than another. B. coli in the peritoneal cavity in the case of ascites may take the form of a diplococcus; in milk or in urine it may develop into a dense network of branching filaments resembling B. anthracis, but these changes in form do not imply any obliteration of the specific character of the organism itself. We have already referred in this connection (vide p. 38) to the analogy of a regiment of soldiers at manceuvres and a mass meeting of miners at the pithead. The various military formations assumed by the first are as characteristic as the concentrically arranged crowd formed by the second—so much so that an observer at a distance might from the appearance of these “zoogleic forms” state with confidence the character of the units composing them although too far away to identify the latter. A crowd of pitmen on strike might, however, march in military formation and a regiment of soldiers at a boxing match take the form of a crowd concentrically arranged—each reproducing, that is to say, the appearance regarded as typical of the other. This would not indicate that the pitmen were changing into soldiers or the soldiers into pitmen. It is true, nevertheless, that the arrangement most frequently observed in one or the other case does indicate a tendency on the part of the individual unit and may, therefore, afford a clue to its identification. The behaviour of a civilian under certain circumstances may furnish evidence of a military training and deserters from the colours are not infrequently recognised by such means. In a similar way, the occasional assumption by the bacillus of diphtheria of clubbed and branched forms, while helping us to identify it, also provides us with a clue to its mycelial ancestry (Kanthack and Andrewes, 1905). Many of the variations exhibited by bacteria do in fact, represent steps in the evolutionary process by which, in the past, they have become differentiated—the individual organisms living over again, as it were, the life history of the race. This would appear to be the explanation of many variations in morphology (Chapter IV). Others again repre- sent the advance along new lines of this same evolutionary process, leading to further specialisation and differentiation. 142 SUMMARY [cH. x This aspect of the subject has been considered at length in the sections dealing with Fermenting Power and Virulence (Chapters V and VI). Since we have no absolute criterion as to what constitutes a “species” amongst bacteria, dissimilarity in the several characters they present is our sole guide to classification. In other words the distinction between a “variety” and a “species” depends simply on less or greater divergence in character. The difference, therefore, between variation and transmutation is one of degree only; or, looking at the matter from another standpoint, we may say that the same degree of deviation from type may be interpreted in one case as variation and in another as transmutation. This will be readily understood if it be borne in mind that the various types or “species” of bacteria which we are able to distinguish have developed from a common stock. In the case of some of them the differentiation dates from a remote past and the specific characters are comparatively fixed. In the case of others differentiation is of more recent date and the newly acquired characters are less permanent and “reversion” in one or other character is more frequent. In yet a third class—the groups of closely allied organisms—the gradual process of differentia- tion is only now taking place and it is not yet clear which characters are of specific value. During the process of evolu- tion, in all its stages, there is a tendency shown on the part of the organism to revert towards the original type. Such reversion in one or more characters, although of no greater significance in the case of one class or another of the three we have described, is likely to be differently interpreted. If differentiation is well advanced, a partial reversion in character will merely present itself as an unimportant variation. If differentiation has not progressed very far, a reversion no greater in degree may confuse the identity of the organism concerned sufficiently to suggest the possibility that trans- mutation has occurred. If the process of differentiation is still incomplete, a reversion in character even smallerin degree may entirely obliterate the faint lines of division that we have been able to trace out. The error of assuming too hastily that OH. x] SUMMARY 143 transmutation has occurred will be prevented by.a proper consideration of, firstly, the biological characters of the organism in question as a whole and, secondly, the question of the stability of the characters which distinguish it. With regard to the first of these questions, we have shown in the sections dealing with Morphology, Fermenting power, Virulence and Pathogenesis (Chapters IV—VII) the danger of relying upon any one of these characters alone for the purpose of identification or of classification. In the case of widely divergent types a single character may sometimes suffice to distinguish one organism from another but even in such a case, if that character is liable under any circumstances to variation, it obviously cannot be trusted as an infallible guide. The pathologist is in the same boat, in this respect, with the ethnologist. Certain “race groups,” e.g. the Teutonic, the Mongolian, and the Negroid, though ‘conceivably derived from a common anthropoid stock, are sufficiently differentiated to be readily distinguished by a single character. For example, the flaxen hair of the German, the matted black hair of the Negro and the straight black hair of the Jap are sufficiently characteristic of their respective race-groups. Such a dis- tinction, however, breaks down between the races within the: groups themselves and other characters must then be considered in addition. In some cases, again, the process of differentiation is still incomplete, individuals approximating now to one and now to another recognised type, and a con- sideration of all the characters may still leave the observer in doubt as to the correct classification. The ethnologist has learnt, moreover, that certain char- acteristics are not to be regarded as racial in character. For example—to return to our previous illustration—the pigtail of the Chinaman and the shaven poll of the Thibetan priest, the flowing locks of an Italian impressario and the tonsured crown of a Romish monk, are not racial characters at all but artificial modifications. They do, however, signify a certain environment and training and this is precisely the case with many of the variations which the pathologist meets with 144 SUMMARY [cu. x amongst bacteria. In other words, a study of such variations in a given case may afford valuable and trustworthy informa- tion as to the source from which the particular strain of organisms has been derived. This subject would repay further investigation. One or two instances may be given here to demonstrate its im- portance. Rosenow (1912-13) found that the ordinary streptococcus pyogenes, if grown in unheated milk, became modified in its morphology, its cultural properties and its virulence. He had previously isolated from several cases of epidemic sore throat a streptococcus which possessed precisely similar modifications in character. The epidemic had been recognised as “milk- borne” but, had its origin been in doubt, the unusual char- acters of the organism concerned would obviously have pro- vided a clue. Ohlmacher (1902) isolated branching filamentous forms of B. coli from the heart’s blood in a case of septicaemia. He quotes various observations to the effect that residence in the biliary passages develops this unusual morphology in B. coli, and he therefore considers that the original source of the systemic infection in this case was in the region of the gall bladder or bile ducts. Moreover the degree to which the modifications persist on subculture is a measure of the time during which the organism was subjected to the modifying influence. This brings one to _ the second question, the stability of the variations produced. Remarkable differences are to be observed in the degree of permanence exhibited by a variation in different cases and it is difficult to decide upon what factors these differences depend. We have already referred to the fact that variations may be either “spontaneous” in character or “impressed” upon the organism by external agencies. Spontaneous variations may be of several kinds and the nature of the variation may itself decide its degree of permanence. 1. Some variations represent an early stage in the life history or are due to imperfect development, and are seen CH. x] SUMMARY 145 in young or backward cultures. We have spoken of the atypical morphology of a young culture of the Klebs-Loeffler bacillus, which renders it difficult to distinguish it from Hofmann’s bacillus (vide p. 42), and of its inability to ferment glycerin and lactose (vide p. 55). Such differences are comparable to the juvenile features and unskilled hands of a class of schoolboys and tend to disappear of their own accord as the strain grows and develops. 2. Other variations represent senile changes or are due to lowered vitality, and are seen in old or worn out strains. The loss of motility, or of pigment production, in an old culture will serve as an example. Such variations are comparable to the slow steps and grey hairs that characterise a party of old men and will tend to become more and more developed unless some external influence intervenes and, by effecting a radical change in the conditions of growth, contrives to rejuvenate the strain. 3. Others again are degenerative in character or are due to atavistic tendencies—such as, for example, the appearance of branched and clubbed forms of the tubercle bacillus. These variations are comparable to some forms of mental impairment in a family, or to defects such as harelip. They may be passed on from father to son and so persist, or they may disappear, but in the latter case they tend to recur ina later generation. 4, Others, finally, are evolutionary in character and repre- sent a higher specialisation on the part of the organism—such as, for instance, the development by B. typhosus, after a long training, of power to ferment lactose, or the acquisition on the part of a feebly pathogenic organism of the quality of extreme virulence. Such changes are analogous to the de- velopment of a national genius for literature or ‘conquest. The more highly specialised a function is the more easily does it become deranged and a character, therefore, of this kind, is readily lost. For example, however permanent other newly acquired characters in bacteria may appear to be, variation in the direction of increased virulence seldom is so and almost invariably proves unstable. It is easy to see how, in every one of the four classes we D. 10 146 SUMMARY [CH. x- have mentioned, two or more variations may be constantly associated. In some cases the association is explained by the fact that both variations are due to lowered vitality. For example, the loss of power to produce pigment may be associated with the loss of power to liquefy gelatin or to grow on certain not very favourable media—all these functions being dependent upon the vitality of the organism. Again, the evolution or higher specialisation of an organism may involve simultaneous modification in two or more direc- tions. These modifications may all represent a casting off of saprophytic characters by the organism in question on its entry upon a parasitic career. For example, a saprophyte may derive its vital energy from the sunlight by means of a pigment, comparable to the chlorophyll of a vegetable cell, or from carbohydrate food through its ability to ferment it. When it becomes parasitic, and in many cases pathogenic, it is cut off from sunlight and must subsist on the body fluids. We may find therefore that the acquirement of virulence is associated with the loss of power to form pigment and to ferment sugars. In the same way, the constant association between two different variations may be due to the fact that the young strains which show them have not developed their adult powers, or to the fact that the variations are both signs of degeneracy or atavism. “Impressed” variations show even greater differences in their degree of permanence. In some cases a variation is only maintained while the influence which caused it continues to act. In others the variation persists for a shorter or longer period after that influence is withdrawn. In others again the variation is apparently permanent and persists under normal conditions of growth indefinitely. We use the expression “apparently permanent” for it is impossible in any case to guarantee the permanence of the characters exhibited by a strain of bacteria. This has been shown both by observation and by experiment. Mention has been made elsewhere (vide p. 14) of a strain of bacteria which after nine years’ cultivation lost its power to ferment maltose, and of another strain which after five years cultivation lost its power to produce pigment. CH. x] SUMMARY 147 Twort found that B. typhosus grown in a lactose medium retained its character as a non-fermenter of lactose for two years before variation occurred. Eyre and Washbourn found that to raise a particular strain of an avirulent saprophytic pneumococcus to full virulence by animal passage, no less than fifty-three successive inoculations were required. Characters which persisted for periods of two, five and nine years, and withstood a series of over fifty passages through an animal body, might well have been regarded as “permanent.” They were, however, only “apparently” so. Certain principles which govern the stability of impressed variations can, however, be discerned. 1. The variation may affect all the members of a strain or only certain of them. In the latter case an apparent reversion is obviously more likely to occur. The rapidity with which this apparent reversion takes place will depend upon the comparative rate of growth of the unaltered organisms and the variants. If the new character is of advantage to the organism it will enable the variants to multiply more quickly and they will gradually get the upper hand. Apparent re- version will not take place as long as the new character continues to confer an advantage upon its possessors but when this ceases to be the case the organisms possessing the new character may disappear and reversion to the original type appear to take place. For example, the acquirement of viru- lence by some members of a non-virulent or feebly-virulent strain, when this is injected into the living body, gives these variants an advantage as long as they are in the body. If the mixed strain is grown on artificial media the advantage is done away with and the unaltered bacteria, other things being equal, have now as good a chance as the variants of increasing their numbers and the variants may disappear. We have used the expression “apparent reversion” for it is evident that, unless every member of a strain acquires the new character, the loss of that new character by the strain may be brought about by the dying out of the variants without a single organism having actually “reverted.” This fallacy can be readily excluded if care be taken at each step to ensure that 10—2 148 SUMMARY . [cH. x the strain of bacteria under observation is a pure one—that is to say, derived from a single organism by the methods sug- gested by Barber and others. 2. The more readily a new character is “impressed” on an organism the longer it is retained—conversely, the more slowly and reluctantly an organism takes on a new character the more easily is that character lost. The behaviour of B. typhosus is a good illustration. This organism can be trained to ferment dulcite in a few days and will then retain the power for many weeks in the absence of that sugar. It cannot be trained to ferment lactose in less than two years and then loses the power in a few days if the lactose is withdrawn. It must be understood that we are here speaking of “im- pressed” variations. In the case of “spontaneous” variations the reverse holds true. Bacteria which vary spontaneously with great readiness often revert with equal facility, while those that are tenacious of their normal characters often prove tenacious of any new character they may spontaneously develop. 3. The longer an organism which has undergone a varia- tion continues to be exposed to the influence which caused it, the longer will the variation persist after that influence has been withdrawn. For example, Rosenow (1912-13) isolated a streptococcus, from a number of cases of general infection, possessing unusual morphological and cultural characters which, however, showed reversion on cultivation outside the body. He found that strains isolated from the peritoneal exudate and blood at a later stage in the disease showed these modifications in char- acter to a greater degree than those isolated earlier in the attack. The behaviour of B. typhosus again illustrates this point. If a strain is grown on dulcite medium it acquires, in a few days, the power of fermenting that sugar and this power is retained for some weeks after the strain has been removed. from the dulcite medium. Reversion then occurs and the power is lost. If however the strain is grown on a dulcite medium continuously for three months the power to ferment CH. x] SUMMARY 149 dulcite is found to persist afterwards, on ordinary media, “permanently.” This observation, based on the results of laboratory experi- ments, provides a clue, as Adami observes, to the nature of the process by which new races of bacteria are developed. In the laboratory organisms can be exposed to certain modifying influences for many months or even years and the new char- acters developed by such means are found to persist for long periods before reversion takes place. In nature agencies which possess the power of modifying the characters of bacteria may exert their influence for an indefinite period and the process of reversion in this case may be indefinitely postponed. In other words the new characters developed may appear to be permanent. A variant, however, may retain its new characters indefinitely and show no tendency whatever to revert under ordinary conditions of growth and yet it may still be capable of reverting immediately under suitable conditions. Examples of this are common in the laboratory and may be found in nature. Laurent describes a decolourised strain of B. ruber which was grown for 12 months at a temperature of 25-35° C., being subcultured 32 times in this period, without once show- ing anytrace of pigment. On lowering the temperature to 18°C. pigmentation at once reappeared. Again, the diphtheria ba- cillus is far removed from its mycelial ancestry but under suitable conditions will still display a partial reversion to a mycelial structure. We do not on this account deny the title of “species” to the diphtheria bacillus, for we recognise that the idea of absolute permanence in character is not essential to our conception of a speciés in the case of bacteria. It is not permanence in character but the degree of resistance to al- teration in character displayed by an organism that determines our opinion of its specific nature. In spite of the many minor variations they display there is exhibited by most species of bacteria a resistance to modification—a “ vis inertia ”—which constitutes true racial stability. We have seen, then, that the difference between variation and transmutation is one of degree alone. It is a question of the extent of the modification and the degree of permanence 150. SUMMARY [cH. x it exhibits. It is no less true that the process of transmutation only differs in degree from the process of evolution. Here it is a question of the rapidity of the change. Let us take by way of illustration the case of a family of ancient lineage, the members of which hold high office in the State and are remarkable for their wealth and erudition. Such a family may have sprung 500 years ago from humble origin and, while the fortunes of one branch have steadily prospered and successive generations have gradually acquired fame and amassed wealth, the original yeoman stock from which it sprang has continued to be represented throughout the centuries, in some corner of the kingdom, by men chiefly remarkable for their deficiency in the riches and learning and reputation for which the others are distinguished. It is con- ceivable that a son of the older and less distinguished branch of the family, seizing a favourable opportunity, might, by the exercise of the same faculties of industry and thrift displayed by the others, raise himself in the space of a single life-time to a position of wealth and power equal to theirs. We can trace the steps by which, in the course of time, a virulent and highly specialised race of bacteria has been evolved from a less virulent and less highly organised race. We find the two races living still side by side. The question arises whether it is pos- sible under unusually favourable conditions for the process of adaptation and specialisation to take place with such rapidity as to suggest a sudden transmutation. The conversion of the saprophytic pneumococcus into the parasitic pneumococcus by Eyre, Leatham and Washburn (vide p. 115) appears to offer an example. These observers describe the virulent parasitic pneumococcus as requiring for its growth a certain reaction and temperature and particular media (blood agar); it would not grow if the reaction were even faintly acid or at a temperature much below 37°C. and rapidly died out on agar or in broth. It would not liquefy gelatin and in broth formed a dust-like deposit. The avirulent saprophytic variety, on the other hand, grew luxuriantly at temperatures ranging from 37° to 20°C., on agar, gelatin, potato or in broth, whether acid or alkaline, slowly liquefying CH. x] SUMMARY 151 gelatin, producing a uniform turbidity in broth, and it retained its vitality for many months; it also exhibited differences in its morphology, “instead of isolated diplococci and strepto- cocci, large masses of cocci and diplococci were found, and forms dividing into tetrads were common.” Nevertheless this avirulent saprophytic pneumococcus could, by a single “ pas- sage” through a rabbit, be converted into a typical parasitic pneumococcus of high virulence. The occurrence of such a remarkable transition would be regarded as more significant if it were not that both organisms bear the same name and are considered—in spite of the many differences existing be- tween them—to be variants of each other. If we consider the possibility of a similar transition in the case of two races of bacteria less closely associated with each other, we find little direct evidence in proof of its occurrence —and this often of doubtful value—but a great deal of cir- cumstantial evidence in favour of the supposition that it may occur. We have discussed at length (Chapter VIII) such a possibility in the case of organisms found in close association in the body, such as Hofmann’s bacillus and the Klebs-Loeffler bacillus, Staphylococcus epidermidis and Staphylococcus pyo- genes, Micrococcus catarrhalis and the meningococcus, and others. Finally, we have discussed in detail (Chapter IX) the re- cords of certain experiments in the course of which bacteria became so changed in character as to suggest that they had undergone transmutation. In the first series of experiments—those of Major Hor- rocks—the results seem to be capable of explanation on other grounds. In the first place adequate precautions do not appear to have been taken to guarantee the purity of the strains at different stages of the experiment. In the second place, many of the changes in character stated to have been observed may _be regarded as examples of temporary variation only, similar to those recorded by many other observers. The second series of experiments—those of Schmitt, and of Miihlens, Dahm and Fiirst, and of the writer—which suggest the occurrence of transmutation between different 152 SUMMARY [cH. x members of the paratyphoid group of bacilli, are open to the same criticism. In the first place, a temporary variation in one character alone—namely in agglutination properties—would sufficiently explain the results obtained. In the second place, these results may have been due to a secondary invasion—in other words, it is conceivable that there may have been a pre- existing but unrecognised infection in the animals utilised for the experiments. This hypothesis we have shown, from the records of other investigators and by analogy with other pro- cesses of infection, to be not improbable; while the writer's experiments further demonstrate the ease with which such a secondary invasion may be overlooked. In none of these experiments, therefore, can the occurrence of transmutation be regarded as proved, nor, on close ex- amination, does its occurrence appear probable. A theory which we propose to discuss in conclusion suggests a via media by means of which organisms might conceivably exchange many of their characters and functions without them- selves undergoing transmutation. This is the Enzyme theory of disease. CHAPTER XI THE ENZYME THEORY OF DISEASE It is impossible to leave this subject without some further mention of a theory, to which passing reference has already been made more than once in the foregoing pages, namely the Enzyme theory of disease. This theory predicates that the results which follow, and are regarded as characteristic of, infection by a certain organism —including both the pathological lesions produced and the train of symptoms observed clinically—are caused not only (if at all) by the activities of the micro-organism itself, but by the activities of ultra microscopic bodies of the nature of enzymes which are associated in each case with a particular bacterial cell in the same way that the ferments of yeast are associated with a particular vegetable cell. If the scattered references to this theory in the foregoing pages be collected together they will be found to constitute a by no means negligible weight of evidence in favour of it. The considerations which lend support to the theory are the following. 1. In the first place, there is the observation that a sapro- phytic organism incapable at one time of giving rise to disease, even after it has invaded the living tissues, may suddenly ac- quire pathogenic powers and give rise in the living body to definite lesions and a definite group of symptoms. Harmless organisms such as the saprophytic pneumococcus, the Micro- coccus catarrhalis and B. colt, for example, may mysteriously acquire the power to produce respectively pneumonia, menin- gitis and enteric fever. On the other hand, virulent pathogenic organisms such as the Klebs-Loeffler bacillus, the meningo- coccus and B. typhosus may as mysteriously become deprived of their power to produce respectively diphtheria, meningitis 154 THE ENZYME THEORY OF DISEASE [ou. x1 and typhoid fever. Eyre and Washbourn (1899) showed in the case of the pneumococcus that such an alteration in character could be brought about, in one direction, by a single passage through an animal and the reverse change with almost equal facility. We have no explanation of the processes upon which such changes in character depend but we know that many of the conditions which bring them about are precisely those which foster or destroy other properties in organisms which we believe to depend on ferment action (vide infra). 2. In the second place, there is the observation that the pathological lesions and clinical symptoms resulting from, and characteristic of, infection by a certain organism may be faith-, fully reproduced as a result of infection by a totally different organism. For example, we have noted (vide pp. 99 et seq.) some of the lesions and symptoms of diphtheria to be caused by the pneumococcus, those of scarlet fever and of influenza by WM. catarrhalis, those of cerebrospinal fever by the Klebs- Loeffler bacillus, by MZ. catarrhalis and by B. typhosus, and those of rabies by the Klebs-Loeffler bacillus. The description of the last example given—a case of rabies due to infection by the bacillus of diphtheria—will bear repetition. It was recorded by Head and Wilson (1899). The diagnosis of rabies was founded on the history and clinical symptoms. “The well authenticated history of a bite on the cheek by an animal, the two months’ incubation period, the onset with extreme pain and numbness in the region of the scar, the development of the characteristic laryngeal and respiratory spasms on attempting to take liquids, the spasm at first being slight but later more pro- nounced and towards the close again feeble or absent, the insomnia, the absence in the beginning of fever which later in the illness became pronounced, the rapid pulse at all stages, the attacks of violent delirium interspersed with periods of calm and complete rationality, the absence of all symptoms pointing towards any other simulating disease and the fatal termination—all serve to make an almost complete picture of rabies.” The Klebs-Loeffler bacillus was isolated from the ventricular fluid and detected in the nerve cells of cH. x1] THE ENZYME THEORY OF DISEASE 155 the medulla. The recognition of this organism was complete and beyond doubt. “Not less suggestive of rabies than the clinical history were the results of subdural inoculations in rabbits with emulsions prepared from the medulla of the patient. There occurred the long period of incubation (20 and 21 days) followed by phenomena similar to those in experimental rabies of rabbits, and other rabbits inoculated subdurally with the medulla of the first rabbits behaved in a similar manner.” 8B. diphtheriae was demonstrated after death in the medulla of the rabbits. By a thorough investiga- tion, full details of which are given, infection by the virus of rabies was definitely excluded. : Such phenomena become intelligible on the supposition that both the lesions and the symptoms of a disease result from the activity of particular enzymes which are usually associated with one particular organism but are capable of being associated, under certain conditions, with an altogether different organism. 3. In the third place, representatives of one specific organism, morphologically and culturally indistinguishable from one another, may give rise in the living body to entirely different lesions and symptoms. Indeed, the contrast between the train of lesions and symptoms produced in one case and that produced in another may be as marked as the contrast between the lesions and symptoms produced by two organisms representing two distinct species. For example, different epidemics of the same disease may present altogether different features. Thus, strains. of B. influenzae, morphologically and culturally indistinguishable from one another, may give rise to epidemics of “influenza” characterised by symptoms resembling in one epidemic a simple coryza, in another epidemic rheumatic fever, in a third typhoid fever, and in a fourth cerebrospinal meningitis. Not only do different epidemics present different types of disease but individual cases occurring in the course of one and the same epidemic, and undoubtedly due to infection by the same organism, may exhibit a totally different train of symptoms. We have mentioned elsewhere, the account given 156 THE ENZYME THEORY OF DISEASE [cu. x1 by Andrewes and Horder (1906) of a number of cases of contagious disease, obviously passed on from one patient to another, of which some presented the symptoms of scarlet fever and others those of puerperal fever (vide p. 98). . Another remarkable instance (recorded by Dunn and Gordon, 1905) has been already alluded to but is of sufficient interest, in this connection, to warrant a second description. They mention an epidemic in Hertfordshire characterised by an extraordinary diversity of symptoms in different patients. In some cases there were sneezing, coryza and the ordinary symptoms of a common cold. In other cases patients com- plained of aches and pains all over and stiff neck, and suffered subsequently from great debility ; such cases had all the appearance of influenza. In others, again, the illness closely resembled scarlet fever; it began with sore throat, rigors, vomiting, headache, fever and rapid pulse, and was ac- companied by a punctate rash at the end of the first 24 hours (followed later by desquamation), the “strawberry” tongue, circum-oral pallor, enlarged cervical glands which in some cases suppurated, and in some patients by complications such as nephritis, arthritis and otorrhoea. A fourth type resembled diphtheria and exhibited a suspicious membrane on the tonsil. A fifth type was notified in some cases as typhoid fever and was characterised by epistaxis, melaena, prostration and, in some cases, it is stated, a positive Widal reaction. Finally, a number of cases, particularly amongst children, resembled cerebrospinal fever and were so diagnosed ; these were characterised by profuse nasal discharge, pain in the back of the neck, headache, photophobia and irritability, dilatation of one or both pupils, persistent vomiting, drowsiness, head retraction, paralysis, coma, and sometimes convulsions and death. Sometimes these widely divergent types were exhibited by the different members of a single family or household struck down by the disease, either simultaneously or consecutively. After a thorough investigation, these observers were convinced that the outbreak of these various types of illness was due to the prevalence and spread of only one disease and not a cH. x1] THE ENZYME THEORY OF DISEASE 157 number of different diseases, and a bacteriological examination of a large number of cases by Gordon showed that the disease was due to infection by an organism closely resembling, if not identical with, M. catarrhalis. Such contrasting groups of symptoms inevitably suggest to our minds that something beside the mere presence of the organism is responsible for them. 4, In the fourth place, one can trace a remarkable resemblance between the conditions which influence the development and the loss of pathogenic power on the part of micro-organisms and the conditions which influence the development and the loss of their power to ferment carbohy- drates. (a) The addition, in small quantities, of an antiseptic— such as carbolic acid—to the culture medium deprives organ- isms growing in it of virulence (vide p. 75). The same agency will destroy the power of organisms to ferment carbohydrates (vide p. 55). (6) The influence of oxygen. Pasteur, 30 years ago, found that the virulence of the organism of chicken cholera was better maintained in the absence of oxygen. Anaerobic growth similarly increases the virulence of the choleraspirillum (Hueppe, quoted Adami, 1892). On the other hand, B. diph- theriae and other organisms become less toxic if deprived of oxygen. The same factor influences the activity of ferments. In some cases the absence of oxygen inhibits their functions, in other cases it appears to augment them. This is exemplified by the sugar splitting ferments associated with bacteria. For example, anaerobic growth may increase the power of the dysentery bacillus to ferment maltose (Torrey, 1905). Andrewes and Horder (1906) mention a strain of streptococcus which failed to ferment lactose under ordinary conditions but did so readily when deprived of oxygen. (c) Changes in temperature. It is characteristic of enzymes that each one has an optimum temperature at which its activities are most effective and also higher and lower limits of temperature beyond which its activities altogether cease. The digestive enzymes in man act most rapidly at the 158 THE ENZYME THEORY OF DISEASE [cu. x1 temperature of the human body—those of cold blooded animals at much lower temperatures. The diastatic ferment of germ barley is most effective at 60°C.—a temperature at which most enzymes are destroyed. The phosphorescence sometimes observed in sea-water is produced by the Micrococcus phos- phorescens through the agency of an enzyme the optimum temperature of which is that of the sea. : The enzymes which are associated with bacteria and bring about the fermentation of carbohydrates show a similar behaviour. We find that a strain of bacteria which will ferment a certain “sugar” at one temperature will not do so at another. For example, Wilson (1910) describes a strain of B. typhosus which at 22° C. would ferment lactose within two days but at 37°C. failed to do so in a month. Coplans (1909) observed certain strains of B. coli which exhibited the reverse phenomenon, fermenting dulcite more readily at 37°C. than at 20°C. The property of virulence in pathogenic bacteria is like- wise governed by temperature. Organisms which are virulent when growing at one temperature lose their virulence when grown at another. For example, B. diphtheriae, B. tetant, B. anthracis and many others (vide p. 74) lose their virulence at temperatures much above that of the body. The fact that no bacterial disease in cold blooded animals is communicable to man may possibly be explained on such grounds. Furthermore, just as the enzymes which ferment carbo- hydrates are destroyed at temperatures much above 60° C., so we find the property of virulence may be completely removed by subjecting an organism to high temperatures, even though the organism itself survives. The tetanus bacillus is deprived altogether of toxicity by growth at 65° C. for one hour (Muir and Ritchie). (d) Exposure to sunlight is another factor which influences both the fermenting power and the virulence of organisms. (e) Finally symbiosis is not without influence. Diseased conditions may result from a mixed infection which neither of the organisms concerned is capable of producing alone. It is said that a dog will not succumb to the infection of tetanus cn. x1] THE ENZYME THEORY OF DISEASE 159 unless it is infected simultaneously with pyogenic cocci. In the same way, processes of fermentation may be brought about by two different organisms growing together in a certain medium which neither can accomplish by itself. For example, neither B. colt nor B. dentrificans alone can reduce nitrates but if allowed to act upon sodium nitrate together they bring about the escape of free nitrogen. 5. There is, furthermore, a remarkable correspondence between the acquirement of virulence by “animal passage ” and the acquisition of fresh fermenting properties by prolonged growth in a medium containing a particular sugar : (a) The virulence acquired by “passage” through a certain animal applies to that particular species of animal; virulence towards another species may be increased at the same time but towards a third species it may actually be diminished. The fresh fermenting power resulting from prolonged growth in a sugar concerns that particular sugar; the capacity of the organism to ferment another sugar may be increased simul- taneously while in respect to a third sugar the fermenting power may be diminished. (b) The method of “passage” is more effective in con- ferring virulence if repeated inoculations are made through a series of animals at short intervals. The prolonged growth in a particular sugar is more successful in developing fermenting power if repeated subcultures are made at frequent intervals on to media containing the sugar. (c) If virulence is readily acquired on “passage” it is easily maintained and is found to persist for a long time on artificial media; on the other hand if it is very slowly developed by “passage” it is quickly lost outside the body. It is so, also, as regards fermenting power. In cases where the property is rapidly developed, by growth on a particular sugar, it is retained for long periods on ordinary media ; on the other hand, where it is very slowly acquired it is found that a return to ordinary media is soon followed by reversion in character. (d) Where virulence has been lost only for a short time by a strain of organisms it is quickly restored by “passage es 160 THE ENZYME THEORY OF DISEASE [cu. x1 the power to ferment a particular sugar, if it has only recently failed, is rapidly regained in the presence of that sugar. 6. Bacterial toxins, again, are considered to be of two kinds—extra-cellular toxins, secreted by the bacterial cell into the surrounding medium, and intra-cellular toxins elabor- ated within the body of the cell and liberated only when the cell itself is disintegrated. The same may be said of the enzymes which ferment carbohydrates. The ferment of yeast, “invertin,” which transforms cane sugar into dextrose and levulose, can be separated from the yeast cell. The breaking up of the dextrose into alcohol and other products is a property of the yeast cell itself and the ferment responsible for this second stage can only be extracted when the actual cell body is expressed (S. Martin, 1904). The ferments of the alimentary canal may be distinguished from each other in the same way. One stage in digestion is brought about in the lumen of the intestine by extra-cellular ferments present in the secretions. Another stage is effected within the actual cells of the intestinal wall by intra-cellular ferments acting upon the foodstuffs as they are absorbed. An emulsion of even a small portion of a glandular organ may possess far more power than its actual secretion, for the former contains the intra-cellular as well as the extra-cellular enzymes. An emulsion of pathogenic bacteria is likewise far more potent than a culture containing the same number of organisms. For example, the smallest fatal dose to a bovine animal of a culture of tubercle bacilli contains 20,000 million organisms. The smallest fatal dose of an emulsion of the bacilli contains only 5000 (Report of English Tuberculosis Commission). 7. In the seventh place, it may be observed that the property of virulence is in many instances associated with the power of producing fermentation. If we study two closely allied organisms, one of them virulent and the other non- virulent, the former will often be found to be the sugar fermenter while the latter has no action in this respect. For example, the Micrococcus catarrhalis is comparatively non-virulent and ferments no sugars; the gonococcus and cH. x1] THE ENZYME THEORY OF DISEASE 161 meningococcus are virulent and ferment sugars. Again, Hofmann’s bacillus is non-virulent and non-fermenting while the Klebs-Loeffler bacillus is virulent and ferments. B. coli communis is a sugar fermenter and readily acquires virulence. We may explain the association between the two properties on the ground that both are examples of adaptation and that an organism which possesses unusual power of adaptability in one particular direction may be expected to show a similar power of adaptability in another direction ; but the associa- tion between virulence and fermenting power lends some support to the supposition that the former may depend upon a process which we have every reason to believe is responsible for the latter, namely ferment action. 8. Bacterial invasion is met, on the part of the body, by measures calculated to destroy the organisms and to counter- act their toxins. These measures consist in the elaboration, by the fixed cells of the body as well as by the leucocytes, of various enzymes (Osler and McCrae). The class of weapon forged by the tissue cells for purposes of defence might, perhaps, be thought to give some indication as to the class of weapon it is designed to meet. 9. Many other functions of bacteria, besides the fermen- tation of carbohydrates, are attributed to ferment action ; for example, the formation of indol, the coagulation of milk (Savage, 1910), the liquefaction of gelatin, the production of pigment (Adami) and the development of agglutinins (Du- claux). Moreover these other functions of bacteria, like their power of fermenting carbohydrates, appear to be governed in many instances by the same conditions which we have already mentioned as influencing their virulence. Thus, the presence or absence of oxygen, high and low temperatures, exposure to and protection from sunlight, the presence of antiseptics, are all conditions which markedly affect the production of pigment by bacteria (Adami, 1892). 10. Many of these ferments are separable from the bac- teria with which they are associated. Twenty-five years ago it was proved (Bitter, 1887, quoted Wood) that the lique- faction of gelatin by bacteria was due to a ferment which D. 11 162 THE ENZYME THEORY OF DISEASE [cu. x1 was independent of the bacteria and survived when the latter were killed by subjection to a temperature of 60°C. Sortinin (cbid.) showed that a culture fluid after it had been passed through a Chamberland filter, which removed all the bacteria, still retained the power to liquefy gelatin. Brunton and McFadyean (1889) found that the gelatin lique- fying ferment could be isolated by suitable solvents, in the same way that the inverting ferment of yeast can be extracted with ether. ; 11. Instances may be cited of chemical processes, taking place in the body fluids, which are invariably associated with the presence of certain micro-organisms but which neverthe- less have been proved to be brought about by the activity not of the organisms in question but of ferments associated with these organisms and yet separable from them. Such an instance is to be found in the action of the micrococcus ureae. In the presence of this organism the urea of the urine is split up with the formation of ammonium carbonate. In the absence of this organism the process does not take place and if the process has begun the removal of the organism at once stopsit. An ethereal extract, however, of the micrococcus ureae has the power of accomplishing all that the presence of the organism itself can effect in this direction. In other words, the results brought about by its presence are due not to its own activities but to those of a ferment “urase ” which is in some way associated with it but which can be dissociated from it without any loss of function. 12, If it were possible to discover a parallel instance of dissociation, not between an organism and its chemical func- tions but between an organism and its pathological functions, the discovery would give great weight to the theory we are discussing. Now such a parallel can actually be traced in the action of the pneumococcus. Rosenow (1912-13) has recently shown that the artificial injection into the body of the toxins manufactured by the pneumococcus may bring about the death of an animal in one of two ways. It may produce an acute bronchial spasm which proves fatal in a few hours. If the dose of the toxin is small the bronchial spasm may not OH. x1] ‘THE ENZYME THEORY OF DISEASE 163 be sufficiently acute to cause death and other symptoms and lesions follow which result in the death of the animal in a few days. He discovered that the particular constituent of the toxin responsible for this bronchial spasm could be removed altogether from a suspension of the organism by the addition of blood charcoal, so that the subsequent injection of the filtered fluid failed to cause the bronchial spasm, although it still produced the other symptoms and lesions and led to a fatal termination in a few days. He, likewise, discovered that, the same constituent of the toxin could—like the sugar- splitting ferment of the yeast cell and the urea-splitting ferment of M. uwreae—be extracted with ether and, further, that if a normal saline solution, to which this ethereal extract had been added, were injected into an animal, the typical bronchial spasm was developed in the complete absence of the organism itself. The force of this analogy is somewhat weakened by the knowledge that this acute bronchial spasm is by no means pathognomic of the pneumococcus, many poisons producing the same result on injection into an animal. The analogy is, however, suggestive. 13. The dissociation brought about artificially in the laboratory by this investigator may be observed to take place naturally in response to certain kinds of environment. Thus, a strain of pathogenic bacteria may lose its power to produce a certain lesion or to cause a certain symptom in the body. Further, the conditions which appear to deprive it of such functions are comparable to those which, we have seen, influence ferment activity. A few examples will suffice. (a) The quality of &éght to which a culture of bacteria is exposed may modify their power to produce pigment. Ex- posure to the ultra-violet rays is found to alter profoundly the lesions and symptoms caused by B. anthracis (Henri, 1914). (b) The presence or absence of owygen influences pigment production and the fermentation of sugars by bacteria. Foa 41890) isolated strains of pneumococci from the lung and from the spinal fluid of a rabbit which had died after inoculation 11—2 164 THE ENZYME THEORY OF DISEASE . [cn. x1 with this organism. The strain from the lung possessed the property of causing, when inoculated into another rabbit, an inflammatory oedema of the skin; the strain from the spinal fluid failed to do so. The strain from the lung, however, when grown anaerobically was deprived of its power to cause this inflammatory oedema of the skin. (ec) Growth in a certain vehicle may alter the fermenting powers of one organism and the pathogenic powers of another. The fermentation properties of a strain of B. coli isolated from cowdung become altered after growth in milk. A milk-borne epidemic of scarlet fever is not infrequently characterised by the partial or complete absence of the usual rash. (d) An analogy may also be traced between the action of chemical substances added to culture media and the effects of drugs administered in disease. For example, the presence of sodium benzoate inhibits the power of B. coli to produce gas from dextrose—one of the most stable and fundamental differences separating B. coli from the typhoid-dysentery group—without in any way affecting its other fermenting reactions. The administration of sodium salicylate in rheu- matic fever eliminates the symptoms of. pain and fever— the two most characteristic symptoms of this disease—with- out apparently affecting any other of its symptoms and lesions in a great many cases. 14. If the foregoing considerations suggest that the symptoms of disease are due to zymotic action they likewise imply that each separate symptom is attributable to the activity of a distinct enzyme. Such a conclusion postulates the existence of innumerable pathogenic enzymes each one concerned in the causation of some particular symptom of disease, and requires us to conceive of different groups or combinations of enzymes associated with different pathogenic organisms and responsible in the case of each organism for the train of symptoms that follow its invasion of the living tissues. Analogy with the sugar-fermenting properties of bacteria. renders such a complex picture of the causation of disease less fanciful than, at first sight, it appears. As we have shown cH. xI] THE ENZYME THEORY OF DISEASE 165 (vide p. 60) it can be proved that not only is the fermentation of different carbohydrates effected by distinct and appropriate ferments but each of the several stages in the fermentation of a single carbohydrate—such as the formation of acids and the production from these acids of gas—is carried out by its distinct and appropriate ferment. Moreover different carbo- hydrates yield on fermentation different acids and each different acid requires to be acted on by a special ferment before it becomes split up into gaseous products. If such a comparatively simple result as the production of acid and gas in various carbohydrate media requires the co-operation of so many distinct ferments, the extremely complex and diverse results of the bacterial invasion of the body would appear to demand proportionately greater complexity and diversity in the zymotic agents causing them. We have seen that the enzymes concerned with the fer- mentation of particular carbohydrates are definitely associated with certain vegetable and bacterial cells but not with others. For example, many yeasts are able to invert sugar but only three yeasts are known which are able to ferment lactose. Proteolytic: ferments are, likewise, associated only with certain vegetable cells, such as the papain. Proteid-splitting and lactose-splitting ferments are associated with certain bacteria of the typhoid-coli group but not with others. It is conceivable that, in precisely the same way, the agencies responsible for certain definite symptoms in disease might be definitely associated with some bacterial cells but not with others. A further suggestion occurs to one at this point. If the enzyme responsible for one particular symptom of a disease can be dissociated from the specific organism of that disease, should we not expect to be able, by suitable methods, to dissociate from the organism not one only but all the enzymes causing the various symptoms of the disease in question ? 15. If such a complete dissociation were practicable it should be possible to accomplish two things; in the first place, to deprive an organism of its power to produce a single one of the symptoms of the disease associated with it and, in 166 THE ENZYME THEORY OF DISEASE [cu. x1 the second place, to reproduce faithfully the complete train of symptoms and lesions characteristic of a disease in the entire absence of the specific organism to which the disease is commonly attributed. Both these results have actually been observed. As regards the first, numerous examples have already been given of virulent organisms, normally capable of giving rise to a complex and characteristic train of symptoms and lesions in the living body (e.g. the Klebs-Loeffler bacillus, B. typhosus) being deprived of their. power to produce a single one of these symptoms or lesions although, in every other respect, retaining their character and properties un- changed (vide “ Virulence,” “ Pathogenesis ”’). It is well recognised, for instance, that infection with B. typhosus may occur without any of the clinical manifestations of typhoid fever. Dudgeon (1908) quotes three cases of patients whose stools contained enormous numbers of typhoid bacilli and whose blood agglutinated these organisms in dilutions of 1 in 200, who nevertheless failed to exhibit a single symptom of typhoid fever. With regard to the second, examples may be cited of diseases associated with the presence of certain bacteria but now generally recognised as being due to “filter passers.” Hog cholera, for instance, is a highly contagious disease associated with a certain bacillus, the “hog cholera bacillus.” A pig suffering from the disease can infect other healthy pigs ; the latter develop the same symptoms and are found to be invaded by the same organism and they are capable, in their turn, of infecting other healthy animals in precisely the same way. It has been shown, however, that a broth culture of the hog cholera bacillus, from an infected animal, after it has been passed through a Chamberland filter—a process which entirely removes any bacilli present—nevertheless retains its power to “infect” a healthy animal with hog cholera, the disease running the same course as usual and exhibiting precisely the same lesions and symptoms. Such a sequence affords a precise analogy to the ex- periment of Sortinin, a quarter of a century ago, which led to his discovery that after a culture of certain bacteria had been cH. x1] THE ENZYME THEORY OF DISEASE 167 passed through a Chamberland filter, a bacteria-free filtrate was obtained which nevertheless retained the power of the original culture to liquefy gelatin. 16. One objection may be urged at this point, namely, that it has not ‘hitherto been possible to separate from any pathogenic organism an enzyme capable of producing, outside the living body, the toxins characteristic of that organism. It is, however, equally impossible in many cases to isolate from bacteria agents which will bring about other of their functions which we recognise to depend on ferment action. Moreover, we have discussed under the head of virulence (vide p. 77) some of the qualities in which artificial media differ from the vital fluids of the body and such differences may well prove an insuperable obstacle to the performance by an enzyme of its usual functions. A pick- pocket may ply his “trade” vigorously in a busy crowded thoroughfare and yet a few hours later, in a workhouse ward, give no sign of his peculiar abilities. In the latter situation certain things are lacking—the incentive which normally stimulates him (that is to say, the “struggle for existence”), the materials he seeks to gain, the conditions essential to his work—and this fact may render difficult if not impossible any display of his customary activities. The enzyme theory of disease is not at the present stage of our knowledge capable of proof. The above considerations, however, lend some measure, if not of certainty at least of probability to the supposition that the organisms associated with certain diseases are not themselves the causal agents of those diseases but merely act as carriers of ultra-microscopic bodies, possibly parasitic in character, which have hitherto eluded detection but which are the real causal agents of the lesions and symptoms produced. 17. If such an hypothesis should ultimately prove to be correct, how would it affect our ideas as to the possibility of transmutation occurring amongst bacteria? Obviously, if it is possible for the enzyme or enzymes which produce a certain disease to become dissociated from the organism to which that disease is commonly attributed and to become attached to some 168 THE ENZYME THEORY OF DISEASE [c#. x1 other organism, the effect, though not the actual process, of transmutation would be brought about. A transference of this kind would present certain diffi- culties. The enzymes—if such be their true nature—of disease would appear to depend, to some extent, for their activity upon the structure and metabolism of the cell body to which they are attached and if they are to be transferred from one organism to another without loss of function the second host must possess those characters in the way of structure and metabolism which are vital to the activity of the enzymes. This implies certain, and possibly rigid limitations. The pro- blem can best be illustrated by analogy with more familiar things. We are able to distinguish at sea, a fleet of fishing smacks, a line of battleships, a couple of pleasure steamers, a solitary four masted barque in full sail. We distinguish these different types of vessels readily from one another by characters analo- gous to the “morphology” of bacteria, that is to say their size, shape, motility and grouping. We have, however, another way of distinguishing them, namely by observing the effects produced by their arrival at a port, analogous to the effects of bacterial “invasion.” The arrival of the fleet of fishing smacks is followed by a rush of people from their houses to the shore (comparable to the exudation of leucocytes), a silvery deposit on the quay-side as they empty their fish, re- placed in a few hours by a brownish membrane as the nets are spread out todry. The train of “symptoms” is invariable and becomes associated in our minds with the entry into port of this type of vesse]. So, too, with the others. The appearance of gunboats may be followed by the destruction of a town (comparable to necrosis). The arrival of the pleasure steamers may be greeted with a display of fireworks (comparable to pyrexia), that of the tall barque with its cargo of spirits may give rise to general intoxication (comparable to delirium). Such a sequence, however, is not invariable. For example, the fishing smacks might be employed in smuggling and land a cargo of spirits, giving rise to intoxication on shore. The gunboats might be employed by Royalty on a pleasure cruise cH. xI] THE ENZYME THEORY OF DISEASE 169 and their arrival be greeted with fireworks. A couple of in- nocent looking steamers might be engaged in piracy and open a destructive fire from their guns. The tall barque might con- ceivably land a cargo of fish. In other words each type of vessel might give rise to a train of events rightly regarded as characteristic of an altogether different type, for the effects they produce depend not on the activities of the ships them- selves, which are merely carriers, but on those of their occu- pants. _ At the same time the function of each different type of vessel, though dependent upon its occupants, ts also to some extent governed by its structure and the equipment it carries (comparable to the structure and metabolism of a micro- organism). A mere exchange of crews would not necessarily effect an exchange of function. For example, a party of fishermen sent to sea in an ironclad would be as unlikely to land a catch of fish as a force of naval officers and seamen embarked in fishing smacks would be to bombard a town. In one respect our analogy fails. Hitherto we have spoken of the enzyme as something grafted on to the micro-organism, in the nature of a parasite, but there is much to suggest in the evidence we have quoted that it is, in reality, a body ela- borated by the organism itself, comparable to one of Ehrlich’s “side-chains.” Such a conception of its nature would go far towards explaining the apparent dependence of the “enzymes” of a particular disease upon a particular organism. But every argument in favour of such a supposition in the case of the enzymes which cause disease applies equally to our conception of the nature of those which ferment carbohydrates. The pur- pose of the arguments here presented has not been to explain the precise nature of these ultra-microscopic bodies but merely to show that the lesions and symptoms of disease may with some confidence be attributed to the action of the same class of body as that to which we unhesitatingly attribute the fer- mentation of sugars. 11—5 CHAPTER XII CONCLUSIONS 1. Variation occurs in every character of bacteria. 2. These variations may be either “spontaneous ” or “im- pressed ” by conditions of environment. —- 3. The recognition of “species” amongst bacteria must, therefore, depend upon a consideration of their biological characters as a whole and upon the stability these characters display. 4. Transmutation differs from variation in degree alone ; it is a question of the extent of the modification and the de- gree of permanence it exhibits. 5. Transmutation differs from evolution in degree alone ; it is a question of the rapidity of the change. 6. The occurrence of transmutation between closely allied organisms in the human body is not capable of proof but is suggested by circumstantial evidence. 7. Supposed instances of transmutation, brought about by experimental inoculation of animals, are shown to rest on in- conclusive evidence. 8. 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