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 Reports of the Committee upon anaerobic 
 
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 Reports of the Committee upon Anaerobic 
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 Report on the Anaerobic Infections ofWounds and 
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 3944 
 
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NATIONAL HEALTH INSURANCE 
 
 MEDICAL RESEARCH COMMITTEE 
 
 REPORTS 
 
 OF THE 
 
 COMMITTEE UPON ANAEKOBIO BACTEEIA 
 AND INFECTIONS 
 
 REPORT ON THE ANAEROBIC INFECTIONS 
 OF WOUNDS AND THE BACTERIOLOGICAL 
 AND SEROLOGICAL PROBLEMS ARISING 
 THEREFROM 
 
'Nb, l&IX^ 
 Medical Research Committee 
 
 {National Health Insurance.) 
 
 The Hon. Waldorf Astob, M.P, {Chairman). 
 
 The Eight Hon. Christopher Addison, M.D., M.P. 
 
 The Viscount Gosohen, C.B.E. {Treasurer). 
 
 0. J. Bond, O.M.G., P.R.C.S. (Hon. Colonel). 
 
 Professor William Bulloch, M.D., P.E.S. 
 
 Henry Head, M.D., P.E.C.P., F.E.S. 
 
 Professor P. G. Hopkins, D.Sc, P.E.O.P., F.E.S. 
 
 Major-General Sir William Leishman, K.C.M.G., C.B., F.E.S. 
 
 Professor Noel Paton, M.D., F.B.S. 
 
 Sir Walter M. Fletcher, K.B.E., M.D., P.E.S. {Secretary). 
 
 15, Buckingham Street, 
 
 Strand, W.C. 2. 
 
 The Conim.ittee upon Anaerobic Bacteria and 
 Infections. 
 
 With a view to the better co-ordination of inquiries into the 
 characters of anaerobic organisms, with special reference to the 
 bacteriology of anaerobic wound infections, the Medical Eesearch 
 Committee invited the following to serve as a special investigation 
 Committee for this purpose : 
 
 Professor William Bulloch, M.D., P.E.S. {Chairman). 
 
 W. B. Bullock, M.D. 
 
 S. E. Douglas, M.E.C.S., L.E.C.P. (Captain I.M.S. retd.). 
 
 Herbert Henry, M.D. 
 
 James McIntosh, M.D. 
 
 E. A. O'Brien, M.D. 
 
 Miss Muriel Eobertson, M.A. {Secretary). 
 
 C. a L. Wolf, M.D. 
 
 {with corresponding members). 
 
.INTRODUCTION 
 
 Among the wound complications which confronted all the belli- 
 gerents in 1914, none was more serious than ' gas gangrene ', whether 
 considered from the point of view of incidence or of mortality. 
 Although this remarkable condition was known and described in 
 pre-antiseptic days, it had not been a disease of any consequence 
 in any of the wars of the twentieth century. The outbreak of the 
 Great War, however, brought it early into prominence, and it was 
 at once recognized as a toxic-infective condition. The unusually 
 high incidence on the Western Front, where fighting took place upon 
 a long cultivated soil contaminated with human and animal excre- 
 ment, led to the belief that the infective agents were soil bacteria, 
 and it was early estabhshed in all the countries interested that these 
 rnicro-organisms were of the anaerobic type. In spite of the accumula- 
 tion of much knowledge on the subject of anaerobes, a study of 
 bacteriological literature before the war shows clearly that' the 
 descriptions published by different writers were for the most part 
 widely divergent, and agreed in only a few instances. Prom the 
 frequency with which Bacillus welchii [B. perfringens) was found, 
 apparently in pure culture, it was considered that this was the chief 
 aetiological agent, and it was actually named ' the bacillus of gas 
 gangrene '. 
 
 A careful scrutiny of the discharges from wounds by more refined 
 
 methods during 1916 and 1917 revealed the fact, however, that gas 
 
 gangrene could not be regarded as an aetiological entity, but could 
 
 be caused by a series of pathogenic anaerobes acting singly or in 
 
 combination with each other or in association with certain well 
 
 defined non-pathogenic anaerobes. By degrees Pasteur's Vibrion 
 
 sepUqv£ was disentangled from the mass of anaerobes, and a number 
 
 of other anaerobes, new to science, were discovered, among which 
 
 may be specially mentioned the highly toxic B. oedematiens. The 
 
 isolation of these bacteria was chiefly the work of MM. Weinberg 
 
 and Seguin of the Pasteuf Institute in Paris, whose conclusions were 
 
 confirmed and extended by English bacteriologists, notably Majo^ 
 
 McNee, Captain Adrian Stokes, Captain Herbert Henry, Captain 
 
 W. E. Bullock (now Gye), with the Forces Overseas, Dr. James 
 
 Mcintosh, working at the London Hospital in the service of the 
 
 Medical Eesearch Committee, and Miss Muriel Eobertson of the 
 
 Lister Institute of Preventive Medicine. Major W. J. TuUoch in 
 
 the laboratories of the Eoyal Army Medical College and the Lister 
 
 Institute made at the same time unusually successful researches into 
 
 the bacteriology of tetanus. Captain C. G. L. Wolf, working for the 
 
 Medical Eesearch Committee, by the application of exact methods 
 
 added important contributions to the biochemistry of anaerobes, first 
 
 at Boulogne and later at Cambridge. In America, Major C. G. Bull 
 
 and Dr. J. L. Stoddard added considerably to our knowledge of the 
 
 bacteriology of anaerobes. 
 
 (5012.) Wt, 47167. 226. 1250. 10/19. O.U.P. 
 
 (3944.) Ps. 2208. Wt. 21317. 309. 1000. 11/20. O.U.P. 
 
 A2 
 
IV 
 
 The advances which were made were the result of applying the 
 most refined and most difficult forms of bacteriological technique, 
 and they depended also upon the early recognition of the fact that 
 many, if not most, anaerobes have an inveterate tendency to live 
 in close association with other anaerobes. This is an association 
 which can only be recognized by the most careful and prolonged 
 scrutiny of what appear to be pure cultures, under diversified and 
 often complicated conditions. To the failure to reaUze this must be 
 ascribed the fact that in the bacteriological literature of the Central 
 Powers the knowledge of gas gangrene was and still remains (1919) 
 in a state of hopeless confusion, and it cannot be doubted that 
 it has been the researches of French and EngUsli bacteriologists 
 that have cleared away the mystery of the aetiological agents pro- 
 ducing gas gangrene. The outcome of this advance in knowledge 
 led the French to test at an early date the prophylactic and curative 
 effects of the serum of animals immunized with pure cultures of 
 specific pathogenic anaerobes. Among the most successful workers 
 in this field must again be mentioned Weinberg and Seguin, and 
 Major C. G. Bull. They were the first to apply the serum treatment 
 in man. In England the earliest attempts to produce sera were 
 those of Mcintosh and O'Brien, the latter of whom, in association 
 with Captain H. Henry and Captain W. E. Bullock, ultimately 
 undertook, with the assistance of the Committee, the preparation of 
 sera in the Wellcome Eesearch Laboratories on a large scale for the 
 British Army. Throughout the course of these inquiries the British 
 workers were greatly aided by French bacteriologists and surgeons, 
 among whom may be specially mentioned Weinberg, Vaucher, 
 Duval, and Chutro. It is a pleasant duty for the Medical Eesearch 
 Committee to record their thanks for the help received from those 
 French workers who with unusual generosity placed their unpub- 
 hshed records in the hands of the British, and kept us in close touch 
 with all the developments in their experience of gas gangrene. 
 
 This wide co-operative effort towards gaining new knowledge and 
 applying it to the production of curative sera was chiefly focussed 
 m this country m the work of the Committee upon Anaerobes which 
 the Medical Eesearch Committee had appointed in March 1917 in 
 the hopes of bringing more closely together the various workers 
 engaged The constitution of this special committee is given at 
 the head of this Eeport. In the autumn of 1918 it seemed certain 
 ti.at if the war continued there would be available large supplies of 
 
 ^w^l P^*f* ''''''^' ^°*^ ^^'^ PJ^eventive and curative use, in 
 which the defence agamst the three chief organisms already men- 
 tioned was to be combmed. Happily the armistice intervened to 
 remove the urgent need for this, and the practical demonstration 
 of the fruits of these long mvestigations must be expected now only 
 in the infinitely rarer occasions of peace. ' 
 
 It appeared to the Medical Eesearch Committee that every effort 
 
 £7ii f T^' ^l^^^^f "^ ""^ ^l^"fy *^« ««ie^tific results^ whS 
 had so far been obtamed The prolonged study of gas gangrene in 
 all the chief countries had led, moreover, to the production of many 
 writings both good and bad, and this gave further reason fo?S 
 preparation of an authoritative and criti^l report uponThe wious 
 
parts of the subject by workers with first-hand knowledge. The 
 special Committee upon Anaerobes were invited accordingly to 
 complete their labours by preparing the Eeport which is now pre- 
 sented. 
 
 At first it was intended to deal completely with the aetiological, 
 experimental, and sero-therapeutic aspects of the gas gangrene 
 problem, and work on these lines had proceeded to a considerable 
 extent, when in 1918 appeared the admirable, exhaustive monograph, 
 entitled La Gangrene Gazeuse, by MM. Weinberg and Seguin. This 
 necessitated a revision of the plans of the Committee, who deemed 
 it inadvisable merely to traverse the same ground as the French 
 writers. It was considered best to concentrate especially on a critical 
 analysis of the experiences of the individual members of the Com- 
 mittee, and to undertake, where necessary, fresh investigations to 
 clear up the doubtful .points and to utilize the practical results 
 gained by the use of the British-made sera in the treatment of gas 
 gangrene. 
 
 The Eeport as it stands is the conjoint work of the members of the 
 Committee. For the sake of completeness, however, several sections 
 were written by others who had special knowledge of certain aspects 
 of the anaerobic and even aerobic microbic complications of wounds. 
 Thus the Committee received a report on ' the chnical features and 
 treatment of anaerobic infection of wounds and gas gangrene ' by 
 Dr. John Fraser, of Edinburgh, who had an almost unique experience 
 of the disease while serving in France. Dr. Alexander Fleming 
 prepared a summary of his extensive researches on the ' aerobic 
 infections of wounds ', while Major W. J. Tulloch gave the benefit 
 of his wide experience on the bacteriology of tetanus. Dr. E. H. 
 Kettle, of St. Mary's Hospital, undertook for the first time an exact 
 inquiry into the finer pathological anatomical changes in human 
 and experimental gas gangrene, which is published in the Appendix. 
 Professor W. Bulloch compiled an extensive bibliography of the 
 literature on anaerobic infections, and personally examined almost 
 all the papers for the purposes of this report, as a help to future 
 investigators who may not have been in touch with much of the 
 medical war literature. 
 
 The Medical Eesearch Committee believe that they are speaking 
 for a wide circle of workers when they express their own cordial 
 thanks to the members of the Anaerobe Committee, not only for 
 their arduous individual work in the laboratory, but even more for 
 the time and effort they have given so ungrudgingly to share their 
 knowledge with others and to contribute in discussion to the common 
 object in view. Thanks are due especially to Professor WilUam 
 Bulloch, who from the beginning has acted as chairman of the Com- 
 mittee, and to Miss Muriel Eobartson, who by kind permission of 
 the governing body of the Lister Institute was able to undertake 
 the difficult duties of secretary, which brought a heavy burden of 
 correspondence with many workers at home and abroad. 
 
 The work of the special Committee was necessarily done in close 
 touch with the Army Medical Service, and here the Committee 
 ■would offer their grateful acknowledgements to the Director-General, 
 A M S , and to many other officers, for the encouragement and direct 
 
 a3 
 
VI 
 
 assistance which made it possible to link effectively the work done in 
 civiHan laboratories at home with that done in the armies and with 
 the practical problems offered by Service needs and conditions. To 
 the Army Medical Department the Committee are indebted for the 
 supply of many data from Medical Case Sheets, which have been 
 important for the study of the sero-therapy of gas gangrene ia the 
 field. 
 
 To the editors and pubhshers of the Journal of Pathology and 
 Bacteriology the Committee are indebted for permission to reproduce 
 a large number of the illustrations given in the plates. Other 
 illustrations are taken from the report by Dr. James Mcintosh, 
 previously published by the Committee (Special Report Series, 
 No. 12). Figures 76, 77 and 78 are reproduced from the beautiful 
 drawings made by Mr. Thornton Shiels from pieparationp by 
 Dr. Mcintosh, and the Committee beheve that these are perhaps 
 the most exact representations yet pubhshed of the infective con- 
 ditions displayed. 
 
 Medical Eesearch Committee 
 15 Buckingham Street, 
 Strand, W.C.2. 
 
 September 1919. 
 
REPORT ON THE ANAEROBIC INFECTIONS OF 
 WOUNDS AND THE BACTERIOLOGICAL AND 
 SEROLOGICAL PROBLEMS ARISING THERE- 
 FROM 
 
 CONTENTS 
 
 FAOE 
 
 iNTEODtrCTIOIf ........... 6 
 
 I. Thb Clinical Ebatubbs and Treatment of Anaerobic Infection oe 
 
 Wounds and Gas Gangbenb . - . . . . 6 
 
 1. General Clinical Considerations ....... 6 
 
 (i) The Type of Wound . 6 
 
 (ii) The General Condition of the Wounded Man .... 7 
 
 (iii) ^he Nature of the Infecting Agent ..... 7 
 
 (iv) The Vascular Supply ....... 7 
 
 (v) The Regions and Tissues Affected ..... 7 
 
 {vi) The Question of Interrupted Drainage fro-in the Wound . . 8 
 
 (vii) The Clinical History of the Wound ..... 8 
 
 2. Varieties of the Bisease ....... 9 
 
 3. Symptoms and Signs of Gas Gangrene ...... 9 
 
 (i) Symptoms ......••■ 9 
 
 (ii) Physical Signs . . . . . ■ ■ . . 10 
 
 (iii) The Group Type ........ 10 
 
 (iv) The Massive Type . ■ . ■ ■ ■ ■ .10 
 
 (v) The Fulminating Type . . ■ . ■ ■ . U 
 
 4. Complications of Gas Gangrene . . . . . . .11 
 
 5. The Diagnosis of Gas Gangrene . . . . . . .11 
 
 6. The Treatment of Gas Gangrene 12 
 
 (i) Preventive Treatment . , . . ■ • .12 
 
 (ii) Treatment hy the Administration of Alkalies ... 12 
 
 (iii) Surgical Treatment ........ 12 
 
 (iv) Methods of Amputation in Oas Gangrene .... 14 
 
 (v) General and Post-operative Treatment 15 
 
 II. Incidence oe Gas Gangbenb 15 
 
 III. BACTBEIOLOffY 1^ 
 
 Introduction ......•■■• 1° 
 
 1. Pathogenic Anaerobes liable to set up Conditions of Gas Gangrene 
 
 with Symptoms of General Intoxication . . . .17 
 
 (i) B. welohii . ^7 
 
 (ii) Vibrion septique : with note on B. chauvoei ... 20 
 
 (iii) B. oedematiens 24 
 
 (iv) B. histolyticus 25 
 
 (v) B. botulinus 26 
 
 (vi) B. fallax 27 
 
 2. B.tetani '. ^* 
 
 (i) Bacteriological account ... ... 28 
 
 (ii) Recent Experimental work on B. tetani . . . .29 
 
 3. AnaerobestheActionofwhiohmaybeAnoillarytotheCondition 
 
 of Gas Gangrene .....••• ^5 
 
 (i) B. sporogenes ^" 
 
 (ii) B. parasporogenes ^^ 
 
 (iii) B. tertius *° 
 
(iv) B. ooohlearius 
 
 (v) B. tetanomorphns 
 
 (vi) B. aero-fetidua 
 
 (vii) B bifermentans 
 
 (viii) B. putrificus . 
 
 (ix) B. sphenoides . 
 
 (x) B. butyricuB . 
 (xi) B. multifermentans tenalbus 
 
 4. Classification of the Anaerobic Bacilli found in Wounds 
 
 (i) Morphology 
 (ii) Gvltural Characteristics 
 (iii) Agglutination Beactions 
 (iv) Toxin-antitoxin Beactions . 
 
 5. Biochemistry .... 
 
 (i) The Mechanism of Anaerobiosis 
 ' (ii) The Biochemistry of certain micro-organisms found in Wounds 
 
 6. Experimental Gas Gangrene .... 
 
 (i) Infection ...... 
 
 (ii) The established disease .... 
 
 7. The Aerobic Infections of War Wounds 
 
 (i) B. mesenterious, B. subtilis, B. myooides groups 
 
 (ii) B. aero-tetanoides 
 
 (iii) B. aero-tertius 
 
 (iv) Staphylococci . 
 
 (v) Streptococci 
 
 (vi) Diphtheroid Bacilli 
 (vii) Goliform Bacilli 
 (viii) B. pyocyaneuB 
 
 (ix) B. proteus 
 
 (x) Gram-negative cocci 
 
 (xi) Miorococous tetragenus 
 
 8. Influence of the Aerobic on the Anaerobic Infection of Wounds 
 
 (i) Influence of Staphylococci and Streptococci on the growth oj 
 
 B. welchii in Milk 
 (ii) Influence of Aerobic Organisms on the growth of B. welchii in 
 
 Serum neutralized vnth Acid, in Serum the antitryplic pouier 
 
 of which has been neutralized with Trypsin . 
 (iii) Influence of Aerobes on the growth of Anaerobes other than 
 
 B. welchii 
 (iv) Influence of B. welchii on the growth of Streptococcus and 
 
 Staphylocooons 
 (v) Effect of a Diphtheroid Bacillus on the growth of Streptococcus 
 
 pyogenes 
 
 9. B6sum6 of the Literature on the Bacteriology of Gas Gangrene, with 
 
 an Account of the Incidence of the various Types of Patho 
 genie Anaerobes ....... 
 
 10. The Isolation of Anaerobic Organisms of Wounds in Pure Culture 
 (i) Material suitable for examination .... 
 
 (ii) MethodiS of Isolation ....... 
 
 A. The separation by mechanical methods of individua 
 
 organisms in a mixture ..... 
 
 B. The selection by appropriate heating of spores coniained 
 
 in a mixture . 
 0. The use of selective media 
 
 D. The separation of a Pathogenic Anaerobe by Animtd 
 
 Experiment 
 
 E. Summary 
 ^iii) The Preservation of Cultures 
 
 PAGE 
 
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 41 
 42 
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 96 
 
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 101 
 103 
 
:5 
 
 B. 
 
 by B. 
 
 welchii. 
 
 vibrion 
 
 Toxins 
 
 IV. Serology . . ... 
 
 1. Toxins and Antitoxins 
 
 (i) Historical Intrcduction 
 
 A. B. welchii 
 
 B. Vibrion septique 
 
 C. B. oedematiens 
 (ii) The Preparation of Toxin . 
 
 A. Toxin of B. welchii . 
 
 ! Vibrion septique . 
 B. oedematiens 
 
 2. The Properties of the Toxins elaborated 
 septique, and B. oedematiens . 
 
 (i) Physical features 
 (ii) Biological characteristics . 
 
 A. The toxin of B. welchii 
 
 B. The toxin of Vibrion septique 
 
 C. The toxin of B. oedematiens 
 
 3. The Symptoms and Pathological Changes produced by these 
 and the Estimation of their Minimum Lethal Dose in Animals 
 
 (i) The Toxin of B. welchii 
 ' A. Mice 
 
 B. Rabbits . 
 
 C. Guinea Pigs 
 
 D. Pigeons . 
 (ii) The Toxin of Vibrion septique 
 (iii) The Toxin of B. oedematiens 
 
 4. The standardization of Antisera . 
 (i) B. welchii antitoxic sera 
 
 (ii) Vibrion septique antitoxic sera 
 (iii) B. oedematiens o»<jif(ia;i6sero 
 
 5. Preparation and Standardization of Anti-gas 
 (i) Introduction .... 
 
 (ii) Preparation of Antitoxins 
 
 A. B. welchii 
 
 B. Vibrion septique 
 
 C. B. oedematiens 
 Methods of estimating th^ value of Ai 
 
 A. Antitoxin of B. welchii 
 
 B. Antitoxin of Vibrion septique 
 
 C. Antitoxin of B. oedematiens 
 The Serum Therapy of Gas Gangrene . 
 
 (i) The Collection of Data 
 (ii) The Serum employed 
 (iii) Analysis of Records . 
 
 A. Deaths 
 
 B. Recoveries 
 (iv) Discussion 
 
 (v) Summary 
 (vi) TMe of Cases . 
 
 (iii) 
 
 gas-gangrene Sera 
 
 6. 
 
 BiBLlOGBAPHY 
 
 Addendum 
 
 Appendix ..... 
 
 The Histopathology of Ga-, Gangrene 
 
 Dbscbiption of Pla.i'ES .... 
 
 gangrene Sera 
 
 PAGE 
 
 104 
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 109 
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 lU 
 
 III 
 
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 146 
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 180 
 
INTEODUCTION 
 
 The serious consequences which may result from the multiplication 
 of anaerobic bacteria in wounds have furnished a medical problem 
 in the present war which, both because of its gravity and of its 
 novelty, has been studied by cHnicians and bacteriologists of most 
 of the belligerent nations. 
 
 The following pages summarize the present state of knowledge of 
 wound anaerobes, of the clinical conditions which they may cause, 
 and of experience in the preparation and the use of antisera. 
 
 The advances chronicled are due to many investigators, who, by 
 means of improvements in technique which have made it easier to 
 obtain pure cultures, have studied the large amount of material 
 which the war has provided. 
 
 I. THE CLINICAL PBATUEBS AND TEEATMBNT OF 
 ANAEKOBIC INFECTION OP WOUNDS AND GAS 
 GANGEENE 
 
 By John Peasbb, M.C, M.D., P.E.C.S. Ed. 
 
 Anabeobio infection with its sequel of gas gangrene has been 
 a common and an extremely serious complication of infected war 
 wounds. 
 
 When the present war began our knowledge of the subject was 
 limited. The condition had been noted as occurring in previous 
 campaigns, and on rare pccasions it had been met with in civil 
 practice, but we possessed httle trustworthy information regarding 
 the pathology and the treatment of the condition. It has been 
 through the medium of expensive and painful experience that the 
 knowledge which we now possess has been gained. 
 
 In the subsequent remarks the anaerobic infection of wounds is 
 dealt with from the point of view of clinical features and treatment. 
 
 1. General Clinical Considerations. 
 (i) The Type of Wound. 
 
 There are certain types of wound which are especially Uable to 
 anaerobic infection and these are wounds which illustrate one or 
 both of two conditions, imperfect drainage and extensive devitaliza- 
 tion and death of tissue with extravasation of blood. 
 
 The first mentioned condition is typically illustrated in the deeply 
 penetrating wounds with a small point of entrance, widespread 
 destruction of the more friable muscles and possibly retention of the 
 infected projectile at the base of the wound. Such wounds are 
 usually produced by fragments of bombs and grenades, by shrapnel 
 bullets, and by small fragments of H.E. shell. The projectile is 
 generally traveUing at a comparatively low velocity and hence the 
 tendency for the missile to be retained. 
 
The second type of wound associated with tissue death in the 
 part affected is usually caused by irregular fragments of H.E.. shell. 
 These fragments are generally travelling at such high velocity that 
 there is widespread tissue destruction not only in the course of the 
 missile but also in a considerable zone of tissue outside the 
 actual track. 
 
 (ii) The General Condition of the Wounded Man. 
 
 It is certain that the general condition of the wounded man has 
 an important bearing on the development of the infection. It has 
 been observed that gas gangrene is more prevalent among fatigued 
 troops, who have undergone long spells in the trenches, than it is 
 among fresh soldiers who have gone direct into action. In a similar 
 way, men who have suffered from severe haemorrhage and shock 
 subsequent to the wound are specially liable to the development of 
 this compUcation. 
 
 (iii) The Nature of the Infecting Agent. 
 
 It is essentially true that the more irregular the projectile fragment, 
 ■the more likely is anaerobic infection to follow. There are certain 
 definite reasons for this statement. (1) An irregular fragment of 
 shell produces widespread devitahzation and tissue destruction. 
 This is well seen when such a wound is studied in comparison with 
 that produced by a rifle bullet. A short range rifle bullet on the 
 other hand may produce as much destruction as a large irregular 
 shell fragment. (2) The more irregular the projectile fragment 
 the more likely is it to carry into the wound pieces of infected 
 clothing. (3) As the majority of H.E. shells burst on percussion 
 with the ground, irregular fragments carry with them into the 
 body of the wounded man portions of soil which in highly fertilized 
 districts may be heavily charged with anaerobes. 
 
 (iv) The Vascular Supply. 
 
 A factor of extreme importance is the question of the blood 
 supply to the part. A dirninished supply of blood to the wounded 
 area predisposes the part to the development of an anaerobic infec- 
 tion. A variety of causes may lead to the interference with the 
 vascularity : the severe haemorrhage which is so often the con- 
 comitant of war wounds results in a general anaemia : the nature 
 of the wound is often such that a tourniquet has been applied and 
 the distal blood supply has thus been arrested : in a more local 
 sense the contusion and tissue destruction which has resulted from 
 the wound leads to a thrombosis in the blood vessels for some 
 extent around the wound. At any rate, in whatever way it may 
 l^ave been caused, an impoverished blood supply to the part is 
 a potent factor in favouring the development of an anaerobic 
 infection. 
 
 (v) The Regions and Tissues Affected. 
 
 Anaerobic infection spreads with great rapidity in muscle and 
 more slowly in areolar tissues. It is interesting to observe that all 
 
muscles are not equally liable to the infection ; for example, it is 
 comparatively rarely that one finds any widespread infection of the 
 muscles of the back. The larger and coarser-fibred muscles, e.g. 
 the glutaei appear to be the most liable to the infection. It is 
 possible that this distinction really depends upon the blood supply. 
 In scalp wounds uncomplicated by compound fracture^ of the skull 
 and in wounds of the face, gas gangrene is a rare complication. 
 
 The serous cavities of the body would appear to offer considerable 
 resistance to anaerobic infection. The disease in these situations 
 progresses as a rule more slowly and is naturally hmited in extent 
 to the region of the infected membrane. Although the infection 
 of a serous membrane is thus for anatomical reasons a localized 
 infection, yet the absorption of toxine from such a focus may yield 
 a progressive intoxication which either by itself or in conjunction 
 with an ensuing anaerobic septicaemia is fatal to the individual. 
 This is particularly true of infection of the pleura. 
 
 (vi) The Question of Interrupted Drainage from the Wound. 
 
 Any factor which interferes with efficient drainage from the 
 wound predisposes to the anaerobic infection. This fact may be 
 illustrated by the type of wound. The deep narrow punctured 
 wound is obviously liable to the infection. A slit-like skin wound 
 may contract, effectively blocking the escape of discharge and at 
 a deeper level there may be considerable retention of infected 
 material. In the same way a foreign body may prevent efficient 
 drainage, and in the early days of the war there were numerous 
 instances in which packing of the wound or insertion of antiseptic 
 pastes acted in a similar way with disastrous results. 
 
 (vii) The Clinical History of the Wound. 
 
 The period of time which elapses before the wound shows signs 
 of infection varies over a wide range. Changes which indicate an 
 anaerobic infection may be apparent within an hour of the injury 
 being sustained ; on the other hand, several days may elapse before 
 distinctive features appear. The changes in the wound which are 
 associated with the infection may be summarized as follows. 
 
 There is a scanty f oul-smeUing discharge composed of broken-down 
 blood clot ; it is of a brownish colour, and mixed with it there are 
 hubbies of gas. The odour of the discharge appears to depend on 
 the type of infecting organism, and on the state of the infection : 
 in the early stages a characteristic smell is absent, later it becomes 
 foul and acrid. The skin around the wound assumes a faint 
 purphsh colour, and there is a local swelling due to the accumulation 
 of brown serous fluid in the subcutaneous tissues. There is a similar 
 infiltration throughout the intermuscular septa and connective tissue 
 planes. Throughout the precincts of the wound there is an infiltra- 
 tion of gas and it extends to further hmits along certain lines— 
 the subcutaneous tissues, the perivascular tissues, and the planes 
 of intermuscular connective tissue. 
 
 Up to this point in the clinical history of the wound, the muscle 
 IS not mfected. When it does become infected, the nomenclature 
 
9 
 
 must be alteted ; it is no longer an anaerobic infection of the wound 
 but the condition of gas gangrene. The muscle is infected in one 
 of two possible ways : 
 
 1. The muscle (usually an injured one) is gradually invaded by 
 the organisms, which starting from the wound make their way 
 towards the extremities of -the muscle until it is totally destroyed. 
 The blood supply of the muscle is intact. In such cases, the line 
 of invasion may be seen and distinct zones of infection recognized. 
 
 . At the actual wound, the muscle is black, friable, and diffluent. 
 Next comes a zone in which the predominant colour is red. Separ- 
 ating the red zone from the healthy muscle is a yellow band, irregular 
 in outline, somewhat raised above the surface and hard to the 
 palpating finger. In the majority of cases, however, no such 
 differentiation into zones is apparent. In these a loss of contractihty 
 is the first thing to be observed, and when this has been established 
 the muscle rapidly changes its colour through varying stages of 
 dirty red and greenish yellow to a dark diffluent mass. 
 
 2. The muscle may undergo what is really a post-mortem infection, 
 for it has already died in consequence of the blood supply having 
 been cut off. In such a case the appearance of the muscle varies 
 from a purplish red to a greenish black diffluent mass. 
 
 Soon after the muscle has been infected, gas can be demonstrated 
 in it, at first as bubbles between the muscle fibres, afterwards in the 
 surrounding areolar tissues. The gas has a deleterious effect in so 
 far as it exerts pressure within the fascial sheath of the limb, con- 
 stricting the blood-vessels and producing that amount of interference 
 with the blood supply which is so favourable to an extension of the 
 infection. 
 
 2. Varieties of the Disease. 
 
 Clinically, several types of the disease may.be recognized. 
 
 1. Localized anaerobic infection in the wound. 
 
 2. Slowly spreading anaerobic infection in the wound. 
 
 3. Gas gangrene of the ' group ' type when a single muscle or 
 a group of muscles is attacked. 
 
 4. Gas gangrene of the ' massive ' type where a whole segment 
 of a limb is involved. 
 
 5. The fulminating type. 
 
 3. Symptoms and Signs of Gas Gangrene. 
 (i) Symptoms. 
 
 The features depend on whether it is early or late. In the early 
 stages, distinctive symptoms are absent but pain is generally marked. 
 The unusual amount of pain probably depends upon increasing 
 pressure within the wound. 
 
 When the infection becomes estabhshed and begins to spread, 
 distinctive signs arise. The pain increases and there is a feehng 
 of numbness in the superficial parts of the limb. Constitutional 
 symptoms begin to make their appearance ; the patient looks 
 distressed and ill ; the lips acquire a cyanotic colour ; the pulse 
 rate is rapid and the temperature rises by three or four degrees. 
 
10 
 
 Vomiting is frequently present. If tKe disease continues to extend, 
 the constitutional symptoms become more marked. The pulse 
 becomes more rapid and is finally running and uncountable ; vomiting 
 becomes more frequent, the extremities are cold and blue and the 
 temperature falls. It is a remarkable. fact that the mind generally 
 remains acute even to the end. In the terminal stages some degree 
 of general icterus may be present. 
 
 Death frequently occurs with such dramatic suddenness as to 
 suggest the occurrence of pulmonary embohsm, although it is to be 
 noted that this has never been found at autopsy. 
 
 (ii) Physical Signs. 
 
 The physical signs differ according to the type of disease which is 
 present. If there is a localized anaerobic infection of the wound, 
 the only distinctive sign may be a foul-smelUng discharge mixed 
 with bubbles of gas. As the disease extends, swelhng of the part is 
 noted and percussion of the swollen area yields a tympanitic note. 
 
 (iii) The Group Type. 
 
 In the ' Group ' type the limb may show no departure from the 
 normal or it may be swollen to a greater or less extent. The skin 
 may be normal in appearance or it may be tense or blanched from 
 the underlying swelling. As a rule, the area around the wound is 
 tympanitic and crepitation may be detected. In a further stage the 
 swelling of the limb increases, the skin acquires a dusky hue and 
 tympanitis and crepitation are more marked. At a still later 
 period, the skin shows mottling with purple patches and finally 
 becomes a greenish yellow. It must be borne in mind that the 
 gangrene of the muscles may be far advanced though covered by 
 a skin which is normal in appearance. In certain cases the skin 
 may show the development of large irregular buUae filled with 
 a blood-stained serous fluid. 
 
 If the muscle is exposed, it presents at first a dry brown appear- 
 ance changing to a dirty red and, finally, it becomes pultaceous, 
 and black in colour with a slimy surface. 
 
 (iv) The Massive Type. 
 
 This type occurs generally in a segment of a hmb from which the 
 blood supply has been cut off by the interruption of the main vessel. 
 There are two groups of features which are met with in this variety 
 of the disease. 
 
 1. The Hmb which is already in a condition of dry gangrene from 
 the interruption of its blood supply suddenly becomes swollen and 
 tympanitic and the patient exhibits the constitutional symptoms, 
 pain, vomiting, rise of temperature, and rapid pulse. 
 
 2. The gangrene, the tympanitis, and the constitutional features 
 manifest themselves at the same time. 
 
 In both varieties, during the early stages the appearance of the skin 
 is that seen m ordinary arterial gangrene, but infection by bacteria 
 causes a more rapid appearance of the signs of decomposition. 
 
• 11 
 
 (v) The Fulminating Type. 
 
 The fulminating type stands out in contrast to all the other 
 varieties. Here it would appear that either the individual powers 
 of resistance are low or the infection is very virulent. The type 
 may appear as a primary manifestation, more commonly it occurs 
 as a secondary feature of the ' Group ' or ' Massive ' type. It is 
 associated .with severe pain, extensive swelling of the affected part, 
 rapid spread of the disease, and severe constitutional features. 
 
 4. CoMPLICATlbNS OF GaS GaNGKBNB 
 
 Apart- from the local spread of the infection and, the gas gangrene 
 toxaemia which is invariably present, there are two complications 
 which may occur : 
 
 1. Gas gangrene septicaemia. 
 
 2. Gas gangrene pyaemia (metastatic gas gangrene). 
 
 Gas Gangrene Septicaemia. Under certain conditions -invasion of 
 the blood-stream with the organisms of gas gangrene occurs ; such 
 an invasion produces the features of gas gangrene septicaemia. 
 It is interesting that the general signs of the condition are by no 
 means so acute as those of gas gangrene toxaemia, but in contrast 
 to the latter, the general appearance is good, there is a swinging 
 temperature, normal in the morning and 102 or 108 in the evening ; 
 there is progressive wasting and this in spite of the fact that the 
 appetite may remain good. In the majority of cases pyogenic 
 organisms eventually gain entrance to the blood-stream and to this 
 super-added infection the patient rapidly succumbs. 
 
 Gas Gangrene Pyaemia. This complication is rare, but a number 
 of instances of its occurrence have been recorded. It generally 
 follows a gas gangrene infection associated with a compound 
 fracture. There is an invasion of the blood-stream and secondary 
 deposits of gas gangrene organisms occur throughout the body ; 
 crepitant swellings containing gas appear in the muscles and sub- 
 cutaneous tissues. These secondary foci of gas gangrene often 
 develop in various parts of the body where the tissues have suffered 
 some slight damage as, from the introduction of a hypodermic 
 injection or an infusion of saline, or they may develop in tissues 
 subjected to prolonged pressure as in the buttock when the patient 
 lies in bed tilted on one hip. The prognosis is extremely bad. 
 
 5. The Diagnosis of Gas Gangrene. 
 
 There are certain conditions which may be mistaken for gas 
 gangrene. Tiiese are : 
 
 (a) Extensive haemorrhage into the tissues, generally from a 
 wound of a large blood-vessel. 
 
 (b) Post-traumatic oedema of the tissues. 
 
 In the case of haemorrhage, the limb while swollen is firm to the 
 touch, dull to percussion and there is an entire absence of the 
 constitutional features which are so typical of gas gangrene infection. 
 The situation of* the wound may give an indication of the source 
 of the haemorrhage ; blood clot is generally escaping from the 
 
12 
 
 wound and X-ray examination demonstrates the absence of gas in 
 the tissues. Pain while present is generally much less severe than 
 in gas gangrene. If the swelling is due to tissue oedema, there is 
 acute pain on palpation, a dull note on percussion, herniation of the 
 underlying tissues through the wound, and absence of constitutional 
 symptoms. 
 
 The diagnostic features of a gas gangrene infection are : pain, . 
 crepitation, resonance on percussion, and, above all, severe constitu- 
 tional symptoms. 
 
 6. The Treatment of Gas Gangrene. 
 
 (i) Preventive Treatment. 
 
 There are certain conditions which undoubtedly favour the 
 development of gas gangrene. They may be summarized as follows : 
 
 (a) Eetention of extravasated blood. 
 
 (b) Interference with the local circulation. 
 
 (c) The presence of masses of devitalized tissues. 
 
 (d) Extensive fractures and comminution of long bones. 
 
 (e) Eetention of wound secretions by dressings, pastes, or packing. 
 (/) Deky in the mechanical cleansing of the wound. 
 
 (g) Eetention of foreign bodies. 
 
 It is, therefore, evident that steps should be taken to avoid any 
 of these predisposing factors. It is of the utmost importance that 
 any constricting influence on the limb should be avoided. 
 
 Cases in which there is any suspicion of gas gangrene occurring 
 should be evacuated to some centre where eflBcient surgical treatment 
 ■can be carried out as rapidly as possible. 
 
 (ii) Treatment by the Administration of Alkalies. 
 
 All patients suffering from this disease should be given alkali by 
 the mouth. If there is vomiting and the general symptoms are 
 inarked the alkah (4 per cent, sodium bicarbonate in normal sahne) 
 should be given intravenously— fifteen to twenty ounces are generallv 
 ■sufttcient- ° -^ 
 
 (iii) Surgical Treatment. 
 
 The surgical treatment will necessarily be guided by the general 
 condition of the patient and the extent of the gangrene Thus 
 a patient who is in good condition can be subjected to an operation 
 for extirpation of the diseased muscles while another patient with 
 
 '^l!rto7:^ifj'^r::^^^^^^ ^--^ --^^*- - ^ad, wiu 
 
 tolttTeSrdSrrs :^ ^'°P*^' '-' '''' '^^"^^^-^ --^-g 
 
 ■ (1) A localized infection. 
 
 (2) Group gangrene. 
 
 (3) Massive gangrene. 
 
 .(4) Fulminating gangrene. 
 
13 
 
 (1) Localized Wound Infection. The most efficient treatment of 
 this condition is complete removal en bloc of the affected part. 
 After primary excision of the wound with skin and fascia, and 
 after prolonging the incision to expose the deeper structures, an 
 attempt is made to remove the whole of the injured and infected 
 tissue en masse together with any foreign material. This leaves a 
 large fresh wound surface which though almost invariably infected is 
 at least easier to sterilize than the original wound. Excision, en Uoc, 
 is often impossible on account of the involvement of such structures 
 as bone, large arteries, or nerves. In such instances dead muscle, 
 and other soft structures which are obviously a source of danger are 
 cut away, after all injured and ragged skin has been cleanly excised ; 
 infected tissues which cannot be removed are carefully cleansed. 
 The wound is then immediately subjected to a process of continuous 
 sterilization by the Carrel-Dakin method. .It is important that the 
 wound should not be closed in any way immediately after operation, 
 even though complete excision has apparently been carried out. 
 The question of secondary suture can be considered when repeated 
 bacteriological examinations of the part show that the proper degree 
 of sterility has been secured. The operative treatment outlined 
 must be carried out with all possible speed — a long operation is 
 most harmful. 
 
 (2) Group Gangrene. In this type of the disease the treatment 
 will vary according to whether the general condition of the patient 
 is good or bad. 
 
 (a) Patient in good condition. The mechanical cleansing of the 
 wound is carried out by the method which has already been 
 described. The affected tissues are exposed by adequate longi- 
 tudinal incisions. The condition of the muscles is carefully 
 observed. All muscular tissue is removed which does not contract 
 or bleed when cut into, or which shows any departure from the 
 normal colour. 
 
 Certain muscles may require to be removed in toto, that is from 
 origin to insertion ; in this way a limb may be preserved which, 
 iudging from the degree of crepitation and tympanitis present, would 
 otherwise have been sacrificed. After excision has been performed, 
 a Carrel-Dakin dressing is applied. 
 
 (h) Patient in had condition. This is most likely to occur in 
 fractures of the long bones. In these cases, amputation at the site 
 of fracture is generally indicated. If on account of the crepitation 
 and tympanitis of the limb, it is thought that a higher amputation 
 is necessary, it is advisable to investigate the condition of the muscles 
 through skin incisions which are made at the level of the injury : 
 in this way it can be ascertained whether the muscles are dead at 
 the level of the proposed removal. 
 
 When gas gangrene affects the tissues of the leg it is seldom 
 justifiable to amputate through the thigh ; in these cases, a guillotine 
 amputation is performed through the head of the tibia or through 
 the knee-joint, the heads of the gastrocnemius muscles being then 
 removed from their attachment to the femur. By this procedure 
 the operative shock is lessened, a painful stump is avoided and an 
 increase of length of limb is gained. 
 
14 
 
 (3) Massive Gangrene. As this type is usually the sequel to injury 
 of the main vessel of the limb, every attempt should be made_ in 
 cases of wounds of the larger blood-vessels to maintain the circulation 
 until at least such a time as the collateral circulation has been 
 estabUshed. This may be done by arteriorrhaphy or by the use of 
 Tuffier's tubes. 
 
 When gangrene has occurred and the limb is obviously dead, 
 amputation is the only possible method of treatment. As regards 
 the level at which the amputation should be done, the condition of 
 the muscles may be accepted as a guide and the limb should be 
 removed at the lowest level of the living muscle. 
 
 In cases rn which there is grave injury such as a shattered joint 
 or a fracture of a long bone, it may be necessary to disregard the 
 extent of the gangrene and amputate at a higher level as dictated 
 by the injury. In massive as in group 'gangrene, amputation 
 through the knee-joint or head of the tibia is to be preferred to 
 amputation through the thigh. 
 
 (4) Fulminating gas gangrene. Here the rate of spread of infection 
 is so rapid that within a few hours not only may the whole Hmb be 
 gangrenous but the process may have extended for some distance 
 on to the trunk. Immediate amputation by the most rapid means 
 is the only treatment which offers any possibility of success. The 
 disease may have extended to such a level that the amputation must 
 be performed through tissues which are already infected. This need 
 not be taken as a contraindication to operation, for if the flaps 
 are stitched back and continuous sterilization by the Carrel-Dakin 
 method is carried out, the progress of the disease becomes arrested 
 in a certain proportion of cases. 
 
 This type of the disease often appears after amputation through 
 the thigh ; in these cases re-amputation at or near the hip is almost 
 uniformly fatal and should only be undertaken with caution. 
 
 In cases in which the disease has spread from the limb to the 
 trunk, high amputation should be carried out, and the infected area 
 above the amputation should be treated by free incision. 
 
 (iv) Methods of Amputation in Gas Gangrene. 
 
 Whenever possible, short flaps of som^ description should be made ; 
 the wound is kept open for several days and is irrigated by the 
 Carrel-Dakin method. Below the knee, where amputation will 
 almost always be of a provisional nature, the guillotine method is 
 often the best. It may be performed through the head of the tibia 
 or through the knee-joint and the cut surface of the amputation 
 stump should be carefully examined. It should be borne in mind 
 that the lesion is not a horizontal one, but owing to certain muscles 
 being infected and others not, the upper level of the disease is very 
 uneven, mounting much higher at one point than another. For 
 this reason the appearance on section of an individual muscle may 
 afford evidence that it is infected, the disease having extended 
 locally above the general level. If such be the case, the individual 
 muscle should be dissected out. In this way the disease is eradicated 
 and length of hmb saved. 
 
15 
 
 (v) General and Post-operative Treatment. 
 
 The usual remedies for the treatment of shock are carried out. 
 Stimulants are given freely with advantage. As all cases are 
 suffering from some degree of acidosis, it is important to administer 
 an alkah. It may be given by mouth (sodium bicarbonate 8 drachms 
 to one pint of water) or if the general condition is poor and there is 
 persistent vomiting, it may be given intravenously (4 per cent. 
 solution of sodium bicarbonate). The serum treatment, both pro- 
 phylactic and therapeutic, is under trial and as yet no definite 
 statement can be made as to its efficiency. 
 
 II. INCIDENCE OP GAS 6ANGEENE. 
 
 During the different phases of the war the incidence of gas gangrene 
 among the wounded varied greatly. The highest figures were 
 always observed during active operations, when the wounded were 
 not collected with the usual rapidity and when treatment was, on 
 that account, delayed. Again, the incidence varied according to the 
 site of the battle area, being most frequent in highly cultivated soils. 
 The figures given below must be looked upon as approximate 
 rather than accurate on account of the difficulty in collecting 
 the necessary information. 
 
 The highest incidence of gas gangrene undoubtedly occurred in 
 the early period of the war about the time of the first battle of the 
 Marne and the heavy fighting in the Ypres salient. No actual figures 
 have as yet appeared, but surgeons in charge of casualty clearing 
 stations in these areas were of opinion that among the wounded the 
 incidence was well over 12 per cent. From that date the condition 
 of affairs improved very markedly in the British army. In the spring 
 and summer of 1918, during heavy fighting, the figure was only about 
 1 per cent. In the first and second armies, among 13,303 wounded, 
 there were 158 cases (1-1 per cent.) of gas gangrene in the forward 
 stations, while for the same period in the base hospitals, out of 
 23,792 wounded, there were 92 cases of gas gangrene (0-39 per cent.). 
 
 The figures given by Bowlby (1919) are very similar in that out 
 of a total of 25,060 patients at certain base hospitals during 1917 
 and 1918 there were only 84 patients (0-83 per cent.) with severe 
 or ' massive ' gas gangrene, while at one base during the great retreat 
 of March 1918 the incidence of gas gangrene among 20,000 wounded 
 was 1-0 per cent. 
 
 Many factors contributed to this reduction, chief amongst which 
 may be mentioned, more efiicient surgical cleansing, earlier evacua- 
 tion of wounded, and prophylactic measures. 
 
 In the French armies the incidence appears to have been rather higher. 
 Ivens (1916) had 107 cases of gas gangrene among 1,694 wounded 
 (6-3 per cent.), while Chalier and Chalier (1) give the figure as 5-4 per 
 cent., and Ombredanne as 13 per cent, (cited by Guermonprez). 
 
 For the German army on the Western front, Wederhake puts the 
 incidence, at 2-2 per cent, and Franz at 2 per cent. Eumpel's figures 
 are more detailed : in one war hospital, from May to September 
 1916, among 8,036 wounded there were 114 cases of gas gangrene, 
 
16 
 
 an incidence of 3-7 per cent. He also gives statistics for one of the 
 armies from the middle of December 1916 until the end of March 
 1917 ; there were 5,921 wounded with 170 cases of gas gangrene, 
 a total incidence of 2-8 per cent., but during an offensive the mcidence 
 rose to 7 per cent. By January 1917, however, following upon more 
 energetic surgical treatment and the employment of anti-gas gangrene 
 serum, the incidence was reduced to 0-6 per cent. 
 
 On the Eastern front, on the other hand, the general incidence was 
 lower, as might have been expected. Out of 5,000 wounded, Wieting 
 observed gas gangrene in 1-43 per cent, of the cases. 
 
 Although the morbidity from gas gangrene was considerably 
 reduced amongst all the belligerents, nevertheless even an incidence 
 of 1 per cent, constituted a serious loss on account of the high 
 mortahty (20-50 per cent.). 
 
 III. BACTEEIOLOGY. 
 Introduction. 
 
 A perusal of the pre-war literature of anaerobic bacteria reveals 
 the state of disorder which prevailed in this branch of bacteriology. 
 First, in the matter of technique there was no method of choice 
 by which surface growths could be easily obtained ; many 
 methods were detailed but none was entirely satisfactory. Further, 
 it cannot be doubted that most of the cultures of the anaerobes 
 described were impure, and consequently the descriptions were 
 inaccurate. This is seen in the case of such a well-known and impor- 
 tant organism as B. oedematis maligni, which is described by von 
 Hibler and later by Fehx von Werdt (1912) as proteolytic. It is highly 
 probable that B. enteriditis sporogenes, Klein, represented a mixture 
 of B. welchii and B. sporogenes. Such errors were numerous, were 
 copied from book to book, and led to sterile controversy which ended 
 in general confusion. 
 
 No attempt will be made in this Eeport to clear up the confusion 
 except where absolutely necessary, nor to put names to the numerous 
 organisms insufficiently described in the older books and journals. 
 
 The errors were due for the most part to the fact that the 
 problems of anaerobic infections were of academic interest and were 
 not thoroughly studied ; and in part to the very great inherent 
 difficulties of the subject. 
 
 The war transformed these problems into questions of high 
 importance and urgency; the sufferings of our men and the 
 grievous losses of our armies compelled professional bacteriologists 
 to attack the subject with renewed vigour and enlisted to their 
 aid colleagues from kindred branches of biological science. The 
 united efforts of all these workers, with their different disciplined 
 experiences, have advanced knowledge, enlarged our comprehension 
 of wound infections, and aided in abating suffering. 
 
 The advances in knowledge may be thus summarized : 
 
 It may be said that it is now not much more difiacijt to obtain 
 surface cultures of such anaerobes as B. tetani or vii)rion sepique 
 than of common aerobes. This fundamental advance is due to 
 improved methods of anaerobic cultivation, among which should 
 
17 
 
 be especially noted the beautiful, adaptation by Mcintosh and 
 Pildes of Laidlaw's method of obtaining anaerobiosis by the use of 
 finely divided platinum or palladium as a catalyst. 
 
 The pathogenic and probably most of the non-pathogenic anaerobes 
 which may infect wounds contaminated with soil, have been isolated 
 and described. The list of these microbes is long, but the majority 
 are apparently non-pathogenic. The principal pathogenic species are 
 B. welchii, vibrion septique, and B. oedematiens. 
 
 These three organisms, acting alone or in combination, are respon- 
 sible for almost all of the acute rapidly-evolving cases of gas gangrene 
 where the whole course of the infection may be run in the space of 
 8 to 48 hours. They may be helped in their pathogenic activity by 
 the so-called non-pathogenic anaerobes and also by aerobes, but our 
 knowledge on this point is not precise. It is certain, however, that 
 they all assist one another. 
 
 B. welchii is responsible for a larger percentage of cases of gas 
 gangrene than either of the other two, though it is now known that 
 the percentage is not so high as it was formerly thought to be. 
 Vibrion septique is commoner than B. oedematiens. 
 
 The role played by the more numerous but less dangerous non- 
 pathogenic anaerobes has not been clearly defined ; they may be 
 responsible for some of the features of gangrenous wounds — such as 
 putrefactive odour, blackening and digestion of tissue — both in the 
 acute cases and in local anaerobic infections. Certain strains of 
 B. histolyticus undoubtedly play a large part in some cases of gas 
 gangrene (Weinberg). 
 
 It should be noted that any one, or possibly any combination, of 
 these organisms may be found in a wound which is not, and does not 
 become, gangrenous. This point will be discussed in the section 
 devoted to experimental gangrene. 
 
 The part played by B. tetani will be dealt with separately. Finally, 
 in some cases, specific names are used in this Eeport for species which 
 are believed to comprise several varieties. 
 
 The anaerobes infecting wounds may, therefore, for practical 
 purposes, be considered under the following headings. 
 
 1. Pathogenic anaerobes liable to set up conditions of gas gangrene 
 with symptoms of generahzed intoxication. 
 
 2. B. tetani. 
 
 3." Wound infecting anaerobes whose action may be ancillary to 
 the causal agents of gangrene and tetanus. 
 
 1. Pathogenic Anaerobes liable to set up Conditions of 
 Gas Gangrene with Symptoms of Gbnbea"l Intoxication. 
 
 (i) B. welchii. Migula, 1900. (Synonyms : Bacillus of Achalme, 
 1891 ; B. aerogenes capsulatus, Welch and Nuttall, 1892 ; B. phleg- 
 monis emphysematosae, Eug. Fraenkel, 1893 ; B. perfringens, Veillon 
 and Zuber, 1898 ; B. enteritidis sporogenes, Klein, 1895). 
 
 B. welchii is a non-motile organism, uniformly Grani-positive in 
 young cultures; in old cultures Gram-negative individuals are 
 frequently met with. 
 
 B 
 
18 
 
 Morphology. The length of the bacilli varies from 4 to 8 /u, and 
 the breadth from 1 to 1-5 /x. In general they are straight and the 
 ends are usually rather square. 
 
 Very short, almost coccal forms, and long filaments may be found 
 under certain conditions of growth. Some strains show curved rods. 
 
 Involution forms showing considerable ' pleomorphism ' (club 
 shapes, filaments, tadpole forms, granular types, &c.) are found, 
 particularly in old cultures upon coagulated serum. 
 
 A capsule may be demonstrated ; it is most conspicuous in rods 
 from the exudate of infected tissues or from media containing serum, 
 but it does not appear to be absent at any time (M. Eobertson). 
 
 The organism has no flagella. 
 
 Spores. Sporing forms of B. welchii are sometimes to be found in ' 
 infected wounds, but are most readily formed in such media as casein 
 broth, alkaline egg broth, and' coagulated serum, all of which are rich 
 in protein and free from fermentable carbohydrates. They are rarely 
 seen in media containing s*ubstances which the bacillus is able to 
 ferment. , Individual strains of the organism vary a good deal in the 
 readiness with which they form spores even in the most favourable 
 media. The spores are relatively large, oval in" shape and with 
 slightly flattened ends. They are subterminal or central in position ; 
 the body of the rod in which they develop disintegrates very rapidly. 
 
 Anaerohiosis. B. welchii does not require very strict anaerobic 
 conditions for its growth. 
 
 Colony. Surface colonies on nutrient agar, serum agar, or glucose 
 agar are circular in contour. In 24-48 hours they may attain a size 
 of 1 to 2 mm. in diameter ; older cultures may become much larger, 
 the edges of the colonies sometimes being crenated. Young 
 colonies (12 hours) are translucent ; they soon become opaque and 
 granular. There is a variety of the colonies of B. welchii -which shows 
 a very thick opaque centre and a granular slightly crenated margin. 
 The latter are of a viscid consistency and tend to adhere to the 
 medium In deep agar shake cultures the colonies are lenticular. 
 
 Cultural reactions. 
 
 Meat medium : Very rapid growth, with the development of a pinkish 
 
 colour ; gas ; and an acid reaction. The meat is not 
 
 blackened, and there are no macroscopic signs of 
 
 digestion. The odour is sour but not putrefactive. 
 Milk medium : Vigorous reaction ; rapid acid clot with evolution of 
 
 much gas, i.e. so-called 'stormy fermentation'. 
 
 This reaction takes place as a rule within 12 to 
 
 48 hours ; spores are not formed. 
 Coagulated serum : No change; no liquefaction. Spores are usually 
 
 formed in this medium ; filaments, granular forms, 
 
 and many involution types may be seen, especially 
 
 in old cultures. 
 Alkaline egg broth : The medium becomes evenly opaque but is not 
 
 precipitated in large flocculi. 
 Gelatine : ^ Liquefied. 
 
 hJ J'^w^'"'"!*''^^ Report the gelatine reaction is recorded upon the result obtained 
 by incubating the inoculated tube at 37°C. for 48 hours, and thereafter coolin" Tby 
 immersion in a beaker of cold water. i.""uu„ is uy 
 
19 
 
 Cultural reactions (continued). 
 
 Broth : Active growth ; medium becomes turbid and opaque ; 
 
 there is evolution of gas; after a period of- time 
 varying between about 2\ to 48 hours the organisms 
 sink to the bottom of the tube. The reaction 
 becomes strongly acid. 
 
 , Substances fermented. Subsrances not fermented. 
 
 Glycerine (variable) Mannite 
 
 Glucose Dulcite 
 
 Laevulose Salicin. 
 
 Galactose 
 Maltose 
 Saccharose 
 Lactose 
 
 Inulin (variable) 
 Starch. 
 
 Animal reactions. — The pathogenicity of the various strains differs 
 considerably, many being non-pathogenic, but under constant condi- 
 tions the virulence of a given strain does not, as a rule, show much 
 alteration. Other strains, however, which have been propagated 
 upon artificial media for long periods of time may show considerable 
 loss of virulence. 
 
 The lethal dose of different strains of B. welchii in broth culture 
 is variable within fairly wide limits for pigeons, guinea-pigs, and 
 mice ; rabbits show a considerable resistance, but they can be 
 infected if massive doses are used. 
 
 Washed bacilli suspended in saline are non-pathogenic. Spores 
 of B. welchii, when washed off serum slopes with saline, fail as a rule 
 to produce any effect whatsoever in the animal, though slight local 
 induration of the limb injected may occur. 
 
 If a lethal dose (0-1 to 1 c.c. according to the virulence and condi- 
 tion of the culture) of a broth culture is injected into the muscles of 
 the thigh of a guinea-pig, an extensive oedema is produced which 
 involves the limb injected and which may spread over the whole 
 abdomen and into the axillary region. The animal usually dies 
 within 24 to 48 hours, but spontaneous recoveries do occur even 
 with doses of a pathogenic broth culture which are lethal for the 
 majority of animals of the same weight. Injection in a guinea-pig 
 may produce a perforating necrosis of the skin and subcutaneous 
 tissue, in which case recovery not infrequently takes place. Sub- 
 lethal doses produce lesions of greater or less severity from which 
 the animal recovers. 
 
 A post-mortem examination reveals an extensive oedema, usually 
 slightly blood-stained, but never to the same extent as with infection 
 by vibrion septique. There may be some gas in the tissues ; the 
 muscles of the injected limb are of a pale pink colour. They are very 
 friable and soft and give the appearance of being digested. The 
 odour is sour but not putrefactive. 
 
 The internal organs in guinea-pigs do not show any conspicuous 
 macroscopic alterations, except the suprarenal glands, which may 
 be of a deep red colour, especially where the death of the animal 
 
 B2 
 
20 
 
 has taken place rapidly. In other cases they may he mottled^or 
 hardly altered at all. If the animal survives until the third day, the 
 suprarenals do not as a rule show discoloration. 
 
 In the case of mice the liver is small and pale in colour, the kidneys 
 and suprarenals are congested, and the bladder is sometimes full of 
 blood-stained fluid. The duodenum is constantly dilated and often 
 pink in colour from congestion of its vessels. 
 
 In all cases the blood-stream is invaded at a relatively early'stage 
 of the infection. 
 
 Microscopic examination. Very large numbers of bacilli are to be 
 found in the muscles at the site of inoculation, and these may also 
 be numerous in the exudate. Spores are not formed in the body_ of 
 animals experimentally infected with cultures of B. welchii. Chains 
 of 3 to 5 elements may be seen on the peritoneal surfaces. Long 
 filaments are never observed. 
 
 Toxin. . A soluble toxin can be demonstrated in young (24 to 48-hour) 
 broth cultures ; the nature and action of this toxin is discussed in 
 another section. 
 
 Agglutination. Agglutinins for B. welchii cannot be demonstrated 
 in the blood of rabbits which have been repeatedly inoculated intra- 
 venously with the organisms. Weinberg claims that horses which 
 receive massive intravenous injections of B. welchii emulsions over 
 a long period of time do sometimes develop agglutinins for the 
 horiiologous strain, but the reaction is usually restricted to this and 
 cannot be applied for diagnosis. . 
 
 As a general rule a good emulsion can be made in saline, although 
 certain strains show a tendency to spontaneous agglutination. 
 
 Distribution in nature. B. ivelchii is a very frequent and widely 
 spread organism. It is present in the intestine of men and animals. 
 It is found in many samples of milk, in earth and dust, and can be 
 cultivated from clothing and practically from any object exposed to 
 dust. 
 
 (ii) Vihrion septique. Pasteur et Joubert (1) (1877). Synonym 
 B. oedematis maligni. Koch. 
 
 This microbe has been the centre of much controversy ; it has, 
 however, become clear that an organism agreeing in, characters with 
 Pasteur's vibrion septique is of frequent occurrence in wounds. 
 Numerous strains have been isolated and the characters of the bacillus 
 are now perfectly well known. Vibrion septique and B. oedematis 
 maligni of Koch are probably identical. Many writers subsequent 
 to Koch, such as von Hibler, G. 0. Jensen, von Werdt, and others, 
 have, however, repeatedly described B. oedematis maligni as liquefying 
 serum and digesting meat with the production of a putrid odour. 
 These reactions do not obtain in pure cultures of undoubted vibrion 
 septique, the inference being that these writers and even certain quite 
 recent workers, such as Conradi and BieUng, were dealing with impure 
 cultures (see p. 93). 
 
 Confusion has also arisen in the tendency to consider vibrion septique 
 as identical with B. chauvoei (bacillus of Eauschbrand). This is 
 undoubtedly an error. B. chauvoei is quite distinct from vibriovr 
 
21 
 
 sepUque, but strains of vibrion septique have been isolated from ■ 
 cases which appeared chnically to be symptomatic anthrax and 
 also from accidental wounds in animals. The most recent discus- 
 sion of this sjabject will be found in the paper of K. P. Meyer (1915). 
 
 Morphology. Vibrion septique is a Gram-positive organism ; it is 
 motile in young cultures and in the exudate from infected animals. 
 It presents a rather wide range of different forms according to the 
 conditions of culture. In broth or in meat medium the organisms 
 appear as rods of varying length somewhat more slender than 
 B. welchii. Spores are readily formed and are usually situated 
 towards one extremity ; central spores are, however, not uncommon. 
 Deeply stained bulb-like types may be present, especially in young 
 cultures. In fluid media containing fresh tissue and on coagulated 
 serum very varied appearances may be seen, such as * navicular ' or 
 ' citron ' types, i. e. pale, citron or boat-shaped bodies with deeper 
 staining points at one or both extremities, deeply staining club- 
 shaped forms, filaments, and bulb-like types often growing in short 
 chains. The navicular forms may be observed in films made directly 
 from infected tissues and blister fluid, &c. 
 
 Cultural Beactiofis. Colony. Vibrion septique is a strict anaerobe. 
 Surface colonies can be obtained on plates under good conditions 
 of anaerobiosis or upon the surface of serum agar slopes. Serum 
 agar containing 1 per cent, of salicin produces a more robust colony 
 than other media. There is always a noticeable tendency for growth 
 to occur in a continuous film instead of in discrete colonies, especially 
 if the agar surface is too moist. The colonies are transparent and 
 faintly opalescent, the contours are smooth or indented, and the 
 edges may be more or less crenated. 
 
 Colonies in deep agar shakes are delicate and branching, the centre 
 of the colony is never very compact or well defined. 
 
 Meat medium : Gas ; colour varying from bright red to pink ; no 
 
 blackening ; odour is rancid but not putrid. 
 
 Milk : Acid and clot ; some gas may be formed : the change 
 
 in the milk is slow ; the clot does not as a rule 
 appear before 3 to 6 days. 
 
 Coagulated serum : No liquefaction. The morphology of the organism 
 is very variable. 
 
 Alkaline egg broth : The medium is rendered more opaque ; there is no clot. 
 
 Gelatine : Liquefied. 
 
 Broth : The medium becomes turbid : after a period of growth 
 
 the organisms settle down to the bottom of the 
 tube, leaving a perfectly clear supernatant fluid. 
 
 Substances fermented. Substances not fermented. 
 
 Glucose Glycerine 
 
 Laevulose Saccharose 
 
 Galactose Inulin 
 
 Maltose Mannite 
 
 Lactose Dulcite. 
 Salicin. 
 
 Animal reactions. Pigeons, guinea-pigs, mice, rabbits, and dogs are 
 all susceptible' to infection with vibrion septique. Individual strains 
 
22 
 
 vary a little in pathogenicity, but the pathogenic quality is retained 
 unaltered over years of subculture. The lethal dose of a given strain 
 remains fairly constant, provided the same growth conditions are 
 repeated.! 
 
 The most infective inoculum for Experimental animals is a 24 to 48- 
 hour culture in glucose, broth with or without a piece of fresh tissue. 
 Ordinary nutrient broth cultures are slightly less infective. 
 
 If a lethal dose (0-01 to 0-5 c.c, according to virulence and condition 
 of culture) of a living culture of vibrion se^tique is injected into the 
 muscles of the thigh of a guinea-pig, the animal dies in 12 to 24 hours 
 with oedema and the development of gas in the tissues. Spontaneous 
 recovery in guinea-pigs which have become infected with vibrion 
 septique does not occur. Sub-lethal doses produce no symptoms 
 whatsoever, and •with.vibrion septique it may be said that for guinea- 
 pigs an infecting dose is a lethal dose. 
 
 On post-mortem examination an extensive blood-stained oedema 
 is observed and a considerable amount of gas is found to have been 
 developed in the areolar tissue around the muscles involved. Pockets 
 of gas are almost invariably seen in the groins and axiUae. The 
 muscles affected have a characteristic very intense deep red colour 
 and are softened, but there is no putrid odour. 
 
 There may be a collection of fluid in the peritoneal cavity and in 
 the pericardium ; this is not as a rule very evident in guinea-pigs 
 which have died in 12 to 24 hours, but is very well-marked 
 in rabbits which have succumbed to the infection after 24 to 
 48 hours. The suprarenal glands may show a variable amount of 
 redness, but this is not so regular or so marked a feature as in guinea- 
 pigs which have died from infection of B. welchii. In mice the local 
 lesion is of the same character as that described for guinea-pigs. 
 The organs do not, however, show any great changes. The adrenal 
 bodies are most frequently affected, and they may show congestion 
 varying in degree from a light pink tp a deep red colour. 
 
 Microscopic examination of the exudates of animals infected with 
 vibrion septique shows very numerous motile rods and usually the 
 characteristic ' navicular ' or ' citron ' types. Swollen club-ended 
 rods and short chains of intensely staining bulb-shaped individuals 
 may be seen. Spore-bearing rods are, however, not as a rule to be 
 found until some hours after death. 
 
 The peritoneal surface of the liver shows long snake-hke filaments 
 which are of diagnostic importance. It was this appearance which 
 led to the erroneous idea that the organism was a vibrio. It may 
 be noted, however, that when dealing with mixed cultures of vibrion 
 septique and other spore-bearing anaerobes, ' citron ' types in the 
 muscles and filaments on the guinea-pig's hver are not alwavs 
 present. -^ 
 
 Toxin. A soluble toxin can be demonstrated in 24 to 48 hour 
 broth cultures. (See later, section IV). 
 
 Tf\-5^i?n''°'''^''i'°'' °*- f 'i?*''''' <^,°'^' °* ^ 'i^i'ig "'^'"o i« notoriously inexact, 
 rlfn'n r,?Tr^-'"'fW misleading With the vibrion septiqw than in most cases. The 
 r/ .nn1iHnn=' ^^^ Pf "bably a iairly constant amount of toxin is required toproduce 
 the tissues 'nfection, i.e. for the proliferation of^the bacteria in 
 
23 
 
 Agglutination. Agglutinins are produced in the blood of rabbits 
 inoculated intravenously with heated and washed cultures of vibrion 
 sejptique. In using monovalent agglutinating sera, it is found that 
 the strains fall into several well-defined serological groups. 
 
 Distribution. Vibrion septique is said to be present in the intestine 
 of men and animals and in the soil of cultivated land ; it is, however, 
 less frequently found in nature than B. welchii. 
 
 B. chauvoei. 
 
 B. chauvoei. This organism is the cause of the disease known as ' Black- 
 leg ', ' Quarter ill,' or ' Blackquarter ', as ' Charbon symptomatique ' by 
 the French and as ' Rauschbrand ' in the German literature. There has 
 been a good deal of confusion in regard to this bacillus, although it has been 
 known and studied for a long period. It was probably with impure 
 cultures of this organism that Grassberger and Schattenfroh (1, 2 and 3) 
 carried out their extensive studies which led to conclusions now recognized 
 as being erroneous. 
 
 The characters of B. chauvoei are very briefly as follows : 
 
 It is a very strict anaerobe. Although it has not so far been recorded as 
 occurring in man (see p. 92), it is very pathogenic for mice and guinea- 
 pigs. Rabbits are relatively insusceptible. 
 
 Morphology. In broth and meat media, clostridial forms and rods with 
 subterminal spores are to be seen ; the spores are of a more elongated oval 
 shape than those of vibrion septique. In serum media or in the presence of 
 fresh tissue, navicular, forms and club-shaped types are produced. The 
 riaviculaf individuals are, however, longer and more slender than those 
 usually seen in vibrion septique. cultures. In infected guinea-pigs the 
 navicular and club-shaped elements are also present, but no long 
 filaments are found on the peritoneal surfaces of the liver. This is quite 
 an important feature in the difEerential diagnosis of B. cjiauvoei and vibrion 
 septique. 
 
 It is frequently stated that B. chauvoei is a Gram-negative organism. 
 In fresh smears from infected tissues and in young cultures the bacillus is 
 definitely Gram-positive . In older cultures Gram-negal ive individuals may, 
 however, frequently be met with. : 
 
 Cultural reactions. 
 
 Meat Medium : Gas ; pinkish colour which may fade ; no putrid 
 
 odour ; no blackening of the medium. 
 Milk : Acid ; clot in 3 to 6 days ; some gas may be evolved. 
 
 Coagulated serum : No liquefaction. 
 Gelatin : Liquefied. 
 
 Substances fermented. Substances not fermented. 
 
 Glucose . ' Glycerine 
 
 Galactose Mannite 
 
 Laevulose ' Dulcite 
 
 Maltose Inulin 
 
 Saccharose Salicin, 
 Lactose. 
 
 It should be noted that B. chauvoei ferments saccharose and does not 
 ferment salicin, whereas the contrary is the case with vibrion septique. 
 Toxin. A specific soluble toxin is produced by B. chauvoei. 
 
24 
 
 (iii) B. oedematiens. Weinberg and Seguin (1), 
 
 This organism, which was discovered by Weinberg and Seguin in 
 1915 and subsequently identified by Legros (3), Vaucher (1)^ Dalyell 
 and others, is closely allied to, if not identical with, B. oedematis 
 maligni II described by Novy in 1894. It is motile under strictly 
 anaerobic conditions, for instance when examined in a sealed capillary 
 tube ; under a cover-slip, however, it is non-motile. In shape 
 B. oedematiens is a stout rod as thick as B. welchii (0-8 to 1 ix) but 
 usually longer. The rods are frequently curved and the- spores which 
 are readily formed in all media are large and oval with slightly 
 flattened ends. They are subterminal or central in position. The 
 morphology of the organism should be studied in young cultures as 
 autolysis sets in very early. There is relatively little variation in 
 the appearances presented by B. oedematiens but short chains and 
 filaments are occasionally formed. 
 
 Cultural reactions. B. oedematiens requires strict anaerobic con- 
 ditions to ensure surface growths. Surface colonies are flattened and 
 tend to be confluent forming a translucent film. In agar shakes the 
 growth is dehcate, resembling snowflakes, but more solid colonies like 
 conventional bursting grenades may also be seen. 
 
 Meat medium : Gas ; pinkish colour which fades quickly ; in some 
 
 samples of meat medium the colour may not change 
 
 very markedly with growth. 
 Milk : After long incubation (10 to 30 days), there is an acid 
 
 reaction and clot, the acid reaction may be seen in 
 
 vigorous cultures after 4 or 5 days, but the clot is 
 
 always delayed. 
 Coagulated serum; no change in medium. 
 Broth : Early flocculation and sinking to the bottom of the 
 
 tube where the organisms rest as a semi-opaque 
 
 cloud (18 to 36 hours). 
 Gelatine : Medium is liquefied. 
 
 Fermentation : Production of acid is feeble. 
 
 Substances fermented. Substances not fermented. 
 
 Glucose Glycerine Mannite 
 
 Laevulose Galactose Dulcite 
 
 Maltose Saccharose Inulin 
 
 Lactose Salicin. 
 
 Animal reactions. The pathogenicity of different strains varies 
 somewhat ; gmnea-pigs, mice, rats, and rabbits are all susceptible ' 
 
 The mjection of 0-25 to 1 c.c. of a 24 hour broth culture into the 
 thigh muscles of a guinea-pig produces death within 24 to 48 hours 
 At the post-mortem examination the muscles immediately at the 
 site of inoculation are found to be red, softened but not diffluent 
 As a rule very httle if any gas develops. There is a spreading 
 gelatinous oedema which is usually quite colourless but if the 
 infection has progressed very rapidly the oedema may be tinged 
 with pmk. The muse es of the abdominal wall are Unaltered in 
 appearance. The baciUi are to be found in numbers only lithe 
 site of inoculation. In general the oedema fluid and the peritoneal 
 
25 
 
 surfaees of the liver show only infrequent individuals. Cultures 
 from heart blood are generally positive, except in cases where the 
 animal succumbs to the effect of the toxin present in the inoculum 
 and not to an actual infection with the bacilli. 
 
 Toxin. B. oedematiens develops an active toxin in broth cultures. 
 (See section IV).' 
 
 Agglutination. Agglutinins can be produced in rabbits by the 
 intravenous injection of washed bacilli. The sera thus obtained, 
 however, agglutinate only the homologous strain. Agglutination 
 "tests must be carefully controlled as many strains show auto- 
 agglutination. 
 
 Distribution. B. oedematiens has been isolated from samples of 
 earth from cultivated areas by injecting small quantities into the 
 muscles of guinea-pigs protected by means of antitoxic sera against 
 B. welchii, B. tetani, and vibrion septique. 
 
 (iv) B. histolyticus. Weinberg and Seguin (12). 
 
 B. histolyticus holds a position that is intermediate between the 
 acutely pathogenic, invading anaerobes and the secondary purely 
 saprophytic forms. 
 
 B. histolyticus is a motile rod very frequently arranged in pairs. 
 It is about 0-5 to 0-8 microns broad and about 3 to 5 microns long. 
 Very young cultures (14 to 16 hours growth) should be examined to 
 obtain information concerning the appearance of this organism. 
 The bacilli degenerate very soon in cultures, and after 24 to 48 hours 
 of growth many Gram-negative individuals may be seen together 
 with fusiform and skittle-shaped involution types. The spores, 
 which are readily formed in all media, are oval and usually sub- 
 terminal ; they are considerably wider than the rod in which they 
 arise. 
 
 Cultural reactions. Surface colonies are delicate and flat ; they 
 have crenated or irregular edges. Colonies in deep agar are arbor- 
 escent or coral like with fine woolly ends to the branches. 
 
 Meat medium : Digested with production of a white deposit of tyrosin. 
 
 Coagulated serum : Liquefied. 
 Gelatin : Liquefied. 
 
 Substances fermented. 
 Glucose 
 Laevulose 
 Maltose. 
 
 Distribution. This organism is difficult to isolate ; it is found in 
 wounds and has been obtained from earth. 
 
 The strains of B, histolyticus vary very greatly in pathogenicity 
 as tested by injection of young broth cultures (16 to 18 hours) into 
 guinea-pigs. Some strains produce very serious lesions culminating 
 in the death of the animal, while others are only very slightly if at 
 all pathogenic. The bacillus is actively proteolytic and digests 
 living tissue (Weinberg and Seguin (15) ). This can be demonstrated 
 by injecting 1 c.c. of a young broth culture of a virulent strain into the 
 thigh of a guinea-pig ; in the course of 12 hours the skin and muscles 
 
26 
 
 become digested and a haemorrhagic liquefaction of the soft, partj 
 of the Umb takes place. This digestion of the, tissue may spread 
 over the abdomen and the animal may die in, the. course of the next 
 12 to 24 hours or it may recover with a more. or less complete necrosis 
 
 of the limb. , ,, i, e-iA 4-^ 
 
 Toxin. A toxin can be demonstrated m broth pultureS .ot 14 to 
 16 hours' growth, but is destroyed if growth is allowed to proceed 
 beyond 18 hours. It is difficult to filter the toxin successfully as 
 it is largely retamed in the candle. The action of the toxin 
 resembles that of vibrion septique in that when injected intravenously 
 (in rabbits) there is practically no incubation period, death occurring 
 within five to fifteen minutes. An antitoxin neutrahzmg this 
 substance has been produced in horses (Weinberg and Seguin). 
 
 (v) B. hotulinus. van Ermengem. 
 
 This bacillus was first described by van Ermengem (1) in 1896 in 
 connexion with an epidemic of meat poisoning in Flanders. It has 
 not been found in wounds, and so far as is known is in no way involved 
 in any aspect of the gas gangrene question. A description of its 
 chief characters is appended here for the sake- of completeness as it 
 ranks among the highly toxigenic anaerobes. 
 
 Although it has not been frequently isolated the recent in- 
 vestigations of E. C. Dickson would indicate that its distribution 
 in nature is wider than was formerly believed. 
 
 Morphology. B. hotulinus is a motile Gram-positive rod, some- 
 what larger than vibrion septique but resembling it in general 
 appearance. The bacilli are usually single but short filaments are 
 not infrequently formed. Spores are not readily produced, but 
 when present are small and oval, usually sub-terminal in position 
 and do not distend the rod to any great extent. 
 
 Cultural reactions. B. hotulinus is difficult to cultivate upon 
 artificial media and requires suitable conditions. The organism 
 will not grow in media the reaction of which is acid. Even the 
 presence of COg inhibits the growth. The best results are obtained 
 by the use of media containing fresh animal tissue or glucose, and 
 growth occurs between 18° C. and 35° C. Good conditions of 
 anaerobiosis are essential. Surface colonies can be obtained upon 
 glucose media. They may attain to 1 mm. in diameter in 24 to 
 48 hours. They are flat, irregular in shape, and of a greyish 
 colour. If they are examined with a hand lens they are found to be 
 irregularly mottled. Madsen described colonies on glucose gelatine 
 as being circular, transparent, and yellowish in colour ; they were 
 coniposed of coarse granules which are seen to be in continual move- 
 ment. The gelatine was Uquefied round the colony. Upon further 
 incubation the colonies increased in size and sometimes reached 
 several millimetres in diameter. They do not assume an opaque 
 feathery appearance. In deep shakes of glucose agar, semi-opaque 
 biconvex or kidney shaped colonies are produced of about 1 mm. 
 in diameter after 24 to 48 hours' incubation. There is usually 
 a central nucleus and older colonies may send out irregular pro- 
 jections. A considerable amount of gas may be produced. 
 
27. 
 
 Meat medium : Poor growth. This medium is not suitable for the 
 
 organism. 
 Milk medium : Growth is scanty- and frequently fails altogether. No 
 
 change in the medium. 
 Coagulated serum : Not liquefied. ' 
 Gelatine : Liquefied. 
 
 Substances fermented. Substances not fermented. 
 
 Glycerine Galactose 
 
 Glucose - Saccharose 
 
 Maltose Inulin 
 
 Lactose Mannite 
 
 Starch. Dulcite 
 
 Salicin. 
 
 Animal reaction. B. botulinus grown in broth, or in broth to which 
 a piece of Hving tissue has been added, produces a characteristic 
 toxin which causes the death of guinea-pigs within 36 to 48 hours, 
 A tenth of a c.c. of such culture injected subcutaneously into a guinea- 
 pig of 250 grm. causes typical signs of botulismus in 36 hours, there 
 is a complete muscular paralysis, dilatation of the pupils, shallow 
 breathing, intense salivation, and the death of the animal supervenes 
 after a short period. The death is mainly due to the action of the 
 toxin. The organism can, however, be cultivated post mortem from 
 the tissues and the blood of the injected animal (Mcintosh). 
 
 Animals very susceptible to botulinus toxin are rabbits, guinea- 
 pigs, mice, cats, and monkeys. Madsen using a filtered toxin found 
 that 0-0015 c.c. killed a guinea-pig in 1 to 2 days and 0-0010 c.c. killed 
 in 4 to 5 days. The symptoms he describes are marked muscular 
 relaxation, greenish discharge from the mouth, aphonia, aphagia, 
 constipation, dilatation of pupils, and great loss of weight. 
 
 (vi) B. Jallax. Weinberg and Seguin (5). 
 
 This is a motile bacillus which occurs in infected wounds and 
 may be found in cases of gas gangrene. It is occasionally present 
 in blood cultures from the patient. When recently isolated certain 
 strains are pathogenic for guinea-pigs (Weinberg and Seguin (9)), 
 this character is, however, rapidly lost upon cultivation. A strain, 
 which had lost its pathogenic properties for mice, killed however 
 when injected along with a small dose of calcium chloride. 
 
 B. fallax is a somewhat slender rod, of about 3-6 microns in length, 
 with rounded ends. It is often slightly curved. Gram-negative 
 elements are frequent and there is altogether rather a feeble capacity 
 to retain the stain. Spores are not readily formed on any media 
 but do occur in small numbers in meat and coagulated serum. They 
 are oval and usually subterminal. 
 
 Growth reactions. On surface plates the colonies are round or 
 crenated- and slightly granular. Deep colonies are lenticular, 
 irregular or bean shaped. 
 
 Meat medium : Gas ; pinkish colour ; no digestion. 
 
 Milk : Acid clot after some days (3-7). 
 
 Coagulated serum : No liquefaction. 
 
 Gelatin : No liquefaction. 
 
28 
 
 B. fallax ferments glucose, laevulose, and maltose. Some strains 
 also attack galactose. Accounts vary somewhat in regard to the 
 fermentations of B. fallax ; Henry considering that in addition to the 
 above sugars, galactose, saccharose, starch, inulin, and salicin are 
 also fermented. 
 
 Toxin. A soluble toxin is produced. 
 
 2. B. Tbtani. Nicolaier. 
 (i) Bacteriological account. 
 
 Bacillus tetani was described by Nicolaier in 1884 and cultivated 
 by Kitasato in 1889. 
 
 It is extremely difficult to isolate in a pure state ; many of 
 the cultures obtained from serum institutes and from laboratory 
 collections being found to contain some other anaerobe in addition 
 to B. tetani. The contaminating organisms usually belonged to 
 the type of B. sporogenes, but oval and round end-sporing bacilli 
 such as B. cochlearius and B. tetanoides, were also found. It is to 
 the impurity of many of the cultures of B. tetani that the great 
 variety in the cultural characters ascribed to this organism is due. 
 (Mcintosh, Eobertson). 
 
 It may be noted that agglutination tests showed that all these 
 laboratory cultures belonged to one serological type (TuUoch (1) ). 
 
 Morphology. B. tetani is a motile Gram-positive rod. In young 
 cultures it is rather a stout bacillus about 4 to 8 microns in length 
 and 0-4 to 0-6 microns in breadth. 
 
 The spores are spherical and always strictly terminal. The 
 bacillary rod becomes thinner after the spore is formed but remains 
 attached to it for some time ; this condition gives the familiar 
 drumstick appearance. In pure cultures in the ordinary culture 
 media spores are not formed as a rule until the 3rd or 4th day. 
 
 Cultural reactions. B. tetani is a very strict anaerobe but surface 
 growths can be obtained on serum agar with or without the addition 
 of glucose under good conditions of anaerobiosis. 
 
 Surface colonies are flat and delicate, sometimes with finger-hke 
 projections. After 48 to 72 hours the centres become raised a Uttle 
 above the surface of the medium and a ground glass appearance 
 may be observed. They are generally small, being not more than 
 1 mm. in diameter. 
 
 In agar shakes (preferably containing glucose) the colonies appear 
 as dehcate filamentous outgrowths spreading from a small central 
 nucleus. They may attain a diameter of 2 mm. after 48 hour's 
 growth. 
 
 Meat medium : Pink colour or no change in medium according to the 
 
 samples of meat ; softening of the consistency of 
 the meat. The odour is characteristic but not 
 putrefactive. 
 
 ^ilk : Poor growth ; no change in medium. 
 
 Coagulated serum : Little or no liquefaction. 
 
 Gelatine : Liquefied. 
 
29 
 None of the following substances are fermented 
 
 Glycerine 
 
 Maltose 
 
 Starch 
 
 Glucose 
 
 Saccharose 
 
 Mannite 
 
 Laevulose 
 
 Lactose 
 
 Dulcite 
 
 Galactose 
 
 Inulin 
 
 Salicin. 
 
 Animal reactions. B. tetani when injected in the form of a broth 
 culture of 3 to 4 days' growth produces death with the characteristic 
 symptoms of tetanic intoxication. This condition is due solely to 
 Ihe absorption of toxin ; the bacilli do not invade the tissues. 
 Washed and heated emulsions of bacilli and spores, that is to say, 
 cultures deprived of their toxin, are not pathogenic. 
 
 Toxin. A characteristic toxin is produced by the growth of 
 B. tetani in broth. 
 
 Agglutination. Washed and heated organisms injected intra- 
 venously into rabbits provoke the production of agglutinins in the 
 serum of the animals injected. 
 
 By means of the agglutination test the strains of B. tetani can be 
 divided into 4 serological groups. This matter along with its 
 bearing on serum prophylaxis is discussed in detail in the following 
 section by Major Tulloch. 
 
 (ii) Recent Experimental Work on B. tetani. 
 By W. J. Tulloch, M.D., Bt.-Majoe E.A.M.C. 
 
 Lecturer in Bacteriology, University of St. Andrews. 
 Member of the War Office Committee for the Study of Tetanus. 
 Prom the Laboratories of the K.A.M. College and the Lister Institute of Preventive 
 
 Medicine. 
 
 The prevalence of tetanus in the war has been studied statistically 
 by many bacteriologists, Lumiere, Chavasse, H. Pribram. In this 
 country the papers published by Bruce (1,2, 3, 4, 5, and 6), byLeishman 
 and Smallman, and by Cummias and Graeme Gibson, show that, 
 roughly speaking, the disease was comparatively common in the 
 ea;rly months of the war, but that its incidence has steadily fallen since 
 the autumn of 1914 when prophylactic inoculation with antitetanic 
 serum was introduced. 
 
 The tetanus bacillus can generally be cultivated from the wounds 
 of actual cases of the disease and is by no means uncommon in the 
 wounds of patients showing no clinical evidence of tetanus. Thus, 
 from 100 wounds of such cases examined, true tetanus bacilli were 
 recovered from 19 at one time or another. The bacilli may persist 
 in wounds for long periods, as is shown by the fact that in one case 
 B. tetani was obtained 882 days after infliction of the injury. 
 
 A brief description of B. tetani has been given above and will 
 therefore not be repeated here. Nor will the generally known and 
 well established facts of tetanus be touched upon, attention being 
 devoted to the new experience gained since 1914 and particularly 
 to the researches which have been conducted on behalf of the tetanus 
 committee of the War Office. 
 
30 
 
 A new technique which has been found of great use in dealing 
 with large numbers of cases of tetanus, or supposed tetanus, is 
 described in the Appendix. 
 
 One of the most interesting facts estabhshed by the study of 
 a considerable number of strains of tetanus bacilli is that there exist 
 at least 4 distinct serological types. Type I is the standard U.S.A. 
 bacillus and is the organism which appears to have been usually 
 employed in laboratories in Europe prior to 1914 for the preparation 
 of antitetanic serum. Types II, III, and IV have been differentiated 
 since. 
 
 The relative, frequency of occurrence of these types, their value 
 as toxin producers, the similarity or dissimilarity of their toxins and 
 •consequently of antitoxins and other evident problems have been 
 studied. 
 
 With regard to the first of these points it should be emphasized 
 that, in all probability, most of the anti-tetanus serum manufactured 
 in England and in America in the first years of the war was obtained 
 by the employment of toxin from Type I of the bacillus. If such 
 monovalent serum be more efficient against infection with the 
 ■corresponding type of bacillus, one would expect that a census of 
 tetanus bacilli found in wounds, from actual cases of the disease 
 among inoculated men, would show a relative preponderance of 
 infections due to Types II, III, and IV. 
 
 Of 100 strains of B. tetani obtained from cases of tetanus among 
 inoculated men, Type I bacillus was found in 41 instances. Type II 
 in 22, Type III in 33, and Type IV in 4. But of 25 strains of true 
 tetanus bacilli isolated from wounds of men who showed no signs 
 of the disease 76 per cent, were of Type I, 12 per cent, of Type II, 
 8 per cent, of Type III, and 4 per cent, of Type IV. 
 
 This very large discrepancy between the percentage of Type I 
 in the two series is possibly due to the greater efficiency of a mono- 
 valent serum against the corresponding type of bacillus. The 
 methods which have been employed in the investigation of this have 
 been both statistical and experimental. On the statistical side an 
 .analysis of the series of 100 cases of tetanus has shown that of 
 91 cases known to have received tetanus antitoxin prophylactically 
 the case mortahty in the men infected with Type I bacillus was 
 lowest. This, together with the figures for the other types of bacillus, 
 is shown in the accompanying diagram (Fig. I), which deals only 
 with the 91 cases definitely known to have received serum prophy- 
 laxis ; in the remaining 9 cases the records are incomplete. 
 
 Figure II shows the mortahty in cases of tetanus occurring" in 
 men moculated prophylactically with antitoxin. The onset of 
 symptoms occurred within 14 days of the infliction of the wound. 
 
 The influence of the antitoxin employed— presumably monovalent 
 Type I— on the percentage occurrence of the different types of 
 B tetani m ' mdifferent wounds' and in wounds of actual eases 
 •of tetanus, is shown graphically in Figure III. 
 
 This statistical analysis suggests that the serological type of the 
 infectmg bacillus may be of importance in relation to the patho- 
 genesis of tetanus in inoculated men. To prove this by such methods 
 -would require an investigation of a very large number of cases of 
 
31 
 
 /O 20 
 
 50 
 
 40 
 
 10 
 
 
 lO 
 
 30 ac 
 
 
 
 
 
 ' 
 
 
 
 1 I 
 
 
 
 
 
 ^■^r~ 
 
 
 
 11 
 
 
 
 
 
 E 
 
 
 
 
 
 
 
 
 
 
 1 BT 
 
 
 
 
 NIL.-Bt 
 
 
 
 
 The Actual mj/nher ofc/eafhs c/ue fo each fi/pe I 
 
 Fia I. 
 
 10 20 30 4-0 
 
 SO 
 
 ^ 'yyyyyyyy. y/yy/z^^y -tt ttttt^ 
 
 ^ZZ'ZZZZZZZZ'ZZZZZZZZITZZZZZI-S. 
 
 r^'TT? ^yy//y7:7. 
 
 ''/////A 
 
 m 
 
 IncA/ence. \WW^/A Death- r<xte 
 
 Fm. II. 
 
 lO 20 30 iO 
 
 60 /O 
 
 
 
 
 
 ■ I 
 
 
 
 
 y777^77777. 
 
 /'/'////'///, 
 
 \////////^ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ■■117 
 
 ^777S nr 
 
 From casM X////^/A From "fnc/iWere/tt" wounef'S 
 
 Fig. III. 
 
32 
 
 the disease. The matter was, therefore, submitted to experimenti 
 inquiry. 
 
 • It may be stated at once that the results obtained by exper 
 mental methods, though they are of considerable general interes 
 are inconclusive as to the special point under consideration. Takin 
 first the antitoxins, it has been shown that a mono-typical antitoxi 
 is equally active in neutralizing toxin of any or of all the types c 
 B. tetani when the test animals are rats, mice, or guinea-pigs. Bu 
 there is some evidence, not conclusive, that monotypical antitoxi 
 sera may under certain circumstances exhibit specific anti-infectiv 
 properties in relation to the types. 
 
 The difi^erences may be bound up in the different types of bacill 
 but it may be said that anti-bacterial sera containing no antitoxi: 
 or negligible quantities of it, do not prevent infection in the experi 
 mental conditions under which one is forced to conduct the investiga 
 tion, nor is there so far, any evidence that anti-bacterial sera o 
 a given antitoxin content afford more adequate protection agains 
 infection, than do pure antitoxic sera of the same antitoxin content 
 
 This statement is made with reserve as anti-bacterial sera weri 
 not available in sufficient quantity to permit of crucial experimenti 
 being carried out. 
 
 Infection in Tetanus. Tetanus is rarely produced by the inocula 
 tion of bacilli or spores deprived of their toxin by washing or, in th( 
 cases of spores, by washing and heating combined. SusceptibL 
 animals withstand very large numbers of the organisms, 2,000 millioi 
 in the case of guinea-pigs and 200 miUion in the case of mice. Th( 
 toxin of the tetanus bacillus as ordinarily prepared is feebly aggres 
 sive, differing in this respect from the toxins of the organisms o: 
 gas gangrene. When the toxin of B. welchii, in sub-lethal doses 
 is inoculated along with washed and heated spores of B. tetani 
 the spores germinate and the animal succumbs to tetanus. Th( 
 toxin of vibrion septique acts in a similar manner though not sc 
 regukrly. Chemical irritants have been used to initiate tetanus 
 infection in animals. Lactic acid is very uncertain in action: 
 trimethylamine in certain concentrations is able to permit the 
 development of tetanus in guinea-pigs but in mice rarely ; saponine 
 will invariably provoke tetanus in guinea-pigs, seldom in mice; 
 calcium chloride, on the contrary, almost invariably induces the 
 development of infection in the latter. 
 
 The degree of tissue destruction with lactic acid, trimethylamine 
 and saponine is considerable ; with soluble calcium salts it is 
 small. 
 
 The quahty of tissue ' debility ' at the nidus of infection is, there- 
 fore, of great importance, and experiment has shown that the 
 degree and quahty of such debihtation is of greater importance tlian 
 the actual number of organisms injected. 
 
 When an insufficient degree of local disturbance is produced in 
 gumea-pigs, typical tetanus, due to infection, occurs, but some of 
 the animals recover and in ^uch circumstance local tetanus has 
 frequently been noted. Cases of local tetanus in experimental 
 animals was also noted, when the degree of tissue debilitation was 
 relatively great, provided, in this instance, that the animals had been 
 
33 
 
 passively imnmnized with anti-tetanus serum before, or soon after, 
 the injection of the ' spore debiUtant ' mixture. 
 
 The importance of the symbiotic factor, i.e. the influence of the 
 products of B. welchin,, &c., in the initiation of tetanus infection is 
 evident. On the one hand it has been shown that the toxin of 
 B. welchii may provide the conditions required by the tetanus 
 bacillus in initiating the disease ; on the other hand it has been- 
 shown that certain mixed cultures from wounds are non-toxic 
 although they contain true tetanus bacilli. From these cultures the 
 tetanus bacillus may be recovered in a sufficient state of purity to 
 permit of its being agglutinated in the presence of type sera. Further, 
 the organisms are not deficient in toxigenic capacity as is shown 
 by injecting into animals the washed spores, derived from such 
 cultures, together with tissue debilitant, when tetanus develops. 
 Moreover, by growing one of the stock cultures of B. tetani in the 
 presence of these mixed growths, the toxigenic capacity of the 
 tetanus culture was greatly reduced. The particular combination 
 of organisms which leads to this depression of pathogenicity is not 
 yet fuUy determined, nor can it be stated whether it is the infectivity 
 or toxigenicity which is depressed, though presumably the latter. 
 
 All these facts show that the conditions, under which tetanus may 
 occur naturally, are very complex, and that when the infection has 
 begun factors may come into operation which increase or diminish 
 the violence of the disease. 
 
 An inquiry into surgical procedures in relation to degree and persistence 
 of Anaerobe Infection of Wounds. 
 
 This investigation, which was carried out by Miss Cayley, indicated 
 that 
 
 (a) None of the antiseptics investigated could be specially recom- 
 mended as valuable for the elimination of anaerobic infection. 
 
 (b) Anaerobic bacilli, even those of pathogenic significance, may 
 persist in wounds until the completion of the process of repair. 
 
 (c) The degree of anaerobic infection of wounds that have been 
 excised is, on the whole, less than in those that have not been so 
 treated. While excision therefore does not eliminate infection it 
 does alter the condition of the wound in such a fashion that the 
 harmful capacity of any anaerobes present is much reduced. 
 
 (d) In examining the influence of brilliant green and other ani- 
 line dyes on growth of anaerobes in vitro, it was found that their 
 activity was very much reduced when the cultures were made by 
 Tarozzi's method. This reduction of activity is more marked than 
 that caused by the presence of serum. This observation has a direct 
 bearing on the application of aniline dyes as antiseptics in surgical 
 therapeutics. 
 
 Technique. 
 
 In making the investigations summarized above, the following 
 technique was used. 
 
 Swabs taken from wounds — whenever possible these were taken 
 from the deeper parts — were emulsified in about 3 c.c. of saline. 
 
 'c 
 
34 
 
 Of this, 1 c.c. was inoculated into ordinary meat water medium 
 and incubated anaerobically— Culture A. This gave an index of the 
 non-sporing anaerobic bacilU present. 
 
 The remainder— 2 c.c— was heated to 80° C for 10-15 minutes 
 or to 60° C for 30^0 minutes. 
 
 Of this 1 cc. was inoculated into a tube of meat water medium 
 and incubated anaerobically— Culture B. This gave a growth of 
 the sporing anaerobes present. 
 
 The 1 cc. which remained was inoculated into a tube of ' selective ' 
 medium designed to give overwhelming growth of endsporing bacilli 
 —Culture C. 
 
 The selective medium was prepared thus : 
 
 Take 1 lb. of chopped meat, add 1 litre of tap water, and boil for 
 30 minutes. Cool to 45° C, make shghtly alkaline to litmus and 
 add trypsin as for the preparation of Douglas' broth ; then incubate 
 in an open vessel for 4-5 days at 37° C. allowing the material to 
 undergo natural putrefaction. Pass the putrescent material so 
 obtained through paper pulp to clear, and make neutral to phenol- 
 phthalein at room temperature. The material is then sterilized by 
 filtration through a Berkfeld and a Doulton candle in series. The 
 medium is stored in sterile flasks under a layer of paraffin to which 
 sodium formate has been added to the extent of 1 per cent, of 
 the total volume of medium. The flasks should be provided with 
 a hooded delivery tube, so that the material may be distributed 
 as required. It keeps fairly well — about three weeks — but should 
 never be sterilized by heat. 
 
 Before use the sterility of the medium should be tested by inocu- 
 lating quantities of from 5 c.c. to 0-1 c.c. into tubes of meat water 
 medium and incubating anaerobically for at least 7 days. When the 
 medium is to be employed for cultures that may ultimately be used 
 for animal inoculation, it must be shown that the medium itself is 
 non-toxic. Before use, fresh sterile rabbit kidney is added to the 
 medium, J^th P^^^t of a kidney being sufficient for 5 c.c. The tubes 
 to which kidney has been added should be used within 3 days. 
 
 Prepared thus, the medium inhibits the growth of B. sporogenes 
 but allows of the growth of B. tetani, atoxic round endsporing 
 bacilli, and certain oval endsporing bacilli. 
 
 Latterly, in place of using meat water tubes for culture- A and B, 
 the following medium has been employed : 
 
 The flesh of one rabbit is chopped, 1 litre of water containing 
 5 grams sodium carbonate is added, and the mixture is allowed to 
 decompose for 16-24 hours at 37° C. This mixture is then examined 
 and the reaction again adjusted to be slightly alkahne to litmus, 
 and 2 per cent, of trypsin added. The flask is now returned to the 
 mcu^ator for a further period of 16-24 hours. The material is then 
 filtered through paper, made slightly acid, and boiled to coagulate 
 protems, filtered agam and neutraUzed. In neutrahzing, the mean 
 of two titrations is taken at room temperature (the specimens for 
 tiie tests having been previously boiled and rapidly cooled) (1) with 
 phenolphthalem and (2) for a-naphtholphthalein, and the requisite 
 amount, of sodmm hydrate is added to the bulk of the medium. 
 Boil again and finally filter. The medium may then be tubed and 
 
35 
 
 autoolaved or autoclaved and stored in bulk. Before use, j-^h 
 part of fresh sterile rabbit kidney is added to tubes of 5 c.c. Tubes 
 which have been stored should be boiled for 30 minutes and rapidly 
 cooled before the kidney is added. The medium should be used as 
 soon as possible after the addition of the kidney. This medium 
 gives very heavy growths of B. tetani and appears to have some, 
 though not marked, selective properties. 
 
 Tubes A, B, and C were examined at two days' intervals and when 
 C showed a growth consisting mainly of endsporing bacilli the 
 culture was centrifuged, washed, and tested by the agglutination 
 technique. Not infrequently culture failed to show growth, 
 although round endsporing bacilli might ultimately develop in 
 tube A or tube B. In such cases A or B or both A and B were 
 heated to 60° C. for 45 minutes, and subcultures made from them 
 into the selective medium. In this way a number of cultures of 
 B. tetani could be obtained, although they failed when directly 
 inoculated into the selective medium. 
 
 The technique of the agglutination test and that of the preparation 
 of agglutinating sera are described fully in papers dealing with the 
 serological differentiation of B. tetani. 
 
 3. Anaerobes the Action op which may be Ancillary to the 
 Condition of Gas Gangrene. 
 
 The less directly pathogenic anaerobes which proliferate in wounds 
 comprise a very large number of diverse types. It is proposed to 
 describe only the more frequent and important species. It is not 
 claimed that even this list is complete ; the forms included are, 
 however, those which have appeared with sufficient regularity to be 
 considered of some definite importance in wound infection. It is ad- 
 mittedly difficult to estimate the role that these ancillary organisms 
 play in gas gangrene and in the very much more frequent local 
 infections caused by anaerobic bacteria. 
 
 Blood culture from the patient occasionally reveals the presence 
 of some of these types, notably B. sporogenes, but the fact that 
 organisms can be cultivated from the patient's blood is not neces- 
 sarily significant when wounds of large area are present. In pure 
 culture they are practically all to be classed as non-pathogenic for 
 laboratory animals. With few exceptions soluble toxins have not 
 been found in cultures of any of these organisms but in no case 
 so far have they been exhaustively examined for this property. 
 
 In mixed cultures they are for the most part, though not 
 invariably, favourable to each other's growth, and it is pre- 
 sumed that they act with an equally successful mutual assistance 
 in wounds. 
 
 It is well-known that it is not until two or three days after the 
 infliction of the wound that the anaerobic flora of this mixed, less 
 pathogenic type, arises. Some of the characters of these infections, 
 such as putrefactive odour, digestion, and gas formation can be 
 explained from the known reactions of organisms isolated from 
 these cases. These sub-acute conditions may show one or several 
 of the pathogenic anaerobes already dealt with and this mixed type 
 
 02 
 
36 
 
 of infection is always a state that may proceed to acute gas 
 eanerene or to acute tetanus. . i,„-4„j 
 
 In wounds in which these sub-acute local infections have subsided 
 and also in late septic wounds without obvious local symptoms ot 
 anaerobic action, many of these bacterial types Imger on especiaUy 
 when sequestra or adherent sloughs are present, multiplymg suffi- 
 ciently to be very readily cultivated from qmte_ small samples of 
 wound exudate. It is not precisely known what, if any, importance 
 these organisms have in weakening the tissue resistance and m delaymg 
 the healing process, but it is probable that their local action is 
 unfavourable to the patient. ^, i, i „„ 
 
 Pathogenic anaerobes may prohferate as apparently harmless 
 saprophytes, but their presence, even as spores m sequestra and 
 in the scars of healed wounds, are a source of great potential danger 
 especially when disturbed by injury or surgical interference. 
 
 (i) B. sporogenes. Metchnikoff. 
 
 This organism was first described by Metchnikoff, 1908, who 
 distinguished two varieties or races ; ' A ' derived from the faeces 
 of healthy persons, and ' B ' derived from persons suffering from 
 chronic cohtis. He distinguished them chiefly by their morpho- 
 logical characters, A being more slendej than B. Both races 
 have filamentous woolly colonies and agree with each other in 
 cultural characteristics. 
 
 B. sporogenes as found in wounds appears to belong to Metchnikoff 's 
 race A. 
 
 It is present in a very large proportion of wounds, and appears 
 in acute cases of gas gangrene as well as in conditions in which 
 the wound is progressing in a satisfactory manner. It has been 
 cultivated from the blood of the patient during hfe, sometimes 
 alone, but more often in association with other bacteria. 
 
 B. sporogenes is widely distributed in nature ; it is frequently 
 found in earth and in practically all materials exposed to dust. 
 It is a common inhabitant of the alimentary canal of man and 
 animals and can be cultivated from a great variety of sources includ- 
 ing milk. The organism is extremely tenacious of life as is shown 
 by its capacity to survive 8 days in a 5 per cent, phenol solution 
 • (Schiitze), or an exposure in a capillary tube to 100° C. for 45 
 minutes. 
 
 The name B. sporogenes is used in a wide sense and the strains 
 designated by this title are capable of further sub-division upon 
 minute characteristics. 
 
 Morphology. B. sporogenes is an actively motile rod of about 
 3 to 7 f^i in length ; it closely resembles vibrion septique in appear- 
 ance and is more slender than B. welehii. Spores are very readily 
 formed in all media, are oval in shape and central or subterminal 
 in position. The range of bacillary forms is more restricted than 
 in the organisms already described ; long filaments being the only 
 variation. The baciUi are Gram-positive, but in old cultures Gram^ 
 negative individuals are found. 
 Cultural reactions. B. sporogenes is an anaerobic organism, but it 
 
37 
 
 does not exact a perfect condition of anaerobiosis ; it grows readily 
 on agar plates in an exhausted cylinder. 
 
 Colonies. Surface colonies of B. sporogenes are characterized by 
 the production of woolly tangled filaments which grow out all round 
 the more solid centre and down into the medium. Young colonies 
 are very transparent and irregular in shape ; later, they become 
 opaque and woolly with a definite raised centre. Old colonies may 
 be yellowish in colour. They have a tendency to grow large and 
 are solid in type. Isolated colonies on thinly sown plates may 
 attain a diameter of several millimetres. Deep colonies are woolly. 
 
 Meat medium : Vigorous growth ; gas ; alkaline reaction ; digestion 
 
 of the medium and blackening ; the extent of the 
 change of colour varies with different samples of the 
 meat medium. Putrid odour. 
 
 Milk : Digestion occurs without the production of a firm 
 
 clot, leaving a somewhat turbid supernatant fluid : 
 the reaction becomes alkaline. 
 
 Coagulated serum : Liquefied and becomes darker in colour. 
 
 Alkaline egg broth : There is a flocculent precipitate or even a soft clot 
 which sinks to the bottom of the tube leaving 
 a clear supernatant fluid ; the clot is digested to 
 greater or less extent. 
 
 Gelatine : Liquefied. 
 
 Sugars : Glucose, laevulose, and maltose are alone fermented. 
 
 Micrdbic association. One feature of the» behaviour of B. sporo- 
 genes calls for particular notice, namely, its extraordinary capacity 
 for persisting in the presence of other organisms. Only bitter 
 experience can convince workers, unaccustomed to the clinging and 
 pervasive character of this bacillus, of the extreme difficulty of 
 dislodging it from the cultures of any other organism with which it 
 has become associated. B. sporogenes will linger unsuspected for 
 weeks or years in a culture of jB. welchii or of vibrion septique, only 
 to appear when some particularly favourable set of circumstances 
 (such as an alkaline protein medium) permits it to obtain the upper 
 hand in the association. Moreover B. sporogenes will fall into 
 a commensal adjustment with some other organism such as B. tetani, 
 B. terti/us, B. cochlearvus or vibrion septique, lending its putrefactive 
 and proteolytic characters to these types and thus leading to great 
 confusion. These mixed cultures will keep a consistent appearance 
 in regard to type of colony and cultural reactions over long periods, 
 and it is. only by ringing the changes over a wide range of media and 
 conditions that the observer has reason to suspect the composite 
 character of the strain which he beheved to be pure. 
 
 This difficulty of freeing anaerobes from one another is not confined 
 to B. sporogenes, but there is no doubt that it is the most frequent 
 and most persistent intruder. The tendency to grow in mixed 
 colonies, the power of cryptic proliferation within the characters of 
 another species without being detectable, and the capacity of mutual 
 adjustment possessed by anaerobes and by B. sporogenes in particular 
 are at the root of nearly all the more serious confusions and dis- 
 crepancies in the literature of this subject. 
 
38 
 
 Animal reactions. In general B. sporogenes cultures are not lethal • 
 for laboratory animals in doses up to 4 c.c. Some strains are, how- 
 ever, capable of producing a putrid perforating gangrene of the limb 
 injected and a small number of strains are described as being defi- 
 nitely lethal in doses of about 1 c.c. and upwards. _ 
 
 The presence of B. sporogenes in combination with B. welchii 
 quite definitely enhances the pathogenicity of the latter and pro- 
 duces a mixed putrefying type of gangrene closely resembling the 
 putrefying gangrene of wounds which was of frequent incidence 
 in the earhest days of the war. B. sporogenes added to a sub-lethal 
 dose of B. welchii produces a virulent and rapid type of gangrene in 
 experimental animals. In this condition an extensive oedematous 
 swelling of the limb is observed a few hours after inoculation. 
 The skin takes on a shiny bhstered appearance, and a curious 
 green colour. The hair is generally shed over the bhstered parts. 
 Death may supervene at this stage. If the animal survive for 
 twenty-four hours the skin over the oedema becomes sodden and 
 perforates, with the production of a foetid sloughing gangrene of the 
 limb. The animal may die after the gangrene has set in or it may 
 gradually recover even in cases where a considerable amount of 
 tissue has become necrosed. The post-mortem examination of 
 a guinea-pig, dead from a mixed infection of B. welchii and B. 
 sporogenes shows the following features. The skin of the abdomen 
 is soft and greenish, and the hair is easily detached if it has not 
 already come out before death ; there is an extensive fluid oedema, 
 blood-stained and oily in appearance, which extends as a rule 
 over the whole abdomen up to the axillary region. Haemorrhagic 
 patches are sometimes to be observed in the subcutaneous tissues 
 remote from the lesion. The site of the inoculation is oedematous ; 
 the tissues are friable and are dark in colour. There is some gas 
 formation ; the odour is foetid and distinct from that produced by 
 B. welchii alone. The parietal peritoneum is purple and the blood- 
 vessels are injected. Lungs, liver, and spleen are unchanged in 
 appearance. 
 
 The injection of B. sporogenes with vibrion septique gives less 
 certain results, but the general effect is the same. 
 
 B. sporogenes therefore cannot be neglected in the gangrene 
 syndrome though it may be difficult or impossible to gauge its effect 
 or importance in any given case. 
 
 Non-specific toxic products of B. sporogenes. The growth of 
 several of the non-pathogenic anaerobic bacilli, in particular that of 
 B. sporogenes, is accompanied by the production of toxic substances. 
 These are for the most part of a non-specific nature and appear 
 to be formed as the result of the breaking down of the food 
 material. _ Without doubt, similar substances are produced by the 
 pathogenic anaerobes in addition to the formation of specific toxins. 
 The exact relationship of these two kinds of products is not very 
 clear, but the non-specific products lead to much of the confusion 
 concerning the presence of the specific bodies. In certain instances 
 their presence together with the great lability of the specific toxins 
 masks more or less completely the presence of the latter. 
 
 The demonstration of the non-specific toxic substances is best 
 
39 
 
 made by growing the bacilli in a meat medium, as suggested by 
 Besson. This is prepared by placing minced meat in a flask 
 with enough water to cover it. Several cubic centimetres of soda 
 (1 per cent.) are added and the medium is then sterihzed at 115° C. 
 for 15 minutes. If this is inoculated before it is completely cold the 
 anaerobes will grow quite well if the cotton-wool plug be replaced 
 by a rubber stopper. 
 
 The maximum toxicity seems to be developed about the sixth 
 day, after which it gets rather less. The fluid is decanted off the 
 meat, the juice expressed, and the two fluids mixed and filtered. 
 
 Toxicity. The toxicity of such a fluid varies considerably, but as 
 a rule from 1 c.c. upwards injected into the peritoneum of a young 
 guinea-pig will produce almost immediate signs of intoxication 
 which are not infrequently followed by a fatal issue. If death does 
 not occur in the course of a few minutes the animal usually recovers 
 completely. After the. injection of a lethal dose the animal imme- 
 diately becomes restless, jumps about, jerking its head upwards in 
 a pecuhar manner, then becomes convulsed and falls over on its side. 
 The symptoms observed are therefore quite distinct from those 
 which follow the intraperitoneal injection of fatal doses of the true 
 anaerobic toxins. 
 
 Nature of the poison. Dale and Barger stated that the poisonous 
 substance present passed over when the media was distilled. An 
 analysis of the distillate convinced them that they had to deal 
 with a volatile poison probably of the nature of an ammonium base. 
 
 A series of experiments by Mcintosh and Pildes suggested that 
 some sulphide — probably ammonium sulphide — played an im- 
 portant part ; the presence of sulphides in the cultures can readily 
 be demonstrated, and the injection of weak solutions (0-5 c.c. of 
 a J per cent, sol.) of ammonium sulphide produces symptoms of 
 immediate intoxication and even death. 
 
 The influence of the growth products of B. sporogenes upon the 
 action of the toxin of B. welchii has been studied in mice (W. E. 
 Bullock and Cramer '(1 and 2)). When a mouse has been injected 
 with a sub-lethal dose of sterile toxin of B. welchii together with sterile 
 filtrate from a culture of B. sporogenes, in an amount of either 
 substance insufficient of itself to produce symptoms, the animal dies. 
 
 Specific toxin. A true soluble toxin has not as yet been demon- 
 strated for B. sporogenes. 
 
 Agglutination. Agglutinins can be produced in the blood of 
 rabbits. So far as is at present known strains of B. sporogenes can 
 be divided into at least two serological groups by means of this test. 
 
 (ii) B. parasporogenes. Mcintosh. 
 
 This organism was originally isolated and described as Type XII 
 by- J. Mcintosh (1917). It agrees with B. sporogenes in all its 
 characters with the exception of two. In the opinion of the com- 
 mittee these differences, which are quite constant, are considered to 
 be of sufficient importance to make it advisable to place the organism 
 in a separate species and the name parasporogenes is adopted. 
 
 B. parasporogenes differs from B. sporogenes in the nature of its 
 
40 
 
 colony ; in agar shakes it is lenticular or slightly irregular in shape 
 but not woolly. 
 
 The second feature which distinguishes B. parasporogenes is the 
 production of specific agglutinins. In morphology and in cultural 
 characters and in all other respects the two organisms resemble 
 each other closely. 
 
 (iii) B. tertms. Henry, 1917 ^ (Eodella's Bacillus III) (von Hibler's 
 
 Bacillus IX). 
 
 B. tertms is a saccharolytic oval endsporing anaerobe. It is found 
 both in early and in late wounds and may be present in severe cases 
 of gas gangrene, or in ordinary wound infections. 
 
 Morphology. B. tertius is sluggishly motile. The rods are slender 
 and about 3 to 5 microns in length, they are Gram-positive when young 
 (12-18 hours), but soon tend to become Gram-negative especially 
 in fluid media. Spores are readily formed, and as stated, are oval 
 and strictly terminal. The morphology of the organism is best 
 studied in cultures 24 to 36 hours old. 
 
 Cultural reactions. B. tertius requires only a moderate degree of 
 anaerobiosis for growth. 
 
 Surface colonies are small (1 mm.) and are rounded or crenated ; 
 when young (24 hours) they are delicate and almost transparent ; 
 examined under a hand lens by transmitted light they have a char- 
 acteristic greenish-blue iridescent appearance not at aU unlike 
 a small opal. On certain media they become more opaque and 
 slightly granular when incubated for two or three days. 
 
 Colonies in agar shakes are small, lenticular or irregular in shape 
 and do not show branching filaments. 
 
 Meat medium : Pink colour ; gas ; no blackening ; no digestion. 
 
 MUk : Acid clot in 3 to 6 days ; some gas. 
 
 Coagulated serum : Not liquefied. 
 
 Gelatine : Not liquefied. 
 
 Substances fermented. 
 
 Substances not fermented, 
 
 Glucose 
 
 Glycerine 
 
 Laevulose 
 
 Inulin 
 
 Galactose 
 
 Dulcite. 
 
 Maltose 
 
 
 Saccharose 
 
 
 Lactose 
 
 
 Mannite 
 
 
 Salicin. 
 
 
 (iv) B. cochleariMs. Douglas, Fleming, and Colebrook (3). 
 
 This organism was first described as type III C. by Mcintosh 1917 
 _ It IS an endsporing bacillus very frequently found in wounds'. 
 Like ii. tertms it may be present both in acute early infections and 
 m late septic cases. It is frequently associated with B. tetani. 
 
 mi^rltg'armS J:Ss'"'" '^ ^^"^ '^"^"^^ '* ^^« ^'^^ '"^^ --* — °" 
 
41 
 
 Morphology. B. cocMearius is an actively motile slender rod ; 
 the length is variable, and there is a tendency to give up the stain 
 in Gram's method. The spores are strictly terminal and are 
 oval when fully developed, giving the organism a spoon-shaped 
 appearance, hence the name cochlearms. There are stages, how- 
 ever, during their growth in which the young spores may be 
 almost spherical. In perfectly pure cultures on ordinary media 
 spores are not readily formed. The morphological features can be 
 well observed in an alkaline digest broth containing fresh tissue, 
 as this medium is particularly favourable to the formation of 
 spores. The examination should be made after 24-36 hours' in- 
 cubation. An impure culture containing a slight admixture of B. 
 sporogenes gives a vigorous growth of B. cochlearius with active 
 sporulation — an instance of stable microbie association between two 
 organisms. Such a culture resembles B. putrificus (Bienstock) 
 and has frequently been designated by this name. It is proteolytic, 
 digests meat and inspissated serum and gives off a putrid odour. 
 It is often extremely difficult to dissociate B. cochleariiis from 
 B. sporogenes in such a mixed culture. 
 
 Cultural reactions. In pure culture B. cochlearius is one of the less 
 vigorous anaerobes. Surface colonies can be obtained upon serum 
 agar plates or serum agar slopes under good conditions of anaero- 
 biosis. They are delicate, glass-clear droplets, sometimes with 
 faintly crenated edges. When colonies show minute opaque spots 
 or opaque centres or have rootlets penetrating into the medium 
 it is a sign of admixture with some other anaerobe, probably B. 
 sporogenes. Colonies in agar shakes are lenticular in shape. 
 
 Meat medium : Very little change in the colour. There is no digestion 
 
 and very little gas. There is a characteristic odour 
 which is not putrid. 
 
 jyClk : Scanty growth ; no change in medium. 
 
 Coagulated serum : Not liquefied. 
 
 Gelatine : Not liquefied. 
 
 None of the following substances are fermented : 
 
 Glycerine Maltose Mannite 
 
 Glucose Saccharose Dulcite 
 
 Laevulose Lactose Salicin. 
 
 Galactose Inulin 
 
 Agglutinins can be produced in rabbits by the inoculation of 
 washed baciUi. 
 
 The inoculation of living cultures of B. cochlearius intramuscularly 
 into guinea-pigs in doses up to 3 c.c. does not produce pathogenic 
 results; 
 
 B. cochlearius is distinguished from B. tertims by its failure to 
 ferment any of the sugars, by its motiHty, characteristic odour, and 
 the production of agglutinins in the blood of rabbits. 
 
 (v) B. tetanomorphus. 
 
 This is the organism described by Mcintosh and Fildes as ' Type IX, 
 B. pseudotetanus '. It closely resenibles B. tetard in its morphological 
 
42 
 
 appearance ; but differs in cultural characters, its power to ferment 
 maltose and glucose, the absence of liquefaction in gelatine and in 
 its, inability to produce the specific toxin. It is frequently found 
 in wounds, especially in late infections, and is often present along 
 with B. tetani. 
 
 Morphology. B. tetanomorphus is highly motile ; the rods are 
 Gram-positive. The spores are spherical, terminal in position, and 
 are formed in all media in the course of 24-36 hours. 
 
 Growfhreactions. B. tetanomorphus grows best in an alkaline medium. 
 When a sample of wound exudate containing B. tetanomorphus is in- 
 cubated in meat medium the organism makes its appearancein numbers 
 about the 3rd to the 5th day, that is to say considerably later than 
 B. sporogenes. The conditions produced in the medium by the 
 growth of B. sporogenes are favourable to B. tetanomorpJius. 
 
 Surface colonies can be obtained upon serum agar slopes and 
 plates. They are deHcate, flat, and have a slightly crenated edge. 
 There is a tendency to grow in a continuous surface film. Colonies 
 in deep agar are small in size and irregular in shape but are not 
 woolly or branched. 
 
 Meat medium : Pink colour ; gas ; no digestion or blackening. (This is 
 the best medium for the growth of B. tetanomorphus.) 
 Milk : No change ; scanty growth. 
 
 Grelatine : No liquefaction. 
 
 Substances fermented. Substances not fermented. 
 
 Glucose Laevulose Lactose 
 
 Maltose. Galactose Inulin 
 
 Saccharose Glycerine 
 
 Salicin Dulcite. 
 
 Ma unite 
 
 Agglutinins for B. tetanomorphus can be demonstrated in the serum 
 of rabbits inoculated intravenously with the bacillus. B. tetand' 
 morphus is not pathogenic for guinea-pigs. 
 
 (vi) B. aerofetidus. Weinberg and Seguin (8). 
 
 Morphology. This is a small slender baciUus about 3-5 fi in length ; 
 it has a tendency to be Gram-negative. Motility is shght. 
 
 Growth reactions. Spores are not readily formed ; they are 
 subterminal. Surface colonies on serum are round and trans- 
 parent and may attain to 1 or 2 mm. in diameter after 24 hours' 
 incubation. 
 
 Deep colonies are small and irregular. 
 
 J^eat : Putrid odour ; change of colour, first reddening and 
 
 then blackening. 
 Milk medium : Clot and gas in 24-48 hours, later, a certain amount of 
 
 digestion. 
 Coagulated serum : Putrid odour ; liquefaction. 
 Gelatine : Liquefied. 
 
 Alkaline egg broth : Soft clot which may be partially digested. 
 
 This organism is not pathogenic in pure culture for guinea-pigS, - 
 
43 
 
 Fermentation. There is some divergence of opinion as to the 
 sugars fermented by this organism. Glucose, maltose, and lactose 
 were, according to Mcintosh, the only sugars fermented, whereas 
 Henry (2) found that laevulose and salicin were also affected. 
 
 (vii) B. bifermentans. Tissier and Martelly. 
 
 This bacillus was first described by Tissier and Martelly in 1902 
 under the name of B. bifermentans sporogenes. It received the name 
 of bifermentans owing to its being the first anaerobe in which a 
 capacity to split both proteins and sugars was definitely recognized. 
 
 B. bifermentans is occasionally found in acute cases of gas gangrene, 
 as well as in late septic cases. 
 
 Morphology. In the non-sporing state it is a stout, non-motile 
 rod closely resembling B. welchii. Individual elements may be very 
 short. Chains are frequently formed. Spores are readily formed 
 in all media ; they are central or subterminal. 
 
 Cultural reactions. Surface colonies are- round or crenated. Deep 
 colonies are lenticular or irregular but without filamentous out- 
 growths. 
 
 Meat medium : Blackened and digested ; putrid odour ; gas. 
 
 Milk medium : Casein is precipitated and later digested. 
 
 Coagulated serum : Liquefied. 
 
 Gelatine : Liquefied. 
 
 Alkaline egg brotli : Soft clot wMcli is later digested to a variable extent. 
 
 Fermentation. Glucose, laevulose, and maltose are alone fer- 
 mented. B. bifermentans is non-pathogenic in pure culture for 
 guinea-pigs. Agglutinins can be produced in rabbits inoculated 
 with the organism. 
 
 (viii) B. putrificus. Bienstock (2). 
 
 This organism was first described by Bienstock in 1899 under the 
 name" of B. puirijicus. He and many subsequent writers consider 
 that it is strongly proteolytic. Experience during the war has shown 
 that many of the cultures of B. putrificus so-called are really mixtures 
 of B. cochleariMS or B. tertius with B. sporogenes. 
 
 The following is a brief synopsis based upon the incomplete 
 descriptions of Bienstock, Salus, Tissier and Martelly, Metchnikoff, 
 Wiircker, Weinberg and Seguin, and others. 
 
 B. putrificus is a slender Gram-positive rod, the spores are oval 
 and strictly terminal giving the drumstick appearance described by 
 Bienstock. 
 
 The cultures are strongly proteolytic; gelatine and serum are 
 liquefied and milk digested with or without the formation of a clot. 
 
 The organism is described as being non-pathogenic (Bienstock, 
 Tissier and Martelly). 
 
 (ix) B. sphenoides. Douglas, Fleming, and Colebrook (3). 
 
 This is a small motile bacillus. Gram-positive when young but 
 rapidly becoming Gram-negative. In the non-sporing state it is 
 
44 
 
 fuisiform in shape and often arranged in pairs placed end to end not 
 unlike a Hoffmann's pseudodiphtheria bacillus. _ _ 
 
 The spore when first formed is subterminal, but as it mcreases m 
 size its position becomes terminal ; it is perfectly round and is 
 of large size, being considerably broader than the body of the 
 bacillus. As the spore reaches its maximum development the 
 body of the bacillus assumes a wedge shape the point being situated 
 at the end opposite to the spore. It was the appearance of the 
 bacillus at this stage which suggested the name sphenoides. As 
 the body of the bacillus degenerates in older cultures, the orgamsm 
 becomes shaped Hke a drumstick, but the end opposite the spore 
 always remains pointed. 
 
 Cultural reactions. Surface colonies on agar attain to a size of 
 about 1 mm. in diameter. They are round and usually have smooth 
 edges ; occasionally, however, slight irregularities are seen. 
 
 Meat medium ; A little gas is formed ; no change is produced in the 
 
 colour. 
 Milk medium : Acid is formed and occasionally a soft clot. 
 
 Coagulated serum : No change. 
 Gelatine : Not liquefied ; good growth. 
 
 Broth : The growth is profuse, an even turbidity being 
 
 produced. 
 
 The fermentation reactions of this organism appear to be some- 
 what variable. Glucose, maltose, galactose, lactose, and saHcin are, 
 however, fermented by the three strains isolated. Two of the 
 strains ferment mannite, saccharose, dextrine, and starch in addition. 
 
 Incidence. It was isolated in pure culture from three cases and 
 was observed in two others in a total of sixty-one (Douglas, Flencdng, 
 and Colebrook (3)). The majority of the patients had had gas 
 gangrene. 
 
 (x) B. hutyricus. 
 
 B. hutyricus is a non-pathogenic bacillus rarely met with in 
 wounds. In 1861 Pasteur (1) described, under the name of ' Vibrion 
 butyrique ', an anaerobe which was capable of producing butyric 
 fermentation. This organism of Pasteur is probably identical with 
 the Clostridium butyricum of Prazmowski, the Bacillus, amyldbacter 
 of Gruber and with the mobile ' Buttersaurebacillus ' of Grassberger 
 and Schattenfroh (1 and 2). 
 
 The literature deahng with the organism is full of confusion and 
 it is almost impossible at the present date to decide exactly with 
 what organisms the above authors worked. The characters of what 
 the committee consider to be B. hutyricus are detailed below from 
 the study of a single strain isolated by Fleming from a wound. 
 
 Morphology. It is a small Gram-positive motile bacillus. The 
 spores are small and oval in shape, usually, central though occa- 
 sionally subterminal in position. The most frequent appearance 
 is that of an elongated Clostridium. 
 
 Cultural reactions. Surface colonies on serum-agar are small, 
 flat, irregular in shape, greyish in colour and semi-transparent. 
 Shake colonies are small irregular lenticular masses. 
 
45 
 
 Meat medium : No digestion ; some gas. 
 
 Milk medium : A firm acid clot appears in 24 tours. 
 
 Coagulated serum : No change in the medium. 
 
 Gelatine : Not liquefied. 
 
 Fermentations. Glucose, maltose, lactose, saccharose, and starch 
 are fermented. 
 
 (xi) B. multifermentans tenalbus. Stoddard (3), 1919. 
 
 This is a non-pathogenic organism recovered from a case of gas 
 gangrene. It is an infrequent organism but is of interest as it shows 
 a morphological resemblance to vibrion septique. 
 
 Morphology. It is a Gram-positive motile bacillus. The spores are 
 central or subterminal. When grown upon egg or serum media, 
 navicular types, filaments, and swollen club-shaped individuals are 
 formed. 
 
 Cultural reactions. Surface colonies, cultivated under anaerobic 
 conditions, may attain a large size (3 to 5 mm.) ; they are round, with 
 slightly irregular edges ; after incubation for several days they 
 become white and opaque and rise up considerably above the surface 
 of the agar. In glucose-agar shakes the colonies are white and 
 opaque, irregular or lenticular in shape with projecting outgrowths. 
 
 Meat medium : G-as ; no obvious digestion ; no blackening ; the odour 
 
 is not putrid. 
 Milk medium : Acid and clot. 
 
 Coagulated serum : No change. 
 Gelatine : Not liquefied. 
 
 Substances fermented. Substances not fermented. 
 
 Glycerine Glucose Mannite 
 
 Maltose Saccharose Dulcite. 
 
 Lactose Inulin 
 
 Eaffinose Salicin. 
 
 4. Classification of the Anaeeobic Bacilli found in Wounds. 
 
 The characters available as a basis for the classification of the 
 anaerobic bacilli can be grouped under the following headings : 
 
 Morphology. 
 
 Cultural characteristics. 
 
 Serological reactions. 
 
 Production of characteristic toxin. 
 Each of these categories deals with a different aspect of the organism, 
 and owing to the difficulties inherent in the study of bacteria no one 
 of these aspects taken by itself furnishes the data for a satisfactory 
 classification. Since, therefore, a combination of these several 
 different series of characters has to be- relied upon, the order and 
 relative value of the different criteria must be considered. 
 
 (i) Morphology. 
 
 The first subdivision is based upon morphology and the next upon 
 cultural reactions. The production of characteristic toxins is a quality 
 
which is valuable in classification where available, and serological 
 reactions form a final series of distinctions which may or may not 
 be of specific value. 
 
 The anaerobes may be divided into three groups upon their 
 morphology : 
 
 (a) Organisms with oval spores which are central or sub-terminal 
 
 in position. 
 (h) Organisms with oval spores which are strictly terminal in 
 
 position, 
 (c) Organisms with spherical spores which are strictly terminal 
 in position. 
 
 (ii) Cultural Characteristics. 
 
 Further divisions can be made upon cultural reactions such as 
 the capacity for decomposing protein, carbohydrates and alcohols. 
 
 All organisms have a certain capacity for attacking proteins and 
 splitting sugars, but these characters have to be sufficiently marked 
 to be readily appreciable under the test-tube conditions of artificial 
 cultivation and have to be somewhat arbitrarily defined for the 
 purposes of classification. 
 
 In the case under consideration proteolytic characters are judged 
 by the capacity of liquefying coagulated serum and of gelatine. 
 In working with the anaerobes the gelatine test has been used in 
 the special manner first employed by Eiineberg and differs from 
 that generally employed in bacteriology. Owing to the unsatis- 
 factory amount of growth of the anaerobes obtained at 25° C, the 
 inoculated gelatine stab is incubated at 37° C. for forty-eight hours 
 under anaerobic conditions, thereafter it is cooled by being placed in 
 a beaker of cold water and the reading taken. In the case of the 
 saccharolytic organisms the fermentation is recognized by a definite 
 capacity for producing acid or acid and gas in sugar-containing media. 
 
 Subsections based upon these characters can be arranged as follows : 
 
 A. Organisms showing proteolytic and saccharolytic properties. 
 
 B. Organisms showing proteolytic properties only. 
 
 C. Organisms showing saccharolytic but no proteolytic pro- 
 
 perties. 
 
 D. Organisms showing no obvious proteolytic or saccharolytic 
 
 properties. 
 
 Subsection A would be subdivided into : 
 
 A 1. Organisms predominatingly proteolytic with, however, a 
 restricted but definite capacity of fermenting certain 
 carbohydrates. 
 
 A 2. Organisms predominatingly saccharolytic possessing, how- 
 ever m addition, slight proteolytic properties as shown by 
 the liquefaction of gelatine. 
 
 The criterion in accordance with which an anaerobe is here classified 
 as a predominatmgly proteolytic organism is the capacity to produce 
 liquefaction of coagulated serum. r j r 
 
 The accompanying table shows the arrangement of the organisms 
 classmed m these terms. 
 
47 
 
 
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52 
 
 (iii) Agglutination Beactions. 
 At a very early period in the history of anaerobic bacteria, it 
 was found that the injection of the bacilli into animals led to the 
 production of agglutinins. Thus Leclainche and Morel prepared 
 a serum which agglutinated vibrion septique in dilutions of 1 in 500 
 and upwards, while Leclainche and Vallee (1) prepared a serum which 
 agglutinated B. chauvoei in dilutions up to 1 in 3,000. These writers 
 claimed that the sera produced were rigorously specific. About the 
 same time the production of specific agglutinins against B. tetani 
 was demonstrated by Courmont. Markoff (1911) showed that aggluti- 
 nation was of considerable value for the differentiation of certain 
 anaerobes, in particular of ' Eauschbrand ', mahgnant oedema and 
 ' Geburtsrauschbrand ' and almost identical results were obtained by 
 K. P. Meyer. 
 
 The more recent researches deaUng with the identification of the 
 various anaerobic bacteria of wounds, has again directed attention 
 to the question of agglutinins. Amongst those who found that the 
 agglutination reactions were sufficiently specific to be of diagnostic 
 value may be mentioned, Gaehtgens, Landau, McLitosh, H. C. Plant, 
 Pfeiffer and Bessau, Robertson and Weinberg and Seguin. Piirth, 
 on the other hand, was much less successful. The consensus of 
 opinion is, therefore, that the agglutination reactions of the majority 
 of anaerobes with the exception of B. welcMi and B. tertius are 
 of great value. According to the above writers the test may be 
 used not only for the differentiation of species but also for the 
 differentiation of sub-species. ' 
 
 Agglutinating sera are best prepared by intravenous injection 
 into rabbits, surface cultures or the centrifugalized deposit of broth 
 cultures suspended in saline should be used for the injection. Eabbits 
 are relatively insusceptible to these emulsions and a whole serum 
 agar slope may be injected. Usually three or four injections made at 
 three or four days interval are necessary to produce a high titre serum. 
 It is of course essential that the cultures used to prepare the 
 agglutinating sera should be absolutely pure. Failure to observe 
 this will lead to endless confusion and worry. 
 
 The agglutination test is best carried out in small test-tubes or 
 m Pasteur pipettes according to Wright's method, immersed in 
 a water bath at 56° C. The .bacterial suspensions should be obtained 
 from young cultures, well washed in saline and diluted to give 
 shghtly opalescent emulsions. As a rule living bacilli are more 
 
 Xr twotr *^°'' ^^^'^ ^^ ^''*- ^'^^^^' ^^^ *^i^«^ 
 
 arenas foHowsf '"^ *''*' '"^ ''^''^ *^ *^' ^"^' ''^^^^^ ^^^^^obes 
 1. B. welchii. 
 Werner (1905) prepared agglutinating sera against four ' Gasbrand ' 
 
 1 in 1,000. No agglutination was obtained Jn ?ross ailutin^tin^ 
 
was agglutinated to 1 in 40, ten strains to 1 in 2Q and ten strains 
 not at all. Weinberg (1918) obtained serum from two horses which 
 agglutinated the homologous strains in 1 in 500 and 1 in 2,000 
 respectively, but he does not state the action of these sera on other 
 examples of the organism. 
 
 Bull (1917) mentions the agglutination and lysis of B. welchii by 
 normal rabbit and guinea-pig sera and also agglutination in immune 
 sera but has given no exact details of his results. Gaehtgens (1917) 
 found he could prepare agglutinins to all his anaerobes with the 
 exception of B. welchii. Pfeiffer and Bessau claimed to have pro- 
 duced a serum against B. welchii which agglutinated the homologous 
 strain in 1 in 40. This serum, however, had no effect on fourteen 
 other strains of B. welchii. Mcintosh (1917) found it impossible to pro- 
 duce definite agglutinating sera against any of his B. welchii strains. 
 
 It may be assumed from the above evidence that the agglutination 
 test is of no value in the diagnosis of B. welchii. 
 
 2. Vibrion sejptique. 
 
 As previously mentioned, this was one of the first anaerobes 
 for which specific agglutinins were demonstrated. Weinberg and 
 Seguin (18) in titrating a monovalent agglutinating serum against 
 nine strains of vibrion septique isolated from human cases found that 
 four of these (including the homologous culture) were agglutinated in 
 a dilution of 1 in 1,000, eight in 1 in 600 and one in 1 in 10 only. 
 The same serum agglutinated a type strain of vibrion seftique from the 
 Pasteur Institute collection 1 in 500 and a stock strain of so-called 
 B. chauvoei from the same source in 1 in 10 only. 
 
 More recent research upon the agglutination reactions of vibrion 
 seftique strains has shown that there are at least three serological 
 types, which while producing different agglutinins are found to be a 
 single group as regards their toxin antitoxin reactions. (Eobertson.) 
 
 3. B. oedematiens. 
 
 Weinberg (18) has prepared rabbit sera which agglutinate the 
 homologous organism 1 in 100, but have no action on other strains 
 of the organism. The organism is a difficult one to work with, 
 because most strains tend to agglutinate spontaneously. 
 
 B. tertius. Attempts to obtain an agglutinating serum from 
 rabbits for this organism have failed. 
 
 4. B.fallax. 
 
 The following table of agglutination reactions is given by 
 Wemberg (18). 
 
 Strains. Serum 1 Serum 3 Serum 4 
 
 rabbit. rabbit. human. 
 
 1 1/500 1/100, 1/50 
 
 2 1/50 1/50 
 
 3 1/500 
 
 4 1/100 
 
 5 
 
 None of the strains of B. Jallax dealt with were agglutinated by 
 sera prepared against B. welchii, V. septique, B. oedematiens, B. 
 sporogenes and B. aerofetidus, nor did the B. fallax sera agglutinate 
 any of the above mentioned anaerobes. 
 
54 
 
 5. B. sjporogenes. 
 
 Metchnikoff in his original article on B. sporogenes described two 
 types but did not mention agglutinins. Mcintosh (1917) by means 
 of agglutination tests found at least three different types. 
 
 The following table illustrates a series of experiments undertaken 
 with four strains of B. sporogenes which were indistinguishable 
 morphologically or culturally (Henry). 
 
 Sera. 
 
 Normal rabbit (5) 
 Normal sheep (1) 
 Normal horse (3) 
 Mcintosh (rabbit) 
 Weinberg (rabbit) 
 Rabbit (against 3) 
 Rabbit (against 4) 
 Rabbit (against 1) 
 Rabbit (against 1) 
 Sheep (against 1) 
 Goat (against 1) 
 F8. 
 T8. 
 M8. 
 8. 
 
 Strains. 
 
 SO 
 
 50 
 
 
 
 
 
 50 
 
 50 
 
 
 
 
 
 500 
 
 500 
 
 
 
 
 
 200 
 
 200 
 
 . 
 
 
 
 4,000 
 
 2,000 
 
 — 
 
 — 
 
 4,000 
 
 4,000 
 
 4,000 
 
 4,000 
 
 — 
 
 — 
 
 8,000 
 
 8,000 
 
 — 
 
 — 
 
 64,000 
 
 32,000 
 
 — 
 
 — 
 
 250,000 
 
 64,000 
 
 — 
 
 — 
 
 2,000 
 
 2,000 
 
 50 
 
 — 
 
 500 
 
 500 
 
 — 
 
 50 
 
 1,000 
 
 500 
 
 — 
 
 50 
 
 1,000 
 
 1,000 
 
 — 
 
 — 
 
 F 8, T 8, M 8 and 8 were German gas gangrene sera prepared 
 apparently by the inoculation into horses of mixed whole cultures 
 of anaerobes. 
 
 A glance at the table shows conclusively that the four strains of 
 B. sporogenes, which were taken at random for the experiment, may 
 be divided into two serological groups, each of which is distinctly 
 demarcated from the other in the possession of the capacity to 
 produce a specific agglutinin. 
 
 An additional experiment with eleven other strains of B. sporogenes 
 showed that of these only two came into the 1-2 group and only 
 one into the 3-4 group. In the case of the remaining eight strains 
 there was no agglutination. One of the latter, a strain isolated from 
 a case of haemothorax was agglutinated by the patient's own serum 
 and by his serous pleuritic exudate, both collected during convales- 
 cence, in a titre of 1 in 2,000. 
 
 The agglutination reaction would appear, therefore, to divide the 
 B. sporogenes group of organisms into a number of sub-groups. 
 
 6. B. tetani. 
 
 TuUoch (1) has shownthat the strains of B. tetani he investi- 
 . gated are divisible on their serological reactions into three groups. 
 
 Summary. 
 
 The application of agglutination as a diagnostic procedure in the 
 case of the anaerobes results in a differentiation which ma^ be said 
 to reveal evidence of tribal rather than of national characteristics. 
 
 Where an anaerobe can be shown to be pathogenic, its capacity 
 to produce a toxin which presents well-defined characters provides 
 an unassailable basis for classification. Hence, in dealing with 
 a group or collection of strains, the individual members of which 
 have m common the property of producing one and the same toxin. 
 
65 
 
 any further division into sub-groupa that results from the apphcation 
 of an agglutination test leads to an unwarranted multipUcation of 
 separate bacteriological types. In this respect the agglutination 
 test as applied to the anaerobes becomes ultraspecific. 
 
 (iv) Toxin-antitoxin Beactions. 
 
 B. welchii, vibrion septique and B. oedematiens produce specific 
 toxins, each of which has its own particular characters, and each of 
 which, when inoculated into animals, gives rise to specific antitoxins. 
 
 A well-washed emulsion of B. welchii may be introduced in 
 large doses into experimental animals without producing infection. 
 The combination, however, of a few washed bacilli with a sub-lethal 
 dose of toxin results in the production of a rapidly fatal gas gan- 
 grene. The toxin, therefore, is endowed with powerful aggressive 
 properties. This aggressive action of B. welchii toxin can be 
 successfully neutralized by B. welchii antitoxin, but not by other 
 antitoxins. One is thus enabled to say quite definitely that if 
 the toxin produced by an unknown anaerobe is completely de- 
 prived of its lethal effect in animals when it is mixed with, for 
 example, B. welchii antitoxin, then the organism in question is 
 B. welchii. A simpler method of demonstrating the same fact 
 consists in mixing a liquid culture of the organism with varying 
 doses of antitoxin before inoculation into animals. If the infection 
 be inhibited with B. welchii antitoxin then the organism is B. welchii. 
 Or again, if it can' be shown that the inoculation of an animal with 
 a B. welchii antitoxin renders it passively immune to infection with 
 a particular organism, then one can be certain that the organism is 
 B. welchii. 
 
 The same is true for vibrion septique and for B. oedematiens ; the 
 toxin of each gives rise to a specific antitoxin which is capable not 
 only of neutralizing the corresponding toxin, but also of inhibiting 
 the infection , of experimental animals by whole cultures of the 
 organisms in question. 
 
 This method of arriving at an exact diagnosis of the nature of an 
 unknown pathogenic anaerobe is applicable both to pure cultures 
 and to mixed cultures. 
 
 The question of toxin-antitoxin reactions is dealt with in detail 
 in the serological section of this Report. 
 
 5. Biochemistry. 
 
 It is to Spallanzani in 1776 that we owe one of the first, if not the 
 very first, real inquiries into the behaviour of hving organisms in 
 the absence of oxygen. 
 
 In a set of experiments which are a model of experimental technique, 
 he exposed to a vacuum animalculae in small tubes, sealed at one 
 end. Similar control tubes were left in the air. The experiments 
 were continued for 24 days, at the end of which time he observed that 
 the animalculae in the anaerobic tubes were dead, while those 
 exposed to air survived. 
 
 Other organisms were not so susceptible to privation of oxygen, 
 for he recounts that certain animalculae lived 35 days in vacuo, 
 
56 
 
 and others died in 14, 11, and 8 days. Some were only able to with- 
 stand oxygen want for 48 hours. 
 
 He was able to observe that motility and the power of reproduction 
 was not totally inhibited by a vacuum, but he believed that depriva- 
 tion of air eventually stopped both these processes. 
 
 He also showed, in the case of some organisms with which ho 
 worked that a partial vacuum was favourable to their development. 
 He thus anticipated by more than a hundred years the work_ of 
 subsequent investigators on oxygen minima. This astonishing 
 investigation appears to have been entirely overlooked by aU the 
 later workers in this field. 
 
 (i) The Mechanism of Anaerobiosis. 
 
 Pasteur in 1861 recognized that certain organisms were able to 
 exist and multiply in the absence of free oxygen. This belief in 
 the possibility of protoplasm continuing to function under strictly 
 anaerobic conditions has always encountered a certain amount of 
 scepticism, but it may be said that the arguments advanced against 
 the original Pasteur conception do not carry great weight with later 
 workers. 
 
 The matter of oxygen tolerance is one of a large number of grada- 
 tions in the animal kingdom, and is therefore analogous to many 
 other processes connected with life and growth. At the upper end 
 of the scale one encounters animals which exist normally in an 
 atmosphere containing twenty volumes per cent, of oxygen, but 
 which can tolerate for long periods pressures of oxygen up to three 
 atmospheres, or concentrations of oxygen as low as seven per cent. 
 Above and below these pressures the animal rapidly succumbs. 
 Other animals, such as Spirostomum, one of the ciliates, are killed 
 by the partial pressure of the oxygen in the atmosphere, but exist 
 normally at one-third of this pressure. 
 
 With bacteria all gradations of oxygen tolerance are foimd. 
 Some species cannot live without a certain partial pressure of oxygen, 
 while others, the facultative anaerobes, are capable of hving in or 
 without the presence of this element. The obligate aerobes are 
 distinguished by the fact that continued absence of oxygen leads 
 eventually to impairment of function and cessation of growth. 
 
 Prom these forms, the transition to one which does' not require 
 free oxygen for its existence is a natural one. In this case oxygen 
 acts as a poison. For this toxic action we have an analogy, higher 
 up in the scale, where a certaiu concentration of oxygen is necessary 
 for hfe but a greater one is fatal. 
 
 Much careful work has been done in establishing the fact that 
 anaerobic bacteria hve in the complete absence of oxygen. To this 
 may be added the consideration that no amount of reserve oxygen 
 present m the cell would suffice to carry on such an intense metabolism 
 as IS often found during the growth of these organisms. 
 
 Whether the optimum growth of anaerobic bacteria takes place in 
 complete absence of oxygen is still a matter for discussion. The 
 elaborate series of investigations which were undertaken by Fermi 
 and Bassu would lead one to believe that a certain small partial 
 
57 
 
 pressure is necessary for intensive growth. These authors made 
 many experiments to determine the conditions under which the 
 strictest anaerobiosis was obtainable. It seems probable, judging 
 from their experiments, that. the ordinary anaerobic technique never 
 gives a medium completely free from oxygen. Another important 
 point is disclosed by their research. While it is possible to obtain 
 growth in the complete absence of oxygen, this is not so abundant 
 as when a very small amount of the gas is present. Whether the 
 small amount has a stimulating action, such as is seen with other 
 poisons, is not known. 
 
 In the case of some anaerobes, the oxygen tension at which growth 
 takes place is very low. Chudiakow has shown that in the case 
 of Bactridium huiyricum the limits are between and 15 mm. 
 At 10 mm. of oxygen there was already a retarded growth. By 
 way of comparison he gives an interesting account of the behaviour 
 of a strict aerobe, B. suUilis. This bacterium can grow at 10 mm. 
 of oxygen, but ceases to do so at 5 mm. 
 
 The question of ' symbiosis ' in the growth of anaerobes is un- 
 doubtedly an important one in soil infected wounds. A certain 
 amount of work has been done, which is not for the present purpose 
 very satisfactory. The subject is a difficult one but ought to yield 
 most useful results if further investigated. 
 
 One of the earlier ideas regarding ' symbiosis ' was that aerobes 
 abstracted oxygen from the medium, and so made conditions 
 suitable for anaerobic growth (Pasteur (2 and 4)). 
 
 Kedrowsky claimed that aerobes elaborate certain substances 
 favouring the growth of anaerobes, and that the absorption of 
 oxygen by the former is not an important process. Scholtz and 
 von Oeitingen criticized Kedrowsky's results and showed that no 
 special substance was necessary. The idea of Pasteur was modified 
 in such a way that von Oettingen came to the conclusion that the 
 aerobe acted as does the presence of reducing substances in a medium. 
 The anaerob'e has an incomplete capacity for oxidation. When it is 
 in company with a micro-organism which is eager for oxygen, the 
 latter acts synergetically and promotes growth. The action of the 
 aerobe is therefore a secondary one. Von Oettingen's technique was 
 ingenious but not beyond criticism. 
 
 The question of the smallest quantity of oxygen necessary for 
 growth was taken up by Beijerinck, who concluded that a very 
 small amount of free oxygen always took part in anaerobic growth. 
 Using his experiments as a basis, he proposed to replace the terms 
 anaerobic and aerobic by micro-aerophilic and aerophilic. 
 
 The experiments of Kiirsteiner, who used B. phosphoreum (Cohn) 
 Molisch, as an indicator for free oxygen seem to point to the con- 
 clusion that even the smallest quantity of oxygen is not at all essential 
 for the vigorous growth of strict anaerobes, and in this respect his 
 results confirm the earlier experiments of Chudiakow. An excellent 
 critical review of this much debated question is given by Omelianski, 
 who does not believe that free oxygen is necessary. Omelianski 
 also points out the important influence of the composition of the 
 mediuin on the degree of anaerobiosis necessary for the growth of 
 individual organisms. 
 
58 
 
 A full discussion on the mechanism of anaerobiosis will be found 
 in the papers of Fermi and Bassu, von Oettingen, Beijerinck, OmeTian- 
 ski, and Kiirsteiner (cf. Bibliog.). 
 
 The chemical reactions which take place durmg the growth of 
 anaerobes are manifold, and may roughly be divided into attacks 
 on carbohydrates and the decomposition of proteins. In the 
 fermentation of carbohydrates certain compounds are formed of 
 great theoretical and technical importance, acetone, alcohols, 
 especially butyl alcohol and acids of the fatty series. The acid 
 encountered in dealing with anaerobes found in wounds is chiefly 
 butyric acid. This is formed according to the reaction 
 
 0,11.^^0,= C3H7COOH + 2H2 + 2CO2. 
 
 This reaction may, however, have many by-products, and quite 
 appreciable quantities of other fatty acids are formed at the same 
 time. Non-volatile fatty acids such as lactic acid are formed in 
 quantity. 
 
 All the micro-organisms so far examined conform in general to the 
 above scheme. The attack on albuminous compounds is a much 
 more complicated affair, and individual differences between the 
 anaerobes are more manifest. 
 
 Eoughly speaking they all exert a certain amount of tryptic 
 action and break down proteins to amino acids and ammonia. 
 The so-called saccharolytic organisms B. welchii and vibrion septique 
 have not this power to any great extent, and therefore one finds 
 the particles in a culture such as cooked meat retaining their original 
 appearance for long periods of time. An analysis shows, however, 
 that a certain amount of digestion has taken place, and it is obvious 
 that such large quantities of gas as are found in cooked meat cultures 
 must have their origin from some type of protein cleavage. 
 
 Apart from purely hydrolytic action whereby albumoses, peptones, 
 and amino acids are formed from the original protein molecule, 
 other processes take place, and it is noteworthy that both reductions 
 and oxidations may be occurring at the same time. It is due to 
 these simultaneous chemical reactions that the whole chemical 
 mechanism of anaerobic metabolism is rendered so difficult to 
 disentangle. 
 
 The chemical processes may be divided into three groups (Ellinger). 
 
 1. Eeduction. Eeplacement of the amino group by hydrogen. 
 In this way phenyl, oxy-phenyl, and indol propionic acids are formed 
 from phenyl alanine, tyrosine, and tryptophane. The general type of 
 equation is the following : 
 
 E.NH^COOH-i-H^ = E.H.COOH + NH3. 
 
 2. The elimination of carbon dioxide with the formation of 
 amines : 
 
 E.NH2C00H= CO2 + E.H.NH2. 
 
 3. The oxidative degradation of amino acids whereby the chain 
 IS shortened. At the same time the amino group is removed. For 
 example, phenyl acetic acid may be formed from phenyl alanine 
 
 E.CH,.CHNH,.COOH + 0, = E.CH,COOH-t-CO, + NH,. 
 
59 
 
 Apparently in these three groups of chemical reactions the reductions 
 take place -with compounds having an aromatic nucleus. The 
 oxidations are principally confined to the aliphatic amino acids. 
 
 Very little indeed is known regarding the properties of individual 
 bacteria for producing definite types of reactions. Experiments to 
 determine this must be done by bacteria of undoubted purity of 
 strain on definite chemical compounds. In the case of anaerobes, 
 few of the experiments which have been hitherto performed will 
 bear scrutiny. 
 
 The chemical mechanism by which the necessary energy is pro- 
 duced for anaerobic metabolism is not an economical one. E. Buchner 
 showed that for B. subtilis it was necessary to use up 4-7 times as 
 much material to produce a given amount of heat when grown 
 under low tension as when the organisms grew in the presence of air. 
 
 In ordinary aerobic metabolism, carbon dioxide is usually the 
 result of the combustion of the carbon atom, the most efficient heat 
 producing reaction into which carbon enters. In anaerobic meta- 
 bolism, associated with the formation of this gas, the carbon dioxide 
 is derived from the breaking down of a carbon chain with the simple 
 elimination of carbon dioxide from a carboxyl group, or the formation 
 of an acid of lower molecular weight, or the production of an amine 
 or ah alcohol. These are reactions associated with comparatively 
 little heat set free. 
 
 (ii) The Biochemistry of certain micro-organisms found in Wounds. 
 
 A certain amount of chemical work has been done during the war 
 on both the obligate and facultative anaerobes found in wounds. 
 Greater attention has been paid to the former class. The main results 
 are to be found in the papers of Wolf and his co-workers (cf. Bibliog.). 
 
 The directions in which biochemical investigations have taken 
 place are two-fold, viz. those on the nature of the toxic products 
 which are formed and those concerned with the growth metabolism 
 of the organisms. 
 
 Previous to the war, most of the chemical work on anaerobes dealt 
 with those concerned in putrefaction. A certain amount of informa- 
 tion was gathered in the studies of the souring of milk and the 
 ripening of cheese. 
 
 Eecently the bacterial chemistry of soils, of silos, and of manures, 
 has contributed not a little to our knowledge of the metabolism of 
 anaerobic bacteria, and it is obvious that this branch of agricultural 
 bacteriology is closely alhed to the biochemistry of wound infection, 
 for it was from the heavily manured fields of France and Flanders 
 that most of the pathogenic anaerobes were derived. The later 
 chemical investigators used pure strains of anaerobes for their 
 researches and they were, therefore, at a definite advantage over 
 earlier workers who were unable to do so. The work of Nencki, 
 Tissier, and others on putrefaction gives a composite picture of the 
 chemical changes taking place in an albuminoid medium under the 
 influence of putrefactive and other anaerobes, while later work 
 assigns to each orgariism a definite function in the process. Hence 
 it is that the foundations of our knowledge of the metaboHsm of the 
 
60 
 
 individual bacteria are based on the methods which have been 
 elaborated for the isolation of pure cultures. . 
 
 Surgeons have come to recognize three important processes taking 
 place in gas gangrene infections which are capable of bemg studied 
 
 biochemically. . , . ,, , • 
 
 The first of these is gas formation, for with it there are obvious 
 physical signs of the type of infection. The production of gas m 
 the tissues is moreover important, for it leads to disturbances ot 
 nutrition, whereby the progress of the infection is materially 
 accelerated. It may be said at once that aU anaerobes are gas 
 formers under suitable conditions ; furthermore that all anaerobes 
 are capable of producing gas in a protein medium like cooked meat. 
 The capacity for gas formation varies with the individual organism, 
 and is, in some cases, largely dependent on the presence of available 
 carbohydrates. 
 
 The second sign of gas gangrene infection is that of proteolysis, 
 as denoted by the breaking down of the tissue and the odour, which 
 is sometimes one of the first diagnostic signs encountered in wounds. 
 
 All anaerobes are proteolytic, in that they are capable of trans- 
 forming a certain amount of protein into ammonia and substances 
 liberating nitrogen when treated with nitrous acid. 
 
 The differences between the individual species are, however, very- 
 great. On the one hand, there are organisms of the type of B. welchii 
 and vibrion septique in which the proteolysis can only be followed by 
 refined chemical methods ; on the other, B. sforogenes and B. histo- 
 lyticus produce as profound a disintegration of protein as that 
 produced by trypsin. Indeed the reaction is a more far reaching 
 one, for with the breaking up of highly complex nitrogenous groups 
 to amino acids, deamination and other types of reactions occur 
 whereby large quantities of ammonia are formed. B. oedematiens 
 is rather more proteolytic than vibrion septique especially if incubated 
 in protein media at 37° C. for a long time. The. proteolytic action 
 has not the practical significance of that of B. sporogenes or B. histo- 
 lyticus, which, under favourable conditions, will transform a damaged 
 muscle into a stinking pulp within 48 hours. B. oedematiens has the 
 power, as its name implies, of producing an alteration in the tissues 
 leading to the exudation of a clear gelatinous oedema. The chemical 
 investigations which have been made on the growth of this micro- 
 organism have, however, not thrown any light on this peculiar 
 change. 
 
 It is worthy of note that the truly proteolytic organisms are not 
 those which produce potent toxins. The attack on proteins, or the 
 synthetic process which gives rise to toxin formation must be of 
 a very special kind, and is not associated with any great protein 
 destruction. Experiments which have been made indicate that 
 toxin formation takes place under certain limited conditions especially 
 with regard to the reaction of the medium. The ' fragility ' of some 
 of the toxins produced is directly due to the action of the hydrogen 
 ions upon them, when the hydrogen ion concentration of the nutrient 
 medium is higher than that which exists at absolute neutrality. 
 
 The third sign is the general toxaemia seen in gas gangrene. 
 Three classes of toxic effect due to the products of bacterial meta- 
 
61 
 
 bolism, have been distinguished. The first is the true toxic effect, 
 and this varies widely both in degree and in nature. 
 
 Some of the anaerobes, e. g. B. sporogenes, produce no toxin. 
 With vibrion sepUque a toxin can. be prepared of which 0-25 c.c. 
 injected intravenously will kill a large rabbit in 5 minutes. B. 
 oedematiens produces a toxin of which 0-003 c.c. subcutaneously 
 will kill a mouse (of 15 gr.) within 24 hours. The details of the 
 toxicity of these products are given in the serological section. 
 
 The second type of product which has been thought to cause toxic 
 symptoms is the ammonia. This is certainly formed in large 
 quantity by organisms such as B. sporogenes and B. histolyticus 
 but it seems improbable that a toxic alkalosis develops as a result. 
 
 The third toxic effect has been assumed to be due to the acids 
 formed during the metabolism of these organisms. This view has 
 occupied a prominent place in the discussion of the toxaemia of 
 gas gangrene. Large quantities of acids, especially volatile acids, 
 are formed in cultures of some of the organisms. Whether in large 
 infections of muscle tissue the amount produced is sufficient by 
 itself seriously to deplete the reserve alkali is a matter which has not 
 as yet been fully demonstrated. A. E. Wright and Fleming have 
 shown that a fall in alkaline reserve takes place and have looked upon 
 this as the result of acid production in the wound itself. There are, 
 however, so many factors concerned in the diminution of reserve 
 alkali that it seems unjustifiable for the present to assume that acid 
 formation in wounds occupies a principal place in the production 
 of the toxaemia. 
 
 Acid production in culture. All anaerobes, so far examined, produce 
 volatile and fixed acids in the course of metabolism. It is highly 
 probable that this is a property of all micro-organisms, in which 
 behaviour they correspond to other forms of cell life. 
 
 As might be anticipated where large amounts of organic acids are 
 formed from amino acid complexes, ammonia is simultaneously 
 produced in the process of deamination and reduction. The am- 
 monia is utilized to preserve the neutrality of the medium, and this 
 is one of the reasons why cultures remain viable in media like cooked 
 meat or alkaline egg, when in fluids containing carbohydrate they 
 are destroyed owing to the formation of acids without the corre- 
 sponding quantity of ammonia. This is quite apart from any 
 question of spore formation. 
 
 In some instances, e.g. the cultures of B. ^sporogenes, the equivalent 
 formation of ammonia and organic acids results in the practical 
 stabilizing of the reaction of the medium. Absolute cessation of 
 growth does not take place for weeks after inoculation. With other 
 organisms, where the acids prevail over the ammonia, and the 
 reaction of the medium progresses in an acid direction, growth stops 
 by reason of the ultimate acidity of the medium. The point at 
 which growth ceases is of theoretical and of possible practical 
 importance. 
 
 Taking a medium of a given constitution and given initial reaction, 
 it is always found that the final reaction at which growth stops is 
 constant for each organism. There are slight differences in the 
 final reaction if the initial reaction is varied, but the variation in 
 
62 
 
 the final reaction, especially when available carbohydrate is present, 
 is so small as only to be discovered when the hydrogen electrode is 
 used. 
 
 The other point which is of practical importance is that growth 
 of these organisms is limited by an acidity which is approximately 
 that of one thirty thousandth normal solution of hydrochloric or 
 sulphuric acid. 
 
 Attention has been paid almost exclusively to the volatile acid 
 formed during growth ; this is so because of the technical difficulties 
 of performing serial determinations of the fixed organic acids. 
 Earlier work has shown that a number of these latter acids are 
 present, but the amount has not been determined. Eecent work 
 (Wolf and Telfer), revealed that in cultures of B. sporogenes and 
 B. welchii on milk and on 2^er cent, glucose peptone broth, 30 per 
 cent, to 50 per cent, of the total acid production consists of non- 
 volatile acids. 
 
 Of the fixed acids, lactic acid undoubtedly forms a considerable 
 part, but other acids, such as succinic, phenylacetic, and p.oxy- 
 phenyl propionic are undoubtedly present. 
 
 All the earlier qualitative work was done with impure cultures 
 and therefore cannot be accepted without reserve. 
 
 The Biochemical Characteristics and their Bearing on the Deter- 
 mination of Species. The work which has been done on the bio- 
 chemistry of these organisms leads one to inquire whether the 
 available data are sufiicient to determine the identity of bacteria 
 which have not been resolved by the usual cultural and morpho- 
 logical methods. 
 
 Harris, in a paper which is now in the press, has made for this 
 purpose a careful study of two organisms about the individuahty of 
 which some uncertainty exists. 
 
 A comparison was made between a typical strain of B. sporogenes 
 and the ' Reading ' bacillus, isolated from a wound by Donaldson. 
 The cultural and morphological features of the two organisms were 
 identical. 
 
 _ The results of the work show that in every chemical character- 
 istic the two organisms were the same within the hmits of present 
 experimental error. In only one respect did they differ. The 
 oxygen tolerance of the ' Reading ' bacillus was low. It is a verv 
 strict anaerobe. The strain of B. sporogenes, however, tolerated 
 large partial pressures oi oxygen in liquid media, although both 
 behaved as strict anaerobes in surface growths. Whether the 
 difference is a real criterion of individuahty it is impossible to say, 
 but It IS certam that a large field of investigation is open to anyone 
 who cares to examine the characteristics of closely related strains 
 by the chemical method. "^ hwdmb 
 
 The Chemical Characteristics of Anaerobic Bacteria. So far 
 a detailed study has been made of those bacteria which are presen 
 m wounds and are considered clinically important These are 
 B. sporogenes B. welchid, vibrion septique, B.hisMykcmZdB 
 oedemaUens. To these has been added a study oTvarious straps of ' 
 B.proteus as accessory organisms in wounds. 
 
 Of the above mentioned, B. sporogenes and" B. histolyticus are 
 
63 
 
 strongly proteolytic. B. welchii and vibrion septique are feebly 
 proteolytic, but attack certain carbohydrates with great vigour. 
 
 B. oedematiens appears to stand by itself. When the quantity 
 of medium is large and the observations are prolonged, it produces 
 ' considerable quantities of gas in glucose and lactose media, and also 
 in cooked meat when available free carbohydrate is not present. 
 At the same time its proteolytic activities are not inconsiderable in 
 media free from sugars. 
 
 Bacillus sporogenes (Metchnikoff). This organism is characterized 
 by the violent attack which it makes on proteins and on carbo- 
 hydrates. Its proteolytic action, especially in a medium which is 
 rich in protein is very great. In a cooked meat medium containing 
 0-87 per cent, of nitrogen, 71-5 per cent, will be changed to ammonia 
 and amino acid nitrogen in the course of 286 hours. 
 
 It forms large quantities of gas from solutions containing available 
 carbohydrates, over 1,000 c.c. per htre being formed from milk. 
 The amount of gas formed from a 2 per cent, glucose peptone may 
 be 1,020 c.c. in the course of 96 hours. 
 
 One of the notable and important features of this organism is the 
 large amount of acids which are formed during fermentation both 
 of carbohydrates and proteins. At the same time the ammonia 
 formed is sufficient to maintain the reaction at the initial level, 
 or one even finds the fermented medium more alkaline than at the 
 start. The reaction resulting from fermentation is never acid enough 
 to destroy the organism. 
 
 Two other features of the growth action of this bacillus are worthy 
 of notice. One is the putrid smell, the cause of which is not known. 
 Volatile sulphides are formed, but these by no means account for 
 the characteristic odour. The second feature is the rapid blackening 
 which takes place in cooked meat. This is probably due to the 
 action of the sulphides on the iron compounds present in the medium. 
 The action can be developed more easily as Henry (1) has shown by 
 the addition of carbonate of iron. 
 
 B. histolyticus. This organism, as the name implies, is supposed 
 to be characterized by its vigorous proteolytic action. This is so, 
 but it does not appear from the experiments which have been 
 carried out that the proteolysis is more rapid or extensive than that 
 obtained with B. sporogenes ; neither is the gas production so 
 vigorous. 
 
 A detailed investigation of the acid production has not been made, 
 but it must be very great, for with a large production of ammonia, 
 the reaction of the medium remains almost constant. The volatile 
 acids formed in a concentrated cooked meat medium reach the high 
 level of a 5 per cent, butyric acid solution. 
 
 One of the characteristics of B. histolyticus is the appearance in 
 cooked meat medium of fine silky crystals. These have not been 
 carefully examined, but they have the naked eye appearance of an 
 amino acid like tyrosin. 
 
 B. oedematiens. This organism has a proteolytic action much 
 less vigorous than that of the two preceding bacilli, but it attacks 
 proteins more readily than either B. welchii or vibrion septique. 
 Its gas forming power is not great, although in milk as much as 
 
64 
 
 750 c.c. of gas may be formed per litre of medium. The volatile acid 
 production is comparatively small. 
 
 Bacillus welchii. This organism is distinguished by its extra- 
 ordinary power for gas formation. This is possibly best shown in 
 a carbohydrate containing medium like milk. In a litre of this fluid 
 as much as 3,800 c.c. of gas, consisting of carbon dioxide and hydrogen 
 have been evolved in 20 hours. In a cooked meat mixture in which 
 carbohydrates were not present 1,072 c.c. have been given off in 
 8 hours. This was an exceptionally vigorous fermentation, but 
 300 c.c. per litre in 10 hours is a very usual amount. 
 
 The carbohydrate consumption can be very large indeed and 
 corresponds to the stormy fermentation in milk which is so charac- 
 teristic of this organism. On one. occasion 13-8 grams of lactose 
 per litre of milk disappeared while 2,714 c.c. of gas were evolved 
 from 1,000 c.c. of the medium. Even in a liquid like alkaline casein 
 344 c.c. of gas per litre have been given off. 
 
 The proteolytic action of the B. welchii cannot be disregarded. 
 In media in which there is a large proportion of amino acids 
 additional amounts are formed. In no case has a definite pro- 
 teolysis been found wanting. Both ammonia and amino acids are 
 produced. 
 
 The volatile acid production naturally varies with the composition 
 of the medium, but in a nutrient mixture like cooked meat the 
 acids formed are only ^ to J^ of what is found with B. sporogenes. 
 This is undoubtedly due to the acids slowing down metaboHsm by 
 reason of their hydrogen ion effect. Whether in a medium more 
 strongly buffered an increased acid production can be effected is 
 not known, but experiments are now being made in this direction. 
 The practical point in connexion with the acid production of B. 
 welchii is, that in cultures in a medium which has a resemblance to 
 muscle, the acids formed are not of the order which one would 
 expect to produce systemic effects. 
 
 The effect of hydrogen ion concentration on the growth of B. 
 welchii has been examined, and it is found that metabohsm ceases 
 at an average Ph. =4-82 in glucose peptone medium. The variation 
 which takes place in the inhibiting concentrations of hydrogen ions 
 due to individual acids are small. The principal volatile acid pro- 
 duced in metabolism is butyric acid. 
 
 Vibrion septique. This organism in its capacity for gas formation 
 in a milk medium is in the same class with B. welchii. On one 
 occasion 2,453 c.c. of gas were evolved from a litre of this fluid. 
 The method of gas production is, however, quite distinct. While 
 with B. welchii this amount of gas may be formed in 24 hours 
 vibrion septique will occupy 218 hours in doing so. The last 1,000 c c' 
 were formed in the particular experiment under discussion between 
 the 168th and the 218th hours. This is certainly a ' late ' fermenta- 
 tion. The proteolytic activity is of the same order as that of 
 B. welchii. It does not appear to form as much volatile acid as 
 B. welchii under similar conditions. 
 
 Bacillus proteus. The biochemistry of this facultative anaerobe 
 was examined because of the possible symbiotic effect it might have 
 on the growth of true anaerobes. All the strains used were isolated 
 
65 
 
 from wounds. None of the strains showed the putrefactive charac- 
 teristics so often attributed to the micro-organism. It is a gas 
 former, especially in meat media. Very moderate proteolysis was 
 exhibited. No indol was produced. The volatile acid production 
 was small. No notable difference was observed in the growth of 
 cultures in the presence of air from those which were grown under 
 anaerobic conditions. 
 
 • 
 
 6. EXPBKIMBNTAL GaS GaNGRBNB. 
 
 A general account of the results, obtained by inoculating animals 
 with broth cultures of wound anaerobes, has been given in the pre- 
 ceding pages. It is proposed in this section to deal with the more 
 general problems in the pathology of gas gangrene and the light 
 thrown upon them by experimental work ; to show how far the 
 accepted facts can be explained and whether the conceptions of 
 the disease which have been formed as a result of general clinical 
 observation and post-mortem experience can be verified by animal 
 experimentation. 
 
 (i) Infection. 
 
 When a culture of a non-pathogenic anaerobe, such as B. sporogenes, 
 is injected under the skin, as a general rule the animal remains in 
 good health and is apparently unaffected. Examination of the site 
 of inoculation,, however, shows that a local effect, generally oedema 
 and congestion, occurs. If films are made and examined, the 
 organisms are seen undergoing extra-cellular lysis and phagocytosis. 
 When the number of bacilli injected is very large the leucocytes 
 emigrate in such quantities as to form what amounts to an abscess, 
 which may lead to necrosis, but which is almost invariably followed 
 by the healing process. 
 
 The same general results follow the inoculation of a saline suspen- 
 sion, free of toxin, of the pathogenic anaerobes. 
 
 It is evident, therefore, that the means by which the body protects 
 itself against these microbes consists in lysis and phagocytosis and 
 that the pathogenic organisms are of themselves non-virulent. 
 
 When a culture of one of the organisms which elaborate toxin is 
 injected — that is to say, when the organisms and their metabolic 
 products are injected together — the result is different. Lysis and 
 phagocytosis do not occur ; on the contrary the organisms multiply 
 and invade the tissues, producing at the same time fresh toxins 
 which the animal is unable to neutralize, and this process continues 
 until death ensues. 
 
 A study of these facts has shown very clearly that the main, 
 indeed all-important, difference between the pathogenic and non- 
 pathogenic anaerobes of gas gangrene, from the point of view under 
 consideration, is this capacity to form a toxin ; and further that the 
 toxins act as aggressins, that is to say, annul the animal defences 
 and enable the microbes to proliferate freely in the tissues. 
 
 Toxin does not stand alone in this respect. Other substances 
 have been discovered which are able to rupture the defensive system 
 of an animal ; their mode of action is different from that of toxin 
 but it is not yet clearly understood. The first and probably the 
 
66 
 
 most important of these substances are the ionizable calcium salts 
 (Bullock and Cramer (1)). When washed and detoxicated bacilli ot 
 B welcUi, for example, are injected together with small doses ot 
 calcium chloride— 21 mg. for a mouse, 5 mg. for a gmnea-pig— under 
 the skin, the animal becomes ill in 8 to 10 hours and almost invariably 
 succumbs within 24 hours to a violent gas gangrene. 
 
 Sterile distilled water in large doses— 1 c.c. for a mouse of about 
 15 grm.— leads to the same result as do calcium salts, though not 
 so regularly, nor yet with the same violence. Certain colloids show 
 a similar behaviour when injected with the spores of vibrwn septique. 
 The most efficient colloid so far examined is siUcic acid ; the less 
 efficient are gelatine, colloidal gold, palladium, and iron. 
 
 These facts, established during the war, call to imnd the work 
 of a number of investigators in which it is claimed that sand, lactic 
 acid, cultures of staphylococci, and other materials may enable 
 washed and heated spores of B. tetani or of vibrion septique to gerimn- 
 ate in animal tissues and set up infection. It has not been possible 
 to confirm these statements. 
 
 Such is the broad outline of the modes of infection in experimental 
 gas gangrene. How far do they explain the occurrence of gas 
 gangrene as observed during the war ? 
 
 "When a man is wounded and the wound becomes gangrenous 
 
 it is clearly not sufficient to infer that this is because the wound 
 
 is infected with a pathogenic anaerobe, since we may with safety 
 
 conclude that toxin, beyond an infinitesiinal amount, is never found 
 
 in mud or soil. It is difficult to see why any wound should go 
 
 beyond the stage already described for non-pathogenic and washed 
 
 pathogenic anaerobes, namely a local gas abscess. The difficulty 
 
 has been explained by the supposition that the nature of the wound 
 
 is such that the infecting organism is able to grow and produce 
 
 toxin. Thus, the presence of large masses of dead muscle and an 
 
 impeded circulation, the condition of shock and depression of 
 
 vitality are all factors which are supposed to explain this paradox, 
 
 that non-virulent organisms may become violently virulent. These 
 
 factors do undoubtedly play an important part in the gangrene 
 
 process. Dead muscle is a good culture medium and in shock the 
 
 circulation is defective and consequently resistance to bacteria is 
 
 weak. Experiment has shown that cold may be an important 
 
 factor in the evolution of the disease. But after giving due weight 
 
 to all these facts and possibilities it cannot be said that they are 
 
 sufficient to explain the occurrence of gas gangrene in the majority 
 
 of cases. Experimentally it has not been possible to elicit with 
 
 regularity a fatal gas gangrene in animals by imitating the conditions 
 
 enumerated. Further, it is a matter of common observation that 
 
 cases of fulminating gas gangrene sometimes occur in men in whom 
 
 the amount of tissue destruction in the wound is small and the 
 
 circulation is not noticeably impeded. 
 
 The minor and comparatively unimportant type of anaerobe 
 infection known as a gas abscess, in which simple incision and free 
 drainage are generally sufficient treatment, corresponds no doubt 
 to the result obtained in animals when whole cultures of the non- 
 pathogenic or suspensions of washed pathogenic anaerobes are 
 
67 
 
 injected into the subcutaneous tissues, except that in the wounded 
 man there is generally a sufficient quantity of necrotic muscle to 
 provide a fermentable pabulum. 
 
 The fulminant cases of gas gangrene which occur in 6 to 72 hours 
 after the infliction of the wound are similar in character to the 
 experimental cases in which either a broth culture of the organism 
 or calcium chloride and the organism have been injected. It is in 
 these cases that cold, fatigue, and shock may play some part in the 
 genesis of the disease ; but the influence of the soluble calcium 
 compounds in the soil contaminating the wound is probably of 
 paramount importance. 
 
 It is a well-known fact that cultivated soil almost invariably 
 contains soluble calcium compounds and that the quantity of these 
 varies from time to time. They are produced by a complicated 
 series of chemical reactions from insoluble calcium salts, notably the 
 sulphate and phosphate, under the influence of bacterial activity, 
 the sun and rain. It is, therefore, probable that the chemical com- 
 position of soil plays a large part in the production of this most tragic 
 disease of war. For a long time it has been known that tetanus is 
 liable to follow wounds contaminated with heavily manured soil. 
 Here also it is now clear that it is not merely the presence of 
 the tetanus bacillus, in no matter what numbers, which determines 
 infection, but that other substances, of which calcium is possibly 
 the chief one, enable the bacillus to live and multiply in animal 
 tissues. 
 
 Whether calcium salts, silicic acid, and water, all constituents of 
 soil, can reinforce each other in enabling organisms to break through 
 the animal defences, has not yet been determined. 
 
 There is at present no reasonable explanation of the mechanism 
 of infection in the rare cases of gas gangrene which occur, generally 
 Brfter secondary operation, weeks or months after the infliction of 
 a wound. 
 
 A further point in connexion with the subject of infection is the 
 steady fall in the proportion of cases of the disease as the war con- 
 tinued. It is a well-known fact and is discussed by Bowlby in the 
 Hunterian Lecture for 1919. An altered condition of the soil, 
 leading to a reduction in the numbers and a diminution of the 
 virulence of the soil organisms, is one of the numerous causes brought 
 forward to account for the decHne. The alteration of the soil which 
 is of direct consequence in this matter is probably the disappearance 
 of calcium salts and of other similar substances, under the conditions 
 described by Bowlby. It is less likely that the organisms lose viru- 
 lence than that they decrease in numbers. 
 
 (ii) The Established Disease. 
 The general features of gas gangrene in men are observed in 
 animals inoculated with broth cultures of the causative organisms. 
 The local condition of oedema and gas production, the rapid invasion 
 of the tissues by the baciUi, the occurrence of septicaemia, and 
 the final toxaemia are all reproduced with striking fidelity. Any 
 differences which exist are easily referable to anatomical or biological 
 pecuharities of the animal employed. Thus, in mice, which are 
 
 B2 
 
very prone to septicaemia, B. welchii invades the blood stream an 
 hour or two after a subcutaneous injection of a pure culture of this 
 organism. 
 
 The striking feature of the disease which distinguishes it from 
 almost all other infections is the rapidity Avith which it may lead to 
 a fatal issue. It is not uncommon for death to occur within 24 hours 
 after the active infection has begun. 
 
 It is indicated elsewhere in this Eeport that the toxins produced 
 by B. welchii and vibrion septique are not very potent ; a killing 
 dose of less than 0-1 c.c. for a guinea-pig being rarely obtained. 
 When such a toxin is compared with that of B. tetani in which 
 the killing dose for the cavy may be as smaU as 0-0003 c.c, it is 
 evident that the potency of the toxins poured iiito the tissues cannot 
 account for the rapidity with which death may follow infection.^ 
 But in tetanus there is no general invasion of the tissues by the 
 infecting organisms, whilst in gas gangrene this is a conspicuous 
 phenomenon. It is probably safe to infer, therefore, that in the 
 latter disease there is a greater multiphcation of the pathogenic 
 organisms and consequently a much greater amount of toxin 
 elaborated. But this does not explain completely the extraordinary 
 fact, that in a disease like tetanus in which the toxin is about a 
 thousand times more lethal than that of B. welchii, death does not 
 often occur sooner than 2 to 3 days after the infection is estabhshed, 
 whilst in gas gangrene due to B. welchii death may occur in 12 to 
 18 hours. 
 
 The basis of these differences has been at least partly revealed. 
 It has been shown (Bullock and Cramer (2)) that exhaustion of the 
 suprarenal glands is of constant occurrence in animals killed either 
 with sterile toxin or with cultures of B. welchii and vibrion septique. 
 This can be most easily demonstrated in mice by means of Cramer's 
 osmic acid vapour method. The suprarenals show congestion of the 
 cortex and a great diminution of the cortical hpoids, while in the 
 medulla there is a complete disappearance of adrenahn. Similar 
 changes have been observed in actual cases of gas gangrene in men 
 It IS the change in the medulla which is of special significance : a 
 diminution of the cortical hpoid occurs in a number of septic con- 
 ditions of diverse origm. The disappearance of adrenahn is not a 
 temporary effect such as may occur after excessive stimulation of 
 Sf ^% I ^""^ *^ gland seems to be unable to re-form new adrenahn. 
 The effect may be suitably described as an ' inhibition ' or ' paralysis ' 
 of the organ. fai.a,±jaia 
 
 inIhe%Ew?W^ °* *^' mode of death in gas gangrene consists 
 hJ ot f f ^^'f'i^ ^"^^ ^^''^^^^ ^^ *l^eir action on the adrenal 
 
 bodies by a variety of factors, some of which are inherent in the 
 
 r^ t W^'^^t^.*^''' are adventitious to it and come into pay only 
 tSL ri V^' T''^^ conditions obtaining in war woundk ^ 
 Ihus It has been demonstrated experimentally that haemorrhaee 
 
 the injection of acid, and exposure to cold lead to a disappe^ance^f 
 
 r^hZ^t:rlt°:::r:'i:\'f^:t^^^ - -i-t^d intravenous^ « 
 
 does not kill under approximXv 24 ifn,',.? same dose mjeotedsubcutaneously 
 
69 
 
 adrenalin from the suprarenal medulla. It has also been shown 
 that these three conditions transform doses of toxin which are 
 non-lethal for a normal animal into lethal doses. 
 
 The bacteria of gas gangrene are very active acid producers and 
 are assisted in this respect by other common wound organisms, such 
 as B. sporogenes, which by themselves are not pathogenic. The 
 production of acid at the site of injection — or in a wound — proceeds 
 therefore concomitantly with that of toxin. Both are continuously 
 being formed in rapidly increasing quantities as the local lesion 
 extends, are absorbed into the circulation and assist each other in 
 exhausting the suprarenal gland, which has already been largely 
 depleted of adrenalin by such extraneous factors as cold and haemor- 
 rhage. The general conception of the. infection which emerges from 
 experimental work is therefore as follows. 
 
 The disease begins, not when a wound has become infected with 
 the pathogenic anaerobes, but from the moment when a group of 
 these bacteria have been enabled to surround themselves with 
 a toxin sufficiently concentrated to abolish the local defences of 
 the tissues. This condition may be brought about by one or more 
 of the substances already enumerated which rupture the primary 
 defences before toxin is produced. The organisms multiply rapidly 
 and invade tissues which are often cut off from an active blood 
 supply or are debilitated by the bacterial poisons. Acids are pro- 
 duced, and these promote the pullulation of the microbes (Wright 
 and Fleming) and assist the systemic toxic effects by acting on the 
 suprarenal bodies, which are also affected by shock, fatigue, cold, and 
 haemorrhage. The consequences which flow from this ' paralysed ' 
 state of the adrenals favour the invading microbes. A vidious circle 
 is established which, unless rapidly, broken, leads to that fulminant 
 character of the disease which Wright has so aptly compared to the 
 progress of an avalanche. 
 
 A general theory of gas gangrene which has found wide acceptance 
 is that it is essentially a disease of muscle, that is to say, that muscle 
 is always affected and generally contains the primary focus of 
 infection, and that unless muscle is involved the classical type of 
 the infection does not follow. This belief finds no support in experi- 
 mental work. It is almost as easy to produce a fatal gas gangrene 
 in animals by subcutaneous as by intramuscular inoculation. 
 Further, when a culture of vibrion septique for example is injected 
 intravenously into a rabbit, if the animal does not die within an hour 
 of the effects of the toxin, it may die in 24 to 48 hours of an infection 
 of vibrion septique. Post-mortem examination shows the bacilli 
 distributed throughout. the body but reveals no special predilection 
 for muscle. It is possible also to produce the disease by intravenous 
 injection of the organism deprived of its toxin. Thus if mice are 
 injected intravenously with washed and heated spores of vibrion 
 septique together with 0-7 mg. of siUcic acid, they die within 24 hours 
 and the bacillary forms of the organism can be seen in large numbers 
 in the heart blood of the animals. 
 
70 
 
 7. The Aerobic Infections of Wab Wounds. 
 Br Albxandkb FLEMnfa, F.R.C.S., Bnq. 
 
 In the early stages of gunshot or shell wotrnds the association of 
 anaerobic and aerobic bacteria is an almost constant one. The 
 primary infection of these wounds, coming as it does from mud 
 and material from the skin of the soldier, gives rise to a growth of 
 anaerobic and of aerobic bacteria most of which, like the anaerobes, 
 have a faecal origin. 
 
 Stokes and Tytler have carried out a series of observations on 
 the bacteria found in recently inflicted wounds on their arrival at 
 a casualty clearing station. In most cases examinations were made 
 within twelve hours of the infliction of the wound, and it was 
 found that out of 365 cultures, 310 showed the presence of aerobic 
 bacteria. In cases where the species of aerobe was identified they 
 obtained the following results : 
 
 Table I. 
 
 Aerobic bacteria found in wounds on admission to a casualty 
 clearing station. 
 
 Organism, 
 Haemolytic streptococcus . 
 NoH-haemolytio streptococcus 
 White staphylococcus . 
 Yellow „ 
 
 Tetragenus 
 Diphtheroids 
 Gram-negative cocci . 
 
 „ „ bacilli 
 
 Oram positive bacilli , 
 Negative .... 
 
 Total number of cultures examined = 165. 
 
 These results should be contrasted with others, obtained in the 
 later stages ot wounds, at a base hospital in France and at a 
 hospital m England (Tables IT and III). 
 
 Number of cultures 
 
 
 obtained. 
 
 Percentage 
 
 30 
 
 18-2 
 
 64 
 
 38-8 
 
 89 
 
 48-5 
 
 23 
 
 14 
 
 22 
 
 13-3 
 
 12 
 
 7-2 
 
 15 
 
 9 
 
 38 
 
 23 
 
 59 
 
 35-7 
 
 23 
 
 13-9 
 
 Table II. 
 
 Common types of bacteria, other than the spore-bearinq anaerobic 
 bac4h, found m wounds at a base hospital in France {FleZnH^. 
 
 Time since 
 
 ■ infliction of 
 
 wound. 
 
 1 to 7 days 
 3 to 20 days 
 Over 20 days 
 
 No. of 
 
 wounds 
 
 examined. 
 
 127 
 66 
 
 27 
 
 Strepto- 
 
 Staphylo- 
 
 Conform 
 
 cocci. 
 
 cocci. 
 
 bacilli. 
 
 102 
 
 40 
 
 37 
 
 51 
 
 16 
 
 18 
 
 24 
 
 19 
 
 19 
 
 Some of these bacilli were obligate anaerobes 
 
 Diphtheroid 
 bacilli.^ 
 
 9 
 21 
 16 
 
On udmission to 
 
 At any period during 
 
 hospital. 
 
 their stay in hospital. 
 
 48 
 
 53 
 
 33 
 
 45 
 
 8 
 
 23 
 
 19 
 
 29 
 
 19 
 
 29 
 
 34 
 
 43 
 
 71 
 
 Table III. 
 
 Common tyj)es oj bacteria, other than the spore-bearing anaerobic 
 bacilli, found, in wounds in a hospital in England^ (Douglas, 
 Fleming, and Colebrook). 
 
 Organism. 
 Streptococcus 
 Staphylococcus 
 B. pyocyaneus 
 B. proteus 
 B. coli type . 
 Diphtheroid bacilli 
 
 Total number of wounds examined = 54. 
 
 The faecal element of the infection tends to disappear. The large 
 anaerobic spore-bearing bacilli become fewer in numbers, and the 
 streptococci which in the primary infection are usually of the non- 
 haemolytic faecal type, become replaced by others which usually 
 conform to the type of S. pyogenes. In a wound, therefore, after 
 about two weeks when the sloughs have largely disappeared and when 
 the ' healthy ' suppuration has become established, the organisms 
 usually found are streptococcus pyogenes, staphylococcus aureus, 
 diphtheroid bacilH, and sometimes B. proteus and B. pyocyaneus. 
 As these organisms are absent in most of the recently inflicted 
 wounds, the question arises what is their source ? There can be 
 little doubt that in most, cases they are hospital infections, and it 
 is probable that they are spread from case to case in the dressing of 
 the wounds, although sometimes,, doubtless, the secondary invaders 
 gain access to the wound from the skin around. On several 
 occasions I have taken cultures from every patient in a ward and 
 have found that only one or two were infected with B. pyocyaneus 
 or B. proteus, but when I took cultures again from the same patients 
 in the same ward a week or ten days later I found that every patient 
 was infected with one or other of these organisms. 
 
 Again, as regards the haemolytic streptococcus (which in wound 
 infections is practically always streptococcus pyogenes), this organism 
 was found in only 18-2 per cent, of wounds on admission to a casualty 
 clearing station (Table I). During the summer of 1918 Captain 
 Porteous and myself examined a large number of wounds (com- 
 pound fractures of the femur) on admission to No. 8 Stationary 
 Hospital and found that only 20 per cent, of these were infected 
 with haemolytic .streptococci where the wounded had been sent 
 immediately to the base after the primary surgical cleansing. In 
 similar cases, however, which had remained in the same base hospital 
 for more than a week, being dressed every day, it was found that 
 over 90 per cent, were infected with haemolytic streptococci. 
 
 We see then that, whereas the primary infection of wounds is 
 a mixed aerobic and anaerobic one, the secondary infection is almost 
 wholly aerobic. This might be expected when it is remembered 
 that the anaerobic bacilh of wounds will in general grow in a 
 serous fluid only when the implantation is a large one, whereas 
 
 =These men remained in hospital in London for an average of something like six 
 weeks, during which time examinations were made of the bacteria about once a week. 
 
72 
 
 the aerobes will grow with much greater readiness, and especially 
 streptococcus pyogenes which is by far the most cominon micro- 
 organism of wounds in their later stages; and the one which persists 
 after all the others have disappeared. According to Sir Almroth 
 Wright's nomenclature, the anaerobic baciUi are of the na,ture of 
 sero-saprophytes, i. e. possessing the power of readily growing out 
 in serum only when that serum has been altered from the normal 
 especially in regard to a lowering of its anti-tryptic power, while 
 organisms like streptococcus and staphylococcus are serophytes, 
 i. e. possessing the power of growing freely in unaltered serum. Sir 
 Almroth Wright's work in this connexion has done much to explain 
 the change in the bacterial flora of wounds. 
 
 Eeference to Table I will show that on examination at a casualty 
 clearing station almost 36 per cent, of wounds contained aerobic 
 Gram-positive baciUi other than diphtheroids, and most of these 
 were spore-bearing baciUi. In the cases I have examined on their 
 arrival at the base I found about one in three to contain these 
 baciUi, which appear to have no pathogenic power, and their im- 
 portance to the bacteriologist lies mainly in their morphological 
 resemblance to some of the important anaerobic bacilli. In the 
 wound it is possible that they have some importance owing to 
 ■their symbiotic action on the anaerobic bacteria. This question wiU 
 ■be dealt with later. 
 
 (i) B. mesentericus, B. subtilis, B. mycoides groups. 
 
 These are the most common types of aerobic spore-bearing bacilli 
 found. They are large, Gram-po5itive, and in a film made from a 
 wound may resemble B. welchii or one of the other large anaerobic 
 bacilh. Frequently very long elements may be seen or the bacilli 
 may be arranged in chains. Central, subterminal, and terminal 
 spores are to be seen, but in most cases the spores are not much 
 broader than the bacillary body. 
 
 (ii) B. aero-tetanoides. 
 
 This baciUus resembles B. tetani morphologically but grows 
 aerobically. It is motUe and ciliated. It gives bluish colonies on 
 agar with flowery prolongations. It is feebly proteolytic and does 
 not ferment any sugar. 
 
 (iii) B. aero-tertius. 
 
 This baciUus I have recovered from wounds in cases admitted 
 to a base hospital in Prance and also at a later stage in England. 
 It bears a remarkable resemblance to B. tertius. It is Gram- 
 positive, motile, and grows on ordinary media both aerobically 
 and anaerobically. The colony on agar resembles that of B. tertiiis 
 closely, being smaU and transparent with slightly irregular edges. 
 It does not digest coagulated egg or serum, and its cultures have 
 no putrid smeU. It does not hquefy gelatin. It is a powerful 
 saccharolytic organism fermenting glucose, lactose, saccharose, 
 clextrm, starch, salicin, mannite, and glycerin, with the formation 
 ot acid and a smaU quantity of gas. 
 
73 
 
 (iv) Staphylococci. 
 
 . In the receiatly inflicted wound it is very common to find 
 staphylococci of the albus variety while the aureus type is rare. 
 These white staphylococci do not differ from the white staphylo- 
 cocci found in open wounds in civil practice. 
 
 Staphylococciis aureus is common in wounds in the later stages. 
 It, is very seldom found in pure culture, being almost invariably 
 associated with streptococci and other organisms. A severe staphy- 
 lococcus infection is always associated with much tissue necrosis. 
 Occasionally it gives rise to a generalized infection. The incidence 
 of staphylodoccus aureus in war wounds seems to be very similar 
 to its incidence in the open suppurating wounds which come into 
 the casualty room of a London hospital. 
 
 (v) Streptococci. 
 
 This group is by far the most important of all the non-sporing 
 bacteria found in wounds, and it is responsible for most of the 
 deaths from sepsis in the later stages of wound infections. There 
 are found in wounds two well-defined types of streptococci — the 
 faecalis type, especially frequent in the recently inflicted wound, 
 and the pyogenes type which is very common in the later stages. 
 Other types occur but much more rarely. 
 
 Streptococcus faecalis. This organism is referred to in French 
 literature as the ' enterocoque '. It is part of the primary infection of 
 the wound and it gradually - diminishes in numbers so that two 
 weeks after the injury it cannot in most cases be discovered at all. 
 It is present in the intestinal contents of man and animals and it 
 is very commonly found in manured soil. Houston and McCloy 
 recovered it on every occasion from mud scraped off the boots of 
 wounded men on their admission to hospital. 
 
 Streptococcus faecalis is a large oval coccus generally occurring in 
 pairs, the individual cocci being often set at an angle to each other. 
 In culture it tends to be ' pleomorphic ', large and small, round and 
 oval cocci being found. It grows well on all the ordinary media. 
 On peptone agar the colony is a little, but not much, larger than 
 that of Streptococcus pyogenes, but when it is planted on Douglas ■ 
 trypsin agar it grows with great luxuriance, the culture resembHng 
 staphylococcus rather than streptococcus. It ferments glucose, 
 lactose, saccharose, mannite, and salicin but not raffinose or inuHn. 
 Usually it does not liquefy gelatin, but some strains have this power 
 as also of digesting coagulated egg and serum. In broth it grows 
 weU, giving an even turbidity, in sharp contrast to the Streptococcus 
 pyogenes culture in this medium. 
 
 This organism, although classed as an aerobe, prefers anaerobic 
 conditions and sometimes when first isolated it will only grow in 
 the absence of oxygen. In glucose broth under anaerobic conditions 
 it grows very rapidly and luxuriantly and forms chains of twenty 
 or more elements. 
 
 One of its most striking characters is its remarkable resistance to 
 certain adverse circumstances. It lives for a very long time in 
 culture. It will resist heating for half an hour at 55° C, and 
 
74 
 
 sometimes will survive after being heated for 10 minutes at 80° C. 
 Its growth is not inhibited by ox bile and it grows well on Drigalski 
 Conradi medium. Houston and McGloy have used heating to 
 55° 0. for half an hour as a means of isolating the organism, and 
 Weissenbach has employed glucose peptone water to which has 
 been added one-tenth of its volume of ox bile to differentiate this 
 organism from 8. pyogenes. In this medium Streptococcus pyogenes 
 is completely inhibited while Streptococcus faecalis grows well. 
 
 Animals inoculated with a vaccine of this organism develop 
 agglutinins to the strain with which they were inoculated but they 
 do not of necessity develop agglutinins to all the other strains. 
 The subject, however, requires further investigation. 
 
 Streptococcus pyogenes. In war wounds as in civil practice this 
 is the most dangerous of all the pyogenic microbes met with, and 
 it is the cause of nearly all the septicaemic conditions occurring in 
 the later stages of woimd infections. During the eight months from 
 April to November 1918, working with Captain Porteous at No. 8 
 Stationary Hospital, we obtained forty-four positive streptococcal 
 blood cultures and the streptococcus in every case belonged to this 
 group. Of the other positive blood cultures which we obtained from 
 woxmded men, two were of B. welchii, one of B. oedematiens, and 
 two of Staphylococcus aureus. These cultures were aU made from 
 cases of septic compound fractures of the femur and the results 
 show the enormous preponderance of streptococcus pyogenes in 
 generalized infections. ; 
 
 Source of the streptococcal infection. As we have seen (Table I), 
 haemolytic streptococci are present only in 18-2 per cent, of the 
 wounds soon after infliction. In the later stages nearly aU the 
 wounds contain this organism. It is usually a hospital infection. 
 Levaditi and Delrez foimd that 54 per cent, of the Enghsh soldiers 
 had streptococci habitually in the epithehal squames of the skin. 
 Among the Belgian soldiers in rest, they foimd that only 12 per 
 cent, had streptococcus in the skin although 62 per cent, showed 
 the presence of this organism on the skin when they were coming 
 out of the trenches. They state also that the wounds among 
 the Belgians were less often contaminated with streptococci than 
 •those among the EnglisTi. It may be then that the infection in 
 some eases is by direct spread from the surrounding skin. It seems 
 probable, however, that the infection is from case to case in the 
 dressing of the wounds. It has long been known that B. pyoayaneus 
 can easily spread from case to case in a ward unless the most rigid 
 precautions are taken. The spread in this case is obvious to the 
 surgeons owing to the colour imparted to the dressings, but unfortu- 
 nately the streptococcal infection does not manifest itself to the 
 naked eye until some disaster hke a spreading celluHtis or a general 
 infection results. In view of Sir Almroth Wright's finding, that of 
 all the microbes found in wounds streptococci will grow most easily 
 m the unaltered serum, it is probable that it is much more easily 
 carried from case to case than B. pyocyaneus. 
 
 Characters of streptococcus pyogenes. In films of pus from wounds 
 this orgamsm occurs either in pairs, or in long chains, which are 
 usually seen to be made up of pairs of cocci. Very frequently the 
 
75 
 
 cocci are intracellular and when so placed can be seen in all stages 
 of disintegration. 
 
 In young cultures the cocci are usually round and nearly uniform 
 in size, but in older cultures they vary very much, and it is common 
 to see one or two elements in a chain very much larger than the. 
 others. The individual cocci may become elongated, pear-shaped,' 
 or almost bacillary in old cultures. 
 
 Streptococcus pyogenes grows on all the ordinary media, and on 
 Douglas trypsin agar forms rounded, grey colonies about 1 mm. in 
 diameter with a very sUghtly irregular margin and a definite dark 
 area in the centre when seen by transmitted hght. In b'roth it 
 grows in woolly masses which settle to the bottom or adhere to the 
 sides of the tubes, leaving the supernatant fluid clear. 
 
 Growth occurs slowly on gelatin at 20° C. It does not Hquefy 
 gelatin or coagulated serum. 
 
 In milk it usually produces acid and a firm clot which later contracts 
 and expresses a clear whey. The clotting of milk by Streptococcus 
 pyogenes is, however, variable and appears to depend very much 
 on the calibre of the tube used in the test. The smaller the tube 
 used the quicker does clotting occur, and sometimes there will be 
 a definite clot in a small tube when even after long incubation no 
 clot will be produced in a large tube. 
 
 Sugar fermentations. The sugar reactions of this organism seem 
 to be variable. Usually it produces acid in glucose, lactose, saccha- 
 rose, and aalicin, but not in mannite, raffinose, and inulin. A certain 
 number of the strains (about 12 per cent.) ferment, in addition, 
 mannite. Apart from this difference, the mannite fermenters and 
 the non-mannite fermenters appear to be the same morphologically, 
 culturally, and serologically. 
 
 Haemolytic power. All strains develop a haemolysin in culture. 
 This haemolysin is very unstable, being rapidly destroyed by keeping 
 or by heating to 60° C. McNee and Macleod showed that filtered 
 broth cultures contained a powerful haemolysin. It was found 
 impossible to produce an anti-haemolysin by the injection of the 
 haemolysin into animals. 
 
 Viability. It usually dies out in a month or less on agar or in 
 broth, but it can be kept alive for a long time without subculture on 
 Dorset's egg medium or Eobertson's meat medium. 
 
 Methods of isolation. This organism, like streptococcus faecalis, 
 in general prefers anaerobic conditions, and a certain number of 
 strains will only grow anaerobically when first isolated, although 
 after one or two subcultures they become accustomed to aerobic 
 conditions. 
 
 Usually an agar plate, preferably incubated anaerobically, suffices 
 for the isolation of this streptococcus. It is often an advantage 
 to use a blood-agar plate as the haemolytic action is then made 
 manifest. The simplest method for this purpose is to pour a plate 
 of ordinary agar and then after it has set to pour over the surface 
 a ■'thin layer of blood agar. Thus the haemolytic action is not 
 obscured hy the opacity of a thick layer of blood agar. 
 
 If B. proteus is present, isolation by simple plating may be diffi- 
 cult owing to the spreading growth of this microbe obscuring' the 
 
76 
 
 streptococcus colonies. In such a case cultures can readily be ob- 
 tained by Wright's pyo-sero culture method or by growing anaero- 
 bically in glucose broth for six hours, diluting and then plating on 
 agar or blood agar. 
 
 Blood culture methods. Douglas and Colebrook have shoAvn that 
 broth contaiping active trypsin is much better than ordinary or 
 glucose broth. To 5 c.c. of broth 0-25 c.c. of trypsin (AUen and 
 Hanbury's) is added and the tubes are incubated to ensure that 
 they are sterile ; 1 c.c. of blood is then added to each tube. In such 
 a medium it has been found that growth occurs with a smaller 
 implantation and is noticeable earlier than when plain broth is used. 
 
 Another method which has given very good results is to put 
 2 or 3 c.c. of blood into sufficient sterile distilled water to produce 
 complete laking. This diluted blood clots slowly and there is plenty 
 of time to carry it from the bedside to the laboratory and deal with 
 it before clotting occurs. The laked blood is mixed with 15 c.c. of 
 melted agar at 45° C. and a plate is poured. This method, in 
 addition to giving merely a positive or negative result, gives an 
 indication of the number of bacteria in the circulating blood. In 
 a case of generalized streptococcal infection following a septic wound 
 the number of streptococcus colonies obtained from 1 c.c. of blood 
 is usually under 100 and often only one or two colonies develop. In 
 one case, however, which was nearing a fatal issue, we obtained as 
 many as 1,000 colonies from 0-25 c.c. of blood. 
 
 It has also been found that Eobertson's meat medium, such as 
 is used for the growth of anaerobes, gives better results than does 
 plain broth in the cultivation of streptococci from the blood, and 
 this medium has the advantage that not only will it yield a growth 
 of streptococci which are indifferent to oxygen, but also of- those 
 which at first are strict anaerobes as well as the obligate anaerobic 
 bacilli if they are present. 
 
 Serum reactions of Streptococcus pyogenes. It has been found that 
 a rabbit injected with a vaccine of Streptococcus pyogenes develops 
 agglutinins to this organism, and that while the serum obtained in 
 this way agglutinated all the strains of Streptococcus pyogenes up to 
 the same dilution, it did not agglutinate other streptococci except 
 ia a few cases, and then only in a very slight dilution. 
 
 A rabbit received first a dose of 1,000 mUUon streptococci sub- 
 cutaneously ; four or five days later 1,500 miUions were injected 
 intravenously, and this was followed by three intravenous doses of 
 8,000 millions at intervals of one week. The rabbit was bled nine 
 days after the last injection. The serum thus obtained was found 
 to agglutinate emulsions of the homologous streptococcus up to 
 a dilution of 1 in 500. o f )^ 
 
 t]^ P^^^*?™™n agglutination tests with Streptococcus pyogenes, 
 diaculty has been experienced in obtaining a good emulsion, 
 and IJouglas has devised the following method for growing fluid 
 cultures of streptococci for this purpose. The medium consists of 
 two parts of broth (Douglas') and one part of serum or hydrocoele 
 Huid which has been heated to 60° C. for 30 minutes. The cul- 
 tures were incubated in a slanting position. In this mixture the 
 Streptococcus pyogenes was found to grow in such short chains 
 
77 
 
 that on shaking up the culture an almost even turbidity was 
 produced. The culture should then be diluted with 0-85 per cent, 
 salt solution to a convenient strength. Twenty-four strains of 
 Streptococcus pyogenes were found to be agglutinated by the serum 
 of a, rabbit inoculated as above with one strain, to the same titre 
 (1 in 500). It was found that the strains which fermented mannite 
 but which otherwise had the characters of Streptococcus pyogenes, 
 were agglutinated exactly as were the non-mannite fermenting 
 strains. 
 
 It appears from these experiments that the mannite fermenting 
 and the non-mannite fermenting strains of Streptococcus pyogenes 
 are, essentially the same. 
 
 Anaerobic Streptococci. In 1915 I described an anaerobic strepto- 
 coccus as being of frequent occurrence in wounds. It was found 
 in 9 out of 12 wounds taken at random. Gottet found a true 
 anaerobic streptococcus in 10 out of 33 wounds. 
 
 This streptococcus was difficult to isolate, as it always occurred 
 in association with Streptococcus pyogenes, and the colonies of the 
 two were identical. It grew in long chains and in old cultures showed 
 very marked involution forms. It did not clot milk, and gave no 
 change of colour on neutral red egg medium {Streptococcus pyogenes 
 always gives a bright red colour on this medium). In shake or stab 
 cultures on glucose agar, growth only occurred in the depths. Only 
 one of my strains was carried through many generations, and it main- 
 tained its true anaerobic characters. The fact that these cocci did not 
 give a red colour on neutral red egg medium indicates that they are 
 a different type from Streptococcus pyogenes, which invariably gives 
 a bright red colour. No serum reactions have been carried out in 
 connexion with these cocci. 
 
 It is unnecessary here to go into the characters of the various 
 other streptococci which have occasionally been found in wounds, 
 as the work which has been done on them during the war has added 
 little to our knowledge of them. It is interesting, however, to note 
 that Malone and Ehea found that in penetrating chest wounds it 
 was common to find streptococci of the type normally to be found 
 in the respiratory tract. 
 
 (vi) Diphtheroid Bacilli. 
 
 Bacilli of this type are more common in the later stages of wound 
 infections (see Tables I, II, and III). They have generally been 
 regarded as of little importance and as leading merely a saprophytic 
 existence. 
 
 True Diphtheria Bacilli. Fitzgerald and Eobertson reported that 
 out of 67 cases arriving in Toronto between May 20 and June 7, 1917, 
 true diphtheria bacilli were recovered in 40. These bacilli were 
 identified both by cultural and inoculation, tests. In some cases, 
 but not in all, the wounds showed the characteristic membrane of 
 a diphtheritic infection. The extraordinary prevalence of this in- 
 fection was partly explained by the fact that one of the nurses who 
 was looking after these wounded men had a slight wound on her 
 finger from which B. diphtheriae was isolated. 
 
78 
 
 Fitzgerald and Robertson's report led to an investigation of the 
 diphtheroid bacilli in wounds in some of the Canadian hospitals in 
 England, and the results of this investigation have been published 
 by Ad ami and others. Out of 306 oases investigated, bacilli were 
 found in four which had the morphological and cultural charac- 
 ters of B. difhtheriae, and of these four, two were found to be 
 pathogenic to animals producing the lesions characteristic of the 
 Klebs-Loeffler bacillus. 
 
 Prior to this, in the latter part of 1916 and the early part of 1917, 
 Douglas Fleming and Colebrook, in an investigation of wound 
 infections at St. Mary's Hospital, found diphtheroid bacilli in 43 
 out of 54 cases at some period during their stay in hospital, and out 
 of these 43 diphtheroid bacilli isolated, five morphologically and 
 culturally appeared to be true diphtheria bacilli. These five strains 
 were inoculated into guinea-pigs, and four of them were found to be 
 virulent. 
 
 It seems clear, therefore, that a certain proportion of the wounds 
 in England were infected with virulent diphtheria bacilH. In our 
 series at St. Mary's Hospital none of the wounds showed any sign 
 of a membrane, and they were all large flesh wounds healing up 
 without any clinical sign of the presence of the bacillus. 
 
 Anaerobic Diphtheroid Bacilli. Some of the diphtheroid bacilli 
 seen in films from wounds, especially in the early stages, are obUgate 
 anaerobes, and we isolated from wounds on a number of occasions 
 a bacillus which we called the ' Wisp ' baciUus, which belonged to 
 this group. It was Gram-positive, non-motile, long and slender, 
 and it arranged itself more or less in the typical diphtheroid maimer. 
 It grew anaerobically on agar or glucose agar in colonies shghtly 
 smaller and more transparent than those of Sireptococmis fyo- 
 ^genes. We were unable to test its fermentative activities, and there 
 seems to be no record of them elsewhere. Cultures on agar rapidly 
 die out. 
 
 In wounds in the later stages it is very common to see large 
 numbers of diphtheroid bacilli in films of the pus, but when this is 
 planted out there is only a very scanty growth. It is possible that 
 some of these baciUi, and especially those which can be seen cram- 
 ming the leucocytes, are really acne baciUi which have gained access 
 to the wotmd from the surrounding skin. The acne bacillus is one 
 of the most common inhabitants of the skin, and in pus or in culture 
 it shows a very definite diphtheroid arrangement. In pus from an 
 acne pustule, also, it is very frequently found in large numbers 
 mside the leucocytes. This bacillus only grows freely under anaerobic 
 conditions when first isolated, and even then it does not appear in 
 culture for three or four days, so that in the ordinary routine of 
 wound examination where plates are made and incubated for 24 
 or 48 hours It would be missed altogether. The acne bacillus is to 
 some extent a seropliyte, and it is not unreasonable to assume that 
 as It IS so common on the skin it would sometimes gain access to 
 a wound in the same way a^ staphylococci or B. pyocyaneus. 
 
 Aerobic mpUhermd Bacdh. There is not complete agreement 
 S?5:,r ^^\ff?^^^. *ph/ervers regarding the characters of the common 
 diphtheroid bacilh found in wounds. At St. Mary's Hospital we 
 
79 
 
 examined 34 strains, and found that they grouped themselves as 
 follows : 
 
 No. of 
 
 Characters. 
 
 Large opaque white or creamy colonies on agar. Short stout 
 segtate bacilli. Only show a few Neisser's granules. 
 Ferment glucose and saccharose with formation of acid. 
 Do not ferment mannite, glycerin, or dextrin. 
 
 As group 1 except that the culture medium becomes a rich 
 tawny brown colour in the presence of oxygen. 
 
 As group 1 except that they ferment mannite in addition. The 
 mannite fermentation was always slow and there was no acid 
 formation for two or three days. 
 
 Bacilli longer than group 1 and growth on agar not so copious. 
 Do not ferment any of the sugars. 
 
 Bacilli have the morphological characters of B. diphtheriae with 
 many Neisser's granules. 
 
 Ferment glucose, glycerin, and dextrin with acid formation. 
 Pathogenic for guirrca-pigs, killing them with oedema at the 
 site of inoculation and with enlarged and hyperaemic adrenals. 
 
 As group 4 except that they were not pathogenic for animals, 
 
 In this series the sugar tests were made in Cole and Onslow's 
 broth, using acid fuchsin as an indicator. None of these bacilli 
 formed acid from lactose with the exception of one strain in Group 4. 
 
 The Canadian observers, using Hiss's serum water medium, 
 found that 16 strains out of 41 fermented lactose. They did not 
 test the fermentative action on mannite. Their results are shown 
 on Table IV. 
 
 Table IV. 
 
 Group. 
 
 No. of 
 Strains. 
 
 1 
 
 15 
 
 la 
 
 4 
 
 2 
 
 7 
 
 3 
 
 3 
 
 4 
 
 4 
 
 4a 
 
 1 
 
 Tt/pe. No. 
 
 Dextrose. 
 
 Lactose. 
 
 Saccharosi. 
 
 Dextrin 
 
 Wound diphtheroid 1 . .6 
 
 A 
 
 
 A 
 
 A 
 
 A 
 
 „ 2 . . 6 
 
 A 
 
 
 A 
 
 A 
 
 
 
 B. diphtheriae (virulent) . . 2 
 
 A 
 
 
 A 
 
 
 
 A 
 
 „ (non-virulent) . 2 
 
 
 
 
 
 Wound diphtheroid 3 (xerosis 
 
 
 
 
 
 
 type) . . . .23 
 
 A 
 
 
 
 
 A 
 
 
 
 B. Hoffmann .... 2 
 
 
 
 
 
 
 
 
 
 
 A = 
 
 acid formation. 
 
 
 
 
 The most common type of diphtheroid bacillus found in wounds, 
 therefore, appears to be an organism of the xerosis type which grows 
 Ijixuriantly on agar, and which ferments only glucose and saccharose. 
 
 The dijjhtheroid organisms generally have been regarded as being 
 of little importance in wounds, and it has been found that wounds 
 could be closed by secondary suture when the number of diphtheroids 
 present was relatively high. Some of the infections with true virulent 
 diphtheria bacilh have apparently resulted in the formation of the 
 characteristic membrane, but this has not been a constant featiire 
 even when virulent diphtheria bacilli were isolated. 
 
 We shall see later that the non-pathogenic diphtheroids have 
 a powerful symbiotic influence on the anaerobic bacilli and on 
 streptococci, and in the connexion they may have some importance 
 in the wound. 
 
80 
 
 (vii) Coliform Bacilli. 
 
 In this group are included all the organisms which in a Gram- 
 stained iilm resemble the B. coli type. Many varieties of these 
 bacilli have been isolated from wounds in all stages, but usually it 
 has been found that they are more common in the later periods 
 than they are in the first few days after the wound is inflicted. Stokes 
 and Tytler (Table I) found that 23 per cent, of the wounds on admis- 
 sion to the casualty clearing station contained these bacilli. Their 
 incidence in the later stages can be gauged from the following 
 table compiled from figures given by Stewart working in Leeds 
 and Fleming working in Boulogne. 
 
 
 Table V. 
 
 
 Time after 
 
 
 PercenUtge incidence of 
 
 infection. 
 
 No. of .cases investigated. 
 
 coliform bacilli. 
 
 
 Stewart. Fleming. 
 
 Stewart. Fleming. 
 
 Under 7 davs 
 
 17 127 
 
 41 29 . 
 
 8 to 20 days 
 
 47 56 
 
 42 32 
 
 Over 20 days 
 
 58 27 
 
 74 70 
 
 Prom this table it is obvious that infection with these bacilli takes 
 place very largely in hospital. It is very noticeable that in a wound 
 there may be on one occasion many coliform bacilli, but when 
 cultures are taken a week later they have disappeared, and later 
 they may be replaced by a coliform bacillus of a different type; 
 With few exceptions they seem to be merely passing saprophytes 
 which do not really infect the wounds but which, probably due to 
 some temporary-favourable condition, gain access to the wound and 
 proKferate in the discharges until such time as the condition which 
 was favourable to their growth is changed. Some experiments by 
 Wilson are very instructive in connexion with coliform infections. 
 This observer planted a clean woimd copiously with a living culture 
 of B. coli from the intestine of the patient and then observed the 
 fate of the organisms. He found that they had nearly all disappeared 
 m 24 hours, and at the end of 48 hours they could not be seen in 
 films or recovered in culture. 
 
 Two very definite types, B. pyocyaneus and B. proteus, occur 
 frequently m wounds, but there are in addition a number of other 
 types more or less definite which are met with. The classification 
 of the cohform bacilli of wounds has been studied by Matthew J 
 Stewart, who isolated 148 strams from 122 wounds and arranged 
 them as shown in Table VT. • * 
 
 
 Table VI. 
 
 \ 
 
 No. of 
 
 Oroup. 
 
 Strains. 
 
 B.coli . 
 
 49 
 
 B. proteus 
 
 29 
 
 B. Morgan, No. 1 
 
 7 
 
 B. faecalis alkaligenes 
 
 1 
 
 Group X . . , ■- 
 
 8 
 
 Group Y . 
 
 26 
 
 B. pyocyaneus . 
 
 24 
 
 Unclassified 
 
 4 
 
 No. of Percentage 
 
 Varieties. Case incidence. 
 
 34 26 
 
 4 24 
 
 2 5-7 ' 
 1 0-8 
 
 3 5 
 
 4 20 
 1 20 
 4 3 
 
81 
 
 The chief characters which have determined the place of the 
 organisms in the above table are given by Stewart. 
 
 B. coll Fermentation of glucose and lactose with or without the 
 
 formation of gas. 
 B. Morgan, No. 1. Fermentation of glucose, laevulose, and galactose 
 only with the formation of acid and gas. 
 
 Formation of indol. 
 
 Voges and Proskauer's reaction absent. 
 Group X. Fermentation of glucose, saccharose, laevulose, galactose, 
 and inosite with the formation of acid but no gas. Non- 
 fermentation of lactose. 
 
 Litmus milk rendered acid and then strongly alkaline. No clotting. 
 
 Motility present. 
 
 Formation of indol. 
 
 Slow liquefaction of gelatin. 
 
 Voges and Proskauer's reaction absent. 
 Group Y. Fermentation of galactose without formation of gas. 
 
 Non-fermentation of laevulose. 
 
 Motility absent. 
 
 Indol negative. 
 
 Voges and Proskauer's reaction absent. 
 B. proteus. Fermentation of glucose and saccharose with the forma- 
 tion of acid and gas. 
 
 Non-fermentation of lactose. 
 
 Eapid liquefaction of gelatin. 
 
 Clotting and bleaching of litmus milk and finally more or less 
 digestion of the clot. 
 
 No formation of indol. 
 
 Voges and Proskauer's reaction absent. 
 B. faecalis alhaligenes. Fermentation of none of the carbohydrates 
 tested. 
 
 Motility present. 
 
 Litmus milk rendered strongly alkaline. 
 
 Gelatin not liquefied. 
 
 No formation of indol. 
 
 Voges and Proskauer's reaction absent. 
 
 It is unprofitable to discuss at length all these different varieties 
 of coliform bacilli as they seem to play little part in wound infections. 
 Stewart in his article deals with them fully. On account of their 
 relative frequency, however, B. -pyocyaneus and B. proteus deserve 
 some further mention. 
 
 (viii) B, pyocyaneus. 
 
 The war has not added much to our knowledge of this organism. 
 The incidence of the bacillus in wounds varies much in different 
 hospitals and in different wards of the same hospital, but in 
 general it seems to be more frequent the longer the wounded man 
 has remained in hospital. 
 
 The colour produced by different strains on agar may vary frora 
 almost jet-black to a very pale green. Some strains hardly produce 
 aay colour. 
 
 F 
 
82 
 
 In somG cases it seems to exercise a definite pathogenic effect, and 
 in some cases a vaccine of this organism has had a remarkable effect 
 in reducing the temperature of a patient whose wound had become 
 infected. A chart showing this is appended. 
 
 (ix) B. profeus. 
 
 This organism, Uke B. pyocyaneus, seems to be in most cases 
 a hospital infection. Its importance to the bacteriologist is greatly 
 enhanced by the fact that it usually spreads rapidly over the 
 surface of the culture medium, thus rendering the isolation of 
 other organisms difficult. It can usually be isolated from the other 
 organisms ])y planting the material into the water of condensation 
 of an apar slope and incubating aeiobically, when after 24 hours 
 
 B. proteus will have spread as a sheet of growth up to the top of the 
 tube, from which situation pure cultures can be obtained. Anaero- 
 bically growth is much delayed, and if it is desired to isolate other 
 organisms from B. proteus, anaerobic methods are of great assistance. 
 
 This organism rapidly hquefles gelatin, and it wiU digest coagulated 
 serum or egg, but its proteolytic activities are not nearly so marked 
 as those of a bacillus like B. sporogenes. Cultures have an unpleasant 
 but not a putrid smell. In trypsinized serum considerable quantities 
 of gas are evolved in the growth of B. proteus. 
 
 Sera of patients suffering from infections of this organism will 
 frequently agglutinate this bacillus in some cases up to a dHutioh 
 of 1 m 100 of the serum. 
 
 An animal inoculated with a vaccine of B. proteus develops aselu- 
 tinins, and It was found that the serum of a rabbit inoculated 
 with a vaccme of one strain agglutinated not only this sti'ain but all 
 the other strams to which it was tested (15 in aU) up to a dilution 
 
83 
 
 of 1 in 20,000 of the serum. Another rabbit, however, inoculated 
 with another strain of B. proteus agglutinated the homologous 
 strain to a dilution of 1 in 5,000, but (3nly three of the other strains 
 were agglutinated by this serum in a 1 in 1,000 dilution, and one 
 out X)i the 15 strains was not agglutinated even in a 1 in 50 dilution. 
 By means of absorption tests the existence of sub-groups of this 
 organism can be demonstrated, but it appears that the differences 
 between these are relatively slight. 
 
 Pathogenicity. In many cases this organism appeared to cause 
 little trouble in wounds, but sometimes it seemed to be the actual 
 cause of much or all of the fever from which the patients were suffering. 
 It has been noted above that the patient's serum in many cases 
 agglutinates the organism, which one would not expect if it were 
 leading only a saprophytic existence. It has been observed that 
 better results have been obtained in the vaccine treatment of wounds 
 with a mixed vaccine of B. proteus and streptococcus than were got 
 by streptococcus alone. 
 
 It is possible that the chief importance of B. proteus in a wound 
 is due to its symbiotic action on the other bacteria. 
 
 (x) Gram-negative Cocci. 
 
 These are present in some of the wounds from the beginning (see 
 Table I) and in the later stages they are occasionally to be found. 
 Some of these Gram-negative cocci, and especially those found in the 
 earlier stages of the wound, are strict anaerobes, and are similar to 
 the Gram-negative cocci that can frequently be isolated from faeces. 
 
 One variety of Gram-negative coccus found in wounds is an 
 obligate aerobe. It is somewhat larger than a staphylococcus, 
 non-motile, and apart from a certain number of diplococcal forms, 
 shows no special arrangement. It grows readily on agar in large 
 white round colonies with a dull surface. It ferments glucose with 
 the formation of acid but it has no action on any of the other 
 sugars. It is not pathogenic for animals, and apparently exists in 
 a wound merely as a saprophyte. 
 
 Wilson and Steer have made use of this organism for growing 
 anaerobic bacilli in symbiosis without any other anaerobic pre- 
 cautions. As this coccus fermented glucose only, they were able to 
 test the sugar reactions of the anaerobes by growing them in open 
 test-tubes containing the sugar broth, which they implanted with 
 a mixture of the anaerobe and this Gram-negative coccus. 
 
 (xi) Micrococcus tetragenus. 
 
 ' Cocci arranged in tetrads were very frequently seen in wounds, 
 especially in the earlier stages. Some of these are obligate anaerobes 
 which grow on agar or glucose agar in minute greyish colonies, 
 i'hese is no evidence to show that these tetrads seen in wounds are 
 the same as the Micrococcus tetragenus of Gaffky. 
 
 Wound infections in the later period o/ the war. 
 
 Wound infections as seen at base hospitals were to some extent 
 different in the later period, of the war from what they were at the 
 
 F 2 
 
84 
 
 beginning. In all probability this wai3 due to the careful surgical 
 cleansing which the wounds received at the casualty clearing station 
 towards the end. In the early stages of the war the wounds came to 
 the base containing large masses of slough in which all the faecal 
 microbes which constituted the primary infection grew and flourished. 
 In the later stages the infections seen at the base were much more 
 limited to the pyogenic cocci and to the bacteria which gained access 
 to the wormds at some period during the stay of the patients in 
 hospital. In particular B. proteus, B. pyocyaneus, Streptococcus 
 pyogenes, Staphylococcus aureus, and diphtheroid bacilU were fre- 
 quently found. 
 
 As a result of the primary surgical cleansing the original infection 
 was apparently much diminished and the conditions were not so 
 favourable for its development, so that it disappeared rapidly 
 except in cases where the primary excision had been incomplete and 
 where considerable sloughs or pieces of necrosed bone had been 
 allowed to remain in the wound. 
 
 8. Influence op the Aerobic on the Anaerobic Infection of 
 
 Wounds. 
 
 By Alexandbb Fleming, F.R.C.S., Estg. 
 
 It is a function of the aerobic bacteria that they can, and prefer to 
 make use of, the free oxygen dissolved in the medium and contained 
 in the air above. This can easily be demonstrated by a very 
 simple technique. 
 
 A tube of broth is planted with an aerobic organism. The cotton-wool 
 plug is pushed a short way down the tube and is saturated with melted 
 vaseline, after which melted vaseline is pom-ed on the top of the plug to 
 a depth of about one centimetre. The tube is incubated, and when the 
 contents have become warmed to the temperature of the incubator the 
 position of the plug is marked on the tube. It will be found that the 
 vaseline is sucked down the tube owing to the absorption of oxygen during 
 the growth of the bacteria. Thus, in a culture of a diphtheroid bacillus 
 isolated from a wound, the plug was ultimately sucked down for such 
 a distance that the volume remaining between the fluid and the vaseline 
 plug was only four-fifths of the original volume. When pyiogallic acid 
 and caustic soda were introduced into this tube there was no further 
 reduction of volume and no blackening of the pyrogallic acid, showing 
 that all the free oxygen had been taken up by the bacteria. 
 
 It can easily be imagined, therefore, that the aerobic bacteria may 
 have a powerful effect in making the conditions in a wound suitable 
 for the growth of the obhgate anaerobes. Methods have long ago 
 been devised for growing the anaerobic bacteria without special 
 anaerobic apparatus in symbiosis with B. subtil-k and many other 
 aerobes, and recently Wilson and Steer have made use of an aerobic 
 bram-negative coccus for the same purpose. 
 
 _ The influence which aerobic bacteria have on the production of 
 mfection by spore-bearmg anaerobes excited much attention even in 
 pre-war days Eoger showed that an inactive strain, of vibrim 
 septique can be rendered pathogenic for a rabbit by injecting it in 
 
85 
 
 association with B. prodigiosus. Vaillard and Eouget stated that 
 tetanus spores -would grow out in the tissues when they were injected 
 with aerobic bacteria which protected them against phagocytosis, 
 and a great deal of work has been done along these lines. It has 
 been common experience during the war that an organism like 
 B. welchi'^ can produce a gaseous emphysema in animals in a much 
 smaller dose if it is combined with some other organism, either 
 aerobic or anaerobic. 
 
 Tissier ((3)-(6)) attributes the spread of the primary anaerobic 
 infection in wounds to the action of the associated aerobic bacteria. 
 He maintains that when the aerobes are harmless saprophytes 
 the anaerobic action is slow ; it is more rapid when the associated 
 bacteria belong to the pyogenic group ; and it tends to become 
 fulminating when the streptococcus is present. The aerobe not only 
 favours the growth of the anaerobe, but it opens a way for it into 
 the tissues. According to this author the commencement of the 
 infection of the lymphatic channels is by streptococcus or staphylo- 
 coccus. 
 
 Douglas, Fleming, and Colebrook (1) have studied the question of 
 bacterial symbiosis in vitro, and they have found that all the common 
 aerobes found in wounds have the power of stimulating the common 
 anaerobes. Not only does the anaerobe grow more rapidly in associa- 
 tion with the aerobe, but in some cases a growth was obtained with 
 an implantation one million times smaller than when the anaerobe 
 was grown alone. They found also that this stimulation of growth 
 was shown as well, when the cultures were made under strict anaerobic 
 conditions, as when semi-aerobic conditions obtained. The various 
 anaerobic bacilli grown in association with B. welchii also exercised 
 a beneficial effect on the growth of this organism and a symbiotic 
 effect was observed in some cases Avhen the different aerobic bacteria 
 were grown together. 
 
 A few typical experiments will indicate the extent of this symbiotic 
 action. 
 
 (i) Influence of Staphylococci and Streptococci on the Groivth of 
 B. welchii in Milk. 
 
 A broth culture of B. welchii was diluted by tenfold steps up to 
 one in a million. 40 c.mm. of each dilution were planted into tubes 
 containing (a) milk 1 c.c, (b) milk 1 c.c.+staphylococcus broth 
 culture 20 c.mm. and (c) milk 1 c.c.+streptococcus broth culture 
 20 c.mm. After 24 hours the tubes were examined and the growth 
 of B. welchii was noted by the formation of the typical clot. The 
 result was as follows : 
 
 Micro-organisms planted. 
 
 B. welchii only 
 
 „ staphylococcus 
 „ streptococcus 
 
 Dilutions of B. welchii culture. 
 
 1/10 
 
 1/100 
 
 1/1,000 
 
 1/10,000 
 
 1/100,000 
 
 1/1,000,000 
 
 GR 
 
 GR 
 
 
 
 
 
 
 
 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR = growth of B. welchii. = no growth of B. welchii. 
 
86 
 
 (ii) Influence of Aerobic Organisms on the Growth of B. welchii in 
 Serum neutralized with Acid, and in Serum the antitryptw 
 power of which has been neutralized with Trypsin. 
 
 In this experiment miniature test-tubes were used, and after the 
 implantations had been made, melted vaseline was poured on the 
 surface of the culture fluid to make a column about 1 centimetre 
 in depth. The index of growth of B. welchii was gas formation 
 evidenced by the vaseline plug being pushed up the tube. 
 
 Dilutions of a broth culture of B. welchii were made as in experi- 
 ment 1 and 10 c.mm. of each of these dilutions was added to 300 c.mm. 
 of serum. The same procedure was repeated with serum infected 
 with each of the aerobic organisms. The results obtained were as 
 follows : 
 
 organisms planted. 
 
 
 
 Dilutions of B. 
 
 welchii culture 
 
 
 1/1 
 
 1/10 
 
 1/100 
 
 1/1,000 
 
 1/10,000 
 
 
 ■hii only 
 
 GR 
 
 
 
 
 
 
 
 
 
 
 , diphtheroid 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 
 , streptococcus 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 
 
 
 coliform bac. 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 
 , B. pyocyaneus 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 GR 
 
 
 1/100,000 
 
 
 GR 
 
 
 GR 
 
 
 
 1/1,000,0( 
 
 
 
 
 GR 
 
 
 The same results were obtained when the alkaline reaction of the 
 serum was neutralized with acid or when its antitryptic power was 
 neutralized with trypsin. Sir Almroth Wright has shown that both 
 of these procedures materially aid the growth of B. welchii in serum, 
 but it appears from this experiment that the symbiotic action of the 
 aerobic organism has nothing to do with its effect on the reaction 
 or the antitryptic power of the serum. 
 
 (iii) Influence of Aerobes on the Growth of Anaerobes other than 
 
 B. welchii. 
 
 (a) The technique employed was the same as in experiment 2. 
 After 5 days' incubation the results were : 
 
 ■organisms -planted. 
 
 
 • 
 
 Dilutions of Broth Culture of B. 
 
 sporogenef. 
 
 
 rogenes only 
 
 „ + streptococcus 
 
 1/1 
 
 
 
 GR 
 
 1/10 
 
 
 
 GR 
 
 1/100 
 
 
 
 GR 
 
 1/1,000 
 
 
 
 GR 
 
 1/10,000 
 
 
 
 GR 
 
 1/100,000 
 
 
 
 GR 
 
 1/1,000,00( 
 
 GR 
 
 GR = growth of B. sporogeiies. 
 
 (j)) Equal volumes of serum were implanted with B. sporogenes 
 and B. tert%us. One tube of each was kept as a control, and to the 
 others were added some diphtheroid baciUi staphylococci or strepto- 
 cocci Melted vasehne was now run on to the surface of the fluid 
 and the tubes were mcubated: The results after 3 and 5 days were 
 as follows : •' 
 
 Associated 
 Micro-organisms. 
 
 None 
 
 Diphtheroid bacillus . 
 
 Streptococcus faecaUs 
 
 Staphylococcus 
 
 B. sporogenes. 
 3 days. 5 days. 
 
 
 
 Growth Growth 
 
 B. tertius. 
 3 days. 5 days. 
 
 
 Growth 
 
 Growth 
 Growth 
 
(iv) Influence of B. welchii on the Growth of Streptococcus and 
 Staphylococcus. 
 
 (a) Two tubes were put up, one containing 1 c.o. of serum planted 
 with 10 c.mm. of streptococcus emulsion, and the other containing 
 1 c.c. of serum planted, in addition with 40 c.mm. of a B. welchii 
 broth culture. After eighteen hours' growth under anaerobic 
 conditions the number of streptococci were estimated by Wright's 
 method. The result was as follows : 
 
 Tube containing streptococcus only =100,000,000 strepto- 
 
 cocci per c.c. 
 
 Tube containing streptococcus and B. welchii=B60,000,000 strepto- 
 cocci per c.c. 
 
 (6) Four tubes, each containing 1 c.c. of serum were taken. Two 
 of them were planted with B. welchii and all four were incubated 
 anaerobically for twenty-four hours. An equal quantity of staphylo- 
 coccus was then planted into one of the previously unplanted tubes 
 and into one of the B. welchii cultures. The other two tubes received 
 an equal implantation of streptococcus. After a further twenty-four 
 hours' incubation aerobically the number of cocci was estimated by 
 plating on agar 10 c.mm. of a 1,000-fold dilution of the culture. 
 The number of colonies of the cocci which resulted was as follows : 
 
 Tube 1. B. welchii and staphylococcus gave about 600 colonies. 
 Tube 2. Staphylococcus only gave 64 colonies. 
 Tube 3. B. welchii and streptococcus gave about 1,000 colonies. 
 Tube 4. Streptococcus only gave 1 colony. 
 
 These two experiments show clearly that B. welchii stimulates 
 the growth of streptococcus and staphylococcus. 
 
 (v) Effect of a Diphtheroid Bacillus on the Growth of Streptococcus 
 
 pyogenes. 
 
 Into each of three small test-tubes 1 c.c. of serum was placed. 
 To the first and second were added 10 c.mm. of a 1,000-fold diluted 
 broth culture of streptococcus, while to the second and third were 
 added 10 c.mm. of a 1,000-fold diluted broth culture of a diphtheroid 
 bacillus. The second tube, therefore, had a mixed infection of these 
 two organisms. The number of living microbes was estimated by 
 plating out a known quantity on agar and counting the colonies. 
 The tubes were then incubated for ten hours at 37 C. and the number 
 of living bacteria was again estimated in the same way. The results 
 obtained were as follows : 
 
 Implantation. Content of living Streptococci Content of living Diphtheroid 
 
 per c.c. bacilli per c.c. 
 
 At time of After 10 hours' At time of After 10 Jwurs' 
 incubation. planting. incubation. 
 
 Streptococcus . . 13,000 2,800,000 — — 
 
 ^*^phtli°eroid t^ . 13,000 120,000,000 2,000 2,600,000 
 
 DipMieroid . . - - 2,000 5,300,000 
 
 This indicates that while the diphtheroid bacillus has a powerful 
 stimulant effect on the .streptococcus the action is not reciprocal. 
 
88 
 
 It seems clear, therefore, from the work of many observers .both 
 in vitro and in vivo, that the aerobic organisms such as are found 
 in septic war wounds have a powerful influence m stimulating the 
 growth of the anaerobic spore-bearing bacilh, and this is manifested 
 not only by an increased rate of growth of the anaerobe, but also 
 by the fact that in association with the aerobic bacteria the anaerobes 
 will flourish in a serous medium or will infect an animal in a very 
 much smaller dose. In gas gangrene the anaerobe has been compared 
 to the high explosive and the aerobe to the detonator. Bullock 
 and Cramer have shown that other ' detonators ' exist in the shape 
 of calcium and other salts, but it seems clear that the aerobic bacteria 
 must not be forgotten in this respect. 
 
 It has been held that this stimulating effect is due to the aerobe 
 absorbing oxygen and so rendering the conditions more favourable 
 for the anaerobe, but although this may in some cases be an important 
 factor it cannot explain the whole of the symbiotic effect observed. 
 The stimulant effect of the aerobe is manifest whether the cultiva- 
 tions are made under perfect anaerobic conditions or whether the 
 oxygen is not rigidly excluded. It has also been shown that the 
 presence of a second anaerobe in a culture wiU stimulate the growth 
 of B. welchii, and if mixtures of anaerobes are injected into animals, 
 the lethal dose is very much diminished, and here there can be no 
 question of the absorption of oxygen. 
 
 9. Ebsume op the Literature on the Bacteriology of Gas 
 Gangrene, with an Account of the Incidence of the 
 Various Types of Pathogenic Anaerobes. 
 
 When cases of gas gangrene began to develop with such remarkable 
 frequency shortly after the outbreak of war, bacteriologists found it 
 necessary to take stock of their knowledge of this group of infections 
 and, it must be confessed, found it largely wanting. Many workers 
 had neglected to apply the tedious methods of technique which was 
 necessary for the study of the few cases which came in the ordinary 
 routine of work and their knowledge was admittedly second-hand 
 and based upon the experience of certain ' classical ' predecessors. 
 
 It was understood that gas gangrene was not an aetiological entity 
 but a syndrome capable of being produced by several bacterial 
 species. Among these were the vibrion septique of Pasteur proved 
 by Brieger and Ehrhchto be a cause of disease in man, B. aerogenes 
 capsulatusot Welch and Nuttall and B. phlegmonis emphysematosae 
 of Eugen Praenkel. Further, it was clear from a study of the litera- 
 ture that a large number of bacteria had been described and named 
 nnd thl^n/''^"'"' ^^''^ ^^^ ^° ^"^y ^^^'^^d points of difference, 
 WP i".! V Ff'? ^^^' Tl^^'^'t *^^* '^^^^^1 of tb^se ne^ varieties 
 
 There iLl.?m ""f S\V^' P°^^*^ «* ^^f^^^^^^e ^e^e illusory. 
 
 uVon solid ^Z2 , ^^""""^^f .of plating out ' anaerobic bacteria 
 seCdlv toThP tT *^^' obtaimng perfectly pure cultures, and 
 rchnen bv in ?>, '1'^' of enriching' the anaerobic flora of a 
 variernerLnTI -"^ ^t'*^' ^""'^ ^^^ia thus causing some 
 variety, perhaps an unimportant one, to assume an undue prondnen.^- 
 
89 
 
 After the outbreak of war, a greater familiarity with the disease 
 and improvements in technique led to an increase of knowledge 
 which in France and Great Britain, if not in Germany, tended to 
 become more stabiHzed. The pronounced characters of B. welchii 
 and the ease with which it can be isolated, naturally led it to a position 
 of prominence although disguised under different names. Fleming, 
 Weinberg, Eugen Fraenkel, Simonds, Henry, and many others 
 found the bacillus in practically all wounds or articles of clothing 
 examined. 
 
 Much confusion has been associated with Pasteur's vibrion septique 
 and Koch's B. oedematis maligni. Early in the war B. sporogenes 
 Metchnikoff was found by many workers such as Eobertson, 
 Dean, Goadby, and Lardennois and Baumel. This microbe, a moder- 
 ately large, motile bacillus with a central or subterminal spore 
 possesses the power of liquefying serum and digesting milk. Such 
 an organism had been described by Jensen and Sand, Kitasato (1), 
 Kerry, Silberschmidt, Theobald Smith, v. Hibler, v. Werdt, and others 
 as B. oedematis maligni of Koch which itself was assumed to be 
 identical with Pasteur's vibrion septique. Long ago, however, Sanfelice 
 (1892) pointed out that an organism which produced the typical 
 lesions of Pasteur's experimental disease was not putrefactive and 
 proteolytic as was then being taught. During the war Weinberg 
 and Seguin, Mcintosh, Sacquepee, and others began to report the 
 presence of a motile, non-proteolytic bacillus in war wounds and from 
 its pathogenic and morphological characters identified it with 
 Pasteur's vibrion septique. An extended study of this microbe has 
 confirmed this view and has shown that it differs entirely from the 
 proteolytic jB. sporogenes, the assumption being that proteolytic 
 cultures of B. oedematis maligni are not pure. In order to clear the 
 confusion as to the real relationship of these non-proteolytic pathogenic 
 organisms a short account of their history is given. 
 
 Unfortunately, neither Pasteur nor Koch left ,any complete bio- 
 logical or cultural description whereby the bacilli called by them vibrion 
 septique and B. oedematis maligni respectively, could be subsequently 
 identified with certainty. In every instance the source of their 
 virus was the animal cadaver. In modern bacteriological text-books 
 it is usually stated that B. oedematis maligni can be readily obtained 
 by inoculating guinea-pigs with garden soil. The accuracy of this 
 statement must, however, be questioned. A great deal of the 
 confusion which exists at the present time as to what is and what 
 is not vibrion septique of Pasteur or B. oedematis maligni of Koch, 
 is probably due to this statement. The majority of the writers who 
 followed upon Koch seem to have been content to regard as the 
 bacillus of malignant oedema any sporing anaerobe obtained from the 
 lesion caused by inoculating soil into animals, with the result that 
 a considerable variety of characters has been assigned to this bacillus. 
 Sanfelice and C. 0. Jensen, however, state that the bacillus of 
 malignant oedema can only be obtained from garden soil with diffi- 
 culty, while Passini never found it there. 
 
 Von Hibler (1) in 1899 described three strains of malignant oedema 
 which all liquefied serum ; later, in his monograph of 1908, he 
 reaffirms that his strains of this bacillus all liquefied serum and 
 
90 
 
 digested milk, as did those of Kitasato and Silberschmidt. The 
 type considered to be the bacillus of malignant oedema by Theobald 
 Smith and by C. 0. Jensen, digested milk. . Divergences of opimon are 
 to be found in regard to the morphology of the bacillus, some descnb- 
 ing the spore as more or less terminal (Eoux, Ohauveau and Arloing), 
 others that it is chiefly central (Jensen and v. Hibler). There is also 
 no agreement as to the fermentation reactions. Theobald Smith 
 states that it may ferment glucose, lactose, and saccharose ; Jungano 
 and Distaso consider that glucose and lactose are fermented but 
 not saccharose, while Bahr found that strains collected from different 
 sources showed varying fermentations. 
 
 In consequence of these researches, characters were attributed to 
 B.oedematis maligni (Koch) which we now know to belong to B. 
 sporogenes, an entirely different organism. The confusion between 
 these two bacterial types is perpetuated even in some of the most 
 modern German writings on the anaerobic bacteria of war wounds^ 
 
 In their elaborate and careful research Ghon and Sachs, assuming 
 that the bacillus of malignant oedema was proteolytic, and finding 
 that the organism which they had isolated was non-proteolytic, 
 described it as a new bacillus of malignant oedema. There can, 
 however, be little doubt now that the bacillus of Ghon and Sachs 
 was the vibrion seftique of Pasteur. Grassberger and Schattenfroh 
 were also of this opinion, but Ghon and Sachs could not agree with 
 them because they believed that the mahgnant oedema bacillus 
 of Koch was proteolytic as described, and they further accepted 
 Koch's statement that the bacillus of malignant oedema was identical 
 with vibrion septique. 
 
 So far as we can ascertain at this distance of time the micro- 
 organisms which were studied by Pasteur and Koch were identical ; 
 they were both highly pathogenic for laboratory animals ; the 
 lesions produced were identical, and smear preparations from the 
 peritoneal surface of the hver of animals which had succumbed to 
 the infection showed the characteristic long thread-forms from 
 which the name vibrion septique was derived. 
 
 To resume the history of these researches during the war, certain 
 workers by the use of selective methods have succeeded in demon- 
 strating the existence of some particular anaerobe and concluded 
 therefrom that the anaerobe in question was one of the principal 
 causes of gas gangrene. In this category may be placed the view of 
 Bugen Praenkel with reference to B. pMegmonis emphysematodes 
 [B welchii), of Conradi and Biehng with their B. sarcemphysematodes, 
 of Wemberg with B. oedematiens and of Sacquepee with B. bellonensis. 
 
 A certain unanimity was, however, reached by French and British 
 bacteriologists and the following tables indicate the incidence of the 
 respective types found : 
 
 Weinberg's results, 91 cases of 
 
 B. ferfringens (B. ivelchii) 
 B oedematiens . 
 B- sf orogenies . 
 B.fallax . 
 Vibrion se^tique 
 
 gas gangrene. 
 
 77% 
 
 34 
 
 27 
 
 16-5 
 
 13 
 
91 
 
 Sacquepee's results. 
 
 B. ferfringens (B. welckii) . . ... 
 Vibrion seftique typical .... 
 Vibrion septique atypical .... 
 B. bellonensis ...... 
 
 Mcintosh's results. Series A. 1914-18, 41 cases. 
 B. welckii ...... 
 
 B.sporogenes (including type XIII of Mcintosh) 
 Vibrion seftique ..... 
 
 Terminal oval sporing bacilli 
 Unidentified types : 
 
 Type No. XVIII .... 
 
 Type No. XIX .... 
 
 Series B. 1918. 52 cases. 
 B. welchii .... 
 
 B.sporogenes (including type XIII) 
 Vibrion septique 
 
 B. oedematiens .... 
 Terminal oval sporing bacilli 
 B. tetanoides .... 
 
 82% 
 28 
 11 
 35 
 
 43-9% 
 36-5 
 19-5 
 17-0 
 
 4-8 
 
 4-8 
 
 67-3% 
 
 38-7 
 
 16-3 
 
 4-0 
 "8-1 
 
 2-0 
 
 Henry's results. 
 B. welchii 
 B. sporogenes 
 B. tertius 
 
 1917. 100 cases. 
 
 3% 
 
 Henry's results. 1918. 50 cases. 
 5. welchii ........ 
 
 Vibrion septique ....... 
 
 B. oedematiens ........ 
 
 Otter pathogenic anaerobes, of which 6% are probably 
 B.fallax . . . . . . . 
 
 48 
 22 
 
 80% 
 
 16 
 
 10 
 
 10 
 
 This last unpublished series of Henry's was derived from material 
 obtained from cases of gas gangrene, sent to the Committee by 
 corresponding members and medical officers serving with the British 
 forces in Prance. It will be seen at once that the results differed from 
 those which he obtained in 1917, but this can be explained by the 
 fact that in his second series an entirely different method of analysis 
 was employed the technical details of which were as follows : 
 
 A series of preliminary experiments had shown conclusively that there 
 was no antagonism between B. welchii, vibrion septique, and B. oedematiens, 
 if these were introduced together into an alkaline meat medium kept under 
 suitable anaerobic conditions. For- example, no evidence was obtained 
 that B. oedematiens, reputed to give feeble growths, could be overgrown 
 in culture either by vibrion septique or by B. welchii. Bach of the three 
 organisms mentioned, either alone or in association with the others, 
 reached its full development in 12 to 18 hours when grown in a meat 
 medium. 
 
 Pieces of infected muscle from the cases of gas gangrene were placed 
 in tubes of meat medium, and the resulting cultures, 18 to 24 hours old, 
 were used for inoculation into guinea-pigs. This ' whole culture ' method 
 appeared to have certain outstanding merits : 
 
92 
 
 (a) The transference of the micro-organisms from man to the g^^iea- 
 pig occupied a very short time, and the loss of virulence was reduced 
 to a minimum. vi v + 
 
 (6) Any advantage of miorobic association would presumably be at 
 a maximum in a mixed culture. 
 
 Of the 50 specimens of muscle, 44 yielded cultures which were pathogenic 
 to 250 grm. guinea-pigs in doses of 1 c.c, death resulting in 12 to 24 hours. 
 
 A potent serum containing antitoxins to B. tetani, B. welchii, and vibrion 
 septique protected guinea-pigs against 33 of these pathogenic mixed 
 cultures. 
 
 In 5 out of 11 cases in which the triple serum yielded no protection, 
 the infecting organism proved to be B. oedematiens, and the infection 
 could be arrested with an anti-oedematiens serum. Of the remaining 
 6 cases 6 yielded anaerobes which were not B. welchii, vibrion septique, 
 or B. oedematiens. Three of these five were probably pathogenic strains 
 of B. fallax. 
 
 A glance at the above tables of results is sufficient to show that 
 there is a considerable degree of uniformity in the findings of the 
 British and French workers. The German Tesults, however, are 
 neither uniform nor do they agree with the above. To judge from 
 the number of polemical articles which have appeared in the German 
 medical press no finality has been reached, and it is apparent that 
 this must be attributed to the fact that they have not yet succeeded 
 in isolating the more important anaerobic bacteria in pure culture. 
 
 Aschoff (3) considered that there were three main types which he 
 called : 
 
 1. ' Gasbrand Group '. 
 
 2. ' Eauschbrand group '. 
 
 3. ' Malignant oedema group '. 
 
 and these were again subdivided into pathogenic and non-pathogenic 
 types : 
 
 Non-pathogenic types. 
 
 B. saccharo-hutyricus 
 
 immobilis 
 B. amylobacter 
 B. saccharo-butyricus 
 
 mobilis 
 B. paraputrificus 
 
 1. Gasbrand 
 
 2. Eauschbrand 
 
 Pathogenic types. 
 Welch-Fraenkel type. 
 
 Conradi-Bieling type, 
 Ghon-Sachs ' bacillus, 
 vibrion septique Pas- 
 teur, Kolmaitype. 
 
 3. Malignant oedema B. putrificus 
 
 Von Hibler's malignant 
 oedema bacillus. Koch's 
 malignant oedema ba- 
 cillus ? 
 
 In a later paper, Aschoff (4) republished the table with slight 
 
 modifications m that the ' Gasbrand group ' was called 'immobile 
 
 butyricus group ; ' Eauschbrand group ' being called the ' mobile 
 
 butyricus group while the ' malignant oedema |roup ' was renamed 
 
 putrificus group . » r 
 
 E. Pfeiffer and Bessau came to the conclusion that there were four 
 main types : 
 
93 
 
 A. Non-putrejying anaerobes. 
 
 1. Bacillus of Fraenkel. 
 
 2. Bacillus of malignant oedema (Koch). 
 
 B. Putrefying anaerobes. 
 
 1 . ' Uhrzeiger ' or clock-hand bacillus. 
 
 2. Par-oedema bacillus. 
 
 Conradi and Bieling, however, considered that gas gangrene was 
 due to one anaerobe, B. sarcemphysematodes, and that the Welch- 
 Fraenkel bacillus, the bacillus of mahgnant oedema, the bacillus of 
 Eauschbrand, &c., were merely different stages of B. sarcemphysema- 
 todes. Two main types were fairly constant, viz. type A, which is 
 a plump, non-motile rod, producing in milk an acid clot, which is 
 later digested, as is coagulated serum ; type B which is thinner, 
 actively motile and affects milk and coagulated serum in a manner 
 similar to type A. They further state that type A can be changed 
 into type B by a few passages on coagulated serum buttype B can 
 only with dif&culty be changed into type A. They also claim that 
 specific immune sera can be produced for each type. Conradi and 
 Belling have evidently taken their ideas from Grassberger and 
 Schattenfroh's conception of the mutability of certain anaerobes, 
 but as in the case of these earlier workers the evidence produced is 
 unconvincing. The work of Conradi and Bieling received a con- 
 siderable amount of confirmation, but other German bacteriologists, 
 Pfeiffer and Bessau, however, strongly opposed their view as they 
 had not found any evidence in its favour. 
 
 Klose (6) states that gas gangrene cannot be considered as due to 
 any single species but must be ascribed to the action of a number 
 of different bacilh. By serological tests he was able to sub-divide 
 the anaerobes isolated, into four types. 
 
 1. Welch-Fraenkel group. 
 
 2. Putrificus group. 
 
 3. Eauschbrand group. 
 
 4. K;^ group (corresponding to Novy's bacillus). 
 
 An analysis of 100 strains isolated from soldiers wounded at Verdun 
 showed the following grouping : 
 
 per cent. 
 
 Group! 34 
 
 2 . 
 „ 3 . 
 „ 4(K,) 
 Unclassified 
 
 24 
 
 32 
 
 6 
 
 4 
 
 Of these strains 36 were obtained by venepuncture. 
 
 As regards these German results the position is not clear, and the 
 discrepancies between the findings of individual workers are con- 
 siderable. The descriptions of the biological and cultural charac- 
 teristic which have been given do not enable us to identify with 
 certainty the several species with which they were working. Their 
 cultures have not been available for study in this country ; it is, 
 therefore, impossible to dogmatize, but reading between the lines 
 
94 
 
 it is possible to suggest an interpretation of the significance of their 
 various names as follows : i. • i 
 
 The Welch-Fraenkel group of the German workers can obviously 
 be identified as B. welchvi. 
 
 The BauscUrand group of Aschoff and of Klose presents greater 
 difficulty. The true Eauschbrand bacillus (B. chauvoei) has not 
 been isolated from war wounds by any British or French worker. 
 Klose, however, states that in the early days of the war it was 
 found that the Eauschbrand serum, prepared at the Hoechst works, 
 agglutinated a considerable number of the strains of anaerobes 
 isolated from wounds. It was, therefore, concluded that they were 
 Eauschbrand bacilh. Later, he tested 12 Eauschbrand cultures 
 of animal, as well as of human, origin and found them to be identical 
 in their agglutinating reactions. 
 
 The description of- these so-called Eauschbrand types by Klose, 
 Zeissler (3), &c., shows that they failed to differentiate the organism 
 from vibrion septique of Pasteur. This failure possibly arose from 
 the fact that the Eauschbrand serum in question (Hoechst) was 
 prepared not by the injection of B. chauvoei but from strains of 
 vibrion septique which had been isolated from animals. It has been 
 conclusively demonstrated by several writers (Markoff, Koeves,' 
 Meyer, &c.) that vibrion septique may infect cattle, horses, and pigs 
 with the production of symptoms similar to those of blackquarter. 
 The supposition that Eauschbrand serum made at the Hoechst 
 works was specific and might thus be used for differentiating B. 
 chauvoei from other pathogenic anaerobes was not upheld by Pfeiffer 
 and Bessau who stated that the clinical concept of ' Eauschbrand ' 
 is not an aetiological entity. Further, H. Landau in a series of 
 agglutination tests showed that the Hoechst Eauschbrand serum 
 did not agglutinate two strains (A and B) of B. chauvoei isolated by 
 Kitt one of the classical authorities on the disease in question. 
 A serum prepared from Kitt's strain B did not agglutinate any of 
 the ' Eauschbrand strains ' said to have been obtained from cases 
 of human gas gangrene. In the Uncertain state of present knowledge 
 it is not possible to dogmatize, but the Committee consider that the 
 existence of B. chauvoei in human gas gangrene is not proven. 
 
 The Putrificus group. The members of this group are proteolytic, 
 liquefying coagulated serum and digesting milk. The pathogenicity 
 apparently varies to a considerable extent. The ' Uhrzeiger ' or 
 clock-hand bacillus of Pfeiffer and Bessau would be included here. 
 In the present state of our knowledge of anaerobic bacteria there is 
 little doubt that the putrificus group corresponds to the sporogenes 
 group of British and French workers. The fact that the German 
 bacteriologists found a certain number of the strains to be pathogenic, 
 a result not observed elsewhere, is explicable on the assumption that 
 •their cultures were contaminated with some of the pathogenic 
 anaerobes. It is only this pathogenic effect which distinguishes the 
 putrificus group from that of B. sporogenes. 
 
 The close unanimity arrived at by the British and French workers 
 
 has been recorded above. The only discordant result is to 
 
 be found in the work of Sacquepee (11) who attaches great impor- 
 
 :iance to an organism which he calls B. hellonensis. From the 
 
95 
 
 descriptions which he has so far pubUshed this organism cannot 
 be identified. 
 
 In conclusion, the Committee are of opinion. that acute gas 
 gangrene may result from at least three types of pathogenic anaerobic 
 bacilli ; these in their order of frequency are as follows : 
 
 1. B. welchii. 
 
 2. Vibrion septique. 
 
 3. B. oedematiens. 
 
 In addition to these, many other anaerobes may be present 
 in wounds. The exact part which these play in the production of 
 gas gangrene is not clear. In pure culture they cannot be regarded 
 as pathogenic for .laboratory animals. The more common forms 
 of these are : 
 
 B. sporogenes. 
 
 B. parasporogenes. 
 
 B. tertius (Hibler IX). 
 
 B. Mstolyticus. 
 
 B. fallax. 
 
 10. The Isolation of Anaerobic Organisms of Wounds in 
 
 Pure Culture. 
 
 In the isolation of anaerobic bacteria no single method has been 
 found which will meet every requirement. It cannot be too strongly 
 insisted upon, that, whatever technique may be employed, pure 
 cultures can be obtained only by the exercise of great technical skill 
 and by the constant application of a critical attention. 
 
 (i) Material Suitable for Examination. 
 
 1 . Exudates from wounds. 
 
 2. Pieces of infected muscle removed at operation. 
 
 3. Fluid from haemorrhagic bullae. 
 
 4. Blood from a vein. 
 
 5. Post-mortem material. 
 
 In the collection of the above material certain precautions are 
 necessary. It is preferable that the bacteriologist should collect 
 the material himself, so that it should be as little contaminated as 
 possible. A sufficiently large sample should be taken to inoculate " 
 several tubes of medium. Further, a Gram-stained smear preparation 
 of the wound exudate or muscle juice should be made, as this 
 is frequently found to give useful indications of the varieties of 
 anaerobes present. 
 
 (ii) Methods of Isolation. 
 
 . Whatever the ultimate procedure adopted, the primary aim is 
 to preserve all the anaerobes present in the sample taken, and for 
 this, some medium as little selective as possible should be used. 
 The alkaline meat medium or broth with a piece of fresh tissue are 
 both- useful for this purpose, and part of the material should be 
 inoculated into one or other of these media, which may be regarded 
 as a repository. Surface growths on serum agar under very good 
 
96 
 
 conditions of anaerobiosis may be made direct from the wound 
 material if this is derived from a relatively uncontaminated source. 
 The early mixed cultures obtained in the meat or tissue medium 
 tubes should also be planted in series on agar slopes or plates and the 
 colonies picked off. 
 
 In addition to the above, a number of methods which are essentially 
 selective in character have been found to be of great utiUty. 
 
 They comprise : 
 
 A. The separation by mechanical methods of the individual 
 
 organisms in a mixture. 
 
 B. The selection by appropriate heating of the spores contained 
 
 in a mixture. 
 
 C. The use of a selective media. 
 
 D. The separation of a pathogenic anaerobe by animal experi- 
 
 ment. 
 
 A. Separation iy Mechanical Methods of the Actual Individual 
 Organisms Present in a Mixture. 
 
 Since the memorable discovery of Pasteur a great deal of attention 
 has been paid to the cultivation of anaerobic bacteria but it is certain 
 that prior to the war a large number of anaerobic species had not 
 been obtained in a pure state. It is easy by some and possibly by 
 all of the innumerable methods, which have burdened bacteriological 
 literature, to cause anaerobes to multiply but few of the apparatus 
 although mostly described as ' simple and effective ' will lead to 
 the production of pure, isolated colonies on the surface of a sohd 
 nutrient medium — a result obtained with ease in the case of aerobes. 
 Some of the factors which militate against success with anaerobes 
 have been repeatedly emphasized in various parts of this Eeport. 
 Of these, cohesion of the material leading to a close association of 
 two or more anaerobes in one and -the same colony is perhaps the 
 most important and it must be further added that progressive 
 dilutions may not necessarily overcome it. The high degree of 
 motiUty of many anaerobes will also cause them to spread as a film 
 oyer the surface obscuring others which may themselves have a pre- 
 dilection to assume isolated colonial form. This is especially prevalent 
 in the presence of excessive moisture. Sometimes the .medium is 
 ' not suited to the requirements of all the several bacteria in a mixture 
 and the spores of some may lie dormant on the surface and may be 
 subsequently picked up along with a well-developed colony and 
 contmue with it in subsequent subculture. These are fundamental 
 difficulties which must be expected and mastered if possible. The 
 cohesion of the material may be overcome to a very considerable 
 extent, as pointed out by Stoddard (1) if the original material and 
 ail the subsequent dilutions are thoroughly shaken with glass beads, 
 sea sand, or even saline solution, surface cultures being then planted 
 out. A medium which will encourage the growth of as many of the 
 anaerobes as possible should be employed. 
 
 However interesting may be the fact that anaerobes can grow 
 under apparently aerobic circumstances it is certain that separate 
 colomfes on sohd media can only be obtained under conditions in 
 
97 
 
 which oxygen is intentionally excluded and for surface growths 
 the exclusion must be as complete as possible. 
 
 The exclusion or removal of free oxygen can be carried out in 
 a number of different ways each having a set of supporters. The 
 general principles involved are : 
 
 1. Cultivation in vacuo. 
 
 ' 2. Deep cultures as recommended originally by Hesse and Liborius 
 and subsequently developed by Veillon and Zuber and by Burri. 
 
 3. Cultivation in indifferent gases of which hydrogen and nitrogen 
 are the chief. 
 
 4. Absorption of oxygen by chemicals such as alkali in the presence 
 of pyrogallol, or in atmospheres deoxygenated by combustion with 
 hydrogen in the presence of palladium as originally suggested by 
 Laidlaw and carried to perfection by Pildes and Mcintosh and 
 Smillie. 
 
 5. Combined methods in which evacuation in the presence of 
 alkaline pyrogallol, or hydrogen in combination with alkaline 
 pyrogallol with or without evacuation are the chief. 
 
 Previous boiling of the medium — ^historically the oldest method — 
 should be effected as far as possible but is inadmissible with many 
 of the modern media containing coagulable protein. 
 
 For some reason, imperfectly understood at present, anaerobes 
 often show a great disinclination to grow as separate surface colonies 
 and but few of the many methods will lead to such a development. 
 It is quite evident that for such surface growths the anaerobiosis 
 must be much more complete than in the case where colonies are 
 embedded in the medium. It would also seem to be proved by the 
 researches of Burri and Kiirsteiner that the anaerobiotic condition 
 should be induced as rapidly and as completely as possible when 
 once the anaerobes are implanted on the artificial nutrient medium. 
 
 The exa,ct technical details of the various anaerobic apparatus 
 should be consulted in the various periodicals and journals devoted 
 to bacteriological literature. It suffices to say that in their extended 
 and fruitful researches Weinberg and Seguin employed deep cultures 
 after the manner of VeiUon and Zuber, Henry utiUzed, in particular, 
 special dishes rendered anaerobic by alkaline pyrogallol while 
 Miss Muriel Eobertson relied principally on thorough removal of 
 oxygen by the air pump. Mcintosh was unusually successful with 
 a simple apparatus in which the air was deoxygenated by hydrogen 
 in the presence of palladium asbestos. In most cases it is found 
 that test tubes are preferable to actual Petri plates for isolation 
 of surface colonies. . 
 
 A microbic emulsion may be diluted down to a pomt at whicn 
 a minute volume sufficiently small to be examined microscopically 
 can be shown to contain only one individual organism. Each minute 
 fraction of the emulsion which contains a soUtary organism will if 
 planted out into an appropriate medium give rise to a culture which 
 is pure because it is derived from one individual. This method of 
 obtaining a pure culture has been practised by a number of workers 
 in the isolation of aerobes, and it has been apphed by several in- 
 vestigators in the case of the anaerobes. The larger size of most 
 anaerobe baciUi, as compared with that of aerobic organisras, 
 
 G 
 
98 
 
 a feature which greatly facihtates a microscopic count, appeared 
 at first sight to be a factor which would enhance the chance of 
 success. It was found, however, that of the sohtary baciUi isolated 
 from a mixture of anaerobes only a very small percentage proved 
 to be viable. This difficulty, taken in conjunction with the other 
 Avell-known disadvantages that are inherent in any micro-inoculum 
 technique, renders the method inapphcable to the isolation of 
 anaerobes on an extensive scale. There are, however, occasions 
 when the procedure in capable hands may prove to be of very great 
 value. (Barber.) 
 
 B. The Selection iy Appropriate Heating 0/ the Spores 
 Contained in a Mixture. 
 
 This method which has frequently proved to be very serviceable 
 in the isolation of anaerobes has been practised with two objects 
 in view. 
 
 1. To separate spores from vegetative forms. 
 
 A small volume of a mixed culture . sealed off in the capillary 
 portion of a Pasteur pipette, is plunged into boiling water for fifteen 
 seconds or subjected to a temperature of 80° C. for twenty minutes by 
 immersion in a water bath. This heating is sufficient to kill off all 
 vegetative forms so that only spores remain. By this means there 
 can be removed completely from a mixed culture (a) non-sporing 
 aerobes such as streptococci, B. proteus and coHform organisms, and 
 (b) non-sporing anaerobes. The latter include B. welchii,. B. fallax 
 and B. aer of etidus when the mixture has been grown in a carbohydrate- 
 containing medium such as milk, glucose broth, &c. 
 
 The heated portion of culture is inoculated into a liquid medium 
 and the resulting subculture is used for plating out. 
 
 2. To separate the more highly resistant spores in a mixture from 
 those that are less resistant to heat. 
 
 Von Hibler (3) practised this method and claimed good results for it. 
 
 The use of heat to destroy non-sporing organisms is of proved 
 value. On the other hand, we have not at the present time enough 
 reliable evidence to determine the utility of the method when it is 
 appUed to the differential separation of one species of spore from 
 another. The almost ubiquitous appearance in cultures of B. sporo- 
 genes the spores of which are particularly resistant to heat would 
 appear to militate against success. 
 
 C. Selective Media. 
 If a mixed growth of anaerobes in a tube containing meat medium 
 be exammed at repeated intervals, its bacterial population will be 
 found to alter from day to day. The organisms which are the 
 first to develop include such types as B. welchii, B. fallax and 
 mbnow septique, all of ^^jhich reach their maximal growth within 
 twenty-four hours of inoculation. After twenty-four hours, these 
 organisms are replaced by others of a totally different character, 
 such as B. sporogenes ; and this second developmental phase in the 
 
 i'^rp'', f '- .f ^^ t^^^^ P^^^^' i^ ^hich end-sporers, of 
 which B. tetani is the most -important example, predominate 
 
99 
 
 The history of such a culture may thus be said to comprise three 
 different periods or epochs, each of which is characterized by its own 
 distinctive flora. The organisms of the first period have been found 
 to be predominantly saccharolytic in character, while those of the 
 second period are proteolytic. In the third period the medium is 
 found to have reached a condition in which it is unfavourable to the 
 growth of organisms of the first and second periods, but in which it 
 is pre-eminently suitable for certain organisms of the end-sporing 
 group. 
 
 Under favourable conditions the same sequence of events can be 
 traced in the bacteriological history of a wound infected with 
 anaerobes. 
 
 The information yielded by further investigation of the facts 
 outlined above has proved of great assistance to the bacteriologist, 
 for on it there is based the whole principle that controls and deter- 
 mines the choice of selective or differential media in the isolation of 
 anaerobes. 
 
 (A) Selective media for the saccharolytic anaerobes. 
 
 The first stage in the isolation of members of this group of anaerobes 
 consists in producing from the original tissue broth or meat culture 
 a subculture which will contain an aggregation of individuals which 
 display some particular biochemical feature. For example, an 
 anaerobe which has the capacity to ferment a particular carbohydrate 
 will grow rapidly in a medium containing that sugar and will be found 
 to outnumber all the organisms that do not ferment it. It is to be 
 noted, however, that this numerical preponderance may be in 
 evidence only at a certain stage in the history of the culture. There 
 are, therefore, two factors to be borne in mind, viz., (1) the presence 
 of a fermentable carbohydrate which will ensure the development of 
 organisms with the capacity to ferment that carbohydrate, and 
 (2) the period in the history of the culture at which the numerical 
 superiority in special individuals exists. 
 
 To ensure a further concentration of these individuals, all that 
 is necessary is to make, in tubes of the same special medium, several 
 subcultures in series, each separated from the preceding parent culture 
 by an appropriate interval. 
 
 "The second stage in isolation consists in making surface growths 
 from the last subculture so as to get separate colonies which can be 
 picked off and grown again in the selective medium. The agar 
 employed for this purpose should be mixed with serum or alkaline 
 egg medium. Where plain agar only is available, the admixture of 
 the material to be inoculated with serum or alkaline egg before 
 spreading it on the agar surface gives very good results. The agar 
 may be used in Petri dishes or as slopes in test tubes, according to 
 the choice of the individual worker. Many of the anaerobes tend to 
 give continuous films of growth on a moist surface and it is therefore 
 desirable to free the medium from condensation water as far as 
 possible. The deep agar shake method which has been practised 
 by many workers, particularly by the French, gives results that are 
 distinctly inferior to the surface growth method. It is, however, 
 ii useful method when surface growths cannot be obtained. 
 
 G2 
 
100 
 
 It is to be noted, that of the many methods which have been 
 devised for obtaining anaerobiosis in the case of surface growths on 
 sohd media, only a very few provide conditions sufficiently perfect 
 to promote development of the more strict anaerobes. The method 
 devised and described by Mcintosh and Pildes (1) has been tested 
 extensively by a number of workers during the war and has been 
 found to give excellent surface growths. The use of a metal or 
 glass cylinder from which the air can be extracted by means of 
 a pump and replaced by an atmosphere of hydrogen, the process 
 being repeated two or three times to ensure the absence of oxygen, 
 provides a method which has been severely tested by Miss Eobertson 
 and others at the Lister Institute with equally good results. 
 
 It is of great advantage to examine surface colonies with a pocjiet 
 lens or under a dissecting microscope. It is only by this means that 
 one can distinguish differences between colonies that look alike to 
 the naked eye. 
 
 A colony which appears to be isolated may be found to be sur- 
 rounded by many others, or it may be seen to have developed in the 
 midst of a continuous surface film, both of which conditions are diffi- 
 cult to detect by the unaided eye. The examination may also reveal the 
 growth of a contaminating organism in a culture presumed to be pure. 
 The distinctive appearance in texture and outline shown by colonies 
 of certain of the anaerobes has been described in that section of 
 this Eeport which deals with the bacteriological features of the 
 individual organisms. 
 
 As specific instances of the application of selective methods in 
 the isolation of carbohydrate-fermenting organisms, the following 
 examples may be cited. 
 
 1. B. welchii. 
 
 B. welchii attains its maximal development in freshly boiled 
 glucose broth tubes in three to six hours. It is thus possible to pass 
 the same material through two tubes between the morning and 
 evening of the same day. The second culture is plated out in the 
 evening and left to grow overnight. Colonies picked off the next 
 mormng may again be passed through glucose broth. The alternation 
 of growth m glucose broth combined with plating out can be repeated 
 as often as is necessary. It has been found that a growth which is 
 reasonably certain to be-pure can be obtained in six days, i. e., after 
 SIX agar platmgs and twelve rapid passages through glucose broth. 
 
 Ma;ltose would seem to be more rapidly and more vigorously 
 fermented by B. welchii than is glucose, so that with a 1 per cent, 
 maltose broth or casein digest medium, it is possible to make from 
 tour to SIX serial replants in the course of a day. 
 
 2. Vibrion septique. 
 
 This organism ferments salicin. The reaction takes longer to 
 develop than does the fermentation of glucose or maltose by B. welchU 
 and does not reach its maximum till twelve to twenty-four hours 
 after inoculation A medium containing salicin may, therefore, be 
 used to concentrate vibnon septique where it is associated with 
 B welcUt and J3. sporogenes for neither of the latter are capable of 
 attacking sahcm. The growth of such a microbic mixture fn 1 per 
 
101 
 
 cent, salicin broth or casein digest for twenty-four hours .followed 
 by plating on to agar containing 1 per cent, sahcin, the process 
 being repeated several times, has been found to yield a good chance 
 of obtaining vibrion s&ptique free from other organisms. 
 
 In both of the instances just quoted organisms other than those 
 specially indicated are likely to be met with. For example, in the 
 isolation of B. welchii by rapid passage through glucose or maltose 
 brothtubes, B. fallax or J3. aerofetidus may be found. Each of these 
 organisms gives rise to colonies which after a little experience can 
 be easily distinguished from those formed by B. welchii. Similarly 
 the salicin method gives a good chance of obtaining B. tertius, but 
 here again the colony can be readily differentiated from that of 
 vibrion septique. 
 
 (B) Selective media for the proteolytic anaerobes. 
 
 The best known member of this group is B. sporogenes. It grows 
 much more slowly than do the saccharolytic anaerobes. If a protein 
 containing medium which is sugar free be inoculated with a mixture 
 of anaerobes which includes B. sporogenes, it will be found that this 
 organism outgrows the others. A medium made with tap water or 
 saline containing bits of coagulated egg white offers no attraction 
 to any anaerobe other than B. sporogenes. Small pieces of fish 
 muscle or of crab muscle may be sutsstituted for the hard boiled egg. 
 An alkaline egg medium is also serviceable in obtaining a concentra- 
 tion of B. sporogenes. Material taken from a culture of two days or 
 more is plated on to agar. After forty-eight hours' incubation colonies 
 are picked off and sown into another tube of sugar free medium. As 
 before, the process is repeated several times, until the .organism 
 appears to exist in pure culture. 
 
 (C) Exhausted media. 
 
 A medium in which B. sporogenes has grown for some time is 
 found to be suitable for the development of B. tetani, B. tetanomorphus 
 and B. cochlearius, but not for the development of the saccharolytic 
 anaerobes or of B. sporogenes itself. It would seem to be selective 
 for the organisms mentioned, and was used by Tulloch for this 
 purpose. 
 
 D. Separation of a Pathogenic Anaerobe by Animal Experiment. 
 
 Guinea-pigs or mice may be used for this purpose. 
 
 1. In normal animals. 
 
 Where a mixture of organisms contains only one pathogenic 
 anaerobe, then this particular organism can be recovered after death 
 from the heart blood of an animal which has succumbed to an intra- 
 muscular inoculation of the mixture. If the pathogenic anaerobe 
 be vibrion septique or B. oedematiens it may be obtained in pure 
 culture from the animal's heart blood. It is to be noted, however, 
 that certain strains of B. oedematiens may kill by intoxication 
 without spreading into the circulating blood stream. Where the 
 pathogenic anaerobe is B. welchii there is often a tendency for other 
 organisms Such as B. sporogenes or streptococci or coliforms to 
 appear in the blood along with B. welchii. 
 
102 
 
 On the other hand, where two or all three of these pathogenic 
 anaerobes are present in a mixture, any one of them or any com- 
 bination of them may be found in cultures made fromheart blood. 
 
 It follows, therefore, that the use of a normal animal for such 
 a passage experiment is not likely to give a rehable result unless oiie 
 can be reasonably certain that only one pathogenic anaerobe is 
 present in the inoculated mixture. 
 
 2. In protected animals. 
 
 A guinea-pig which has been passively immunized by inoculation 
 with antitoxic sera against B. welchii, vibrion sejptigue or B. oedema- 
 tiens is capable of successfully resisting infection by the corresponding 
 organism. For example, an animal adequately protected against 
 B. welchii will never be found to develop a fatal B. welchiA infection 
 though it may readily succumb to vibrion septique or to B. oedema- 
 tiens if either of these be present in the inoculum. The same holds 
 good for vibrion septique and B. oedematiens antitoxic sera, the im- 
 munized animal being afforded absolute protection against infection 
 by the corresponding organism but not against other pathogenic 
 anaerobic organisms. 
 
 Further, it has been possible by using appropriate mixtures of 
 these antitoxic sera to protect animals against a combination of any 
 two or of all three of these organisms. 
 
 One may thus construct in a guinea-pig a sort of filtering mechan- 
 ism which will retain or hold up certain organisms by inhibiting 
 their development in the animal, and which will at the same time 
 allow the growth and subsequent passage into the blood-stream of 
 any pathogenic anaerobe against which no specific protection has 
 been induced. This method was actually used in determining the 
 pathogenic anaerobes present in specimens of muscle taken from 
 fifty cases of acute gas gangrene in Prance. A combined serum 
 containing 4,750 units of welchii antitoxin and 5,000 units of septique 
 antitoxin per 10 c.c. was given intraperitoneally in a dose of 4 to 5 c.c. 
 to guinea-pigs of 250 grm. weight, twenty-four hours before the 
 moculation of the injecting dose of mixed culture. The latter 
 consisted m each case of 1 c.c. of culture mixed with an equal volume 
 of the same serum and left in contact with it for one hour at room 
 temperature before mtramuscular inoculation. Of the protected 
 gmnea-pigs which succumbed none died with a B. welchii or vibrion 
 septique infection. The organisms which were recovered from the 
 heart blood of these animals proved in every case to be B. oedematiens 
 or some other different anaerobe. The full results wHl be discussed 
 m detail elsewhere. This animal filtration method has been given 
 an extensive trial and it can be safely recommended as providing 
 
 Pn[w if r^'^fv ''^^^^^^ ^''^^ ^ ™^*^^^ «f anaerobes a purl 
 culture of B. welch%%, vihnon septique or B. oedematiens 
 
 in a case of urgency, three guinea-pigs or mice nroteeted with 
 
 appropriate combinations of the three antlera (TeTchrvSrfon lep 
 
 StTromth?; r 'I "^Y \' '"^^"^^*^^ -^*^ th^ wound Sarle 
 n esent Zv bP^S. ■. ^ ^\'F^'^' °* *^^ pathogenic anaerobis 
 over n gh^ ^ '^'^ ^^ *^' ''™1* «^*^i^e<i ^^ tbe animals 
 
103 
 
 E. Summary. 
 
 The above outline of the several methods which may be employed 
 in the isolation of the anaerobes must be taken as affording no more 
 than general indications in regard to the methods that are most 
 likely to yield success. The individual researcher will develop them 
 along the lines of his own particular need and will find that they 
 are ca,pable of modification and adaptation in a number of useful 
 directioiis. An active and resourceful manipulation of the methods 
 of isolation available is rendered necessary by the great variety in 
 the problems presented. 
 
 The worker who sets out to obtain pure cultures of the anaerobes 
 essays no easy task. There are fortunate occasions when he may be 
 able to plant out the original, material directly on to plates, as, for 
 example; when he is dealing with a post-mortem heart blood or with 
 pieces of infected muscle taken at some distance from a wound. 
 In the majority of instances, however, he is faced with material that 
 is grossly contaminated, and in deahng with it the following points 
 should be constantly borne in mind. 
 
 1. Growth in a special selective medium is merely preparatory to 
 and must alternate with plating out on serum agar. The anaerobic 
 conditions employed must be rigorous enough to give good surface 
 growth of the more exacting anaerobes. 
 
 2. The criterion of purity of a culture is based entirely on its 
 consistent behaviour when it is grown on a large range of different 
 media and when its cultural reactions are observed over a long 
 period of time. 
 
 , 3. The worker need never hesitate to admit the impurity of one of 
 his cultures when 'definite proof of the same is produ'ced before him, 
 for the very best bacteriologists have been deceived by the anaerobes. 
 It is only by the exercise of constant vigilance and the maintenance 
 of a keen critical attention in regard to the possibility of contamina- 
 tion that he can escape these pitfalls. 
 
 (iii) Preservation of Cultures. 
 
 A few general directions are given for the preservation of cultures 
 when isolated. 
 
 The desirable conditions under which to keep stock cultures are 
 those in which the organism will form spores which will retain their 
 vitality over long periods. As a general rule, media containing 
 fermentable sugar and milk media should be avoided as the acid 
 reaction produced by many anaerobes is unsuitable for the prolonged 
 maintenance of the culture. 
 
 The vast majority of the anaerobes may be subcultured on to meat 
 medium, coagulated serum, or broth with a piece of coagulated 
 egg white, and the tubes sealed in a blow-pipe after forty-eight to 
 seventy-two hours' growth. The sealed cultures can be stored in 
 the dark at the temperature of the laboratory for more than a year 
 without losing their vitahty. When the sealed tubes are opened they 
 should be subcultured on to meat medium, a large inoculum being 
 used. The strain should then be grown on milk, meat medium, 
 coagulated serum and gelatine, and the sugar reactions tested so as 
 
J 04 
 
 to assure the worker that the culture is pure and typical. It is often 
 at this period when a resting culture is once more subcultured after 
 a long interval, that contaminations are found in a supposedly pure 
 culture. 
 
 Certain anaerobes require special treatment. 
 
 B. welchii is best preserved in alkaline egg fluid or on coagulated 
 serum as spores are more certainly formed in these media than in 
 the others in use. 
 
 B. Jallax is an organism which is apt to die out in stock cultures, 
 spores are not formed readily and it is not always a prolific grower. 
 It is best kept in alkaline meat medium or coagulated serum and it 
 should be incubated for at least three days before the tubes are 
 sealed. Cultures should be made in triplicate and the strain should 
 be subcultured every four to six months. 
 
 B. cochharms strains are also easily lost. Meat medium of an 
 alkaline reaction, broth with a piece of living tissue or coagulated 
 egg white, are the best media for stock cultures. In pure culture 
 this organism is not a prolific grower. 
 
 IV. SEEOLOGY. 
 
 1. Toxins and Antitoxins. 
 
 (i) Historical Introduction. 
 A. B. welchii. 
 
 Since the appearance in 1892 of the classical article by Welch and 
 Nutta,ll on the gas bacillus, the study of the pathplogy of infection 
 by this organism has attracted the attention of many workers. The 
 exact significance of the pathological changes that are induced in 
 the animal body as the result of infection have been variously 
 interpreted. 
 
 Eugen Fraenkel (1) looked upon the disease as being an intoxication 
 due to the absorption of decomposition products formed in the tissues. 
 Much the same view has been advanced in the course of the war 
 by Conradi and Bieling (1) who considered that the various acids 
 produced by the fermentation of carbohydrates were responsible 
 for the tissue necrosis, and that this necrosed tissue favoured the 
 development of saprophytic anaerobes, the resulting putrefactive 
 products when absorbed giving rise to intoxication. Kamen in 1904 
 t^i that 8-day old cultures contained a haemolytic substance 
 
 riSoullnn '/''^'\ f "'°^'*^"^'/ ^^ ''''' and decided that 
 although strong toxic substances could not be produced in vitro thev 
 were probably formed in the animal body ^ 
 
 Passim (1) found that the toxic substances in filtrates from old 
 culures survived a temperature of 100° C. for 15 rninutes and 
 further, that hey possessed no antigenic properties. ' 
 
 McCampbell m 1909 attributed the lesions produced in animals 
 
105 
 
 Stewart and West in 1916 came to the same conclusion. Of 
 others who have investigated the toxic substances present in filtrates 
 of B. welchii mention may be made of Korentchevsky, Herter, 
 Simonds (1), Costa, and Troisier (3), and Ouranoff. 
 
 In none of the papers cited, however, is there any adequate 
 denaonstration that the toxic substances referred to possessed anti- 
 genic properties. 
 
 Eosenthal (1) appears to have been one of the first to attempt 
 the preparation of a serum against B. welchii. He inoculated horses 
 with gradually increasing doses of culture. The resulting antiserum 
 proved to have only slight protective properties. 
 
 In 1916 Weinberg (6) reported that he had been successful in 
 preparing an antibacterial serum to B. perfringens, the horse being 
 immunized with washed bacilli. This serum, in a dose of 0-005 c.c, 
 neutralized one lethal dose of culture in guinea-pigs. There can be 
 little doubt that this serum was really antitoxic but the difficulties 
 experienced by Weinberg in obtaining a good toxin derived from 
 B. welchii militated against any successful demonstration by him 
 of its antitoxic value. The first clear exposition of B. welchii toxin 
 is to be found in the communications of Bull and Pritohett (1). 
 These workers proved that the necrosing and haemolytic properties 
 of B. welchii filtrates were due to the presence of a real exotoxin, 
 which could be destroyed by heating to between 60° and 70° C, 
 and which was capable of stimulating the production of antitoxin 
 when inoculated into animals. They further showed that this 
 antitoxic serum when employed under proper conditions inhibited 
 and arrested infection with B. welchii. 
 
 B. Vibrion se-ptique. 
 
 In contradistinction to our knowledge of B. welchii antitoxin, 
 which is very recent, our information in regard to immunization 
 against vibrion septique infection dates back to the publication of 
 a communication by Eoux and Chamberland in 1887, Duensch- 
 mann 1894, Leclainche and Morel 1901, M. Nicolle, Cesari and 
 Baphael 1915, Eaphael and Prasey may all be cited as yielding proof 
 of the production of protective sera against Pasteur's organism. 
 
 0. B. oedematiens. 
 
 This organism was described for the first time in the course of 
 the war by, Weinberg and Seguin ((1) (2)). These observers have 
 accurately defined the properties of the specific toxin, and have 
 succeeded in producing high-value antitoxic sera in horses. There 
 is no proof that the organism described in 1894 by Novy as B. oede- 
 matis maligni II, and later by Migula as B. Novyi, produced toxin 
 although Novy (p. 224) suggested that this was likely from the 
 scarcity of bacilli in the oedematous areas. 
 
 (ii) The Preparation of Toxin. 
 A. Toxin of B. welchii. 
 
 . . Bull (1) produced B. welchii toxin by growing the organism in 
 glucose broth containing large amounts of sterile rabbit muscle. 
 The aseptic removal of muscle from a rabbit presents considerable 
 
106 
 
 .echnical difiaculties. To eliminate this particular ' complication 
 has been the aim of all those who have since dealt with the problem, 
 and more particularly perhaps of those workers who have had to 
 prepare toxin on a sufficiently large scale for the immunization 
 of horses. 
 
 The preparation of toxin of B. welchii is beset with even more 
 difficulties than is the production of the toxins of diphtheria and 
 tetanus. There are so many variable factors to be considered in 
 the process and many of these factors are so difficult of control that 
 the issue can never be accepted as a certainty. With repeated 
 experiment, however, and a fuller understanding of the essentials 
 to success it becomes possible to establish the in vitro conditions 
 which assure the probabihty of a fortunate result. 
 
 The points which appear to be of special significance may be 
 considered under the following headings : (a) the medium ; (b) 
 the organism ; (c) the inoculum ; (d) the conditions of growth ; 
 (e) filtration. 
 
 (a) The Medium. — ^Experience has shown that the best medium 
 for use on a large scale is a muscle broth which is prepared as follows : 
 
 1. A mixture of minced horse muscle and tap water, in the propor- 
 tion of 1 lb. muscle to 1 litre water, with 0-5% common salt, is boiled 
 in a cauldron for 1 hour. 
 
 2. The cooked meat is then removed and, to the broth, after 
 filtration there is added 1 per cent, of peptone and 0-2 per cent, of 
 glucose. 
 
 3. The broth is then heated tiU the peptone is dissolved, and 
 sufficient caustic soda is added to make it shghtly alkaline to litmus. 
 
 4. It is then boiled for half an hour and filtered to remove the 
 precipitate. 
 
 5. Into a series of 4-Utre bottles (double Winchester quarts) 
 there are placed about 2 handfuls of the cooked meat and 4 litres of 
 broth. 
 
 6. The bottles are plugged with cotton wool and autoclaved for 
 3 hours at a pressure of 22 lb. per square inch. 
 
 7. The end reaction of medium so prepared varies from 8 per cent, 
 to 4 per cent, acidity when tested with phenol phthalein as the 
 mdicator. 
 
 PeptoTie appears to be a necessary constituent for the formation 
 of toxin but it IS to be noted that the commercial brands differ 
 considerably m value. 
 
 Glucose has a powerful influence in promoting the toxicity of a 
 filtrate, although its action in this respect must not be looked on 
 as specific. It has a direct effect in encouraging the growth of 
 B welchu and an indirect beneficial effect in that after fe^entation 
 It provides the degree of acidity (phenol phthalein titration) that 
 appears to be essential to the formation of toxin 
 
 « SZfTrl^^ !v'^ •' •^*'*,^°'' °* ^ ^- "^'^^^ filtrate depends to 
 carbohVdS ? "^T '"^'f'^^fr^oi the medium in fermentable 
 statement b«; W 7if V^' ^''* to demonstrate this clearly and the 
 statement has been fully borne out by .the findings of other workers 
 Startmg with a sugar free medium b4 showed that the end Reaction 
 taken at intervals from the first to the thirteenth day ofTnocuSon 
 
107 
 
 lay between 2 per cent, and 2-5 per cent, acidity. By increasing the 
 amounts of glucose in the muscle broth he found that the acidity 
 rose, until, with a content of 3 per cent, in glucose an acidity was 
 reached of 8 per cent. He further demonstrated that, although 
 the presence of glucose in amounts up to 1 per cent, had little un- 
 favourable influence on the toxicity of the filtrate, yet glucose in 
 large amounts had a decidedly deleterious effect on toxin production. 
 There is thus a distinct relationship between the end reaction of 
 a filtrate and its toxicity. 
 
 Experimen.ts conducted with 0-2 per cent, glucose muscle broth 
 media in which the initial reaction has been arranged so as to vary 
 from 8 per cent, acid on the one hand to 3 per cent, alkali on the other, 
 have suggested that although the end reaction (after the growth 
 of B. welchii) comes out at practically the same figure, viz. an acidity 
 of 4 per cent, to 5 per cent., yet toxin production is most marked in 
 media which were originally acid. It would seem therefore that 
 B. welchii prefers an acid medium for the production of toxin and that 
 if the organism be grown in an alkahne medium it expends its 
 energies on the fermentation of the glucose present at the expense 
 of toxin formation. Experiments dealing with this matter are still 
 in progress. 
 
 The buffering of a medium with sodium phosphate has not, up to 
 the present, materially influenced the production of toxin. 
 
 Fresh muscle. — The influence of fresh muscle in encouraging toxin 
 formation is unquestionable. It has not been found necessary, how- 
 ever, to use the large volumes recommended by Bull. A small 
 piece of muscle about the size of a hazel-nut is sufficient for the 
 purpose when introduced into a 4-litre bottle of medium. The 
 muscle may be replaced by a few c.c. of a freshly prepared filtered 
 muscle extract. The evidence as to the exact nature of the substance 
 in muscle which is responsible for this beneficial effect is as yet 
 incomplete. 
 
 (&) The Organism. — There are considerable differences among the 
 strains of B. welchii in regard to their toxin-producing capacity. 
 Only a relatively small number appear to be good toxin producers 
 and it is probable that further research is necessary to find a con- 
 sistently good and reliable strain giving high-value toxin. Bull's 
 strain, 61 7D, has been found to give very good results in the hands 
 of other 'workers. There are, however, several strains available at 
 the present time that are quite as good. The capacity of any given 
 strain for producing toxin may be enhanced by increasing its viru- 
 lence. This can be done by repeated animal passage, preferably 
 through pigeons which are more convenient animals for this purpose 
 than guinea-pigs. The inoculation of a lethal dose of culture into 
 the pectoral muscles of a pigeon produces an illness which is fatal 
 in 6 hours or less. The pectoral region in pigeons is readily accessible 
 and portions of infected muscle can be easily removed under aseptic 
 precautions. An emulsion of infected muscle can be introduced 
 straightway into a pigeon without any intervening culture and thus 
 one may carry out several rapid passages in a short space of time. 
 
 It is to be noted that the virulence of a strain may increase by 
 passage up to a certain point and then suddenly fall. This rhythmic 
 
108 
 
 rise and fall in virulence has been noted several times. An organism: 
 of ebbing virulence may be set aside in a culture tube in the hope 
 that when next it is used- for passage the tide may have turned, 
 A feature which all strains of B. welcUi seem to possess at some 
 period or other of their growth in artificial media, is the tendency 
 to produce a sticky gelatinous material, the exact nature of which 
 is as yet uncertain. The phenomenon as it occurs in liquid cultures 
 of B. welchii, and the peculiar characters of the surface colonies 
 of an organism which is thus affected have been already noted. The 
 tendency to produce this sticky material in no way interferes with 
 the toxin producing capacity of an organism, but it does place a very 
 formidable barrier in the way of successful filtration. On the 
 assumption that an organism in this state might be ' sugar-sick ', 
 attempts have been made to rid it of this ailment by long cultivation 
 in carbohydrate-free media, but without any appreciable result. 
 Miss Lacey has found, however, that if a sticky organism be washed 
 repeatedly and kept in normal saline solution for a week in the ice 
 chest, it loses the habit of forming glutinous cultures, 
 
 (c) The Inoculum. — The inoculation of a medium may be conven- 
 iently carried out by introducing a piece of infected pigeon muscle 
 taken from a bird that has died as the result of infection. This method 
 has been adopted during the war by the staff at Brockwell Hall 
 in the preparation of toxin in large quantities, and has been found 
 to yield consistently good results. The titre of the toxin may be 
 still further improved by introducing a large dose of the weak broth 
 culture (100 c.c. to a 4-litre bottle) obtained from the preceding 
 pigeon passage. 
 
 {d) The Conditions of Growth. — It is not necessary to adopt special 
 anaerobic methods in growing B. welchii for the purpose of producing 
 toxin. The meat medium already mentioned, if freshly autoclaved or 
 re-steamed just before use, offers the conditions suitable for growth. 
 The inoculum for each 4-htre bottle consists of a small piece of infected 
 pigeon muscle plus 100 c.c. of culture. The cultures are allowed to 
 mcubate at 37°-38° C. for 16 to 24 hours and then filtered. Cultures 
 yield just as good toxin formation if grown at 42° C. 
 
 (e) Filtration. Cultures are passed through layers of paper pulp 
 m large porcelain cylinders until the filtrates are quite clear and 
 translucent. The material is then passed through Berkefeld filters, 
 after which it is tested for sterility. It should be stored in a cool 
 place away from the light. 
 
 B. Vihrion septique toxin ^ 
 B. oedematiens toxin S 
 
 The conditions which seem to determine the formation of these 
 toxins are identical with those which obtain for B. welchii toxin. 
 ihe same meat broth medium may be employed, the inoculum 
 consisting as before of infected muscle plus culture 
 
 Miss Eobertson has been able to prepare very high titre vihrion 
 septique toxm using a plain broth or glucose broth medium which 
 ?.n.nW. 1 ^"I f •.*'' ^ .^t<=tion of Ph. 7-8 to 8-0, and which has been 
 inoculated with bits of infected liver taken from guinea-pigs dead of 
 vibrion septique infection. ^^ 
 
109 
 
 Lastly, it cannot be too strongly emphasized that the factors 
 which govern toxm formation in vitro are still beyond our control. 
 However careful and exact one's preparations may be, the result 
 appears still to be a matter of hazard. 
 
 2. The Properties op the Toxins elaborated by B. welchii, 
 
 ViBRION SEPTIQUE, AND B. OEDEMATIENS. 
 
 (i) Physical Features. 
 
 1. These toxins are all thermolabile and may be destroyed bv 
 heating to 70° C. for 30 to 60 minutes. 
 
 2. B. welchii toxin is reported by Bull as being non-dialysable. 
 No similar evidence is as yet available in regard to the toxins of 
 vibrion septique and B. oedematiens. 
 
 Each of the toxins mentioned depreciates considerably in potency 
 if passed repeatedly through Berkefeld candles and it may be con- 
 cluded therefore that each is hkely to be non-dialysable. 
 
 3. All the three toxins are very susceptible to low concentration 
 of acid. B. oedematiens would appear to be thg most sensitive and 
 vibrion septique toxin the least vulnerable, while the toxin of B. 
 welchii would seem to occupy an intermediate position in this respect. 
 
 In each case the toxin appears not to be precipitated out of solution 
 by contact with acid but to be actually destroyed. There is no clear 
 evidence that it is converted into toxoids. 
 
 B. welchii toxin is said to be readily destroyed by free chlorine. 
 
 (ii) Biological Characteristics. 
 A. The toxin of B. welchii. 
 
 1. It is haemolytic both in vitro and in vivo. The in vivo action 
 is best seen in animals which have had an intravenous inoculation, 
 though it can also be demonstrated constantly in mice which die 
 as the result of an intramuscular dose of toxin. 
 
 . Bull relates the case of a rabbit which received a dose of 1 c.c. 
 intravenously at 10 a.m. and which had then a red corpuscle count 
 of 5-4 millions. A progressive destruction of red cells occurred, 
 until, 7 hours after inoculation, the count was only 1-0 million 
 per c.mm. Also, a pigeon (quoted by Bull) which gave an initial 
 count of 4-28 millions red corpuscles was found d^ hours after inocula- 
 tion to have no more than 0-8 million red cells per c.mm. in its blood. 
 
 The haemolytic action of the toxin is not due to the presence of 
 acids, for it can be demonstrated equally well with toxins that have 
 been carefully neutralized. 
 
 Haematuria is a constant finding in mice ; it is less frequent in 
 rabbits and in guinea-pigs. 
 
 2. It is necrotic. This action is best seen in the case of muscle, 
 though it can be demonstrated also in skin by intracutaneous 
 inoculation. The muscle becomes oedematous, friable, and necrosed 
 so as ultimately to form a difHuent pulp. 
 
 3. It produces a marked serous oedema which may be found in 
 large quantities in the subcutaneous tissues. This oedema fluid 
 
110 
 
 when collected is found to be almost free from cells. It clots readily 
 and yields a clear serum which is often of a bright yellow colour. 
 In mice, as a rule, the fluid is blood-stained and gives after centri- 
 fuging the spectrum of oxyhaemoglobin. 
 
 4. It stimulates involuntary muscles. A rabbit which has received 
 an intravenous dose of toxin exhibits violent intestinal peristalsis 
 accompanied by diarrhoea. A similar reaction, though less common, 
 occurs in mice and in guinea-pigs. The vomiting which, occurs in 
 pigeons and which may be such a marked feature of the terminal stages 
 of gas gangrene in man is probably explicable on the same grounds. 
 
 5. Washed surface growths of B. welchii do not produce infection in 
 laboratory animals even when inoculated in large doses. It has been 
 found also that the washed bacilli separated out of a liquid culture 
 are, by themselves, non-infective. If, however, an emulsion of washed 
 bacilli be mixed with a sub-lethal dose of toxin, then there can be 
 induced a typical gas gangrene infection which is rapidly fatal. 
 The toxin therefore stimulates the development of B. welchii in the 
 animal body and in this respect has aggressive properties. 
 
 Only a very small number of bacilli under these circumstances 
 is requisite for initiating infection. On the other hand the amount of 
 toxin that is necessary for the combination to be successful is a con- 
 siderable fraction of the lethal dose. 
 
 It is probable that there are two contributory factors involved in 
 the mechanism of the aggressive reaction, viz. (a) the necrosis of 
 tissue which need not necessarily be looked upon as specific (Bullock 
 and Cramer (1)) ; (b) the antiphagocytic property of the toxin which 
 is specific. Both these effects can be completely inhibited by 
 antitoxin. 
 
 6. The toxin has antigenic properties. 
 
 (a) By repeated injection of toxins into animals it is possible 
 to set up an active immunity. 
 
 (b) Under the same circumstances an animal produces an anti- 
 serum which can be shown to possess the following properties. 
 
 I. It neutralizes toxin in vitro, so that a mixture of toxin with 
 the proper proportion of antiserum is innocuous when inoculated 
 into animals. 
 
 Such an antiserum is capable of neutralizing any B. welchii toxin. 
 
 II. The antiserum when inoculated into animals produces a passive 
 immunity to B. welchii toxin and also to B. welchii infections. 
 Protection against the lethal effect of toxin was found by Bull to 
 persist for two weeks after the giving of serum. 
 
 The prophylactic use of the serum against actual infection was 
 clearly demonstrated by Bull in the following experiment. Guinea- 
 pigs which had received a small dose of serum were found on the first 
 day a,fter inoculation to be capable of resisting at least 150 minimal 
 lethal doses of culture. On the fifth day they survived 300 lethal 
 ^0^?^' oi^^*"e eighth day 60 doses, and on the eleventh day 20 doses. 
 
 ill. ihe serum can be used therapeutically to treat the disease 
 when this is already established. Experiments by BuU and others 
 have proved that, if large guinea-pigs be used, a small dose of serum 
 given 24 hours after the inoculation of a lethal dose of culture ensures 
 complete recovery. 
 
Ill 
 
 B. The toxin of vibrion septique. 
 
 This toxiii is Kaemolytic, myolytic, and oedema-producing, but to 
 a less degree than B. welchii toxin. On the other hand, it does not 
 kill laboratory animals when given subcutaneously or intramuscularly 
 except when inoculated in very large doses. Its lethal effect is best 
 demonstrated by intravenous injection. 
 
 Its antigenic properties are in general the same as those outlined 
 for B. welchid, with this important difference. An antitoxic serum 
 to vibrion septique does not appear to protect if it is given to an 
 animal after the infection is established. 
 
 C. The toxin of B. oedematiens. 
 
 The distinctive feature of this toxin is the extensive and heavy 
 gelatinous oedema it produces both in muscle and in subcutaneous 
 tissues. It kills in small doses and gives rise in horses to an antiserum 
 which is relatively more potent than those so far obtainable against 
 B. welchii toxin or vibrion septique toxin. In this respect it more 
 closely resembles the toxin produced by B. tetani. 
 
 3. The Symptoms and Pathological Changes produced by 
 THESE Toxins and the Estimation of their Minimal Lethal 
 Dose in Animals. 
 
 (i) The Toxin of B. welchii. 
 A. Mice. 
 
 Intramuscular inoculation of a multiple (5 to 10) of the lethal dose 
 is followed by urgent dyspnoea and death with convulsions in 2 to 
 3 hours. In these animals at post mortem, the local lesion may be 
 very slight or absent, and the appearance of the viscera is normal. 
 With a smaller multiple of the lethal dose (2 to 5) death occurs in 
 6 to 8 hours. With one lethal dose there is produced an illness which 
 lasts for about 48 hours. 
 
 The progressive oedema which develops at the site of inoculation 
 within a few hours and which extends rapidly upwards into the 
 subcutaneous tissues of the abdominal wall is easily palpable. The 
 inoculated limb is held stiffly with the toes firmly flexed. In 24 to 
 48 hours the animal refuses food, loses weight, and has a staring coat. 
 There is a moderate degree of diarrhoea, and haematuria is frequent. 
 The limb and trunk muscles become paretic so that the animal 
 totters in attempting to walk. Dyspnoeic periods with violent 
 and laboured respiratory movements are an almost constant feature 
 of the illness in its later phases. Ultimately the animal succumbs 
 with a flaccid paralysis of all its limb muscles. 
 
 The post-mortem findings in such an animal are as follows : A 
 blood-stained oedema fluid is found extending from the site of 
 inoculation into the opposite groin and upwards into both axillae. 
 The muscles of the inoculated limb are pale, friable, and necrosed, 
 but are never reduced to the liquid grumous state that is such 
 a marked feature in cases of actual infection. The bladder is usually 
 distendedwith urine which is deeply stained with blood. The kidneys 
 
112- 
 
 are reddened and engorged and bulge from their capsules when cut 
 into. The liver may show fatty degenerative changes either m 
 patches or diffusely distributed throughout the whole viscus. There, 
 may be an increased amount of fluid in the serous cavities, more 
 particularly in the pleura and in the pericardium. The duodenum 
 and part of the small intestine are distended with hquid contents 
 which are heavily bile-stained. The rectum is often filled with gas 
 and semi-fluid contents. The suprarenals may be reddened and the 
 spleen may be much enlarged but both these changes are inconstant. 
 The titration of B. welchii toxin as carried out by the intra- 
 muscular inoculation of mice is exemplified in the following tabulated 
 experiment which was undertaken to estimate the potency of the 
 toxins produced by five different strains of the organism. 
 
 strains. 
 
 B. 85 
 
 B. Petrie . 
 B. King . 
 B. Gorman 
 B. Quirk . 
 
 1-0 
 
 + + 
 + + 
 + + 
 + + 
 + + 
 
 Amounts of toxin ex. 
 0-8 0-6 0-5 0-4 0-3 
 
 ++ +— — 
 
 ++ ++ +— 
 
 ++ ++ ++ — 
 
 0-2 
 
 . lethal 
 ' dose. 
 
 0-5 C.C. 
 1-0 CO. 
 0-6 CO. 
 1-0 C.C. 
 0-3 c.c. 
 
 = death within 48 hours. 
 
 — =recovery. 
 
 Two animals were used in every case. 
 
 Subcutaneous inoculation of B. welchii toxin into mice produces 
 an illness which is exactly similar to that which follows on intra- 
 muscular injection. 
 
 The intravenous inoculation of mice with lethal doses of the same 
 toxin would seem to give irregular results. Death may occur in 
 a few minutes or after many hours, so that it becomes by no means 
 easy to determine the exact minimal lethal dose. 
 
 B. Babbits. 
 
 One minimal lethal dose of toxin given intravenously to a full- 
 grown rabbit produces death in 2 to 2J hours. A smaU percentage 
 of rabbits may survive for a longer period, but as a rule, if death does 
 not occur within 3 hours, the animal recovers completely. With 
 multiples of a minimal lethal dose death may follow in J hour. In 
 this case the symptoms are those of muscular excitation and restless- 
 ness, with marked dyspnoea and convulsive seizures ending in death. 
 
 The inoculation of one minimal lethal dose is followed immediately 
 by a period of dyspnoea ; but this is usually transitory and passes 
 off m a few minutes. Usually no symptoms manifest themselves 
 for 1 to 11 hours. The animal then becomes restless and changes his 
 position frequently. It is attacked with recurring periods of 
 breathlessness, which, though at first slight, soon become longer in 
 duration and severer in degree. At the same time it develops 
 muscular tremors and paresis.. It hes down, slightly to one side, 
 with all its hmbs fully extended. The intestines can be seen in 
 violent peristaltic movement under the flaccid muscles of the belly 
 wall. It defalcates frequently and may have actual diarrhoea 
 with haematuria. The head is thrown back into the nape of the neck 
 in a position of opisthotonos, and the nose is raised high so as to 
 
113 
 
 relieve air-hunger. Towards the end, the flaccidity of the muscles 
 becomes absolute. It can no longer raise its head, nor can it 
 recover its position when this is altered by the observer. The 
 respiratory embarrassment becomes accentuated. Periods of shallow 
 rapid breathing give way to laboured efforts, separated by longer 
 arid longer phases of apnoea. Finally there occur a few convulsive 
 spasms and the animal is dead. 
 
 With the exception of a small amount of blood-stained fluid in 
 the peritoneal cavity, there is very little to be found that is charac- 
 teristic when these animals are examined post mortem. There may 
 be collections of fluid in the pleural and pericardial sacs. The urine 
 is at times blood-stained, and exceptionally the serum may be tinted 
 with haemoglobin. The rectum may have liquid and gaseous con- 
 tents. Otherwise no naked-eye changes are to be observed. 
 
 C. Guinea-pigs. 
 
 The illness which results in guinea-pigs after the intramuscular 
 inoculation of B. welchii toxin resembles in its main features that 
 already described in mice. The post-mortem appearances are much 
 the same, except that the effects of haemolysis are often less obvious, 
 while reddening and enlargement of the suprarenals are probably 
 more common. Numerous instances of the titration of B. welchii 
 toxin by this method can be found in the experimental data recorded 
 by Bull and Pritchett and by De Kruif and his colleagues. 
 
 The intravenous inoculation of guinea-pigs does not appear to 
 afford a reliable means of titration, because it is found difficult to 
 fix a time limit for the recording of a lethal effect. 
 
 D. Pigeons. 
 
 Pigeons succumb to a lethal dose of toxin, given intramuscularly, 
 in from 4 to 8 hours. A multiple of the lethal dose may produce 
 death in 3 hours. When introduced by the intravenous route the 
 toxin kills more rapidly (Bull and Pritchett). 
 
 The titration of a stable B. welchii toxin, precipitated out of 
 solution by absolute alcohol, is given in the subjoined table, the 
 numbers given representing milligrammes of the dried product. 
 
 Mice 
 
 Guinea-pigs' 
 
 Rabbits 
 
 Pigeons 
 
 intramuscular. 
 
 intramuscular. 
 
 intravenous. 
 
 intramuscular. 
 
 3-0+ + 
 
 10+ 
 
 50+ 
 
 5-0+ 
 
 2-0+ + • 
 
 5+ + 
 
 30+ 
 
 2-5+ + 
 
 1-0+ + 
 
 3+- 
 
 20+ + 
 
 2-0+ — 
 
 0-8 
 
 2-5 
 
 15- 
 
 1-5 
 
 . 0-5 
 
 1-0 
 
 10— 
 
 10— 
 
 0-5 
 
 
 
 
 m.l.d.=l-0 
 
 m.l.d. =5-0 
 
 m.l.d. =20 
 
 m.l.d. =2-5 
 
 (ii) The Toxin of Vibrion septique. 
 
 . It: would seem to be so difficult to prepare a vibrion septique toidn 
 which when inoculated subcutaneously or intramuscularly will kill 
 the animals that are commonly used for experiment, that most 
 observers have had recourse to intravenous injections. Where mice 
 and guinea-pigs are inoculated by this route, the problem of setting 
 a satisfactory time limit in estimating the significance of a positive 
 
114 
 
 result presents just as great a difficulty as that which occurs with 
 B. welcUi toxin under similar circumstances. On the other hand 
 where rabbits are used, there is less uncertainty in this matter because 
 these animals either die in a few minutes or recover completely. 
 
 The illness in rabbits has many features in common with that 
 which follows the injection of B. welchii toxin. There are, however, 
 certain characteristic points which distinguish it from the latter. 
 
 1. The incubation period varies from a few seconds up to 20 or 
 30 minutes. 
 
 2. The clinical course of the illness is more fulminant in its mani- 
 festations. The disease tends to be convulsive rather than paralytic 
 in character. Within a few seconds or minutes of receiving an intra- 
 venous injection, a rabbit may suddenly leap from the cage, perform 
 a series of violent convulsive movements about the floor and drop 
 dead in a corner of the room. Or again, the course of the illness may 
 exactly resemble that produced by B. welchii toxin, except that it 
 is compressed into a few minutes instead of being spaced over an 
 hour or more. 
 
 These animals show no special pathological changes post mortem. 
 Exceptionally, where death has been delayed for several hours, small 
 amounts of blood-stained fluid may be seen in the serous cavities. 
 
 In view of the necessity for testing large numbers of toxins during 
 the war and the difficulty of obtaining rabbits in sufficient numbers 
 for this purpose, there has been elaborated at the Wellcome Physio- 
 logical Research Laboratories a method of titrating vibrion septique 
 toxin which depends on the estimation of the minimal oedema 
 causing dose in mice. It has been found possible to determine this 
 amount of toxin with sufficient accuracy to give reliable comparative 
 values in the standardization of different antisera. The unit of 
 vibrion septique antitoxin has been designated as that amount, of 
 serurn which, when mixed for one hour at room temperature with 
 10 minimal oedema-causing doses of toxin, will completely inhibit 
 the production of oedema in the animal which is inoculated with 
 the mixture. The method, however, is one that necessitates constant 
 practice, and the results are open to wide differences in interpretation 
 which depend entirely on the experience of individual observers. 
 
 (iii) The toxin of B. oedematiens. 
 
 This toxin kills laboratory animals when given intravenously, 
 subcutaneously, or intramuscularly. It can be very easily titrated 
 by the intramuscular injection of mice, the final readings in regard 
 to a positive or negative result being recorded at the end of 48 hours 
 as m the case of B. welchii toxin. 
 
 Animals which succumb to B. oedematiens toxin show the same 
 massive gelatmous oedema that characterizes a fatal infection by 
 this organism. The muscles at the site of inoculation are much 
 swollen with oedema fluid. They are pale, and often occupied by 
 
 rCionSf>?™ haemorrhages, but"^ are not necrosed^ Smafl 
 ettusions into the serous sacs are common 
 
 tox?n^:S"out ifmic?"""*^ the .titration of a B. oe5.nu.ti.ns 
 
115 
 
 Toxin LE 235 
 
 c.c. 
 
 0-01 + + 
 
 0-005 + + 
 
 0-002 + + 
 
 0-001 + + 
 
 0-0005+ + 
 
 0-0002+ + 
 
 0-0001 
 
 4. The Standaedization of Antishea. 
 (i) B. welchii antitoxic sera. 
 
 Bull estimated the titre of his antitoxic sera by determining the 
 smallest amount of serum that was requisite to neutralize one minimal 
 lethal dose of toxin in a 250 grm. guinea-pig or a 300 grm. pigeon. 
 This amount of serum he has defined as the unit of antitoxin. 
 
 Where large numbers of different sera have to be tested this 
 method becomes a very expensive procedure. To obviate the diffi- 
 culty, the Wellcome Physiological Laboratories have used mice in 
 place of guinea-pigs and pigeons. As has been already indicated the 
 minimal lethal dose of B. welchii toxin can be readily estimated in mice. 
 For testing purposes at the W.P.E.L. the unit of antitoxin has been 
 fixed as being twice the amount of serum that is sufficient to neutralize 
 the lethal effect of two minimal lethal doses, the mixture of toxin and 
 serum being left in contact for one hour at room temperature before 
 inoculation. The results obtained by this method are claimed to yield 
 a figure-in unitage which within reasonable limits is identical with that 
 obtained when the same sera are tested according to Bull's method. 
 
 A. Titration of B. welchii toxin LW. 218. 
 
 
 0-4 + + 
 
 
 
 
 0-3 + + 
 
 
 
 
 0-25+ + + + 
 
 
 
 0-2 +-- 
 
 — — 
 
 
 
 0-15 
 
 
 
 
 0-1 
 
 
 
 
 0-1 
 
 
 
 
 m.l.d. =0-250.0. 
 
 
 B. Titration of antisera 
 
 against two minimal lethal doses, viz 
 
 0-5 c.c. 
 
 SEBA 
 
 
 
 115B 
 
 25V 
 
 14b 
 
 810 
 
 0-1 + + 
 
 + + 
 
 
 
 
 
 0-01 + + 
 
 + + 
 
 
 
 
 
 0-005 
 
 
 
 
 
 0-004 
 
 
 
 
 
 0-003 
 
 
 + - 
 
 
 0-002 
 
 
 + - 
 
 
 0-001 + + 
 
 + + 
 
 + + 
 
 
 
 0-0008 
 
 
 
 
 
 0-0006 
 
 
 
 
 
 0-0005 
 
 
 
 h 
 
 0-0004 
 
 
 
 + + + + 
 
 0-0002 
 
 
 
 + + 
 
 O-OOOl 
 
 
 
 + + 
 
 Unitage 
 
 
 
 125 
 
 1,000 
 
 + = death in 48 hours. 
 
 — = 
 
 = recovery. 
 
 
 H2 
 
 
 
116 
 
 The previous record shows an evaluation of some sera thus 
 tested. The sera designated 115B, 25V, and 14b were German 
 anti-gas gangrene sera captured on the Western Front, while 810 is 
 a single horse serum prepared by Weinberg. The latter in addition to 
 B. welchii antitoxin contains antitoxin to vibrion septique and to 
 B. oedematiens. 
 
 (ii) Vibrion septique antitoxic sera. 
 
 French workers have defined as the unit of vibrion septique anti- 
 toxin the smallest amount of serum which will neutralize the fatal 
 effect of one lethal dose of toxin given intravenously to a full-grown 
 rabbit, the mixture of toxin and serum being kept at room tempera- 
 ture for one hour before inoculation. 
 
 The following record illustrates the titration of Weinberg's serum 
 810 by this method. 
 
 A. Titration of toxin. 
 
 20 CO. 
 1-5 e.c. 
 1-5 c.c. 
 1-0 C.C. 
 1-0 C.C. 
 
 + 7 min. 
 + 15 min. 
 + overnight 
 recovery 
 recovery 
 
 B. Titration of serum against 5-0 c.c, i 
 doses. 
 
 serum. 
 0-01 
 0005 
 0-004 
 0-004 
 0-003 
 0-003 
 0-002 
 0-001 
 
 e. 3 certain minimal lethal 
 
 result. 
 recovery 
 recovery 
 recovery 
 recovery 
 + 20 mins. 
 + overnight 
 + 8 min. 
 + 3 min. 
 
 The serum then is found to neutralize in such a way that 0-004 
 gives complete protection against 3 m. 1. d., i. e. it contains approxim- 
 ately 750 units. ^^ 
 
 0-1 
 
 0-01 
 
 0-005 
 
 0002 
 
 0-001 
 
 0-0008 
 
 0-0006 
 
 0-0004 
 
 0-0002 
 
 0-0001 
 
 0-00008 
 
 0-00006 
 
 0-00004 
 
 0-00002 
 
 0-00001 
 
 (iii) B. oedematiens antitoxic sera. 
 
 25V 115B 124 
 
 + + — 
 
 ++ ++ ++ 
 
 ++ ++ ++ 
 
 ++ ++ ++ 
 
 ++ ++ ++ 
 
 5 
 
 + =doath in 8 hours. 
 
 410 
 
 + + 
 + + 
 
 + + 
 
 411 
 
 + — 
 + + 
 + + 
 
 + + 
 + + 
 + + 
 
 50 250 
 
 — =reoovery. 
 
 810 
 
 + ■+- 
 -f + 
 + + 
 
 S.OOO' 
 
117 
 
 "Weinberg has defined as the unit of antitoxin the smallest amount 
 of serum which will completely neutralize 100 minimal lethal doses 
 of toxin in a mouse, the mixture being kept for one hour at room 
 temperature before inoculation. The accompanying record illustrates 
 an experiment in which various sera were titrated against a test 
 dose consisting of 50 minimal lethal doses of toxin. 
 
 5. Preparation and Standardization of Anti-gas gangrene 
 
 Sera. 
 
 (A Report by the Staff of the Wellcome Physiological Research Labora- 
 tories). 
 
 (i) Introduction. 
 
 The investigation into the possibility of making anti-gas gangrene 
 serum was first undertaken at the request of the Medical- Eesearch 
 Committee, and the immunization of horses was commenced in 
 October 1917. Dr. Mcintosh on behalf of the Committee, supplied 
 suspensions of surface growths, and also broth cultures of the various 
 anaerobes considered to be concerned in the production of gas 
 gangrene. He added solutions of iodine to these cultures to produce 
 attenuation. Two horses were immunized with these cultures over 
 a period of some months. At one stage, when the horses had been 
 under immunization for several months, and supplies of the bacterial 
 suspension were not available, B. welchii toxin was injected. Prior 
 to this time, the B. welchii antitoxic titre of sera from these two 
 horses was low. What potency the sera of these horses possessed when 
 issued for field trial in Prance in March 1918 was, therefore, the 
 result of immunization with cultures and toxin in the case of B. welchii, 
 and of cultures only in the case of vibrion septique, and other organisms. 
 •In January 1918, Major Carrol Bull of the tfnited States Army had 
 demonstrated in London the power of B. welchii antitoxin to neu- 
 tralize the toxin of B. welchii. Bull presented a culture and a sample 
 of antitoxin to the Laboratories. B. welchii toxin was rapidly 
 prepared in large quantities for injection into horses. Antitoxin 
 of sufficiently high titre to protect animals against large doses of 
 living culture of B. welchii was available for field trial in March 1918. 
 
 Meanwhile, evidence of the importance and frequency of the 
 occurrence of vibrion septique was accumulating. At a War OfBce 
 conference in March 1918 the pathologists present agreed that this 
 organism was frequently present in wounds and was the cause of 
 severe and fatal cases of gas gangrene occurring in soldiers. Work 
 was immediately begun at the Laboratories with the aim of producmg 
 an antiserum to vibrion septique. Cultures of vibrion septique were 
 presented by Dr. Mcintosh, Miss Eobertson, and- Capt. Henry, 
 and samples of toxin for testing purposes by Miss Eobertson. Large 
 quantities of toxin were made at the Laboratories for injection into 
 horses, and by May 1918 a serum had been produced which was of 
 sufficiently high titre to protect an animal, which had received some 
 hours previously 5 c.c. of serum, against many lethal doses of toxin 
 or culture. Some of the horses yielding this serum had already been 
 immunized with B. welchii. Thus, a double serum contammg 
 
118 
 
 antitoxins to both B. welchii and vibrion septique became available, 
 and was ready for trial in May 1918. , , ,, 
 
 B. oedematiens toxin made by Captain Henry from cultures 
 isolated by him, became available in quantity m June 1918. Serum 
 of satisfactory protective value, as judged by results obtamed with 
 laboratory animals, was produced in September 1918. 
 
 The aim throughout the whole of the work was to produce as rapidly 
 as possible a serum which, when injected into a soldier immediately 
 after the infliction of a wound, might protect him against the onset 
 of gas gangrene. 
 
 It was decided at an early date to include the recognized pro- 
 phylactic dose of tetanus antitoxin in the prophylactic gas gangrene 
 serum. The laboratory staff commenced in February 1918 the 
 preparation of T.W. serum, i.e. one containing sufficient antitoxins 
 to both B. tetani and B. welcJiii to protect a laboratory animal against 
 many lethal doses of cultures of these two organisms. 
 
 T.W. serum was produced in quantity in March 1918. T.V.W. 
 serum, containing antitoxin for B. tetani, B. welchii, and vibrion 
 septique, was produced in August 1918. T.V.W.B. serum, containing 
 antitoxin also to B. oedematiens became available only about the date 
 of the armistice in November 1918. 
 
 It may here be remarked that in 1918 almost the whole of the work 
 was directed to the production of antitoxic, as opposed to anti- 
 bacterial sera. It early became evident that the practical difficulties 
 of preparing, in the very large quantities required, the ' whole 
 cultures ' of bacilli to a constant standard of pathogenicity, so that 
 the injections into horses would proceed smoothly and safely, were 
 so great that the production of sera for field trial in large quantities 
 would inevitably have been delayed for many months. It was found 
 that antitoxins of high titre, when injected into animals, gave them 
 complete protection against subsequent injection of many lethal. 
 doses of living cultures. It was, therefore, considered advisable 
 to concentrate the main effort on the production of purely antitoidc 
 sera while investigating the methods of preparation >and evaluation 
 of antibacterial sera. The termination of the war came before the 
 experimental work with antibacterial sera was very far advanced. 
 
 From the foregoing account it is clear that the sera actually 
 tested m the British Army in France and used prophylactically and 
 curatively were the first results of an attempt to produce three new 
 immune sera. The nature of the toxins were only obscurely under>- 
 stood and very serious difficulties both practical and theoretical 
 had to be encountered. The sera were definitely not the final and 
 most potent products possible, it is not even now known what titre 
 should be considered to be a useful minimum. In judging the results 
 therefore of the serumtherapy it must be borne in mind that the 
 tests were made at the earhest possible moment and that rightly 
 considered no really adequate therapeutic trial has been made of 
 these sera. 
 
 , ^ ,,.. (}^) Preparation of Antitoxins. 
 
 A. B. welchii. 
 
 ToKm.-Ordinary peptone meat broth or tryptic digestlbroth 
 contaming 1 per cent, of glucose, was inoculated with a 9*-^onv 
 
119 
 
 culture of B. welchii. The addition of sterile fresh muscle or of 
 autoclaved meat seemed on the whole to be of advantage. The 
 inoculated broth was kept at 37° for 24-30 hours, and then filtered 
 through kieselguhr candles, carbohzed and kept in a cool place. 
 It was not difficult to produce large quantities of toxin of which 
 0-2 c.c. to 0-3 CO. would, when injected intramuscularly, kill a 250- 
 grm. guinea-pig or a 20-grm. mouse. 
 
 It was not found necessary to use any special methods for securing 
 anaerobiosis : the broth was not covered with paraffin. The ino- 
 culum was added to the broth a few hours after it had left the auto- 
 clave and had cooled to about 40° C, 
 
 Immunization of the Horse. 
 
 The processes of immunization correspond almost exactly to those 
 in common use for the preparation of diphtheria antitoxin. B. welchii 
 toxin was injected intramuscularly at intervals of a few days, a repre- 
 sentative series of doses being 1 c.c, 2 c.c, 4 c.c, 8 c.c. at intervals 
 of 2 or 3 days. Ten weeks later the dose of toxin reached 400 c.c. 
 and the antitoxic value of the horse's serum was 1,000 to 2,000 units. 
 With the average horse a titre of 1,000 units was attained in about 
 8 weeks ; at this point the animal was bled. 
 
 Serum containing 6,000 units per c.c. was obtained from several 
 horses, but'as the object of the work was to produce the maximum 
 quantity of serum with a titre of 1,000 units in a given time, no 
 opportunity was taken of continuing the immunization without 
 bleeding to ascertain the highest titre attainable by this method. ' 
 
 B. Vibrion septique. 
 
 Toxin. — The method of making toxin was similar to that described 
 for B. welchii. The inoculum was either a portion of a culture or 
 the infected breast muscle of a pigeon injected the day previously 
 with a culture of vibrion septique. 
 
 The toxin produced in large quantities was sufficiently potent to 
 kill a rabbit within ten minutes when a dose of 0-5 c.c. was injected 
 intravenously. 
 
 Immunization of the Horse. 
 
 The methods closely resembled those used for the production of 
 antitoxin to B. welchii, though the rate of increase of dosage was 
 slower. An average horse would produce in 8 to 12 weeks serum 
 containing 1,000 units of vibrion septique antitoxin per c.c 
 
 0. B. oedematiens. 
 
 Toxin. — ^By using the method above described in the B. welchii 
 section, toxin was produced in large quantities of which 0-0001 c.c, 
 injected subcutaneously, would kill a 20-grm. mouse within 
 24 hours. The toxin is apparently rather unstable, the toxicity 
 rapidly decreasing. Whether this old weak toxin is a good antigen 
 is not at present known with accuracy. 
 
120 
 
 Immunization of the Horse. 
 
 The methods in use are exactly comparable with those in common 
 use for the preparation of antitetanic serum. An average horse in 
 3 months would yield a serum containing 5,000 units of B. oedematiens 
 antitoxin per c.c. 
 
 (iii) Methods of estimation of the value of anti-gas gangrene sera. 
 
 A. Antitoxin of B. welchii. 
 
 The unit in use was based upon a serum supplied by Bull which 
 bore a label indicting the unitage: Bull used as his unit the quantity of 
 serum which neutralized an amount of toxin equal to one lethal dose 
 for the pigeon. We found that this quantity of serum was twice the 
 amount necessary to neutralize twice the lethal dose for a mouse. The 
 unit arrived at in this way was adopted for the titration of B. welchii. 
 
 Toxins used for standardization would in a dose of 0-1 c.c. to 
 0-25 c.c, when injected intramuscularly, kill a mouse within 24 hours. 
 
 Serum, was produced containing 5,000 units per c.c. 
 
 B. Antitoxin of Vibrion septique. 
 
 The toxin produced by vibrion septiqiie differs from that produced 
 either by B. oedematiens or B. welchii in that it does not cause death 
 when inoculated intramuscularly or subcutaneously into mice. It 
 can, however, be readily titrated by intravenous inoculation into 
 rabbits, a high value toxin producing death in 3-15 minutes with 
 a dose of 0-5 c.c. or less when tested by this method. 
 
 A delayed reaction in which death occurs after 8 to 24 hours 
 takes place with lesser doses. This delayed reaction is unsatisfactory 
 from the point of view of a test as it is difficult to adjust the dose, and 
 individual animals show a variable resistance. Since the intravenous 
 test became impracticable when the work was carried out on a large- 
 scale it was necessary to substitute some other method of titration. 
 
 It was found that 0-05 c.c. of toxin injected intramuscularly into 
 a mouse produces a recognizable oedema. This reaction is used to 
 test the potency of the serum. The test dose of toxin used is ten 
 times the amount, i. e. 0-5 to 1 c.c. according to the strength of 
 the toxin. This test dose is equivalent to an intravenous lethal 
 dose for the rabbit. A small number of antitoxins were titrated 
 by : (1) the mouse-oedema method ; (2) intravenous injection into 
 mice ; (3) intraperitoneal injection into mice ; (4) intravenous 
 injection into rabbits. The results agreed sufficiently closely to 
 justify the temporary use of the ' mouse-oedema method '. 
 
 0. Antitoxin of B. oedematiens. 
 
 The unit of antitoxin (which was already in use by Dr Weinberg 
 at the Pasteur Institute) is that quantity of serum which when 
 added to 100 minimal lethal doses of toxin injected intramuscularly 
 mto a mouse will render the toxin inert and prevent the death of 
 the animal. Sera were produced containing 20,000 units per c.d. 
 We should hke to take this opportunity to express our thanks to 
 Dr. Wemberg for his courtesy in sending us samples of his sera on 
 several occasions. 
 
6. The Serum Therapy of Gas Gangrene^ 
 (i) The Collection of data. 
 The treatment of cases of gas gangrene by means of antisera was 
 first carried out in British military hospitals in France during the 
 spring months of 1918. Eeports on the results obtained by the use 
 of these antisera were compiled by a number of E.A.M.C. officers 
 and forwarded to the Medical Eesearch Committee. These reports 
 form the basis of the following summary. 
 
 The accompanying table (Table I) indicates the number of cases 
 recorded by each observer, together with the number of deaths 
 occurring in each series. 
 
 Table I. 
 
 Reported by 
 
 Barling ....... 
 
 Brenan ...... 
 
 ElUs 
 
 Hope ....... 
 
 MoEwen 
 
 McNee 31 
 
 Stokes ....... 23 
 
 Tytler 17 
 
 Wyard ........ 2 
 
 Totals 89 
 
 No. of 
 cases. 
 
 1 
 9 
 1 
 1 
 
 No. of 
 
 4 
 
 1 
 
 3 
 
 1 
 
 1 
 
 12 
 
 16 
 
 11 
 
 1 
 
 50 
 
 (ii) The Serum employed. 
 The sera which were used in Prance were prepared by the staff 
 of the Wellcome Physiological Eesearch Laboratories at Heme 
 Hill. The titre of each serum, i. e., the number of units of B. welchii 
 antitoxin and of vibrion septique antitoxin contained in each c.c. 
 of serum is set out in Table II. 
 
 Table II. 
 
 
 B. welchii 
 
 V. septique 
 
 antitoxin 
 
 antitoxin 
 
 units. 
 
 units. 
 
 2,000 
 
 — 
 
 
 
 1,000 
 
 300 
 
 
 
 1,000 
 
 300 
 
 
 
 500 
 
 300 
 
 
 
 500 
 
 300 
 
 
 
 2,000 
 
 — 
 
 
 
 2,500 
 
 1,600 
 
 
 • 
 
 1,000 
 
 3,000 
 
 G2 . 
 
 G3 . 
 
 G17 
 
 G18 
 
 G19 
 
 G22 
 
 G44 
 
 G53 
 
 It is to be noted that in the titration of the above sera the unit of 
 B. welchii antitoxin is taken as being twice the amount of serum 
 requisite to neutralize two minimal lethal doses of toxin in the mouse. 
 This method of measurement has been found to yield a result which 
 within reasonable limits is identical with the figure obtained by 
 Bull's method of titration. 
 
 The unit of vibrion septique antitoxin is estimated as being the 
 amount of serum which will completely neutralize 10 minimal-oedema- 
 causing doses of toxin in the mouse. This procedure yields a much 
 Ijigher figure than that obtained by the French method of standardiza- 
 tion, in which the antitoxin unit is taken as the amount of serum 
 which will neutralize one intravenous minimal lethal dose of toxin 
 in a full-grown rabbit. 
 
122 
 
 The sera designated as G3, G17, G18, and G19 were prepared by 
 the inoculation into horses of mixed cultures of anaerobes which had 
 been attenuated with iodine. These attenuated mixed cultures 
 were furnished by Mcintosh. The resulting sera proved to have 
 a low antitoxic value. The amount of vibrion septique antitoxin did 
 not pass beyond the 300 unit level, while the titre of B. welchii 
 antitoxin remained in the neighbourhood of 100 units per c.c. It 
 was only after treating these, same horses with B. welchii toxin that 
 the content in antitoxin as set out in Table II was reached. The sera 
 refeiTed to are claimed by Mcintosh to have distinct advantages 
 over purely antitoxic sera in that they contain antibacterial bodies. 
 
 The production of sera by the inoculation of attenuated whole 
 cultures of anaerobes was found to present considerable diBficulties 
 when carried out on a large scale, and the method was abandoned 
 in favour of inoculation with sterile toxins. The sera designated 
 G2, G22, G44, and G53, thus prepared, were therefore purely anti- 
 toxic sera with no antibacterial content. 
 
 None of the sera issued for use in the field contained antitoxin 
 to B. oedematiens. 
 
 The sera designated B. in section (vi) were B. welchii antitoxic 
 sera prepared by Bull in the Eockefeller Institute and had a titre 
 of B. welchii antitoxin varying from 750 to 1,800 units per c.c. 
 
 (iii) Analysis of Becords. 
 
 The serum therapy of cases of gas gangrene became available at 
 a period in the history of the war when it had already been clearly 
 demonstrated that the disease was amenable to rigorous surgical 
 procedures. It so happened then that antisera came to occupy 
 two separate roles in the treatment of gas gangrene. 
 
 1. Where the focus of the disease could be extirpated or otherwise 
 effectively dealt with by the surgeon the use of serum became 
 an adjuvant to surgical measures. If, for example, symptoms of 
 toxaemia persisted after complete removal of all anaerobe infected 
 tissue, recovery could be hastened by the giving of serum. Or 
 agaia, the giving of serum to a man suffering from serious toxaemia 
 might improve his physical condition sufficiently to permit of opera- 
 tion. In either instance, the beneficial effect of the serum was due 
 to the neutralization of toxin which had already reached the circula- 
 tion. The serum was not called upon to combat the infection. 
 
 2. On the other hand, the serum came to be used in desperate 
 cases, m which, for various reasons, surgical intervention was 
 inadequate to cope with the disease. The task assigned to the serum 
 in this particular class of case was a very severe one, for it had to 
 deal not only with toxin in the circulation and in the infected tissues, 
 but it had also to arrest the actual spread of infection and finaUy 
 to overcome it. • ■' 
 
 ..f^^/ !f ''■^^ '^''!' ""^ ?^'^'' *^^ '^*^^^* f^at^J^es of which have been 
 firllvIL''' section (yi), can be best analysed by considering, 
 wbS'.i. A ^^7^ich death occurred ; and secondly, the casts 
 beneficSr ""' ^^"'^ *^' ''™^ ^^' ^""^^'^ *« ^^^^ been 
 
A. Deaths. 
 
 These may be classified as follows : 
 
 1. Oases m which the wounded man recovered from gas gangrene 
 and in which death may be reasonably attributed to some other 
 conditions, such as a streptococcal septicaemia or a pneumonia. 
 
 The following cases in section (vi) come under this category : 
 
 Case number. Day of death. Cause of death. 
 
 38 10 Lobar pneumonia. 
 
 45 10 Lobar pneumonia. 
 
 73 8 Broncho-pneumonia. 
 
 63 9 Streptococcal septicaemia. 
 
 74 6 Streptococcal septicaemia. 
 88 21 Streptococcal septicaemia. 
 56 — Streptococcal meningitis. 
 
 All these cases had undoubted gas gangrene at the outset," and in 
 each the disease was arrested by a combination of surgery and serum. 
 Although it is impossible to ignore the influence of gas gangrene in 
 establishing such secondary infections in a wounded man, yet it 
 is obvious that these cases should not be looked on as failures in serum 
 therapy. 
 
 2. Cases in which a fatal issue is determined by the presence of 
 an anaerobe other than those anaerobes against which the serum has 
 been prepared. 
 
 The serum therapy of gas gangrene was started at a time when 
 there was still lacking much information as to the nature of the 
 anaerobes that caused the disease in wounded men. It was known 
 that B. welchii could be isolated from the majority of human cases. 
 It was known too that vibrion septique could be found in a certain 
 number of cases, but no figures from British sources were available 
 as to the percentage incidence of this anaerobe in wounds. Further, 
 
 B. oedematiens, which Weinberg claimed to have demonstrated in about 
 one-third of his cases, had not been recorded by British workers, with 
 the solitary exception of the case reported by Miss Dalyell. 
 
 It became therefore a matter of prime importance, not only to 
 determine in what percentage of cases of gas gangrene a welchii- 
 septique antitoxic serum could be expected to give beneficial results, 
 but also to investigate the cause of death in all instances in which 
 liberal and fair trial of the serum proved unsuccessful. The first 
 part of the investigation was completed, and provided a census of 
 the anaerobes causing gas gangrene in man. It showed that in 
 about 25 per cent, of all cases of gas gangrene the disease was due 
 to anaerobes other than B. welchii and vibrion septique, i. e. in one- 
 quarter of the cases a welchii-septique serum could not be expected 
 to combat the infection. The second part of the investigation, 
 which presumably would have yielded evidence complementary to 
 the first part, broke down owing to the difficulty of procuring from 
 Prance pieces of infected muscle from unsuccessfully treated cases. 
 
 Of the 87 serum treated cases only 5 were proved to contain patho- 
 genic anaerobes, infection by which in guinea-pigs could not be 
 arrested by a welchii-septique serum ; and there can be no doubt, 
 on the evidence available, that this number would have been con- 
 siderably augmented if a thorough investigation had been possible. 
 
124 
 
 Oases 48, 54, and 89 contained pathogenic strains of B. Jallax. 
 Case 84 contained B. oedematiens, and an unidentified pathogenic 
 anaerobe was isolated from Case 12. 
 
 The details of Case 54 as reported by Stokes are as follows : 
 
 Sa. Multiple wounds of thigh. Leg discoloured, crepitations round 
 ankle. Oedema up to groin. Pulse very bad. 
 
 28. 4. 18. Operation. Multiple incisions. Calf muscle good. Thigh 
 muscles heavily infected. Given serum 30 c.c. Gr 17 and 30 c.c. G 22. 
 
 29. 4. 18. Vomiting stopped. Feels better. Pulse better. Infection 
 has not spread but crepitations obvious round knee and thigh. Later 
 the foot became black and gangrenous. Given 60 c.c. of serum. He 
 again felt better after the serum. In the evening again given 60 c.c. of 
 serum. Amputation through upper third of thigh. Stood the operation 
 well. He could not have done so 24 hours previously. 
 
 30. 4j 18. In the morning fairly well but pulse very weak. Given 
 40 c.c. G 17. In the evening became delirious and died about midnight. 
 There was no recurrence of gangrene in the stump. 
 
 Blood cultures. 
 
 28. 5. 18. B. Jallax. 
 
 29.5.18. B.fallax. 
 
 29.5.18. {evemng) B.fallax. 
 
 30. 5. 18. (after amputation) negative. 
 
 The notes of Case No. 12 reported by Ellis are as follows : 
 
 18. 10. 18. Wounded in right leg and ri^ht arm. 
 
 19. 10. 18. Admitted to hospital. Through-and-through wound of 
 anterior and external part of upper thigh. Also through-and-through 
 wound of right knee-joint. Whole thigh tense, oedematous and tympanitic 
 anteriorly up to Poupart's Hgament. No crepitation. OfEensive, efEerve- 
 scent fluid bubbles from wound in knee-joint. 
 
 Operation. Very incomplete excision of wound. Muscles discoloured 
 but contractile, bleeding muscle reached in some places. Knee-joint 
 flushed with saline and ether and closed. General condition fairly good, 
 colour good, no vomiting, pulse small volume but not exceedingly rapid. 
 Under anaesthetic pulse rapidly ran up to 100. 
 
 Serum G 53, 100 c.c. in 1,000 c.c. salt solution intravenously. 
 
 20. 10. 18, 3.30 p.m. Has been perfectly clear mentally until the last 
 few hours. He is now slightly delirious. No vomiting, colour bad, sweating, 
 intense thirst, pulse bad. No spread of gas to abdominal wall, but the thigh 
 is more extensively involved and malodorous. 
 
 Serum G 53, 100 c.c. in 1,000 c.c. saline intravenously. 7.0 p.m. death. 
 
 Autopsy. 3 hours after death. 
 
 Anterior muscles of thigh all putrid, posterior muscles full of gas. Gluteal 
 and abdominal muscles not involved. 
 
 Bacteriological examination. 
 
 Muscle-direct smear, very numerous Gram-positive thick bacilh many 
 showing spores. Spores oval, mostly subterminal but many central 
 
 Meat culture— orgamsm described and B. sporogenes 
 
 Blood culture, 9 hours before death, gave the organism described as 
 occurring m direct smear from muscle. 
 
 Bemarks. 
 
 Very severe case of gas gangrene, not benefited by serum. Sporing 
 anaerobe m blood culture. i" "S 
 
125 
 
 In a letter accompanying cultures whicli were sent to the Medical 
 Kesearcli Committee, Ellis says : 
 
 ' TMs is my first real failure with the serum. I should have laid down 
 a serum barrage in the thigh and should have repeated serum at 9.30 
 instead of 3.30. However, personally I do not think that in this case the 
 serum was active.' 
 
 3. Cases in which the serum was given either in too small amounts 
 or at too late a period in the course of the disease. 
 
 Experience with the antisera of which we have most reliable 
 information, viz., those against tetanus and diphtheria, has shown 
 conclusively that they must be given in large doses and at the earliest 
 possible stage in the development of the illness. In the case of 
 diphtheria, Behring and many other workers have demonstrated 
 that with efficient serum treatment in the first two days of the disease 
 the death-rate is reduced to about 7 per cent. The use of serum 
 after two days gives less favourable results, and after the fifth day 
 it is found to exercise little or no beneficial effect. The good fortune 
 which attends the early serum therapy of diphtheria depends to a very 
 large extent on the ease with which the condition can be diagnosed 
 clinically, for the sore throat, which is such a well-marked clinical 
 feature, attracts attention to the probable existence of the disease. 
 Less happy results have been obtained in the treatment of tetanus, 
 because here the disease may develop when it is least suspected, 
 and a clinical diagnosis becomes possible only when grave symptoms 
 are already manifest. It is for this reason that antitetanic serum 
 best demonstrates its efficacy when it is given prophylactically. 
 Now, both in diphtheria and in tetanus the march of events is 
 measured in periods of days. In gas gangrene, on the other hand, 
 the disease may rush relentlessly to a fatal issue in the course of 
 a few hours, so that the wounded man may be practically moribund 
 by the time that serum treatment becomes available. 
 
 In searching through the records collected in France one finds 
 a. large number of instances in which serum was first administered 
 when the wounded man was really dying. It can be legitimately 
 claimed that under these conditions the serum had no chance of 
 effectively combating the disease. 
 
 These moribund cases may be divided into two groups. 
 
 Group A. 
 
 Acutely fulminating types of the disease where death occurred 
 within three days. The following may be taken as typical samples : 
 
 Duration of life 
 
 
 
 in hours after 
 
 No. of case. 
 
 Dai/ of death. 
 
 giving of serum. 
 
 17 
 
 1 
 
 G 
 
 31 
 
 1 
 
 9 
 
 ?39 
 
 1 
 
 10 
 
 11 
 
 2 
 
 4 
 
 43 
 
 2 
 
 11 
 
 55 
 
 2 
 
 7 
 
 57 
 
 2 
 
 5 
 
 58 
 
 2 
 
 2 
 
 64 
 
 2 
 
 6 
 
 65 
 
 2 
 
 8 
 
 10 
 
 3 
 
 6 
 
 49 
 
 3 
 
 6 
 
126 
 
 Group B. 
 
 Late cases of gas gangrene in which death occurred after the third 
 day. 
 
 
 
 Duration of life 
 in hours after 
 
 of case. 
 68 
 70 
 69 
 4 
 
 Day of death. 
 
 5 
 
 6 
 
 8 
 
 11 
 
 giving of serum. 
 4 
 6 
 
 4 
 6 
 
 Group A were treated in forward medical units and represent 
 a type of case which is unavoidable under modern conditions of 
 warfare. Group B were base hospital cases and might conceivably 
 have benefited if serum had been given earher. 
 
 B. Recoveries. 
 
 Of these there are 38 cases out of a total of 89 serum treated 
 patients. They may be divided into three groups : 
 
 1. Cases in which surgical treatment consisted in thorough 
 
 cleansing of the wound, together with removal of foreign 
 bodies, excision of infected muscle, &c. 
 
 16 cases. 
 
 2. Cases in which it was necessary to amputate a limb. 
 
 17 cases. 
 
 In groups 1 and 2 there are many instances in which beneficial 
 effects are ascribed to antisera, J)ut in the absence of large statistics 
 it is impossible to decide whether surgery plus serum accomplished 
 more than surgery along could have done. 
 
 3. Cases in which operative procedures were insufficient to arrest 
 
 the disease, or in which the focus of infection was inaccessible. 
 5 cases. 
 
 Of these 4 were reported by Ellis and 1 by Tytler. It is to be noted 
 that both workers gave large and repeated doses of serum, that the 
 serum BlUs used for his series -was the best of the sera sent out to 
 France, and that he used it locally in and around the infected tissues. 
 
 One of the cases treated by Ellis and Tytler 's case are reported 
 in full. 
 
 Case No. 13. St. 
 
 Multiple shell wounds, both legs and thighs. Double fractured femui. 
 G.S.W. scalp. Wounded morning 17. 10. 18. 
 
 19. 10. 18, 3.30. Patient in very bad condition, delirious and wildly 
 excited ; colour definitely yellow. Pulse, bad quaUty, 140. Left thigh 
 gangrenous with green discolouration below upper third. Left leg greenish 
 black and absolutely cold to the knee. Very marked oedema and crepita- 
 tion over whole thigh anteriorly extending up to groin with marked 
 tympany on percussion, and marked reddening of the skin and bronzing. 
 Posterior thigh muscles in upper third soft, glutei soft. Patient inoperabk. 
 100 c.c. serum in 1,000 c.c. salt solution intravenously and 30 c.c. intra- 
 muscularly in gluteal adductor muscles and in anterior abdominal wall. 
 
 10.00. Remarkably improved, now perfectly clear mentally, pulse - 
 112. Gas not spreading or very shghtly, some increase in skin redness 
 especially over glutei. Oedema and crepitation if anything diminished 
 
127 
 
 Spinal novocaine anaesthesia amputation in middle third of thigh immedi- 
 ately above line of green discolouration. Adductors and vasti discoloured, 
 contraction sluggish, other muscles appear fairly healthy. Collapse 
 following amputation, pulse and respiration ceased, patient practically 
 dead. Blood transfusion 700 c.c. with dramatic improvement. 
 
 15.30. Sleeping quietly, pulse fair volume, 116. 60 c.c. serum intra- 
 muscularly (40 c.c. in pectorals, 20 c.c. in thigh and buttocks). 
 
 19.30. Blood transfusion 800 c.c. 
 
 20. 10. 18. General condition good, full strong pulse, rate 112. tempera- 
 ture rising, sweating. Mental condition absolutely clear. The area of 
 skin redness is spreading and now extends some distance up left abdominal 
 wall and up back over lumbar muscles. There is, however, no oedema or 
 tenderness and the thigh feels quite soft though tympanitic. There is 
 a bleb on anterior abdominal wall just above the area of redness. 15.50. 
 80 c.c. serum intramuscularly (60 c.c. pectorals and 20 c.c. in abdominal 
 wall and back). 
 
 23.00. Vomiting since about 15.00, now vomiting almost continuously. 
 Wound dressed, whole stump swollen with superficial crepitation, muscles 
 all red, healthy and contractile except adductors, ends of which show 
 superficial gangrene, beneath this muscle discoloured, soft, oedematous 
 and non-contractile. 50 c.c. serum in 500 c.c. salt solution intravenously. 
 
 21. 10. 18, 22.00. Much improved, vomiting eased ofi through the night 
 and to-day he has not vomited at all. Colour good. Evening temperature 
 99-6, pulse 90. General condition excellent. The redness over anterior 
 abdominal wall and lumbar muscles has now entirely faded. Wound 
 dressed, healthy except for adductors which are absolutely pulped, spongy, 
 and full of gas. Dead muscle dissected away without anaesthetic. Other 
 leg multiple wounds dressed, fractured femur put up in Thomas. 60 c.c. 
 serum intramuscularly. 
 
 Bacteriological Examination. 
 
 Muscle 19. 10. 18, 8.30. Direct smear, non-sporing anaerobe, morpho- 
 logically resembUng B. welchii. Fairly numerous diplococci culture, 
 anaerobes probably B. welcMi and B. sporogenes. 
 
 Muscle, 20. 10. 18, 23.00. Direct smear. Predominant organism a very 
 large, very broad strongly Gram-positive bacillus with rounded ends, 
 some show tendency to oat shape. Spore forms are fairly numerous and 
 are chiefly subterminal but some are central. There is also a long 
 slender bacillus with round terminal spore. Fairly numerous cocci in 
 pairs and short chains. 
 
 Bemarhs. 
 
 Desperate case, successfully treated. Large doses required and recur- 
 rence of signs of intoxication and local spread of gas when serum was not 
 pushed, immediate improvement with increased dosage. All officers who 
 have seen this case consider the recovery extraordinary and agree that 
 they have never seen anything like it before. 
 
 Case No. 76, Ke. 
 24. 4. 18. G.S.W. Buttock. 
 
 27. 4. 18. Admitted. P. 104, T. 103. 
 
 28. 4. 18. X-ray shows large F.B. in left back. Vomiting. T. 104, 
 P. 100. 
 
 29. 4. 18. Operation. Wound excised and excision entended up over 
 crest of ileum, which was sUghtly shattered. Large F.B. removed from 
 body of psoas. Flavine gauze drainage. Probable gas infection. 
 
128 
 
 Exploratory incision througli left rectus abdominis shows no lesion 
 of bowel or peritoneum but some serous fluid. 
 
 Vomited once, 3 p.m. 8.0 p.m. T. 99, P. 120. 
 
 Urine shows considerable albumin and a good number of granular casts. 
 
 30. i. 18. Condition fair. Not much sleep, a.m. T. 102, P. 120. 
 
 No vomiting, p.m. T. 101, P. 114. 
 
 1. 5. 18. Better night, but complains of pain in side, a.m. T. 97, P. 104. 
 
 No vomiting, p.m. T. 103, P. 128, K. 32. 
 
 Urine shows trace of albumin and occasional granular casts. 
 . 2. 5. 18. Condition not so good. Very restless. No vomiting, a.m. 
 T. 99-4, P. 78, E. 30. 
 
 Left scrotum and inguinal canal show marked well demarcated swelling 
 and tenderness. 
 
 Operation. Inguinal canal opened. Coverings of cord contain pus and 
 gas. Vessels of cord thrombosed throughout. Testicle and cord stripped 
 out of scrotum. Incision extended upwards to abdominal wall and cord 
 followed up retroperitoneaUy. Large abscess between ileo-psoas fascia 
 and peritoneum extending into pelvis to base of bladder and upward in 
 loin to former site of F.B. in psoas muscle. Deferential vessels are natural 
 from internal inguinal ring to base of bladder. Spermatic vessels throm- 
 bosed as far as renal vessels and perivesicular tissue is oedematous. Defer- 
 ential vessels tied ofi at brim of pelvis and spermatic vessels at lower pole 
 of kidney. The testicle and cord as removed are completely gangrenoiu? 
 and show definite gas. Cultirres from this tissue yield streptococcus and 
 B. welchii. 
 
 3. 5. 18. Slept fairly well. Vomited in morning, a.m. T. 98, P. 120, 
 E. 30. p.m. T ?102-6, P. 112, E. 28. 
 
 2.30 p.m. 20 c.c. serum (W.G. 22) intravenously. Dressed. Showed 
 some reaction with chill 30 minutes later. 
 
 20 c.c. serum (W. 22) intramuscularly. 
 20 „ „ 
 
 4.30 p.m. 
 
 6.30 „ 
 
 7.30 „ . 20 
 
 9.0 „ 20 
 
 10.0 „ 20 
 
 11.0 „ 30 
 
 2.0 p.m. T. 103-4 P. 126 E. 26. 
 
 6.0 p.m. T. 101-2 P. 90 E. 30. 
 
 10 p.m. T. 102 P. 128 E. 30. 
 
 4. 5. 18. Poor night. Very restless, irrational. Bowels moving con- 
 stantly. Vomited after liquid food in morning. Seems distinctly improved 
 generally, however. 
 
 12 noon 10 c.c. serum (W.G. 22) intramuscularly 
 
 2.0 p.m. 10 „ „ 
 
 6.0 p.m. 10 „ 
 
 9.0 p.m. 10 „ „ 
 
 Slept at intervals during the afternoon. 
 No vomiting. 
 
 Takes noiirishment better. 
 
 2.0 a.m. 
 
 T. 
 
 101 
 
 P. 
 
 120 
 
 E. 30. 
 
 6.0 a.m. 
 
 T. 
 
 102-4 
 
 P. 
 
 118 
 
 E. 28. 
 
 10.0 a.m. 
 
 T. 
 
 102 
 
 P. 
 
 120 
 
 E. 28. 
 
 2.0 p.m. 
 
 T. 
 
 102 
 
 P. 
 
 120 
 
 E. 32. 
 
 6;0 p.m. 
 
 T. 
 
 102-4 
 
 P. 
 
 120 
 
 E. 36. 
 
 10.0 p.m. 
 
 T. 
 
 101-8 
 
 P. 
 
 118 
 
 E. 30. 
 
5.5.18 Eested more quietly. Slept at intervals 
 of nourishment and has not vomited. 
 1.0 a.m. 
 6.0 a.m. 
 8.0 a.m. 
 
 Took a fair amount 
 
 10 c.c. serum (W.G. 22) intramuscularly. 
 
 12 noon. 
 
 2.0 p.m. 
 
 4.0 p.m. 
 
 7.0 p.m. 
 10.30 p.m. 
 
 2.0 a.m. 
 
 6.0 a.m. 
 12 noon. 
 
 6.0 p.m. 
 lOp.m 
 
 10 
 
 10 
 
 10 
 
 10 
 
 10 
 
 10 
 
 T. 
 
 T. 
 
 T. 
 
 T 
 
 T 
 
 1044 
 
 100 
 
 100-8 
 
 104 
 
 103-7 
 
 P. 
 P. 
 P. 
 P. 
 P. 
 
 112 
 114 
 106 
 130 
 
 E. 
 E. 
 E. 
 E. 
 E. 
 
 32. 
 30. 
 26. 
 26. 
 
 Vomited after food— 4.30 p.m. and 6.30 p.m. 
 
 5. 18. Good night. Slept fairly well. Condition about the same. 
 
 Takes nourishment well, 
 infection 
 
 Wound rather dirty externally. No evident gas 
 
 10 c.c serum (W.G. 22) intramuscularly. 
 T. 102-4 P. 120 E. 28. 
 T. 97-8 P. 104 E. 26. 
 T. 100 P. 106 E. 30. 
 
 9.0 a.m. 
 2.0 p.m. 
 6.0 p.m. 
 10 p.m. 
 
 For two days following this the patient was in about the same condition, 
 the morning temperature each day being down to normal and the evening 
 temperature up to 104, with P. 110 to 140. No evidence of local gas 
 infection spreading or of gas intoxication. Looks Hke a streptococcal 
 infection. 
 
 9. 5. 18. General condition the same or better, 
 a.m. T. 98, P. 108, E. 36. p.m. T. 101, P. 120, E. 32. 
 
 10. 5. 18. 6 a.m. T. 101-2, P. 116, E. 30. 
 12 noon T. 97-8, P. 112, E. 32. 
 
 Patient had 150 c.c. serum in first 10 hours from commencement of 
 treatment. In the following 24 hours he had 40 c.c, and in the next 
 24 hours, 90 c.c, i. e. 280 c.c. in all. Considering the extensive gas gangrene 
 of the testicle and cord, its extension as far as the renal vessels, the size 
 of the wound made in removal and the patient's general poor condition, it 
 seems reasonable to ascribe his improvement in large part to the serum. 
 At the time of the second operation the gas infection was well established 
 and there was considerable infection in the retroperitoneal tissues of the 
 iliac fossa. He was considered clinically an almost hopeless case. He now 
 has a tremendous open wound, which when he lies on his opposite side 
 exposes the muscular wall of the abdominal cavity from the bottom of 
 the pelvis up to the lower pole of the kidney. The surfaces of this are 
 covered with necrotic exudate but there is no evidence of deep infection. 
 For the past week he has had the appearance of a streptococcal infection, 
 but the last two days has definitely improved and now appears to have 
 a fair chance of recovery. Since the first day's serum treatment he has had 
 nothing to suggest spread of gas infection or intoxication. The morning 
 after the second operation the patient had the appearance of gas intoxica- 
 tion and in the opinion of his surgeon was a true gas intoxication. 
 
130 
 
 (iv) Discussion. 
 The serum therapy of gas gangrene has presented a problem of very 
 great complexity. The reasons for this may be thus briefly outlined. ^ 
 
 1. Gas gangrene is a polymicrobic disease. The chief orgamsmf| 
 responsible for the condition are, in the order of their frequence and 
 of their relative importance, B. welchii, vibrion septique, B. oedema- 
 tiens, and JB. fallax. Each of these organisms, either by itself or in 
 combination with the others, has been found to be the principal- 
 pathogenic agent in fatal human cases. In addition, however, to 
 these arch-criminals in the bacteriological syndrome, there are a 
 certain number of anaerobes and of aerobes, non-pathogenic in 
 themselves, which act as aiders and abettors in the production of 
 infection. Not only is the number of possible bacterial combinations 
 enormous, but it is also impossible for the bacteriologist, except 
 after prolonged investigation, to determine the special combination 
 of organisms which is responsible for the disease in any individual 
 case. It follows therefore that, whereas in the case of diphtheria 
 and tetanus we have to deal with a specific and well-defined entity, 
 in the case of gas gangrene we are faced with a disease which is much 
 more complicated, so that a successful therapy can be undertaken only 
 with a serum that is polyvalent. The requisite constituents of such 
 a serum are dealt with in the summary which concludes this report. 
 
 2. The nature of the disease is such that it may be difficult to 
 introduce the serum into the general circulation or to bring it into 
 contact with the infected tissues. 
 
 (a) The constriction of the peripheral veins, which is a marked 
 feature in certain cases of gas gangrene toxaemia may render it 
 difficult to carry out intravenous inoculation. Also, the depressed 
 state of the circulation reduces to a minimum the chance of absorption 
 when the serum is given subcutaneously or intramuscularly. 
 
 (6) The infected tissues may be cut off from the general circulation 
 owing to mechanical damage or occlusion of the vessels resulting 
 from the wound itself. Or again, the vascular supply may be seri- 
 ously reduced because of the oedema which is such a characteristic 
 feature of the pathological process. 
 
 The fortunate results reported by EUis,were, we beheve, due in large 
 measure to the inoculation of serum into and around infected tissues. 
 
 The short incubation period in man, and the ruthless rapidity with 
 which the disease progresses when it is once estabhshed, are further 
 factors which mihtate against successful treatment with serum. 
 
 3. Of the anaerobes which produce gas gangrene in man, B. 
 oedematuns is the only organism which yields a toxin comparable 
 m valency to that given by B. diphtheriae or B. tetani. The toxins 
 of B. welcUi and of vii)ri<>n septique, when tested on laboratory 
 meSioned'^^ extremely low value in comparison with those just 
 
 l,nLf ^^ ^^ accepted as a general principle in the immunization of 
 Sirectlv S f^^^f '^^\'^' ^^'' "* the resulting antiserum varies 
 ThefroblrJf? 7 ''' ? ^""T ?' *°^°^^ °* *he material inoculated. 
 sepUque sera is identical with the problem of producing better toxin. 
 
131 
 
 (v) Summary. 
 
 1. Our information in regard to diphtheria and tetanus is based 
 on countless laboratory experiments and a vast clinical experience 
 extending over three decades. In contrast to this, our knowledge 
 of the anaerobes is limited to investigations which have extended 
 over a few months and which were frequently undertaken in un- 
 favourable conditions. In this respect the work on anaerobes may 
 be said to be still in its infancy, so that the serum therapy of the 
 disease, as practised in the last stages of the war, was no more than 
 an experiment. 
 
 In view of the numerous factors which mihtate against success and 
 which have been dealt within various sections of this report we consider 
 that the results obtained in France have exceeded expectations. 
 
 2. The sera which were issued for use in British hospitals in France 
 contained antitoxin to B. welchii and vibrion septique only. Labora- 
 tory experiments have shown that it is necessary to include antitoxin 
 to B. oedematiens and B. fallax in addition, and the clinical reports 
 from France add corroborative evidence to this finding. 
 
 The value of antibacterial substances in a gas gangrene serum is 
 as yet not clearly established. 
 
 3. A gas gangrene serum should be given at the earliest possible 
 stage in the illness. It is probable that prophylactic inoculations, 
 if practised with suflBciently large doses, would give results which 
 cannot be obtained when the serum is used therapeutically to check 
 the already established disease. 
 
 The production of an active immunity in man by the inoculation 
 of carefully adjusted toxin-antitoxin mixtures is suggested as 
 a procedure which might considerably curtail the incidence of the 
 disease and diminish its morbidity rate. 
 
 (vi) Table of Cases. 
 
 Abbreviations, etc., used in the table giving details of cases treated 
 with serum. 
 
 Figures inserted in brackets in front of letterpress in the columns 
 headed ' Surgery ' ' Serum ', ' Eesult ', and ' Bacteriology ' indicate 
 days after admission. 
 
 In the columns headed ' Serum ' the figures following on those 
 in brackets indicate the number of cubic centimetres of serum 
 given. The letters I.V., I.M., S.C. indicate intravenous, intra- 
 muscular, or subcutaneous. A letter followed by a number indicates 
 the batch of serum employed. 
 
 For instance (6) 50 I. V. G17 iadicates that on the sixth day after 
 admission 50 c.c. of serum were given intravenously, the batch of 
 serum being G17. 
 
 Other abbreviations occurring in this column are A.T.S. which 
 indicates antitetanic serum, and when this is followed by a number, 
 e. g. 750, it gives the number of units in the dose administered A.T.S. 
 +W indicates antitetanic? serum with the addition of antiwelchii 
 serum. 
 
 In the column headed ' Eesult ' the sign + indicates the death of 
 the patient, the figures in brackets, as mentioned above, giving the 
 number of days after his admission to hospital ; the sign — indicates 
 that the patient recovered. 
 
132 
 
 Case. 
 ■1. Fe. 
 
 2. Pi. 
 
 3. Jo. 
 
 4. Ey. 
 
 5. Co. 
 
 6. Ch. 
 
 7. Sm. 
 
 8. McI. 
 
 9. Uh. 
 
 10. Bo. 
 
 11. Co. 
 
 12. Le. 
 
 13. St. 
 
 U. Sa. 
 
 Reported hy. 
 
 Barling 
 Barling 
 Barling 
 
 Barling 
 
 Ellis 
 Ellis 
 EUis 
 EUis 
 
 Ellis 
 
 Ellis 
 
 Ellis 
 EUis 
 
 Ellis 
 
 Hope 
 
 Lesion. 
 
 Bight leg and left thigh. 
 
 Left shoulder and left 
 foot. 
 
 Bay of 
 onset of 
 
 gas 
 gangrene. 
 
 Left thigh. 
 
 Left thigh, left shoulder, [ 1 
 right arm. ! 
 
 Right thigh. 1 
 
 Buttock. I 1 
 
 i 
 
 Thigh and hand. i 1 
 
 Thigh and thorax. 
 
 Right knee. 
 
 Right leg and right 
 arm. 
 
 Both legs and thighs. 
 
 Neuk and shoulder 
 
 1 
 
 Multiple wounds thigh [ 1 
 and leg. I 
 
 Left thigh and right leg. 
 
 13 
 
 (1) Right leg cleaned up. 
 
 (2) Left thigh cleaned up, removl 
 
 of foreign body and bitB ^ 
 clothing. 
 
 (2) Foot amputated, foreign boi 
 removed from shoulder. 
 
 (6) Multiple incisions left arm and 
 
 shoulder. ■ 
 
 (1) Femoral vessels tied. 
 
 (2) Amputation and excision of 
 
 muscle. 
 
 (7) Excision of part of femoral veil 
 
 necrosed muscle removed. 
 
 (1) Wound cleaned, foreign bodyl 
 
 removed. 
 (4) Left hip explored. 
 
 (8) Left thigh opened up. 
 (11) Amputation left arm. 
 
 (1) Excision of wound and infected 
 muscle. 
 
 (1) Foreign body removed and in- 
 fected muscle removed. 
 
 (1) Amputation. 
 
 (1) Amputation. 
 
 (1) Amputation, reourrencein stump. 
 
 ' (1) Amputation. 
 
 I (1) Amputation. 
 (1) Excision of wound and muscle. 
 
 (2) Amputation. 
 
 (1) Excision of wounds, thyro-hyoid 
 membrane sutured. 
 (13) Ribs excised, stinking haemo- 
 thorax drained. 
 
20 I.V. G 3. 
 
 50 I.V. G 17. 
 
 40 I.M. G 3. 
 
 60 1.V. G 17 
 10 I.V. G 22. 
 
 60 I.M. G 53. 
 
 60 I.M. G 53. 
 60 I.M. 6 53. 
 
 60 i.m; G 53. 
 10 I.V. G 53. 
 
 80 I.M. G 53. 
 40 I.M. G 53. 
 40 I.M. G 53. 
 40 I.M. G 53. 
 
 80 I.M. G 53. 
 20 I.M. G 63. 
 
 100 I.V. G 53. 
 
 60 I.M. G 53. 
 
 100 I.V. G 53. 
 100 I.V. G 53. 
 
 100 I.V 
 30 I.M. 
 60 1.M. 
 80 I.M. 
 60 I.M. 
 60 1.M. 
 
 . G53 
 G53. 
 
 A.T.S. 
 A.T.S. 
 50 I.V. 
 
 500. 
 750. 
 G17. 
 
 (6) + 
 
 (7) + 
 
 (7) + 
 
 (11) + 
 
 (3) + 
 
 (2) + 
 (2)_+ 
 
 B. welchii, B. aporogenes, and strep- 
 tococci. 
 
 (16) + 
 
 Wound gave B. welchii and 
 tococoi. 
 
 (2) Thigh gave B. welchii, B. sporo- 
 genee, and streptococci. 
 
 (2) Haemothorax gave B. welchii, 
 B. sporogenes and strepto- 
 cocci. 
 
 (7) Haemothorax gave B. welchii 
 and streptococci. 
 
 Muscle gave B, welchii. 
 
 (3) Blood gave B. welchii, B. sporo- 
 genes and streptococci. 
 
 Blood culture gave a sporing anae- 
 robe. Muscle gave the same 
 anaerobe together with B. sporo- 
 
 Musole gave B. welchii, B. sporo- 
 genes, and streptococci. 
 
 Bemarl-s. 
 
 Much relieved by serum ; it 
 ought to have been repeated. 
 
 Death 7 hours after serum. 
 
 Serum had no effect on 
 or local condition. 
 
 Death a few hours after opera- 
 tion and the giving of serum. 
 
 Cured by serum. Fatal prog- 
 nosis given by surgeon. 
 
 Cure following serum therapy in 
 spite of fatal prognosis. 
 
 Death 6 hours after serum. 
 
 Death 4 hours after serum. 
 
 Antitoxic sera prepared against 
 B. welchii, vibrion septique, and 
 B. oedematiens did not protect 
 guinea-pigs against this anae- 
 robe. (Henry). Quoted in full 
 in Section VI of report. 
 
 A welohk-septique antitoxic se- 
 rum protected a guinea-pig 
 against the muscle culture 
 (Henry). Quoted in full in 
 Section VI of Report. 
 
 Anaphylactic death. 
 
134 
 
 Case. 
 15. Sto. 
 
 Beported by. 
 
 McEwen 
 
 16. Ta. I McNee 
 
 17. Li 
 
 18. McL. 
 
 19. Co. 
 
 McNee 
 
 * 
 
 McNee 
 McNee 
 
 20. Ho. McNee 
 
 21. MoD. i McNee 
 
 22. Co. I McNee 
 
 I 
 
 23. Ev. ; McNee 
 
 24. Eok. : McNee 
 
 25. Do. i MoNee 
 
 26. Ea. j McNee 
 
 27. Pr. i McNee 
 
 28. Da. i McNee 
 
 i 
 
 29. Ki. i McNee 
 
 30. Pe. McNee 
 
 31. Cu. ! McNee 
 
 32. Ro. ; McNee 
 
 33. Ha. McNee 
 
 34. An. j McNee 
 
 Lesion. 
 
 Day of 
 onset of 
 
 gas 
 gangrene. 
 
 Thigh. 
 
 Right shoulder and arm. 
 
 Both thighs, feet, 
 
 wrist, &c. 
 Right forearm. 
 
 Thorax. 
 
 Abdomen and both 
 thighs. I 
 
 Shoulder, back and 
 both buttocks. 
 
 Arm and thigh. 
 
 Left arm. 
 
 Left knee and ankle. 
 
 Knee. 
 
 Leg, posterior tibial' 
 vessels torn. [ 
 
 Left arm. 
 
 Right thigh, left hand [ 
 
 and back, left shbul- ! 
 
 der. t 
 
 i 
 
 Right leg and thigh. 
 
 Right buttock. 
 
 Thigh and leg. 
 
 Arm, brachial artery- 
 divided. 1 
 
 Thigh and leg. 
 
 Right leg. 
 
 (2) Ligature of femoral ve 
 
 excision of muscle. 
 
 (3) Amputation. 
 
 (1) Wound cleaned and mm 
 cised. 
 
 (1) Wounds cleaned. 
 
 (1) Wound cleaned. 
 
 (2) Amputation. . 
 
 (1) Aspiration. 
 
 (3) Eib resection. 
 
 (1) Wounds cleaned. 
 (3) Further operation. 
 
 (1) Shoulder, disarticulation. 
 
 2 I (1) Wounds cleaned. 
 j (2) Amputation. 
 
 1 I (1) Infected muscle removed. 
 
 1 I (1) Wound cleaned and in 
 I muscle removed. 
 
 2 (^) Amputation, 
 (stump) I 
 
 ' Wounds cleaned up, ampt 
 
 I later. 
 
 1 
 1 
 
 1 
 1 
 
 1 
 3 
 1 
 
 (1) Excision of muscle. 
 
 (1) Excision of muscle. 
 
 (1) Excision of wound. 
 
 (1) Cleaning of wound and ex( 
 
 of muscle. 
 
 (2) Further excision. 
 
 (1) Excision of muscle. 
 
 (I) Wound cleaned up. 
 (3) Amputation. 
 
 (1) Excision of muscle. 
 (3) Amputation. 
 
 (1) Amputation through knee. 
 
Serum. 
 
 12) 10 I.M. G 19. 
 
 + 
 
 (1) 25 B. 1400. 
 
 (2) + 
 
 (1) 30 G 3. 
 
 (1) + 
 
 (1) 20 B. 725. 
 
 - 
 
 (3) 40 B. 1100. 
 40 B. 1100. 
 
 (5) + 
 
 (1) 20 B. 1175. 
 (3) 20 6 2. 
 
 (4) + 
 
 40 G 2. 
 
 
 (1) 50 B. 1175. 
 (2)20B. 1175. 
 (3) 20 B. 1175. 
 
 (3) + 
 
 (2) 30 B. 1175. 
 
 (3) 40 B. 1175. 
 
 - 
 
 (1) 40 B. 1400. 
 
 
 (3) 40 B. 1400. 
 
 
 (1) 40 G 3. 
 (1) 40 G 3. 
 
 - 
 
 (2) 40 G 2. 
 
 20 B. 
 
 ;i) 50 G 2. 
 [2) 30 G 2. 
 
 (1) 40 B. 1175. 
 
 (2) 100 G 3. 
 
 [1)A.T.S.+ W. 
 [2)20B., 
 
 [1) 50 G 2. 
 
 [2) 50 G 3 . 
 
 ;i) 50 B. 1175. 
 
 :i)A.T.S.+ W. 
 
 ;3) 80 B. 725. 
 [i) 80 B. 1400. 
 
 ;i) A.T.S.+ W. 
 ;i)40G2. 
 
 Sesult. 
 
 (3) + 
 
 Bacteriology. 
 
 Remarks, 
 
 'I Anaphylactic death. 
 
 Death few hours after sornm. 
 
 Haemothorax fluid gave pure B. 
 
 P. M. oedema fluid gave B. wehhii, 
 B. sporogenes, and ? B. oedema- 
 tiens. 
 
 Died 9 hours after operation and 
 serum. 
 
 Small prophylactic dose of serum 
 only given. 
 
 A welchii-septique antitoxic se- 
 rum protected mice against 
 whole culture (Henry). 
 
136 
 
 .Bo. 
 
 , Pi. 
 . Th. 
 
 Wa. 
 Wo. 
 Sm. 
 
 Ma. 
 Po. 
 
 Or. 
 
 We. 
 . Br. 
 
 Ba. 
 Mo. 
 
 Po. 
 Re. 
 
 MoNee 
 
 MoNee 
 
 MoNee 
 MoNee 
 
 McNee 
 MoNee 
 McNee 
 
 McNee 
 Stokes 
 
 Stokes 
 
 Stokes 
 Stokes 
 
 Stokes 
 Stokes 
 
 Stokes- 
 Stokes 
 
 Thigh and pelvis. 
 
 Arm and thorax. 
 Thigh. 
 
 Thorax. 
 
 Knee and thigh. 
 
 DayoJ 
 onset of 
 
 gas 
 gangrene. 
 
 1 
 
 (Thigh) 
 
 1 
 
 Lesion. 
 Thigh and elsewhere. 
 
 Multiple. 
 
 Right arm. 
 Multiple. 
 
 Left thigh. 
 
 Left leg. 
 
 Left arm, brachial 
 artery and median 
 nerve torn. 
 
 Arm. 
 
 Thigh and buttock glu- 
 teal artery torn, sci- 
 
 . atic nerve damaged, 
 fractured pelvis, rec- 
 tum torn. 
 
 Leg. 
 
 Arm, trunk, and right ' 2 
 
 Surgery. 
 
 (1) Amputation of thigh- and excisi 
 of other -wounds. 
 
 (1) Amputation left leg. 
 (1) Wounds cleaned up. 
 
 (3) Amputation right arm and 1 
 leg ; other wounds excif 
 and foreign bodies remove( 
 
 1 
 
 (1) Amputation. 
 
 1 
 
 (1) Amputation. 
 
 2 
 
 (1) Wound cleaned, artery tied, ai 
 
 nerve sutured. 
 
 (2) Amputation. 
 
 1 
 
 (1) Disarticulation at the shoulde 
 
 2 
 
 (1) Wounds excised, foreign boi 
 removed. 
 
 (2) Circular amputation, somen 
 crosed muscle left. 
 
 (1) Partial amputation at field ai 
 
 bulance. 
 (3) Amputation. 
 
 Too ill for operation ; forei( 
 bodies and bits of bone i 
 moved. 
 
 1 (1) Wound cleaned upandhaem 
 
 thorax aspirated. 
 
 1 Inoperable, pulseless, and delirio 
 
 on admission. 
 
 (3) Amputation. 
 
r 
 
 Serum 
 
 Result. 
 
 Bacteriology. 
 
 Bemarks. 
 
 1) 20 LV. B. 
 
 2) 30 LV. B. 
 
 3) 30 LV. B. 
 
 — 
 
 
 ... 
 
 2) 20 LV. B. 
 
 2) 30 LV. B. 
 
 3) 30 LV. B. 
 
 — 
 
 
 
 1) 20 B.' 
 
 2) 30 B. 
 
 - 
 
 
 
 3) 20 B. 
 
 4) 30 B. 
 6) 30 B. 
 
 (10) + 
 
 
 Death from lobar pneumonia. 
 
 
 
 
 1) 30 B. 
 
 + 
 
 
 Death 10 hours after operation. 
 
 1) 20 LV. B. 
 
 - 
 
 
 
 2) 30 B. 
 
 — 
 
 
 
 1) 30 B. 
 
 2) 40 LV. G 18 
 
 and G 19. 
 
 !) 30 LV. G 19. 
 !) 40 LV. G 19. 
 1) 50 LV. G 19. 
 
 t) 20 LV. G 19. 
 i) 20 LV. G 19. 
 
 ) 20 LV. G 19. 
 30 S.C. G 19. 
 10 locally. 
 
 ) 20 LV. G 18. 
 30 S.C. G 18. 
 
 ) 60 LV. G 3 
 and G 22. 
 
 ) 30 LV. G 17. 
 
 ) 50 S.C. G 18. 
 
 (10) + 
 
 (3) + 
 
 (2) + 
 
 (3) + 
 
 Blood culture gave streptococcus. 
 Wounds gave B. welchii, B. spo- 
 rogenes, and streptococci. 
 
 Blood culture sterile. Wound gave 
 B. welchii, B. sporogenes, another 
 welchii-like anaerobe and strepto- 
 cocci. 
 
 Wound gave B. welchii, strepto- 
 cocci, and staphylococci. 
 
 (1) Blood culture nil. 
 
 (1) Wound gave liirion septique 
 
 and B. welchii. 
 
 (2) P.M. heart blood gave vibrion 
 
 septique and B. welchii. 
 
 (1) Blood culture nil. 
 (1) Wound gave B. welchii, B. fal- 
 lax, and B. sporogenes. 
 
 (1) Blood culture gave pure B. 
 
 fallax. 
 (1) Blister fluid gave B. fallax and 
 
 B. vxlchii. 
 
 Haemothorax fluid gave pure cul- 
 ture of B. welchii. 
 
 (3) Blood culture nil ; wound gave 
 
 B. welchii, B. sporogenes, and 
 another anaerobe not iden- 
 tifled. 
 
 Death 1 1 hours after operation. 
 
 Death due to lobar pneumonia. 
 
 Died 1 hour after serum. 
 
 Died 6 hours after serum. 
 
138 
 
 Case. 
 [. Pr. 
 
 !. To. 
 
 I. Fo. 
 
 Br. 
 
 Du. 
 
 Co. 
 
 ■ Sy. 
 
 Wa. 
 
 Ca. 
 
 Ha. 
 
 Ha. 
 
 Reported by. 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 
 Lesion. 
 
 Right thigh, left thigh, 
 and right groin. 
 
 Dayo} 
 onset of 
 
 gas 
 gangrene. 
 
 1 
 
 Multiple wounds both j 
 legs and right arm. \ 
 
 Both thighs and right 
 arm. ] 
 
 Thigh. 
 
 Leg and thorax. 
 
 Arm and head. 
 
 Ijeft femur and right 
 thigh. I 
 
 Thigh. 
 
 Multiple wounds of 
 both thighs. 
 
 Thigh. 
 
 Buttock. 
 
 Buttock. 
 
 Surgery. 
 
 (1) Wounds cleaned, femoral v 
 
 tied. 
 (12) Amputation for streptocoq 
 infection of knee. 
 
 (2) Wounds excised and forei 
 
 bodies removed. 
 
 (1) Wounds cleaned and forei 
 
 bodies removed. 
 
 (2) Amputation of left leg. 
 
 (1) Multiple incisioiiB. 
 
 (2) Amputation. 
 
 (1) Wounds cleaned up. 
 
 (2) Excision of infected muscle. 
 
 (1) Amputation of arm. 
 
 (2) Head operation. 
 
 (1) Amputation left thigh. 
 
 (2) Excision muscles right thigh. 
 
 (1) Excision of wound and ligatu 
 
 of femoral vein. 
 
 (2) Amputation through thigh. 
 
 No operation. 
 
 (1) Excision of infected muscle ai 
 removal of foreign body. 
 
 (1) Excision of muscle andremovi 
 
 of foreign body. 
 
 (2) Further excision of muscle. 
 
 (1) Foreign body removed and 
 wound track excised. 
 
Serum 
 
 35 I.V. G 19. 
 
 15 S.C. G 19. 
 
 (3) 25 I.V. G 19. 
 25 S.C. G 19. 
 
 (1) 20 I.V. G 19. 
 
 30 S.C. G 19. 
 
 (3) 40 S.C. G 19. 
 
 (1) 30 I.V. 6 17. 
 30 I.V. G 22. 
 
 (2) 90 I.V. G 17 
 
 and G 22. 
 
 (2) 60 I.V. G 19. 
 
 (2) 40 S.C. G 22. 
 
 (2) 60I.V. G22. 
 
 (2) 60 I.V. G 44. 
 
 (1) 60 LV. G 44. 
 
 (2) 60 IV. G 44. 
 
 (1) 80 LV. G 22. 
 
 40 S.C. G 22. 
 
 (3) 60 I.V. G 22. 
 60 S.C. G 22. 
 
 (2) 60 I.V. G 22. 
 
 (3) + 
 
 (2)+. 
 
 (2) + 
 
 (2) + 
 
 (2) + 
 
 (2) + 
 
 (2) + 
 
 Bacteriology. 
 
 Blood culture nil ; wound gave B. 
 wdcMi, B. sporogenes, and another 
 anaerobe not identified. 
 
 Blood culture gave B. welchii and 
 B. sporogenes ; wound gave B. 
 welchii, B. sporogenes, strepto- 
 cocci, diphtheroids, and coliforms. 
 
 (2) Blood culture gave B. welchii. 
 
 (3) Blood culture negative. 
 
 (2) Wound gave B. welchii and 
 other anaerobes. 
 
 ( 1) Blood culture gave B. fallax. 
 
 (2) Blood culture gave B. fallax. 
 
 (3) Blood culture negative. 
 
 (1) Oedema fluid gave B. welchii 
 
 and B. fallax. 
 
 (2) Blood culture gave B. welchii. 
 
 Cultures from the arm gave B. 
 sporogenes, B. welchii, and pro- 
 teus. Cultures from head gave 
 B. welchii and B. sporogenes. 
 
 (2) Blood culture negative. 
 
 (2) Heart blood taken at post mor- 
 tem gave B. welchii. Cul- 
 tures from wound gave B. 
 welchii, B. sporogenes, strep- 
 tococci, and a coliform ba- 
 cillus. 
 
 (2) Blood 
 
 culture negative ; B. 
 and B. sporogenes 
 isolated from the wound. 
 
 (1) Cultures from the wound gave 
 B. welchii, B. sporogenes, and 
 a non-pathogenic tetanus- 
 like bacillus. 
 
 (1) Blood culture negative. 
 
 (2) Post-mortem heart blood gave 
 
 B. welchii and streptococci ; 
 the wound yielded B. welchii, 
 B. sporogenes, and strepto- 
 
 B. 
 
 Cultures from wound gave 
 welchii and B. sporogenes. 
 
 (1) Blood culture gave an aber- 
 rant B. welchii. Wound 
 ' cultures gave this aberrant 
 bacillus, B. welchii and B. 
 sporogenes. 
 
 Bemarke, 
 
 Restless, pulseless, and pallid 
 2 hours after serum. Death 
 7 hours after serum. 
 
 Late death from meningitis. 
 Streptococci and staphylococci 
 only found in cultures made 
 from the meningeal exudate. 
 
 Death 5 hours after serum. 
 
 Death 2 hours after serum. 
 
 Gas gangrene arrested by serum 
 
140 
 
 'ase. ' Reported by. 
 
 Stokes 
 
 Stokes 
 
 Stokes 
 Tytler 
 
 Tytler 
 
 Tytler 
 
 Tytler 
 
 Tytler 
 
 Tytler 
 
 Tytler 
 
 Tytler 
 
 Tytler 
 
 Left leg and shoulder. 
 
 Bight thigh and but- 
 tock, right arm, and 
 head. 
 
 Head. 
 
 Left shoulder and back. 
 
 Day of 
 onset of 
 
 gas 
 gangrene. 
 
 Surgery. 
 
 (1) Excision of infected muscle. 
 
 (1) Excision of muscles. 
 
 (2) Eurther excision. 
 
 No operation. 
 
 (2) Amputation of leg. 
 (5) Transfusion with blood. 
 
 (2) Excision of infected muscles. 
 
 (2) Amputation left thigh. 
 
 (2) Wound excised. 
 (6) Thigh incised. 
 
 (5) Bullet removed from back, 
 haemothorax fluid aspirated. 
 
 (2) Amputation. 
 
 (5) Excision of muscle from stump 
 and buttock. 
 
 (2) Excision of wounds and removal 
 
 of foreign bodies. 
 
 (3) Excision of buttock muscles. 
 
 (2) Wound excised, skull trephined,, 
 foreign body removed. 
 
 Extensive muscle excision. 
 
 'ol. Tytler Thorax. 
 
 • Aspiration followed by rib resection. 
 
141 
 
 Serum. 
 
 1) 60 I.V. G 22. 
 
 2) 60 I.V. G 22. 
 40 S.C. G 22. 
 
 2) 60 I.V. G 22. 
 
 5) 10 I.V. G 3. 
 
 4) 20 I.V. G 17. 
 20 I.M. G 17. 
 
 5) 20 I.V. G 22. 
 20 I.M. G 22. 
 
 8) 20 I.V. G 22. 
 
 6) 20 I.V. G 22. 
 40 I.M. G 22. 
 
 5) 20 I.M. G 17. 
 
 6) 20 I.M. G 17. 
 
 3) 20 I.V. G 22. 
 20 I.M. G 22. 
 
 2) 20 I.M. G 19. 
 
 3) 40 I.M. G 19. 
 
 4) 20 I.M. G 19. 
 20 I.M. G 22. 
 
 5) 40 I.M. G 22. 
 
 6) 40 I.M. G 22. 
 
 7) 40 I.M. G 22. 
 
 3) 10 I.V. G 3. 
 20 S.O. G 3. 
 20 S.G. G 3. 
 
 4) 20 S.C. G 3. 
 
 2) 20 I.V. G 22. 
 
 20 I.V. G 22. 
 
 20 I.V. G 22. 
 
 10 T.V. G 22. 
 
 10 I.V. G 22. 
 
 10 I.V. G 22. 
 ?otal 90 c.c. in 
 
 24 hours. 
 
 Besidt. 
 
 (9) + 
 
 (2) + 
 (2) + 
 
 (5) + 
 (8) + 
 
 (6) + 
 
 (3) + 
 
 (8) + 
 
 (6) 
 
 (14) + 
 
 Bacteriology. 
 
 B. welchii and two types of B. 
 sporogenes isolated from the 
 wound. 
 
 (2) Blood cxilture gave B. welchii 
 and streptococci. 
 
 Wound gave B. welchii and strepto- 
 cocci. 
 
 (4) Blood culture negative ; wound 
 gave pure B. welchii. 
 
 Wound gave B. welchii and strepto- 
 cocci. 
 
 Wound gave B. welchii and strepto- 
 cocci. 
 
 Haemothorax fluid gave B. welchii. 
 Heart blood post mortem gave 
 B. welchii. 
 
 Smear from wound showed B. 
 welchii, but cultures were nega- 
 tive. 
 
 (3) Blood culture gave B. welchii 
 
 and a streptococcus. 
 Wound at post mortem gave B. 
 welchii and a streptococcus. 
 
 (2) Wound gave B. welchii. 
 
 (4) Wound gave B. welchii and 
 
 streptococci. 
 (6) Wound gave B. welchii and 
 streptococci. 
 Heart blood post mortem gave 
 B. welchii. 
 
 (3) Muscle excised at operation 
 
 gave B. welchii and strepto- 
 cocci. 
 
 (4) Wound gave B. welchii, strepto- 
 
 cocci, and some spore-bearing 
 bacilli. 
 (6) Heart blood post mortem gave 
 B. welchii and a haemolytic 
 streptococcus. Post-mortem 
 cultures from pericardial 
 fluid and liver yielded pure 
 cultures of haemolytic strep- 
 tococci. 
 
 Remarks. 
 
 Gas gangrene arrested by serum ; 
 death from streptococcal septi- 
 caemia. 
 
 Death a few hours after serum. 
 Died 8 hours after serum. 
 
 Death 4 hoiu-s after serum. 
 
 Death J hour after serum. 
 
 Death 6 hours after serum. 
 
 Death 3 hours after serum. 
 
 Death probably resulted from 
 streptococcal septicaemia. 
 
142 
 
 Case. 
 Ke. 
 
 Reported hy. 
 
 Tytler 
 
 01. Wyard 
 
 Con. 
 
 Qu. 
 
 Pu. 
 
 Cu. 
 
 Wyard 
 
 MoNee 
 
 MoNee 
 
 McNee 
 
 McNee 
 
 Pro. 
 
 Ip. 
 
 Brenan 
 
 Tytler 
 
 Tytler 
 
 Lesion, 
 
 Day of 
 
 onset of 
 
 gas 
 
 Left back. 
 
 Back, neck, and tongue. 
 
 Left buttock, groin, and 
 pelvis. 
 
 Buttock. 
 
 Right elbow ; brachial 
 artery and veins di- 
 vided. 
 
 Thigh. 
 
 Both thighs; also chest 
 with haemothorax. 
 
 Haemothorax. 
 
 Left buttock. 
 
 Multiple wounds. 
 
 (3) Wound excised and large forei] 
 body removed from pso 
 muscle. 
 
 (1) Wounds excised and cleaned. 
 
 (1) Wound laid open and infecte 
 muscle excised. 
 
 (1) Wounds cleaned ; vessels tied 
 
 ulnar nerve sutured. 
 
 (2) Amputation. 
 
 (1) Excision of muscle. 
 (2^ Excision of muscle. 
 
 (2) Resection of rib ; evacuation of 
 haemothorax fluid ; removal 
 of foreign body and bits of 
 clothing from the limg. 
 
 (1) Amputation right thigh. 
 
 (2) Excision wound left thigh. 
 
143 
 
 Serum. 
 
 )20I.V..G22. 
 120 I.M. G 22. 
 ) 40 I.M. G 22. 
 ) 90 I.M. G 22. 
 
 ) 40 I.V. G 18. 
 
 60 I.V. G 19. 
 
 1) 20 I.V. G 19. 
 
 I) 20 I.M. G 17. 
 ■) 100 I.V. G 17. 
 
 ) A.T.S+ W. 
 I) G 44. 
 
 40 B. 
 
 I) A.T.S+W. 
 J) 20 G 44. 
 
 I) 20 G 44. 
 
 (8) + 
 
 (3) + 
 
 30 B. 
 
 W 100 I.V. G 44. 
 
 5) 30 I.M. G 22. 
 
 6) 30 I.M. G 22. 
 6) 20 I.V. G 53. 
 
 (2) + 
 
 (5) + 
 
 (14) + 
 
 (6) + 
 
 Culture from gangrenous testicle 
 gave B. welchii and streptococci. | 
 
 Cultures from wound gave no 
 growth of anaerobes. 
 
 Muscle from fost mortem showed 
 ? vibrion septique. 
 
 Cultures from muscle gave B. wel- 
 chii, B. sporogenes, streptococci, 
 and another anaerobe. A wel- 
 ohii-septique antitoxic serum did 
 not protect a guinea-pig against 
 the mixedmuscle culture(Henryf. 
 
 Cultures from muscle gave B. wel- 
 chii, B. sporogenes, a round end- 
 sporer, and streptococci. A wel- 
 chii-septique antitoxic serum pro- 
 tected a guinea-pig against the 
 mixed muscle culture (Henry). 
 
 gave B. wel- 
 , round end- 
 ■sporer, and 
 
 A welchii- 
 serum pro- 
 against the 
 
 (Henry). 
 
 Cultures from muscle , 
 chii, B. sporogenes, a 
 sporer, an oval end 
 another anaerobe, 
 septique antitoxic 
 tected a guinea-pig 
 mixed muscle otilture 
 
 Cultures from muscle gave B. wel- 
 chii, B. sporogenes', a round end- 
 sporer, an oval end-sporer, an- 
 other anaerobe, streptococci, and 
 a conform bacillus. A welchii- 
 septique antitoxic serum pro- 
 tected a guinea-pig against the 
 mixed muscle culture (Henry). 
 
 Cultures from haemothorax fluid 
 gave B. welchii and B. sporo- 
 genes. A welchii-septique anti- 
 toxic serum protected a guinea- 
 pig against this mixed culture 
 (Henry). 
 
 Culture from infected muscle gave 
 B. welchii and B. oedematiens. A 
 welchii-septique antitoxic serum 
 did not protect a guinea-pig 
 against this mixed culture 
 (Henry). 
 
 Culture from infected muscle gave 
 B. welchii, streptococci, and an 
 unidentified anaerobe. A wel- 
 chii-septique antitoxic serum pro- 
 tected a guinea-pig against this 
 mixed culture (Henry). 
 
 Semarks. 
 
 Gas gangrene checked by serum. 
 Case reported in full in Section 
 VI. 
 
144 
 
 Case. Reported hy. 
 
 i. She. Tytler 
 
 Du. 
 
 Tytler 
 
 Coo. Tytler 
 
 Me. 
 
 Tytler 
 
 Day of 
 onset of 
 
 Lesion. 
 
 gas 
 gangrene 
 
 Multiple, arm. 
 
 3 
 
 Thigh. 
 
 2 
 
 Buttock, torn gluteal 
 artery. 
 
 2 
 
 • 
 
 
 Swrgery. 
 
 Thigh and buttookB. 
 
 (3) Amputation. 
 
 (2) Wounds cleaned up. 
 
 (1) Wound cleaned,' gluteal arte 
 tied. 
 
 (1) Wounda cleaned. 
 
 (4) Excision of hamstring muscle 
 
140 
 
 Serum. 
 
 J) 60 I.M. G 22. 
 3j 20 r.V. G 22. 
 
 2j 100 I.M, G 44. 
 
 75 20 I.V. G 22. 
 1) SO I.M. G 22. 
 3) 90 I.M. G 22. 
 )J 20 I.M. G 22. 
 I) 20 I.M. G 22. 
 J) 20 I.M. G 22. 
 
 t) 100 I.M. G 22 
 80 I.M. G 44. 
 3) 40 I.M. G 44. 
 
 Result. 
 
 (21) + 
 
 (9) + 
 
 Bacteriology. 
 
 Culture from infected muscle gave 
 B. welchii and streptoeocoi. A 
 welchii-septique serum protected 
 a guinea-pig against this mixed 
 culture (Henry). 
 
 Cultures of material from the 
 wound gave B. welchii, strepto- 
 cocci, and an unidentified anae- 
 robe. A welchii-septique serum 
 protected a guinea-pig against 
 this mixed culture (Henry). 
 
 (5) Blood culture gave B. welchii 
 and streptococci. 
 
 (9) Blood culture gave strepto- 
 cocci only. 
 
 A welchii-septique serum did not 
 protect guinea-pigs against mixed 
 muscle cultures from this case ; 
 nor did a high-titre oedematiens 
 serum. An organism was ob- 
 tained in pure culture from the 
 heart blood of guinea-pigs which 
 had succumbed to infection by 
 mixed muscle cultures. This 
 organism is believed to be a 
 pathogenic B. fallax (Henry). 
 
 Remarks. 
 
146 
 
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 ^ Br:: \ll^'\llTZ isl ^^^P^'^^^-°-- ^-*-^- ^^^- Wohnschr., 
 
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 Baetoli, F., L'infezione gasosa delle ferite di guerra. Polidin., Eoma, 
 
 1916, 23, sez. prat., 1493. 
 Bashford, E. F., General pathology of acute bacillary gangrene arising in 
 
 gunshot injuries of muscle. Brit. J. Surg., Bristol, 1916-17, 4, 562. 
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 J. Am. M. Ass., Chicago, 1914, 62, 1153. 
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 Wchnschr., 1918, 65, 175; 212. 
 
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 Bbijeeinck, (1) Les orgariismes auaerobies obligatoires ont-ils besoin 
 
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 1899, Ser. II, 2, 397. 
 
 Beijeeinok, (2) Phenomenes de reduction produites par les microbes. Ibid., 
 
 1904, Ser. II, 9, 131. 
 Bbitzke, Zur Frage der Uebertragbarkeit des Gasbrandes. Berl. Min. 
 
 Wchnschr., 1918, 55, 1143. 
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1.50 
 
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151 
 
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153 
 
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153 
 
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 Feaenkel, Eugen, (10) Die blutschadigende Wirkung des Fraenkelschen 
 
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 Feaenkel, Eug. und Wohlwill, Das Zentralnervensystem bei Gasbrand. 
 
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 Feaenkel, Eugen, und Zeissler, (10) Die Differenzierung pathogener 
 
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 Feankau, Deummond and Neligan, The successful conservative treatmisnt 
 
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154 
 
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ibi 
 
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i6» 
 
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 310. 
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 71. 
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 L2 
 
164 
 
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 RoccHi, Giuseppe, Sulla gangrene gassosa. Riforma rned., ^apoll, 1917, 
 
 33, 387. .. . , o.. ,• . ,, 
 
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 Ztschr.f. Hyg. u. Infektionskrankk, Leipz., 1902, 39, 201. 
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 Rodella, A., (3) Ueber die Bedeutung der streng anaeroben Faulnis- 
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 1910,68,1044. , 
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 RoTJX et Chambeeland, Immunite contre la septicemic conferee par des 
 
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 Rumpel, Bericht iiber die praktischen Erfahrungen mit Serumbehandlung 
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 1918, Heft 68, 35. 
 Rumpel, Klose, von Wasseemann, etc., Gasodemschutz und -bekampfucg. 
 
 Beitr. z. Min. Chir., Tubing., 1918, 113, 76. 
 *RuNEBBEG, B., Studien iiber die bei peritonealen Infektionen appendicu- 
 laren Ursprungs vorkommenden Sauerstofftoleranten, sowie obligat 
 anaeroben Bakterienformen, mit besonderer Beriicksichtigung ibrer 
 Bedevitung fiir die Pathogenese derartiger Peiitonitiden. Arb. a. d. path. 
 Inst. d. Univ. Ilelsingfors, Berl., 1908, 1, 271. 
 Rupp, Ueber einen Fall von Gasgangran mit Metastasenbildung. Miinchen. 
 
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 Sacquepee, (1) Le bacille de I'oedeme gazeux malin. Compt. rend. Soc, de 
 
 biol. Par., 191,5, 78, 316; 540; 588. 
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 Le B. oedematiens et la gangrfene gazeuse. Ibid., 1915, 78, 547. 
 Sacquepee, (3) Une modalite de la gangrene gazeuse; Toedfeme gazeux 
 malin et son agent pathogene. Bull, et mem. Soc. de chir. de Par., 1915, 
 n.s. 41, 1132. 
 
 Same title. Presse med., Par., 1915, 23, 183. 
 Sacquepee, (4) Sur la gangrene gazeuse. Septicemic gazeuse et oedfeme 
 
 gazeux malin. Ibid., 1915, 23, 218. 
 Sacquepee, (5) La septicemic gazeuse et I'ced^me sazeux malin. Ibid.. 
 
 -1915, 23, 227. 
 Sacquepee, (6) Demonstration exp^rimentale des lesions des gangrenes 
 gazeuses. Ibid., 1915, 23, 242 
 
165 
 
 Sacquepee, (7) Sur la gangrene gazeuse. Bull, et mem. Soc. med. d. Mn. 
 
 de Par., 1915, 3e s., 39, 965. 
 Sacquepee, (8) Sur le bacille de I'oeclfeme malin. Gompt. rend. Soc. de bioL 
 
 Par., 1916, 79, ,11 5. 
 Sacquepee, (9) Etudes sur la gangrene gazeuse; le bacille de I'asdfeme 
 
 malin. Ann. de I'Inst. Pasteur, Par., 1916, 30, 76. 
 Sacquepee, (10) Recherches sur la gangrene gazeuse des plaies de guerre. 
 
 Presse med., Par., 1916, 24, 194. 
 Sacquepee, (11) Sur le Bacillus bellonensis (ancien Bacille de I'oedfeme 
 
 gazeux malin). Preparation de serums specifiques ; quelques propii^tes 
 
 essentidles des serums. Gompt. rend. Soc. de bid., Par., 1917, 80, 850. 
 Saoquep^ie, (12) Preparation d'un serum theiapeutique iinti-bellonensis. 
 
 Presse med.. Par., 1918, 26, 17. 
 Sacquepee, (13) Principes dune methode de trjiitement serolherapique 
 
 antitoxique de la gangrfeue gazeuse. Ibid., 1918, 26, 18. 
 Sacquepee, (14) Recherches sur la gangrfene gazeuse des plaies de guerre. 
 
 lUd., 1918, 26, 197. 
 Sacquepee, (15) Traitemeut serotherapique de la gangrfeue gazeuse. Gompt. 
 
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 1918, 18. 
 Sacquepee, (16) Etudes bactetiologiques sur les plaies de guerre (Blessures 
 
 osteo-musculaires des membres). J . de physiol. et de path, gen., Par., 1918, 
 
 17, 621. 
 Sacquepee, (17) La flore initiale habituelle et la Acre de passage dans la 
 
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 Sacquepee, (18) Quelques precedes d'isolement des bacteries pathogenes. 
 
 Signification pathog^nique des resultats dans la gangrene gazeuse. Ibid., 
 
 1918, 81,529. 
 Sacquepee, (19) Sur le traitemeut preventif et curatif de la gangrene 
 
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 Sacquepee, de Laveegne et Dbhoenb, Infections k, Proteus dans les plaies 
 
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 Salus, G., Zur Biologie der Eaulnis. Arch. /. Hyg., Munchen u. Berl., 
 
 1904, 51, 97. 
 Sanselicb, Untersuchungen uber anaerobe Mikro-organismen. Ztschr.f. 
 
 Hyg. u. Infektionshrankh., Leipz., 1893, 14, 339. 
 Saktoet et Spillmann. Sur la bacteriologie de la gangrfene gazeuse. 
 
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 ScHATTENFEOH und Geassbeeger, (1) Ueber Buttersauregahrung. Arch. 
 
 f. Hyg., Munchen u. Berl., 1900, 37, 54. 
 SciiATTENPEOH u. Geassbeegbe, (2) Ueber Buttersaurebacillen und ihre 
 
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 1033. ,. , 
 
 ScHATTENFEOH u. Geassbeegee, (3) Die Beziehungen der unbeweglichen 
 
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 ScHLOSSBBEGBE, Die Differenzierung der anaeroben Gasodembakterien. 
 
 /6i(i., 1919,66, 348. ' _ ,^. „^ , , 
 
 SCHMID, Tod durch Gasembolie bei Gasphlegmone. Wten. klin. Wchnschr., 
 
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 *ScHOCK, Drei Falle von Gasbdem beim Pferde. Berl. tierarxtl. Wchnschr., 
 
 1918, 34, 392. . , . , . , , 
 
 SCHOLTZ, Ueber das Wachsthum anaerober Baktenen bei ungehmderten 
 
 Luftzutritt. Ztschr.f. Hyg. u. Infektionskr., Leipz., 1898, 27, 132. 
 SchOnbaube, Ein Beitrag zur Frage der Anaeiobeusepsis beim Gasbrand. 
 
 Wien. klin. Wchnschr., 1917, 30, 110. 
 
166 
 
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 419. 
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 Segen, Ein Beitrag zur Prophylaxe der Gasphlegmone und des Tetanus. 
 
 Wien. klin. Wchnschr., 1917, 30, 16. 
 Sbhet, Der Gasbiand. Med. Klinik, Berl., 1916, 12, 1056 ; 1081. 
 EB Sejoue, Contribution a I'etude de la septic6mie gazeuse. Bull, et mem. 
 
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 Seltee, Zur Aetiologie der Gasphlegmone. Deutsche med. Wchnschr., Bar]. 
 
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 Senechal, Etude histologique sur un cas de gangrene gazeuse au debut ; 
 
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 SiLBEESCHMiDT, Bakteriologischcs uber einige Falle von ' Gangrene fou- 
 
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 Silbbestein, Gasbrandtoxine und Antitoxine. Wien. klin. Wchnschr., 1917, 
 
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 SiMONDS, J. P., (5) Eecherches sur la presence de spores de bacteries 
 
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167 
 
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168 
 
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169 
 
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170 
 
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 552. 
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 1916, 79, 116. 
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 79, 581. 
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 1916, 79, 1028. 
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171 
 
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 Weight, A. E., (2) Des conditions qui gouvernentledeveloppementdubaoillede 
 
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172 
 
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 ADDENDUM 
 
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 Duval et Vauchee, Premiers resultats des essais systematiques de sero- 
 
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 Got: Bl.f. Schweiz. Aerzte, Basel, 1918, 48, 438; 479 ; 508. 
 *IliTZ u. "ScHLOSSBEEGEE, Ueber die Wirkung chemischer Mittel auf 
 
 Gasbrandbakterien in vitro und in vivo, Arh. a. d. Inst. f. exp. Therapie, 
 
 Frankf. a. M., 1919. Heft. 7. 
 EouviLLOis, GuiLLAUME-Louis [et al.], Traitement de la gangrtoe gazeuse 
 
 par I'emploi des serums antigangreneux (m^thode de Saoquepee). BvM. 
 
 et mem. Soc. de chir. de Par., 1918, 44, 1226. 
 *ScHLossBBEGEE, H. , Die Hamotoxine der Gasbrandbakterien. Arb. a. d. 
 
 Inst. f. exjj. Therapie, Frank, a. 51., 1919, Heft 6 and 7. 
 
173 
 
 APPENDIX 
 
 THE HISTOPATHOLOGY OF GAS GANGRENE 
 
 By E. H, Kettle, M.D. Lond. 
 
 A SYSTEMATIC study of the histopathology of any infection should 
 proceed along certain definite lines, and sliould include investiga- 
 tions into 
 
 1. The distribution and dissemination of the organisms in the 
 body. 
 
 2. The changes taking place at the site of infection. 
 
 3. The lesions produced in the other tissues and organs of the 
 body. 
 
 A. From the action of circulating toxins formed by the organisms. 
 
 B. As the result of the presence of the organisms. 
 
 4. Reactions taking place in various organs in the form of — 
 
 A. Attempts to combat the infection. 
 
 B. Changes accompanying the process of healing and repair. 
 Although the anaerobic bacilli do not lend themselves very readily 
 
 to investigation along these lines, I have adopted this scheme as 
 a working basis since it is desirable to bring these organisms into 
 comparison with other pathogenic microbes. 
 
 For several reasons, the material derived from cases of gas- 
 gangrene in the human being are often unsatisfactory. The lesions 
 due to the presence of anaerobic bacilli in the body are essentially 
 degenerative, and it may be impossible to distinguish them from 
 similar changes due to other causes; and another source of con- 
 fusion is the jpost-inortem degeneration which must elapse between 
 the death of the patient and the autopsy. Further, there is frequently 
 a coexisting infection with other organisms which again complicates 
 the analysis of the histological findings. The human material to 
 which I had access suflPers in a varying degree from these defects, and, 
 in addition, the greater part of it was quite uncontrolled by clinical 
 or bacteriological data. 
 
 The lack of satisfactory human material may be compensated for 
 tor some extent by the use of laboratory animals. Rabbits, guinea- 
 pigs, rats and mice, can all be infected by subcutaneous or intra- 
 muscular inoculation, but it is not easy to reproduce the disease as 
 it occurs in man. Either the dose is too small and the inoculatiouH 
 fail to take efiect, or the local lesion is so severe that the animals 
 die rapidly from toxaemia before the disease becomes generalized. 
 Similarly, I have found that intravenous inoculation with cultures- 
 
174 
 
 of the bacilli produce death without the formation of localized 
 lesions. Of the experimental material which had been placed at 
 my disposal, very little was of value, and, under these circumstances, 
 this investigation is incomplete in many particulars. In respect to 
 section 4 of the scheme my observations are insuflBcient to admit 
 of conclusions being drawn, and I shall therefore omit any dis- 
 cussion of this part of the problem. 
 
 1. The Distribution and Dissemination of the organisms in the body. 
 
 The severity and extent of the lesions at the site of inoculation 
 have so dominated the clinical picture that gas-gangrene has come 
 to be looked upon as a local disease of muscle. This, liowever, 
 is not correct. The organisms are not confined to the neighbourhood 
 of the wound but are distributed widely throughout the body, 
 producing lesions which are identical with those occurring in muscle. 
 It must be recognized, of course, that the bacilli may proliferate 
 extensively in the body after death, and that their distribution in 
 tissues obtained at autopsy is not always representative of their 
 ante-mortem dissemination. But when due allowance is made for 
 this, an examination of human and experimental material shows 
 that septicaemia is of common occurrence. 
 
 In human material, organisms have been found in association 
 with definite lesions in the heart muscle, the liver, the kidney, the 
 spleen, the lymph glands, the supra-renal glands, and the meninges 
 of the brain. Apart from lesions, I have found organisms widely 
 distributed in veins and capillaries. In many instances this dis- 
 semination must be regarded as a post-mortem phenomenon, but 
 cases remain in which the only possible explanation is a terminal 
 septicaemia. 
 
 In animals, probably because of the rapidity with which they 
 succumb to toxaemia, septicaemia is uncommon. I have, however, 
 been able to demonstrate organisms in the circulating blood and in 
 the bone marrow of animals killed at varying times after intra- 
 muscular inoculation. 
 
 Dissemination of the organisms occurs in three ways : 
 
 (a) By direct extension in loose areolar and connective tissue. 
 
 (b) By growth along lymphatic vessels. 
 
 (c) By invasion of the blood-stream. 
 
 (a) Dissemination by direct extension in loose areolar and con- 
 nective^ tissue. This is probably the most important mode of 
 dissemmation since it is the way in which infection spreads from 
 the primary wound till the whole limbs and even the trunk become 
 involved. The muscles are only of importance in defining the path 
 ot infection masmuch as the areas enclosed by the epimysium and its 
 prolongations, the perimysium, form, for all practical purposes, 
 v?"^?i- i''' ':'^f".°el3 la which the organisms can advance. The 
 bacilli flourish in loose areolar tissue and tend to spread along 
 tissue spaces and connective tissue planes with great rapidity. 
 
 {b) Dissemination by groivth along lymiAatic vessels. The bacilli 
 grow very readily within the lymphatic vessels as distinct from the 
 
175 
 
 tissue lymph spaces, a condition of permeation occurring which 
 IS very similar to that seen in the spread of a malignant growth 
 
 Lj-mphatic permeation by the bacilli can be demonstrated in tlie 
 neighbourhood of the site of inoculation in animals, and I have also 
 tound It taking place in human material. A particularly strildno- 
 instance was seen in a deep cervical gland of a cbild who died o'f 
 gas-gangrene of the arm. Areas of the gland showed early localized 
 degenerative changes, and a main afferent lymphatic vessel was 
 plugged with a thick column of bacilli. 
 
 (c) Dissemination by invasion of the bloodstream. This is not 
 so easy to demonstrate. Occasionally, in the local lesion, a few 
 organisms can be detected in the capillaries or veins, but this 
 invasion of the blood-stream is quite overshadowed by the luxuriant 
 growth of the organisms in the tissues and the lymphatics. Never- 
 theless, it would appear that visceral lesions are due to a blood-borne 
 infection. Bacilli may frequently be demonstrated in the sinusoids 
 of the liver, even though they may be absent in other oi-gans ; and 
 I have found them in considerable numbers in the capillaries of the 
 glomeruli. I have never, however, found bacilli in the lungs under 
 conditions in which post-mortem change could be excluded ; and 
 I have never seen pulmonary lesions which could be ascribed 
 to them. 
 
 2. The changes taking place at the site of infection. 
 
 The changes in muscle following infection with anaerobic bacilli 
 have been fully described, and it is unnecessary to deal at any 
 length with this aspect of the questiou. It is important, however, 
 to realize that the infection of muscles is purely an accidental 
 phenomenon. Anaerobic bacilli have no specific action on muscles, 
 nor do they find in them any substances which are especially 
 necessary to their metabolism. It is in deep penetrating wounds 
 that the organisms thrive best, and in the limbs such wounds involve 
 muscle. Wounds of the thoracic or abdominal viscera are fatal, or 
 receive operative treatment, from considerations othei' than the 
 occurrence of gas-gangrene, but were these organs of less immediate 
 importance to the life of the individual there is no reason to suppose 
 that the incidence of gas-gangrene would not be as high in them as 
 it is in wounds of muscle. 
 
 The characteristic lesion of gas-gangrene is necrosis brought 
 about by the toxins elaborated. by the organisms. The changes are 
 most obvious in the muscle fibres which pass through the stages of 
 cloudy swelling, and gradual loss of striation, to coagulation necrosis 
 and solution. The most highly organized structures, the muscles, 
 nerves, and the epidermis and its derivatives are affected earliest, 
 but eventually the blood-vessels and the connective tissues are also 
 destroyed. The organisms, which are usually present in enormous 
 numbers, lie in the connective tissues, and do not invade the other 
 structures except, perhaps when necrosis is far advanced. In an 
 infected muscle they tend to remain within the limits of the 
 epimysium ; and they extend to neighbouring muscles by spreading 
 in the subcutaneous tissue, o'r the deep connective tissue trabeculae. 
 
176 
 
 The most striking feature of the lesion is the entire lack of any 
 inflammatory reaction. Some proliferation of the sarcolemma nuclei 
 can sometimes be seen in front of the advancing margin of the 
 infection, but this is all. The muscle fibres are absolutely quiescent, 
 and, moreover, there is a complete absence of wandering cells. In 
 the subcutaneous and connective tissues, on the other hand, there 
 occurs a leucocytosis which may be very pronounced ; the leucocytes 
 are of the polymorphonuclear variety, and are actively phagocytic. 
 There is little or no reaction of the fixed connective tissue cells. 
 
 The blood-vessels show no very characteristic lesions. In advanced 
 cases the constituents of tlieir walls lose their staining reactions 
 and undergo necrosis in common with the rest of the tissues, but in 
 the early stages they present no constant changes. 
 
 Thrombosis of veins and capillaries frequently occurs but is not 
 invariable. I have not been able to detect any alteration in the 
 endothelium to account for this, but there is clear evidence of a 
 toxic destruction of red blood corpuscles which in itself would tend 
 to cause conglutination thrombi. In advanced lesions haemorrhage 
 and haemolysis frequently occurs. 
 
 I have not been able to come to definite conclusions as to possible 
 diiferences in the lesions produced by the various anaerobic bacilli. 
 B. oedematiens and V. sejitique appear to give rise to greater 
 oedema and more advanced vascular changes than B. wdchii, and 
 haemorrhage and thrombosis appear to be pai-ticularly associated 
 with V. septique, but my observations on this point are not 
 conclusive. 
 
 3. The lesions produced in the other organs and tissues of the body. 
 A . From the action of circulating toxins. 
 
 The finer details of cell degeneration are often obscured by post- 
 mortem changes ; experimental material is thus particularly 
 necessary for this portion of the investigation. By compai-ing the 
 tissues of animals, killed some twenty-four hours after being infected 
 with cultures of anaerobic bacillij with human organs obtained at 
 autopsy, it is possible to arrive at some idea of the changes which 
 usually take place. It was my intention to work also with animals 
 which had been inoculated with toxins alone, but I was forced to 
 abandon this line of research since most of the material at my 
 disposal proved to be infected with pyogenic cocci. 
 
 The Blcod is afiected very considerably by the toxins of the 
 anaerobic bacilli. Thrombosis is common, not only in the primary 
 locus, but also in the smaller vessels throughout the body, the 
 thrombi being of the hyaline, conglutination type. A varying 
 degree of haemolysis seems to occur quite early in the infection, for 
 the spleen olten contains a large quantity of haemosiderin, the 
 granules lying tree in the pulp, or within macrophages and poly- 
 nuclear leucocytes. In advanced cases granules ot" pigment may be 
 found in quantities in the endothelial cells lining'^capiUaries and 
 veins, in the cells of the hver, and in the renal epithelium, especia^Uy 
 m the cells of Henle's loops. I have not been able to m^ke any 
 observations on the condition of the cells and haemoglobin content 
 of the circulating blood during life, and an examination of the bone 
 
177 
 
 marrow m a few cases has not revealed any process of regeneration. 
 In some cases the contents of the vessels in the organs examined 
 suggests a slight leucocytosis, but this condition is not constant. 
 The organs are frequently congested, but there is nothino- to show 
 that this reaction is at all specific. ° 
 
 The Liver. The liver cells in human organs constantly show 
 cloudy swelling which may be extreme, and occasionally some fatty 
 degeneration. The fat has no distinctive distribution, and neither 
 of these changes can be looked upon as specific. 
 
 In experimental animals the hepatic lesions are much more severe. 
 Within twenty-four hours of subcutaneous or intramuscular in- 
 oculation the liver cells show extreme changes. There is no fatty 
 degeneration, but the cytoplasm becomes entirely disorganized and 
 appears to undergo partial solution, nothing but an irregular 
 granular debris remaining within" an unusually prominent cell wall. 
 The nuclei tend to become hypochromatic but are not otherwise 
 affected. Similar changes occur in animals inoculated intravenously 
 with cultures of bacilli, and in one mouse which lived some forty- 
 eight hours after intravenous inoculation there was, in addition, 
 advanced coarse fatty degeneration. 
 
 The Kidney. Here again the changes are constant and severe. 
 The convoluted tubules are chiefly affected. Fatty degeneration 
 never occurs, but the cells undergo advanced cloudy swelling 
 and very often disintegrate. The mitochondria become swollen and 
 irregular in size, and no longer maintain their normal longitudinal 
 arrangement. The cell protoplasm shows an increased affinity for 
 acid dyes, and in some of the experimental animals the appearances 
 almost suggest poisoning with mercury or uranium, so severe are 
 the lesions. There is little change in the nuclei beyond a diminution 
 in the amount of chromatin. It is not uncommon to find coagulum 
 or granular debris in the tubules, and though there is no definite 
 change in the glomeruli, Bowman's capsules sometimes contain 
 a little coagulum. The cells of Henle's loops and the collecting 
 tubules are only slightly affected. 
 
 'The Spleen. The most constant change in the spleen is due to 
 the altered blood condition, and consists of oedema, occasional 
 thrombosis in the capillary vessels, and pigmentation. Some of 
 the organs have been congested but in others the pulp is empty. 
 An increased number of macrophages has been noted in several 
 specimens, and sometimes these cells are multinucleated. 
 
 In two specimens there is a very interesting change in the 
 Malphigian corpuscles. They are considerably hypertrophied and 
 exhibit a central proliferation of endothelial cells, the result being 
 a condition which strongly recalls the germinal node of the lymph 
 gland. The change appears to be analogous to that which occurs 
 in scarlet fever and diphtheria. In some of the nodes the endo- 
 thelial cells seem healthy, but in others there is a considerable 
 degree of nuclear fragmentation and degeneration of the cytoplasm ; 
 and. occasionally poly nuclear leucocytes can also be detected among 
 the cells. This condition appears to be a reaction to the toxaemia 
 alone, for I have not been able to demonstrate the presence of 
 bacilli. 
 
 M 
 
178 
 
 Dymph glands. These are congested, but d-o not exhibit amy 
 reaction comparable with that seen in* the- spleen, 
 
 Musele. Slight fatty degeneration sometimes occurs intheheart 
 muscle, but I have found no change in the voluntary muscles apart 
 from the presence of bacteria. In one pigeon I found advanced 
 fatty degeneration of tlie pectoral muscles into which the toxin of 
 5. welchii had been injected some hours previous to death. 
 
 The Supra-renal glands. My material was unsuitable for the< 
 demonstration of adrenalin, and I have not detected any specifici 
 changes in such glands as I have been able to examine. 
 
 The Thyroid gland. My material is insufficient to allow of 
 definite conclusions being drawn, but in one case the parenchyma 
 cells show advanced degeneration and desquamation, with an 
 almost complete disappearance of the colloid substance. 
 
 B. As the result of the presence of the organism. 
 
 Muscle. I have frequently examined portions of the- voluntary 
 muscles from different parts of the body in human and experimental 
 cases of gas-gangrene but I have never found in them localized 
 lesions, or toxic changes indicating any specific action of the 
 anaerobic bacilli. 
 
 In one case a lesion was present in the heart muscles, the muscle 
 fibres had lost their striafcion and presented the typical 'ground 
 glass ' appearance, and their nuclei no longer stained. Numbers of 
 bacilli were lying free among the desquamated fibres. There was 
 a complete absence of any cellular exudate. 
 
 Liver. The liver appears to be a favourable site for the develop- 
 ment of lesions. My material contains numerous examples, and 
 I have found typical lesions in the liver of a child who died forty 
 hours after an injury to the arm in which gas-gangrene developed. . 
 
 The organisms reach the liver by the blood-stream, and can be 
 seen lymg free in the sinusoids. I have never seen definite bacterial 
 emboli, nor are the bacilli ingested by the endothelial or parenchyma 
 cells, but isolated organisms appear to be held up mechanically, and 
 eventua ly small colonies are developed. In a wide zone arouid 
 these CO onies the liver cells are killed. The" process appears to be 
 extremely rapid; and of the nature of a coagulation necrosis. At 
 the periphery of the lesion the cells may show cloudy swelling, and 
 the nuclei may be hypochromatic or pycnotic, but for the most part 
 they are peculiarly quiescent. The cells suddenly lose their staining 
 power, but their outlmes are retained, and their nuclei can still be 
 distinguished and should fatty. degeneration happen to be present, 
 
 lln / f^ ,r?- '*' ^ ^' ""^^'^ ""*• 0"ly i^ t^« centre of the 
 «nTLl ' ' disintegrate. Here they undergo partial solution 
 dWp^T T^'^'^'^t into irregular masses of copulated and semi- 
 S^hl J T-'" ^t""'^^' proliferate extensively in the centre 
 
 them X'^'J^^'^ f^J'"^''^^^ *^" ^^^^ ««"«' but not invading 
 contain ?T"*^™'''' ''^ t^^f periphery of the lesion the rinusoids maf 
 anv celhJr ™ono and polynuclear leucocytes, but there is never 
 devplni ! .t^' '^^"^ *^^ ^"'^°^- I*^ the late stages gas 
 
 of fh^^!;nPPr'''°^'^ under pressure, for the cells at the.pfriphfry 
 of the vacuoles are compressed. Most of these gas vacuoles are 
 
179 
 
 empty, but occasionally they contain cell debris and bacilli ; and 
 ba'ciUi are always found in numbers at their margins. 
 
 The bile ducts and supporting tissues of the organs show no 
 change unless they are involved in an unusually large focus when 
 they undergo degeneration in a similar way. 
 
 The Kidney. Lesions have been found in the kidney in a few 
 cases. In all essentials they are exactly similar to the hepatic 
 lesions. The presence of colonies of organisms is accompanied by 
 widespread degeneration, and, as might be expected, the changes 
 are first seen in the cells of the convoluted tubules. As in the 
 livei-, gas bubbles develop in late stages. 
 
 The Spleen. Here the lesions are not uncommon and are of the 
 usual type, though the appearances are complicated by the vascular 
 and toxic changes which have been described. It is noteworthy 
 that even here there is never any cellular exudate, and the bacilli 
 are never phagocytosed. 
 
 Lymphatic glands. Infection of the lymphatic glands is probably 
 much more common than is thought. The organisms reach the 
 gland by permeation along the lymph vessels, and give rise to 
 the usual quiet necrosis. Here, again, the lack of any reaction 
 is very striking. 
 
 The Supra-renal glands. In one case I found a lesion which 
 conformed in every respect with the lesions in other organs. 
 
 Nervous System,. The peripheral nerves in the local lesions show 
 changes similar to those in the muscles and other tissues. I have 
 not found lesions in the central nervous system, but in one case 
 there was a condition of cerebral meningitis, apparently due to one 
 of the anaerobic bacilli. The lesion, in this case, had the character 
 of an ordinary acute meningitis. The membrane was congested 
 and covered with a polynuclear leucocytic exudate, and there was 
 active phagocytosis of the bacilli by the leucocytes. Many bacilli 
 were present in the vessels of the underlying cerebral cortex, but 
 the nerve cells showed no change. 
 
 h 2 
 
180 
 
 DESCRIPTION OF PLATES 
 
 1. B. welchii. Film preparation, 18-liour-old culture on alkaline egg. (Henry, 
 
 Pig. 36). 
 
 2. B. welchii. Film preparation, 3 days' growth on alkaline pieat, showing short 
 
 cocooid forms. (Henry, Fig. 37.) 
 
 3. B. welchii. Film preparation from infected haemotliorax fluid showing strepto- 
 
 bacillary form. (Henry, Fig. 38a.) 
 
 4. B. welchii. Filamentous and chain forms grown on alkaline egg agar for 24 
 
 hours. (Henry, Fig. 38b.) 
 
 5. B. welchii. 24-hour culture on alkaline egg agar to show involution forms. 
 
 (Henry, Pig. 39.) 
 
 6. B. welchii. 2 days' growth on human serum to show spore formation. (Henry, 
 
 Pig. 40.) 
 
 7. B. welchii. 24-hour culture in starch broth to show spore formation. (Original.) 
 
 8. Vibrion sepiigue. 24-48 hour meat culture, x 1500. (Robertson, B. M. J., 
 
 Fig. 1.) 
 
 9. Vibrion septiqm. Noguchi tube culture showing rods, spores, and a 'citron' 
 
 type. X 1500. 
 
 10. Vibrion septique. Post-mortem specimen from muscle of guinea-pig ; citrons 
 
 and club-shaped types are shown, x 1500. 
 
 11. Vibrion septique. From liver of guinea-pig showing 'citron', 'bulb' and oval 
 
 forms. X 1500. 
 
 12. Vibrion septique. From liver of guinea-pig showing long filaments, &e. xlOOO. 
 
 13. Vibrion septique. ' Citrons ' from haemorrhagic blister fluid of patient suffering 
 
 from gas-gangrene, x 1500. 
 
 14. Vibrion septique. 48 hours' growth on serum agar, x 1000. (Mcintosh, Plate I, 
 
 Fig. i). 
 
 15. Vibrion septique. Noguchi culture. X 1000. (Mcintosh, Plate I, Pig. 5.) 
 
 16. Vibrion septique. Smear from peritoneum of a mouse. x 500. (Mcintosh, 
 
 Plate I, Fig. 6.) 
 
 VI , 1&. Vibrion septique. Broth cultures, x 1000. (Mcintosh, Plate I, Figs. 7 and 8.) 
 
 19. B. oedematiens. Egg broth culture. (Henry, Fig. 45a.) 
 
 20. B. oedematiens. From surface colony. (Henry, Fig. 45b.) 
 
 21. B. chauvoei. Four days' broth culture, x 1000. (Mcintosh, Plate II, Pig. 1.) 
 
 22. B. cJmmoei. 24-hour Noguchi tube, x 1000. (Mcintosh, Plate II, Fig. 2.) 
 
 23. B. clmmoei. Smear from muscle of guinea-pig. x 1000. (Mcintosh, Plate II, 
 
 24. B. histolyticus. 16 hours' growth in alkaline meat, x 1500. (Original). 
 
 25. 26. B. botulinus. Broth culture, x 1000. (Mcintosh, Plate III, Figs. 4 and 5.) 
 
 27. B. sporogefks. Noguchi tube culture, 3 days' growth, x 1000. (Mcintosh, 
 
 Plate IV, Fig. 4.) ^ 
 
 27a. B. sporogenes. 48 hours' agar. (Mcintosh, Plate II, Fig. 4). 
 
 28. B. tertius. 5-day-old colony on alkaline egg agar, x 1300. (Henry, Fig. 42.) 
 
 29. B. cochlearius. Broth culture, x 1000. (Mcintosh, Plate II, Fig. 9.) 
 
 30. B.tetanomorphus. 48 hours' growth on agar, x 2000. (Mcintosh, Plate II, Fig. 4.) 
 
 31. B. tertius. (Original.) 
 
 32. B. cochlearius. (Original.) 
 
 33. 34. B. tetanomorphus. 48 hours' growth on agar. (Original.) 
 
 35. B.fallax. Broth culture two days old. x 1300. (Henry, Fig. 43.) 
 
 36. B. aerofetidus. 3 days' culture in alkaline egg broth, x 1300. (Henry, Fig. 44.) 
 
 37. B. bifermentans. 48 hours' growth meat medium, x 1500. (Original.) 
 
 38. B. sphenoides. 48 hours' growth starch broth. (Original.) 
 
181 
 
 39. B. memi^s. 48 hours' growth potato broth. (Original.) 
 
 40. J ^t^^. Broth culture, x 1000. (Mclnto.h, Kate II, Fig. 8.) 
 
 42 J If. St°"-l----''l'^aline egg agar.' x6. (Hen ;, Pig. ^2.) 
 
 42. B u^emn 48.hour growth colony in agar. (Mcintosh, Plate V, Fig 1 r 
 
 43. ^.^<i.^„fe.„.. 3 days old colonies on egg agar. x6. (Henry, Fig. 34.) 
 U. Bmstdytuue. 8 days old colonies on egg agar, x 6. (Henry, Fig 35 ) 
 4u. r^hr^on septzque. 24 hours' culture. (Mcintosh, Plate VI, Fig 3 ) 
 
 46. Vibrion septi^. 24 hours' surface colony. (Mcintosh, Plate VII, Fi. 2 ) 
 
 47. B. sporoge,ies. Surface colonies 3 days old. x 6. (Henry, Fi» 28 ) "' 
 
 48. 5. ^-^-- ^^^-^ace^coW : egg agar. Slow-growing coronie's 2 days old. 
 
 49. B. teriius. Surface colonies on egg agar, x 6. (Henry, Fig. 29.) 
 
 50. B./aUax. 3 days old colonies : egg agar, x 6. (Henry, Pig. 30.) 
 
 51. B. aerofilidus. 3 days old colonies : egg agar, x 6. (Heni-y, Fig. 33 ) 
 
 52. B. wdchii. 24 hours' growth on serum agar slope. (McIntosh| Plate XII, Fig 5 ) 
 
 53. B.^orogenes. Growth on serum agar slope 1 week. (Mcintosh, Plate XII, 
 
 54. B. teriius. 48-hour growth on serum agar slope. (Mcintosh, Plate XIII, Fig. 5.) 
 
 55. VibHm septique. 4 days' growth on serum agar slope. (Mcintosh, Plate XIII, 
 
 56. B. welchii {StTa.m 82). Milk culture, 24 hours old, to show stormy fermentation 
 
 JVote the disappearance of the cream layer, the disruption of the casein clot, 
 and the displacement of shreds of clot upwards through the paraffin layer. 
 
 57. B. welchii (Strain Pasteur). Alkaline meat culture, 1 week old, and showing 
 
 reddening of the meat. Contrast this with the normal meat in Plate XXVII, 
 
 58. B. welchii (Strain 82). Culture in alkaline egg, 5 days old. Note the diffuse 
 
 opacity due to fine friable clot. There is no shrinkage and no sign of 
 digestion. Contrast this with normal alkaline egg in Plate XXVII, Fig. 21. 
 
 59. B. sporogenes (Strain Pasteur). Milk culture, 1 month old. To show undis- 
 
 turbed cream layer and complete digestion of the milk. 
 
 60. B. sporogenes (Strain Pasteur). Culture in alkaline milk, 1 week old. To show 
 
 digestion and blackening. 
 
 61. B. sporogenes (Strain Harwood). Culture in alkaline meat, 1 week old. To show 
 
 marked blackening of the upper layers of meat. 
 
 62. B. sporogenes (Strain Pasteur). Culture in alkaline egg. To" show cylindrical 
 
 coagulum which has undergone gradual shrinkage and partial digestion. 
 
 63. B. fertius (Strain 68). Milk culture, 6 weeks old. To show solid clot fissured 
 
 by gas bubbles. 
 
 64. B. tertius (Strain Benson). Culture in alkaline meat, 14 days old. To show 
 
 pinking of meat and commencing bleaching on the surface. 
 
 65. B.fallax (Strain Trucol). Milk culture, 1 month old, showing strong solid clot 
 
 disrupted by bubbles, and with orange colour at the surface. 
 
 66. B. fallax (Strain 92). Milk culture, 5 days old, showing separation into a turbid 
 
 whey and a soft friable clot which is slightly disrupted by gas. There is no 
 dislocation of the cream layer. Bubbles have collected on the air surface of 
 the paraffin layer. 
 
 67. B. aero/elid'us (Weinberg). Culture in milk, 36 hours old, and showing disrup- 
 
 tion of casein clot by gas. The fermentation is not so vigorous as, and 
 develops later than, the reaction given by B. welchii. 
 
 68. B. aerofetidus (Weinberg). Growth in alkaline meat, 7 days old, showing 
 
 reddening of the meat and slight blackening of the upper layers. 
 
 69. B. oedematiens (Strain Domange). Growth in milk, after 4 months' incubation, 
 
 to show slight separation of whey from subjacent casein flocculi. 
 
 70. B. oedematiens (Strain Domange). Culture in alkaline meat, after 15 days, to 
 
 show pinking of meat and extensive bleaching from surface downwards. 
 
 71. B. histolyiicus (Weinberg). Milk culture, 2 days old, showing complete digestion, 
 
 with no disturbance of the cream layer. 
 
 72. B. histolyiicus (Weinberg). Culture in alkaline meat, 4 days old, to show 
 
 digestion and formation of glistening white balls of tyrosin. 
 
183 
 
 73. Ordinary milk under paraffin. 
 
 74. Alkaline meat medium under paraffin. 
 
 75. Alkaline egg medium under paraffin. 
 
 76. Lesions produced in a guinea-pig by the injection of a culture of Vibrion s^tique 
 
 into the muscles of the thigh. The subcutaneous tissues over the abdomen 
 and thorax are the seat of a clear, deep red, sero-sanguinolent oedema in 
 which there are extensive collections of gas bubbles. The abdominal muscles 
 and inguinal fat are intensely congested. 
 
 77. Lesions produced in a guinea-pig by the injection of a culture of BaciUus ■welchii 
 
 into the muscles of the thigh. In comparison with those of V. septique the 
 oedema is only slightly blood-stained, the formation of gas is usually less 
 marked, and the muscles themselves are pale and looked bleached. 
 
 78. Lesions produced in a guinea-pig by the injection of a culture of Bacittus 
 
 oedematiens into the muscles of the thigh. The characteristic feature is a clear 
 colourless gelatinous oedema, while the abdominal muscles are for the most 
 part unaltered. In general, gas formation if present at all is trivial. 
 
 (Pigs. 56-75 from Henry (Figs. 1-9 and 11-21).) 
 
 Printed under the authority of His Majebtv's Stationery Ofpioe 
 By Frederick Hall, at the University Press, Oxford. 
 
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PLATE VI 
 
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PLATE IS 
 
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PLATE X 
 
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PLATE XI 
 
 Fig. 37 
 
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fLATE XII 
 
 Fig. 40 
 
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PLATE XIII 
 
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PLATE XIY 
 
 Fig. 48 
 
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PLATE XV 
 
 Fig. 50 
 
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PLATE XVI 
 
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Figure 78 
 
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