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Characteristics of certain strains of Clostridium Welchii, with special reference to their toxic components Todd, Marjorie Doreen 1941

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CHARACTERISTICS OF CERTAIN STRAINS OF :REFERENCE TO THEIR TOXIC COMPONENTS by Marjorie Doreen Todd A Thesis Submitted i n Partial Fulfilment of the Requirements for The Degree of MASTER OF ARTS i n the Department of Bacteriology and Preventive Medicine The University of British Columbia May* 1941 ACKNOWLEDGMENT I wish to express my a p p r e c i a t i o n t o Dr» D*G.B« Duff f o r the advice and assistance he has given during the course of t h i s work. INTRODUCTION I t was f i r s t i n 1865 that a study of anaerobic "bacteria was begun by Pasteur, marked by h i s discovery of B a o i l l u s butyricus.(1) This discovery l e d t o the i n v e s t i g a t i o n of a new group of b a c t e r i a whose complexity and confusing nature have not yet been mastered. This essay i s p a r t i c u l a r l y concerned w i t h a study of one of the pathogenic anaerobes of the gas gangrene group, C l o s t r i d i u m w e l o h i i . B o t t i n i (2) i n 1871 was the f i r s t worker to demonstrate the i n f e c t i o u s nature and t r a n s m i s s i b i l i t y of gas gangrene. I t was not t i l l 1892 that Welch and N u t t a l l (3) i s o l a t e d Cl» w e l o h i i ? naming i t at that time B a c i l l u s aerogenes capsulatus* There followed a r a p i d succession of independent i s o l a t i o n s of the same organism by d i f f e r e n t workers i n various c o u n t r i e s . I r a e n k e l (4) of Germany i n 1893 i s o l a t e d B», phlegmonis emphysematosae. I n France, V e i l l o n and Zuber (5) i s o l a t e d B a c i l l u s p e r f ringens i n 1898, This name i s f r e q u e n t l y used f o r the organism at the present time* From about 1900 u n t i l the time of the Great War there f o l l o w e d a d e f i n i t e l a c k of i n t e r e s t i n the study of the anaerobic b a c t e r i a , probably clue to the d i f f i c u l t y at that time of obtaining and maintaining pure cultures*, By 1915 the Great War had stimulated an i n t e n s i v e study of the anaerobes of the gas gangrene group. This group i s e s p e c i a l l y important because of the "power of the organisms t o produce exotoxins which are i n j u r i o u s t o l i v i n g t i s s u e . 01. w e l o h i i i s probably the most important organism of the gas gangrene group, f o r i t i s 01. w e l o h i i that was responsible f o r 85 per cent of the cases of gas gangrene during the Great War ( 6 ) . B u l l and P r i t c h e t t (7) i n 1917 i n America were the f i r s t t o obta i n 01. w e l o h i i t o x i n . Some of the d i f f i c u l t i e s of producing a potent t o x i n were recognized by them. Henry (8) i n 1935 recognized two elements i n the t o x i n ; hemolysin and l e t h a l t o x i n , the l a t t e r of which he c a l l e d c y t o l y s i n , Prigge (9) has found 01. w e i c h i i t o x i n t o contain two components corresponding to those described by Henry* Walbum and Reymann (10) have described another t o x i c element which was only produced i n media containing glucose. This t o x i c component was found to k i l l mice instantaneously upon intravenous i n o c u l a t i o n , and was not n e u t r a l i z e d by perfringens a n t i t o x i n . Several workers, Ouranoff (11), Ford and Williams (12), Wuth (13) and Mason and Glenny (14) considered the hemolysin and l e t h a l t o x i n t o he i d e n t i c a l . There i s s t i l l at the present time much confusion r e l a t i n g to the composition of Gl*- w e l c h i i t o x i n . I t was therefore w i t h a vie?/ toward obtaining evidence i n d i c a t i n g the s i m i l a r i t y or d i f f e r e n c e betwe the various components of CI, w e l c h i i t o x i n that t h i s work was undertaken. 4. S t r a i n s of 01. w e l o h i i used during the course of t h i s work* Lab: stock s t r a i n of the Department of Ba c t e r i o l o g y and Preventive Medicine, U n i v e r s i t y of B r i t i s h Columbia, L i v e r ; i s o l a t e d at the U n i v e r s i t y of B r i t i s h Columbia i n May 1940 from a gangrenous l i v e r . PB6H: received from the United States P u b l i c Health Service, I s o l a t e d by B u l l and P r i t o h e t t , SR18: received from the United States P u b l i c Health Se r v i c e , I s o l a t e d by Robertson, 11850; received from Dr. 1.0. H a l l , Colorado, I s o l a t e d i n June 1958 from a case of gas gangrene." 18558; received from Dr, I.e. H a l l , Colorado. I s o l a t e d i n March 1940 f o l l o w i n g a burn which required amputation. 5. Media used f o r c u l t i v a t i o n of CI, w e l c h i i and f o r t o x i n production. Semi-solid i n f u s i o n agar: f o r maintaining stock c u l t u r e s . Robertson's meat medium (15); f o r spore-production. Reed's broth (16): f o r hemolysin production. Reed's medium w i t h the a d d i t i o n of-1*5% agar and 10% sheep's blood: f o r p l a t i n g Glucose peptone beef i n f u s i o n broth (17). to which was added 10% d r i e d ground meat from which the f a t was extracted: f o r l e t h a l t o x i n production. 6 9 Physiology and C u l t u r a l C h a r a c t e r i s t i c s of CI, w e l c h i i The s t r a i n s of 01* w e l c h i i studied agreed w i t h the c l a s s i c a l d e s c r i p t i o n s of the species. They were gram p o s i t i v e , spore-forming rods which v a r i e d considerably i n morphological c h a r a c t e r i s t i c s under d i f f e r e n t c o n d i t i o n s . The older c e l l s were i r r e g u l a r i n t h e i r s t a i n i n g p r o p e r t i e s , many c e l l s being gram negative. The most rapid, growth of c u l t u r e s occurred i n medium containing glucose, c u l t u r e s i n Reed's broth being a c t i v e i n as l i t t l e as two hours. Spores were only produced i n an a l k a l i n e medium containing no glucose. The use of p y r o g a l l i c a c i d and potassium hydroxide or of p a r a f f i n o i l seals was found t o be unnecessary t o ob t a i n good growth i n Reed's broth or Robertson's meat medium cu l t u r e s v M,L»D, The Minimal L e t h a l Dose of whole Reed's broth c u l t u r e s i n the vegetative s t a t e was found c o n s i s t e n t l y to be 0,2 ccV, f o r mice. 7. Attempts to obtain colonies on p l a t e s . 'No success was obtained when the plates were incubated i n carbon dioxide or nitrogen atmospheres. Al s o an unsuccessful attempt was made to obtain colonies using aerobic a, s u b t i l i s on the top.of the p l a t e . The Mcintosh-ifildes j a r was used without success f o r p l a t i n g , but broth and litmus m i l k c u l t u r e s could be s u c c e s s f u l l y c u l t u r e d i n the j a r . A desiccator containing potassium hydroxide and p y r o g a l l i c a c i d was used, and some colonies were obtained on the p l a t e s . A s e r i e s of p l a t e s containing ferrous ammonium sulphate as a reducing agent was set up as fo l l o w s : 20°/o ferrous ammonium sulphate was added to 10 cc amounts of medium i n the f o l l o w i n g q u a n t i t i e s ; 0.05, 0.1, 0.25, 0.35 and 0.5 cc. 01. w e l c h i l was sown on the p l a t e s , which were incubated i n an evacuated d e s i c c a t o r . The 0.1 cc. amount was found to be the most e f f e c t i v e f o r obtaining colonies on the p l a t e s , but .this method was not nearly as e f f e c t i v e as the procedure described below. Vancouver i l l u m i n a t i n g gas blown through an anaerobic j a r was found to provide i d e a l conditions f o r p l a t i n g out 01. w e l c h i i . T h i s method was f i r s t developed i n t h i s laboratory i n conjunction with the p l a t i n g 8. methods c a r r i e d out by Mr. V.J. Freeman i n h i s studies on V i b r i o n septique. A s e r i e s of p l a t e s was set up to determine the minimum length of time i t was necessary t o blow gas through the oar. I t was found that as l i t t l e as one minute s u f f i c e d to obtain good growth on the p l a t e s a f t e r f i f t e e n hours incubation at 57°C. However, allowim the gas to blow through the anaerobic Jar f o r as long as s i x t y minutes i n no way a f f e c t s the a b i l i t y of the c u l t u r e s to form i s o l a t e d c o l o n i e s on the surface of the plates„ 9 Types of colonies observed on Reed's s o l i d and Reed's blood agar p l a t e s , incubated i n i l l u m i n a t i n g gas, l v Smooth, convex, white, 2-4 mm. diameter. Smears show short rods. This type of colony i s g e n e r a l l y considered to be c h a r a c t e r i s t i c of ClV w e l c h i i . (18) 2, F a i r l y large (5mm.), smooth, of milky appearance w i t h dense centre. Smears show long rods, 3, I r r e g u l a r colonies w i t h f i n e l y granular edge, brownish i n colour. Many tspores present i n smears. 4, G l i s t e n i n g , c l e a r , smooth rwith the appearance of water drops. T y p i c a l c e l l s i n smears, but the organisms could not be c u l t i v a t e d i n Reed's medium. Colony types 2, and 3, observed correspond c l o s e l y to variants described by Orr (19) and by McGaughey (20), While a d e t a i l e d study of CI. w e l c h i i v a r i a n t s was not conducted, the d i f f e r e n t t y p e s obtained i n d i c a t e d that 01. w e l c h i i i s subject to wide v a r i a t i o n and that the d i f f e r e n t types of colonies were not due to ^ contamination. However only minute d i f f e r e n c e s i n hemotoxin production by the d i f f e r e n t v a r i a n t s were observed, i n contrast to the gross d i f f e r e n c e s as noted 10. "by Orr (19) and by MoGaughey (30). Hemolytic e f f e c t of c u l t u r e s on blood p l a t e s . I f the p l a t e s are incubated 18 to 20 hours there i s a narrow zone (2 mm.) of c l e a r hemolysis around the c o l o n i e s , surrounded by a second zone which i s wider (5 mm.) and quite opaque and dark. These two zones are always found on incubating any of the s i x s t r a i n s used. I f the p l a t e s are placed i n the r e f r i g e r a t o r overnight a second opaque zone i s l a i d down, terminated at i t s outer ed^e by a dense band, having the appearance of red c e l l s having been p i l e d up at the edge. Upon f u r t h e r incubation the band c l e a r s and a f u r t h e r opaque zone i s l a i d down i f the p l a t e i s placed i n the r e f r i g e r a t o r again. No f u r t h e r completely c l e a r zone i s formed a f t e r the f i r s t i n c u b a t i o n . I f the p l a t e s are l e f t at room temperature to incubate, only the narrow c l e a r zone i s formed. An opaque zone i s then l a i d down i f the p l a t e i s placed i n the r e f r i g e r a t o r s (See F i g . l ) The hemolytic e f f e c t s produced by 01. w e l o h i i c u l t u r e s are not e x a c t l y l i k e those produced by any other organism on blood p l a t e s , so f a r as i s known. 11 . I SR12 s t r a i n i l l u s t r a t i n g zones produced by CI. v r e l c h i i on blood p l a t e a f t e r 18 hours in c u b a t i o n , followed by an overnight period of r e f r i g e r a t i o n and a second period of incubation and r e f r i g e r a t i o n . 12. JfiK. 2 PB6H s t r a i n i l l u s t r a t i n g successive zones produced on a blood p l a t e a f t e r a number of a l t e r n a t e periods of incubation and r e f r i g e r a t i o n . 13, Hemolytic e f f e c t of t o x i n on blood p l a t e s When b a c t e r i a - f r e e t o x i c broth is placed i n a hole cut i n a blood p l a t e and the p l a t e incubated ... . . . . . . . . Vj^  t- . eighteen hours, a c l e a r c o l o u r l e s s zone i s formed i n the agar about the hole* I t i s to be noted the p e r i p h e r a l opaque zone accompanying s i m i l a r bands of c l e a r hemolysis i n the case of colonies incubated eighteen hours, i s absent. However, i f the t o x i n - c o n t a i n i n g p l a t e i s r e f r i g e r a t e d overnight f o l l o w i n g the incubation p e r i o d , a p e r i p h e r a l opaque zone then appears. Upon a second incubation of the p l a t e t h i s zone ev e n t u a l l y becomes c l e a r , i n contrast to the opaque zones produced by c o l o n i a l growth, which p e r s i s t through a l t e r n a t e r e f r i g e r a t i o n s and incubations« (Note that c e r t a i n minor changes may occur i n c o l o n i a l opaque zones,) The hemolytic e f f e c t of t o x i n on blood-plates i s s i m i l a r to the alpha prime hemolytic e f f e c t produced by some Streptococcus cu l t u r e s on blood p l a t e s ( 2 1 ) . There i s no change i n hemolytic a c t i o n of t o x i n i n the t e s t tube by a l t e r n a t e periods of hot and oold. (See p..-z7 ) 14. Optimal Conditions. ..for Production of Toxin As w i l l be discussed l a t e r , CI, w e l c h i i grown i n various kinds of f l u i d media produces t o x i c f a c t o r s which manifest themselves as fo l l o w s : 1, Hemolysis (on sheep and r a b b i t c e l l s ) . 2, L e t h a l a c t i o n (mice). 5, Dermo-necrotic a c t i o n (guinea p i g s ) . I t i s the purpose of t h i s s e c t i o n to record the optimal conditions under which these f a c t o r s are produced and to discuss t h e i r i n t e r - r e l a t i o n s , I . F actors R e l a t i n g t o 01, w e l c h i i Hemolysin. A. Optimal Conditions f o r Production of Hemolysin. I t i s necessary before a d e t a i l e d d i s c u s s i o n of the optimal conditions f o r hemolysin production to designate the manner i n which hemolytic t i t r a t i o n s were c a r r i e d out. The nemotoxin t i t r e was taken as the l e a s t amount of t o x i n which hemolysed 50% of 2% r a b b i t red blood c e l l suspension (16). The end-point as determined by Stewart! (17) i s the l e a s t amount of t o x i n required to produce complete hemolysis of 0,5 cc, of 5% red blood c e l l suspension. 15. The r e s u l t s are recorded I n the f o l l o w i n g manner: •Complete hemolysis 4+ P a r t i a l hemolysis 5+ 50$ hemolysis 50$ S l i g h t hemolysis 1+ No hemolysis 0 ( i ) Influence of Medium.on Hemolysin Production. Reed's "broth (16) has proved an e x c e l l e n t medium f o r the production of hemolysin. T i t r e s up to 1:20,000 have at times been obtained (see F i g . 3 ) , with average r e s u l t s i n the neighbourhood of 1:2560 to 1:5000. I n remarkable contrast to t h i s , glucose peptone beef i n f u s i o n broth (17) containing meat p a r t i c l e s (a type of medium much used f o r Cl» w e l o h i i i n v e s t i g a t i o n s ) , y i e l d e d sometimes s l i g h t hemolysin and o f t e n no measurable amount whatever. These r e s u l t s are i l l u s t r a t e d i n Table 1, 16 Table 1, Hemolytic a c t i o n of toxins produced i n d i f f e r e n t types of media. Exp. 1 PB6H s t r a i n /&> J - * J - ' •iont-Kol Reed' s broth 4v 4+ 4^ 4+- 4^ 3i- 3<- 50% 0 Beef i n f u s i o n 3+ 0 G 0 G 0 0 0 0 SR12 s t r a i n Reed's broth 4+- 4h 4t 3'- 5+ li- 0 0 0 Beef i n f u s i o n 3+- 0 0 0 0 0 0 0 0 Exp. 2 PB6H s t r a i n Reed's broth 4-v 4+ Beef i n f u s i o n 1-t- 1-H L- S~ i_ / - . > i J - J - £W//». "%0 -S» . ^ 3 .^ < r t ? v f7^0 CO/?/* 4+ 4+ 4i- 3*- 3+- 50% H- 0 0 0 0 0 0 0 0 0 0 0 0 0 Exp. 3 PB6H s t r a i n — R a b b i t c e l l s Reed's broth 44- 4f- 4+- 4-h/4+- 4/- 4h 4+- l-i-. 0 Beef i n f u s i o n 3+ 3f 1+ 1+ 0 0 0 0 0 0 PB6H s t r a i n — S h e e p c e l l s Reed's Broth 4f 4+- 4-f- 4-t- 4+- 4i- 4+- 3+- 1+- 0 Beef i n f u s i o n 0 0 0 0 0 0 : 0 0 0 0 17. The production of hemolysin i n Reed's "broth can be increased by s u b s t i t u t i n g Witte's peptone f o r the Bacto peptone i n the medium. This f a c t i s i n accord w i t h the r e s u l t s obtained by Borthwick i n 1937 (22), and i s i l l u s t r a t e d by the r e s u l t s i n Table 3. Tablp P * E f f e c t of peptone on hemolysin production. Exp* 1 PB6H s t r a i n • ; .• Reed's broth k h k & At- ^ •ffe / Bacto peptone 4+ 4- 4+ 50$ 0 0 0 0 0 Witte's peptone 4* 4» .4- 4+ 4^  4+- 4 50$ 0 Exp. 2 PB6H s t r a i n Reed's broth Bacto peptone 4^  4+ 4»- 4t, 4+ 4+ 4+- 50$ 0 0 0 Witte's peptone 4- 4<- 4 4 - 4- 4+- 4- 4+- 3+50$ 0 Expv 3 PB6H s t r a i n Reed's broth Bacto peptone 4+- 4+ 4+ 4f 4*- 4- 3' 3+- 0 0 W i t t e 1 s peptone 4+ 4+ 4+ 4+ 4+- 4+- 4+- 3/- 50$ 0 18. ( i i ) Influence of Time on Hemolysin Production. The change i n the amount of hemolysin produced "by the d i f f e r e n t s t r a i n s a v a i l a b l e was followed over a period of two to s i x t y hours. For each s t r a i n used, an inoculum c o n s i s t i n g of 3% of a Reed's broth c u l t u r e was sown i n t o two 100 cc. f l a s k s of Reed's b r o t h . The cult u r e s were then incubated at 37°C. Fi v e cc, of cu l t u r e were withdrawn a f t e r the f o l l o w i n g periods of incubation: 2,4,6,8,12,18,20,22,24,26,30,40,44,48,52.and 56 hours. The q u a n t i t i e s removed were centrifuged and a hemolytic t i t r e was performed on each t o x i c supernatant f l u i d . The r e s u l t s are recorded i n F i g , 3. I t i s to be noted that the maximum hemolysin production occurs i n from 12 to 18 hours. This i s s l i g h t l y l e s s than the time recorded by Reed (16). Reed does not mention any marked decrease i n hemolysin present a f t e r 20 hours in c u b a t i o n , w i t h an increase again at a l a t e r p e r i o d , as seen i n F i g , 3, This marked decrease, followed by a marked increase i s e x h i b i t e d by a l l s t r a i n s of CI,welohii studied except s t r a i n 11830, I n the case of s t r a i n s SR12, L i v e r and PB6H, the i n i t i a l drop i n hemolysin i s t o about the same l e v e l at the same time. These three s t r a i n s a l l produce 19. hemolysin to a high t i t r e , i n contrast t o s t r a i n s 11850j Lab *and 12358 which only produce a small amount of hemolysin.. The f i n a l gradual decrease i n amount of hemolysin produced does not occur t i l l a f t e r a p e r i o d of 30 t o 40 hours i n c u b a t i o n , ( i i i ) Influence of pH on Hemolysin Production At the same time as hemolytic t i t r a t i o n s were c a r r i e d out on t o x i c broth over a p e r i o d of from two t o s i x t y hours, the pH of the t o x i c broth was a l s o determined i n order to obtain a record of changes i n pH during the same per i o d . The r e s u l t s are recorded i n F i g . 3, There i s a r a p i d drop i n pH f o r a l l c u l t u r e s f o r about the f i r s t f i v e hours, followed by f l u c t u a t i o n s i n pH from about pH 6,4 to 7,0, The f i r s t ' d r o p compares w i t h the i n i t i a l r i s e i n hemolysin production. The average pH f o r a l l s t r a i n s used, at the p o i n t of maximum hemolysin production i s 6.4 to 6,6, I f the a c i d i t y of the medium becomes too g r e a t , the hemolysin y i e l d i s l e s s , and so the medium was buffered so that the pH would not f a l l below 6,0 to 6,2 (16), 20. The optimum pH f o r hemolysis has "been recorded as 6.0 to 6.5 hy Walbum and Reymann (10). This f a c t i s i n agreement w i t h the r e s u l t s obtained above at the point of maximum hemolysin production. A f u r t h e r s e r i e s of experiments, shown i n Table 3, a l s o Indicates the optimum pH f o r hemolysin production t o be 6.4. Table 5. Optimum pH f o r hemolysis. Exp. 1 PB6H t o x i n .U ia J- +>• J _ SCO J U J - Co" r pH 6.4 4^ 4+- 4f 4f- 4f 4i 4+ •4^ 0 7.0 4V 4-h 4+- 4^ 4f 5-*- 3'- 0 0 7.4 4+- 4-t- 4* 4+- 4^ 4i- 4^ 50$ 0 0 7,8 4^ - 4* 4+- 4f- 44- 4+- 4i- 4 + 50$ 0 Exp, 2 PB6H t o x i n pH6,4 4f 4*- 4t- 4+- 4* 4l- 4(- 4* 4^ 0 7,0 4+- 4+- 4+ 41- 4+ 4+ 4+ 3+ 50$ 0 7.4 4f 4+- 4f 4'- 4+ 4+- 4+- 50$ 1+ 0 7.8 4f 4+ 41 4+ 41- 4+ 44- 3i 50$ 0 21. B. D i f f i c u l t y ' w i t h F i l t r a t i o n . o f Toxin. There i s a considerable l o s s of potency of hemolysin and l e t h a l t o x i n by f i l t r a t i o n methods, S e i t z and Berlcefeld f i l t e r s were used i n an endeavour to obtain potent t o x i c f i l t r a t e s . There i s a great l o s s of hemolysin by f i l t r a t i o n w i t h S e i t z f i l t e r , but only •a small amount of hemolysin i s l o s t by f i l t r a t i o n with J"ena glas s f i l t e r , as may be seen i n Table 4. Table 4 : E f f e c t of f i l t r a t i o n on hemolysin. PB6H s t r a i n . & fa, & w & • & ^ ^ £ -%Sf%i 0 Toxin centrifuged 4f 4+ 4f 4^ 4+- 4+- 4+ 4+ 4f 4+- 3+ 3t-Se.itz f i l t e r e d 4<- 4+ 4f 50% 0 0 0 0 0 0 0 0 Jena glas s 4i- 4* 4+- 4+- 4+' 4+- 4-h 4+ 3+- 50$0 0 0 f i l t e r e d I t was thought that by changing the pH of the S e i t z f i l t e r pad or of the m a t e r i a l to be f i l t e r e d (that) more success might be obtained. A s e r i e s of b u f f e r s o l u t i o n s was made up to the f o l l o w i n g pH's: 6.0, 6.4, 6.8, 7.2, 7.6, 8.0 22. A quantity of t o x i c broth was adjusted to each of the. above pH's and the m a t e r i a l was f i l t e r e d through the S e i t z pad at the same pH. No success was obtained from t h i s experiment. Next an attempt was made to d i s s o l v e the absorbed t o x i n out of the f i l t e r pad by washing the pad with 0.3N sodium carbonate s o l u t i o n , using a volume equal to one-half the volume of t o x i c broth being f i l t e r e d . The washings were n e u t r a l i z e d w i t h HC1 and d i l u t e d w i t h an equal volume of s a l i n e . A very s l i g h t amount ..of t o x i n was recovered, but the method was not considered to be s u c c e s s f u l . I t was found that hemolysin and l e t h a l t o x i n would pass through a Jena, gla s s f i l t e r #5 unaltered to any considerable extent. Unfortunately the f i l t e r was l a t e r broken, and other f i l t e r s . o f the same type were not a v a i l a b l e . I t was therefore f o r t h i s reason that throughout the course of t h i s , work the supernatant f l u i d from thoroughly c e n t r i f u g a t e d c u l t u r e s was used as a source of t o x i n . Centrifugated m a t e r i a l i s not always e n t i r e l y s a t i s f a c t o r y , e s p e c i a l l y f o r animal i n o c u l a t i o n s , f o r sometimes a f t e r three hours c e n t r i f u g a t i o n at high speed,spores w i l l remain i n the supernatant. I t i s to be emphasized that c e n t r i f u g a t i o n 33, i s d e f i n i t e l y not the method of choice f o r obtaining t o x i n , p r i m a r i l y because of uncertainty as to s t e r i l i t y . However under the circumstances t h i s was the only means by which t o x i n s of high potency could be obtained. Results obtained from centrifugated toxins d i d not appear however to become confused by p o s s i b l e p a r a l l e l i n f e c t i o n s . Mice inoculated w i t h potent centrifugated t o x i n died i n from four to twelve hours without gross postmortem changes, whereas mice inoculated with l i v i n g Gl» w e l o h i i c u l t u r e s i n the vegetative state d i d not commonly die u n t i l eighteen to twenty-four hours, at which time they uniformly e x h i b i t e d marked oedema and hematuria, C. Attempt to P r e c i p i t a t e Toxin w i t h Ammonium Sulphate The t o x i n could not be s u c c e s s f u l l y p r e c i p i t a t e d from Reed's broth with ammonium sulphate, apparently due to the presence i n the medium of the l a r g e amount of g e l a t i n . Because of t h i s f a c t , d r i e d t o x i n s could not be prepared. This i s contrary to the f i n d i n g s of Reed (16), who claims that t o x i n s produced i n Reed's broth can be s a t i s f a c t o r i l y p r e c i p i t a t e d w i t h ammonium sulphate and d r i e d . 24. D. S t a b i l i t y of Hemolysin, Potent hemolysin i s very unstable. I f the t o x i c broth containing hemolysin i s . l e f t at room temperature f o r more than 48 hours without drying, the hemolytic f a c t o r i s destroyed. The same e f f e c t i s observed i f the t o x i c broth i s l e f t at r e f r i g e r a t o r temperature. Table 5 i l l u s t r a t e s the l o s s i n time of hemolytic a c t i o n of the t o x i c b r o t h . FP-pl?, ,5,*, E f f e c t of temperature on s t a b i l i t y of hemolysin. , SR12 s t r a i n Reed's bi?oth n & 4s & i«> k?o %y-&<> ikip> cSi Eresh-: t o x i n 4 4 4 4 4 4 4 4 50% 1 0 0 0 48 hrs.room temp. 4 4 4 4 5 50% 1 0 0 0 0 0 0 72 hrs.room temp. 4 4 4 5 1 0 0 0 0 0 0 0 0 25, E, Influence on Hemolysin of Reduction w i t h Sodium Thiosulphate. I f the t o x i c "broth i s reduced with 0*1$ sodium thiosulphate by p l a c i n g i n a vacuum desic c a t o r f o r one-half hour, there seems to be very l i t t l e change i n hemolytic power, as seen by the r e s u l t s i n d i c a t e d i n Table 6, This f a c t i s i n agreement with the f i n d i n g s of Stewart (17), However the f a i l u r e of sodium thiosulphate to a c t i v a t e 01, w e l o h i i hemolysin i s i n contrast to the e f f e c t of sodium thiosulphate oh Streptococcus hemolysin(23), ffa—e E f f e c t of sodium thiosulphate on hemolysin. PBBH s t r a i n -k & ' •<£> & t«° &° ife> Centrifugated t o x i n 4+ 4+ 4+- 4=+- 4+- 3+- 50$ 1+- 0 0 0 Reduced t o x i n 4+ 4-t- 4+- 4+ 4-t- 4+- 3+- 50$ • 0 0 26. IP. E f f e c t of Hemolysin on Sheep and Rabbit C e l l s Several p a r a l l e l hemolytic t i t r e s were c a r r i e d out using sheep and r a b b i t c e l l s . The hemolysin seems to have the same e f f e c t on both types of c e l l s , only very s l i g h t d i f f e r e n c e s i n the end-point being demonstrated. These r e s u l t s correspond to those obtained by Stewart (17), and are given i n Table 7, 1 x c '* E f f e c t of hemolysin on sheep and r a b b i t c e l l s . Exp, 1 PB6H s t r a i n J- i- i. j_ go j-3=-° / x S1XO ' S«-''"'<=, Rabbit c e l l s 4+ 4+ 4+ 4+ 4+- 4^ 4+ 50 fo 1*-1+ 0 0 Sheep c e l l s 4^ 4+ 4+ 4:4-4+ 4+ 50% 0 0 0 0 0 Exp, 2 PB6H s t r a i n Rabbit c e l l s 4+- 4*- 4+ 4+ 4+ 4+- 3+- 50% 0 0 0 Sheep c e l l s A& 4+- 4+ 4+ 3+ 50%1+- 0 0 0 0 Exp, 3 PB6H s t r a i n Rabbit c e l l s 4+- 4+ 4<- 4+- 4+- 4<- 4+- 3+- 3+ Sheep c e l l s : 4+- 4+- 4*- 4+ 4+- 4+ 3t 1+ 0 0 0 27. G. Attempt t o Demonstrate Hot-Gold L y s i s with CI., w e l o h i i Toxin There i s apparently no demonstration of 'hot-cold' l y s i s by incubating hemolytic t i t r a t i o n s f o r 01. w e l o h i i t o x i n at 37°C. f o r one hour, followed by r e f r i g e r a t o r temperature overnight. I n f a c t there i s no change at a l l i n the hemolysis i n the tubes a f t e r exposure to cold.- Results to demonstrate t h i s f a c t are set out i n Table 8« Table 8 9 E f f e c t of temperature on l y s i s of r a b b i t erythrocytes by hemolysin. PB6H s t r a i n 4+ 4+- 4*- 4- 4* 4* 4t 4+ 4*- 3'- 3+- 50$ 0 L i v e r 4+- 4*- 4->- 4i- 4 f- 4<- 4+- 4+ 4'- 4 f 3+- 3+- 0 11830 4+ 4 - 4+- 4- 4f- 4<- 4- 3f-50$ 1+- If- 0 0 12358 4+- 4*- 4+- 4i 4+ 4+- -4*- 4'- 3*-50$ 1+- 0 0 Exp. 1 There i s no change i n any of these tubes a f t e r overnight exposure to r e f r i g e r a t o r ^ temperature. Exp 8 2 Lab s t r a i n 8°C. Az\ 4h 4fc 3+- 3^ 50$ 1+- 0 4+ 4t- 4f 5f 3(- 50$ If- 0 0 0 Exp. 3 Lab s t r a i n 8°C. 4+- 4+- 4t- 5<- 0 0 4-t- 4+- 4+- 3+-. 0 0 28 I I . Optimal Conditions f o r Production of L e t h a l F a c t o r . Influence of Medium, Glucose peptone beef i n f u s i o n broth to which was added 10$ d r i e d ground meat (17), proved to be the best medium f o r production of the l e t h a l factor,, C u l t i v a t i o n of 01. w e l c h i i i n t h i s medium r e s u l t e d i n a t o x i n having mouse M.L.D.'s of from 0.1 to 0.15 co. Reed's medium, so suc c e s s f u l as a source of hemolysin, proved a poor producer of the mouse l e t h a l f a c t o r , since amounts i n excess of 0.5 ec. were required to exert the minimal l e t h a l e f f e c t . 29 I I I . Dermo-neorotic Factor as Demonstrated by „Guinea P i g Skin Reactions, A dermo-necrotic f a c t o r which, can be demonstrated by intradermal; i n o c u l a t i o n i n t o the back of a guinea p i g , i s present i n supernatant from centrifugated Reed's broth c u l t u r e s . Also an apparently s i m i l a r dermo-necrotic f a c t o r i s present i n glucose peptone beef i n f u s i o n broth c u l t u r e s . With supernatants from both media, and with d i f f e r e n t CI, w e l c h i i s t r a i n s , the minimal dermo-necrotic dose remained very c o n s i s t e n t l y at the l e v e l of 0.05 t o 0,10 cc. 30. TOXIN-ANTITOXIN REACTIONS. As discussed p r e v i o u s l y (p.14) the three ways i n which CI. w e l c h i i t o x i n can he demonstrated are by hemolytic t i t r a t i o n s , mouse l e t h a l t e s t s and dermo-necrotic a c t i o n on guinea p i g s k i n . These f a c t o r s a s . w i l l be demonstrated i n the f o l l o w i n g section,can a l l be n e u t r a l i z e d by perfringens a n t i t o x i n as supplied by Connaught Lab o r a t o r i e s , Toronto, The standard u n i t of perfringens a n t i t o x i n i s that amount which w i l l n e u t r a l i z e 10 M.L.D. of perfringens t o x i n (24), I n 1920, the Bengston u n i t was the amount which would n e u t r a l i z e 1000 M.L.D.-, but i n 1930 the o f f i c i a l u n i t f o r measuring the potency of the a n t i t o x i n s was changed to one one-hundredth the former standard, as the unitage of a n t i t o x i n s by the former method f e l l below 5 per cc« N e u t r a l i z a t i o n of Hemolytic E f f e c t of ..Toxin on Blood P l a t e s <, A very c l e a r - c u t demonstration of the n e u t r a l i z a t i o n of Cl» w e l c h i i t o x i n by i t s s p e c i f i c a n t i t o x i n can be shown by the a c t i o n of t o x i c broth and perfringens a n t i t o x i n on a blood p l a t e . 31. The a c t i o n of t o x i c broth on blood p l a t e s has already been discussed (p. 13), The n e u t r a l i z a t i o n of the t o x i n by perfringens a n t i t o x i n i s demonstrated i n F i g . 4. I n one hole i n the blood p l a t e , normal serum was placed f o r c o n t r o l . I n the centre hole the t o x i n was introduced and the perfringens a n t i t o x i n was placed i n the t h i r d h o l e . The zones described on page 13 can be c l e a r l y seen i n the agar around the hole containing t o x i n , and they are i n no way i n h i b i t e d by the normal serum i n the adjacent,hole on one s i d e . However on the opposite side the d i f f u s i o n of the t o x i n i s completely i n h i b i t e d by the a n t i t o x i n i n the hole, thus demonstrating the n e u t r a l i z a t i o n of a hemolytic f a c t o r of 01« w e l o h i i t o x i n by i t s s p e c i f i c a n t i t o x i n i n v i t r o . N e u t r a l i z a t i o n of Hemolysin by i n v i t r o Hemolytic T i t r a t i o n s . The hemolysin produced by 01. w e l o h i i ( s t r a i n s PB6H, SR12, 11830, and 12358) i n Reed's broth i s n e u t r a l i z e d by perfringens a n t i t o x i n . The hemolysin i s a l s o n e u t r a l i z e d by r a b b i t a n t i t o x i n #797 and #798 l o c a l l y prepared against supernatant from Reed's medium c u l t u r e s of PB6H s t r a i n , (Appendix I ) 32. Nsrma.1 5erv«t 4 I l l u s t r a t i n g n e u t r a l i z a t i o n of rB6t± s t r a i n t o x i c broth by perfringens a n t i t o x i n . 33. I n t e s t i n g t i t r a t i o n methods f o r obtaining the end-points of n e u t r a l i z a t i o n s of hemolysin by a n t i t o x i n , s l i g h t v a r i a t i o n s were found when the method was used (common i n the l i t e r a t u r e ) of varying amounts of t o x i n i n the presence of constant amounts of a n t i t o x i n . More consistent r e s u l t s were always obtained when the amount of t o x i n was h e l d constant and the a n t i t o x i n v a r i e d from tube to tube. No explanation appears a v a i l a b l e as to the cause of t h i s , but the added r e l i a b i l i t y i s nevertheless present, and t h i s method of t i t r a t i o n was consequently adopted. An example f o l l o w s : Standard a n t i t o x i n .05 .10 ,15 .20 ,25 .30 .35 .40 ,45 (Units) Toxin (cc.) .50 ,50 ,50 .50 .50 .50 .50 ,50 .50 Saline (cc.) .45 .40 ,35 .30 ,25 ,20 ,15 .10 .05 The standard a n t i t o x i n was d i l u t e d so that 1 u n i t was contained i n 1 cc. The end-point was taken as the tube showing 50% hemolysis of 2$ suspension of r a b b i t erythrocytes. I t was found necessary to allow the t o x i n -a n t i t o x i n mixtures to stand one-half hour at room temperature before the a d d i t i o n of the red blood c e l l s i n order to obta i n consistent and accurate r e s u l t s . The M.H.D. and L.H, doses so obtained are recorded i n Table 9,while the r e s u l t s of the i n d i v i d u a l t i t r a t i o n s are given i n Appendix IT, 34, N e u t r a l i z a t i o n of L e t h a l Toxin by Mouse P r o t e c t i o n Tests.- L The mouse l e t h a l t o x i n produced i n glucose peptone beef i n f u s i o n broth i s n e u t r a l i z e d by standard perfringens a n t i t o x i n , a s observed by mouse-protection t e s t s . The M.L.D. of t o x i n f o r PB6H s t r a i n i s 0,1-0.15 For SR12 s t r a i n the M.L.D. i s 0.1 co. The amount of mouse l e t h a l t o x i n produced i n Reed's broth i s very s m a l l , the lowest M.L.D, being obtained was 0,3 co, Procedure f o r Mouse Protection. Tests. Mixtures of t o x i n and a n t i t o x i n were made up as f o l l o w s and allowed t o stand f o r one-half hour before intravenous i n o c u l a t i o n : A n t i t o x i n ( u n i t s ) 0.1 0,2 0.3 0.4 Toxin (cc.) 0.5 0.5 0.5 0.5 Saline (cc.) 0,4 0.3 0.2 0.1 Two mice were used f o r each t e s t , the end-point being chosen as the dose causing the death of 50% of the animals w i t h i n three days (25)*". 35, I n the teste performed w i t h PB6H s t r a i n , 0,3 cc, of t o x i n - a n t i t o x i n mixture were inoculated intravenously i n t o each mouse, and 0,15 cc, t o x i n were used f o r c o n t r o l . Using SR12 s t r a i n , 0,2 cc. of t o x i n - a n t i t o x i n mixture were inoculated and 0.1 cc. t o x i n was used f o r c o n t r o l . The r e s u l t s expressed as M.L.D. and L+ doses are set f o r t h i n Table 9. Results of i n d i v i d u a l t e s t s are given i n Appendix I I . N e u t r a l i z a t i o n of Dermo-neorotic Factor, shown by Guinea P i g Skin Tests. The dermo-neerotic f a c t o r present i n 01. w e l o h i i t o x i n produced from our s t r a i n s i n both Reed's and glucose peptone beef i n f u s i o n broth can be n e u t r a l i z e d by perfringens a n t i t o x i n . The dermo-neerotic a c t i o n of the t o x i n i s demonstrated by intracutaneous i n o c u l a t i o n Into the de p i l a t e d back of a guinea p i g . The t o x i n - a n t i t o x i n mixtures were made up as f o l l o w s and allowed to stand one-half hour before i n o c u l a t i o n i n t o the animals: A n t i t o x i n (units) 0.1 0.2 0.3 0.4 Toxin (ce.) 0.5 0.5 0,5 0,5 Saline (cc.) 0.4 0,5 0.2 0.1 36 The t o x i n - a n t i t o x i n mixtures were inoculated i n 0,2 cc.-amounts, and 0.1 cc. t o x i n c o n t r o l . The n e c r o t i c r e a c t i o n of the t o x i n appeared a greenish-yellow colour, accompanied "by marked oedema i n s e v e r a l instances. Smith (25) claims that the guinea p i g s k i n t e s t s are u n s a t i s f a c t o r y "because of the v a r i a t i o n i n s e n s i t i v i t y of d i f f e r e n t parts of the guinea pig's s k i n . On t h i s account several t e s t s were performed using three sets of s i m i l a r i n o c u l a t i o n s on d i f f e r e n t parts of the guinea pig's hack, to determine the p o s s i b i l i t y of v a r i a t i o n i n s u s c e p t i b i l i t y of d i f f e r e n t p a r ts of the s k i n . As f a r as could be determined from the experiments performed there seemed to be l i t t l e d i f f e r e n c e i n s u s c e p t i b i l i t y of the d i f f e r e n t areas. I n many cases the t o x i n had a tendency to d i f f u s e over a f a i r l y l a r g e area. This i s an agreement with the f i n d i n g s of Smith (25), Necrotic r e a c t i o n s of 5 mm, diameter or greater were found to be s i g n i f i c a n t . Reactions of 2mm, diameter or l e s s were considered to be n o n - s p e c i f i c . The 3 mm, r e a c t i o n i s s l i g h t l y lower than t h a t taken by Smith, who recorded rea c t i o n s of 5 mm, diameter and greater to be s i g n i f i c a n t . The r e s u l t s expressed as M.N.D. and L,N, doses are recorded i n Table 9, while r e s u l t s of i n d i v i d u a l t e s t s . a r e given i n Appendix I I . 3 7 . Synopsis of Constants of four t o x i n s . Strain - Daie. Recd SPEC u . H-GP6C r i - L D . /?<•«/ GPEC i W SPBt R?e4 &PBT LN-Res/ CPBT LN L+ T T H PRfcH In-U-Hn AS--!-1*! J.-1-W 7-a-w .?-A-¥ir •/ a<s° 3,0 V " •?6Lo • / -/ SIM 1.*/. MS in •l5Vc 2,5" 7-f-fI y afo '/ /•66 1-f-vt ••J -o5 o (17 ° t 10 -3<c. '/ • / z-s o SAI2 , 13-3-41 it" 31-85 J %£• \>5 . / 3-3 '/ m •5" 33 • a © + • (0. .(5<c •I > s >3Z 3.1-f-W .5- •15 J o ' / 5 z-s •AT f 3- o •5" SV-o IZ35-Z 3-/H.-HO 38. P r e c i p i t i n Reactions with T o x i n - A n t i t o x i n Mixtures-» So f a r as we hare been able to a s c e r t a i n , no record e x i s t s of p r e c i p i t i n r e a c t i o n s between CI. w e l c h i i f i l t r a t e s and a n t i t o x i n , and no attempt seems to have been made to e s t a b l i s h p o s s i b l e p a r a l l e l i s m between pr e c i p i t i n o g e n s and t o x i c f a c t o r s . The p o t e n t i a l i t i e s of the p r e c i p i t i n or f l o c c u l a t i o n r e a c t i o n therefore seemed worth'' i n v e s t i g a t i n g . When the supernatant from c e n t r i f u g a t e d c u l t u r e s of Reed's b r o t h i s mixed i n varying q u a n t i t i e s w ith the standard perfringens a n t i t o x i n , a c l e a r l y v i s i b l e , f i n e l y granular p r e c i p i t a t e i s formed,, Several complete Dean-Webb t i t r a t i o n s were c a r r i e d out, but i t was very d i f f i c u l t to d etermine the optimal d i l u t i o n of a n t i t o x i n , f o r the r e s u l t s of the t i t r a t i o n s were not c o n s i s t e n t . I n some cases the optimal d i l u t i o n of a n t i t o x i n was 1/10, while i n one instance the best p r e c i p i t a t i o n r e s u l t e d from the use of 1/80 a n t i t o x i n , A good p r e c i p i t a t i o n r e a c t i o n cannot be obtained w i t h supernatant from glucose peptone beef i n f u s i o n medium. The glucose peptone beef i n f u s i o n broth appears p e r f e c t l y c l e a r before i n o c u l a t i o n with 01. w e l c h i i , but the medium i s a l t e r e d during the growth of the organisms so that a f t e r three hours c e n t r i f u g a t i o n at high speed 59. the supernatant contains a f i n e l y granular substance which becomes confused, with- the p r e c i p i t a t e obtained i n the presence of a n t i t o x i n . The r e s u l t s of the p r e c i p i t i n r e a c t i o n s , showing v a r i a t i o n i n end-points>are recorded i n Tables 10 and 11, Any p r e c i p i t i n o g e n that i s present i n the t o x i c supernatant from centrifugated Reed's broth culture i s completely absorbed by a c t i v a t e d charcoal so tha t no p r e c i p i t a t e forms when the absorbed t o x i n i s mixed w i t h a n t i t o x i n . However, i f r a b b i t erythrocyte absorbed t o x i n (see p,42) i s set up against a n t i t o x i n , the p r e c i p i t a t e formed i s considerably increased over the p r e c i p i t a t e formed with non-absorbed t o x i n - a n t i t o x i n mixtures. The r e s u l t s of t h i s experiment are recorded in.Table 11, 40. Table 10. T o x i n - a n t i t o x i n p r e c i p i t i n r e a c t i o n s , using d i f f e r e n t d i l u t i o n s of a n t i t o x i n . PB6H s t r a i n Reed'.s broth Toxin (0.5cc.) Exp. 1 1/10 ?- 10 ZO i so - + - + - - _ # + - _ + — + + •ft + + - - + + + + 1/20 — — + — — — _ - + + - - - - - - — — - • - - - - - -— f + + - + - - -1/40 — Jh. — + — + _ - + •+ A. + + a. + - -— + + A. J . + + -1/80 — — — — - •m- # + ± - -m. - - -- -jw- - » # # + - - -Exp. 2 Exp. 3 1/10 * + + 4* 4 - - - - — * +.- - - - - -# 4- - - - - -1/20 - - — — J. i -J- + 1/10 *• 4> - + # # J . 4, + - - - -+- # # + •L + + - -4 •* + 7- -# •m- •Hf * ml* t 1/20 - - - + + - — - - - - - - - -+ Am + + # 4, + - - - - -41. Table 11. E f f e c t of absorption by r a b b i t erythrocytes on t o x i n , i n d i c a t e d by p r e c i p i t i n r e a c t i o n . PB6H strain,Reed's broth Toxin(0,5< co) Exp. 1 A A n t i t o x i n 1/10 MHO - — t _ _ Toxin unabsorbed - - - « - 4 - - 4- 4 — — — 4 4 _. 4 4 4 - 4 4 -Rabbit c e l l f * + f- BO + _ _ „ absorbed t o x i n - • •tf - 4> — - 4 4 fl- fr. 4 - 4 - - 4 4 ±. -— uff. Wl 1« m *f •H ± - 4 •f 4_ -A n t i t o x i n 1/80 4 4 an M Toxin unabsorbed - _ - - 4 4 — - - - — - - - 4 4 4 4 — — — + 4 4 4 4 — - - _ Rabbit c e l l 4 •OB O B absorbed t o x i n f * f W - - — - - - — -«• • t t - — - - - -f •» 4" 4- 4 + 4 $ - — _ tt t (« » f 4 4 -Exp. 2 A n t i t o x i n 1/80 — — — — — — Toxin unabsorbed + 4 4 - - - - 4 — 4 4 + t - - - 4 4 4 4 Rabbit c e l l • = >;".o 'Ate i-H-t- 4* + _ _ t mm absorbed t o x i n IW fr t t + 4- i - - -mi If + f t f •f i t t — Exp. 3 A n t i t o x i n 1/80 — + 4- _ _ 4 4 4 Toxin unabsorbed - 4 4 t t f - - 4 4 4 Rabbit c e l l _ _ _ _ — f + _ _ _ _ _ _ absorbed t o x i n t t 4 - - - - -+• + •i t 4 — -- - - - - - 4- 41 fr 4 4 4 - -m 4 + - HI «l MA m ¥ 4 -42. Absorption of Hemolysin by Rabbit or Sheep Erythrocytes. The hemolysin as produced i n Reed's broth can be completely absorbed by r a b b i t or by sheep erythrocytes. As l i t t l e as 5.0cc. packed washed c e l l s would completely absorb a l l the hemolysin from 2.0 cc. t o x i c supernatant. A r e c i p r o c a l hemolytic t i t r a t i o n •was set up against both types of c e l l s . The r e s u l t s are given i n Table 12,' Table 12, R e c i p r o c a l absorption and hemolytic t i t r a t i o n . Toxin Rabbit c e l l absorbed *" ^ y"° /l° y"° c o n t r o l t o x i n v s. r a b b i t c e l l s 0 0 0 0 0 0 0 0 4+ Rabbit c e l l absorbed t o x i n v s . sheep c e l l s 0 0 0 0 0 0 0 0 Sheep c e l l absorbed 0 0 0 0 0 , 0 0 0 t o x i n vs. r a b b i t c e l l s Sheep c e l l absorbed 0 0 0 0 0 0 0 0 4* t o x i n vs. sheep c e l l s . 43. E f f e c t of Erythrooyte-Absorbea Toxin on Guinea P i g SMn I f the hemolysin i s completely absorbed from the t o x i n with sheep or r a b b i t erythrocytes, and 0.1 cc. of the absorbed supernatant i s inoculated intracutaneously i n t o the back of a d e p i l a t e d guinea p i g , a t y p i c a l n e c r o t i c s k i n r e a c t i o n of about 25 mm. diameter r e s u l t s . This method of separating hemolysin and n e c r o t i c t o x i n has not been discussed i n the l i t e r a t u r e as f a r as could be determined. Further d i s c u s s i o n of t h i s r e a c t i o n w i l l be found on p . ^ . DISCUSSION. The c h i e f aim of the t o x i n and t o x i n - a n t i t o x i n t i t r a t i o n s recorded here has been to attempt to discover what, i f any, p a r a l l e l i s m or antigenic i d e n t i t y e x i s t s between three d i f f e r e n t t o x i c manifestations given by 01. w e l o h i i supernates, namely hemolytic e f f e c t , guinea p i g dermo-necrotic f a c t o r , and mouse intravenous l e t h a l dose. The present attempt was stimulated by the somewhat con t r a d i c t o r y reports of Glenny and colleagues,(27), (who claim p a r a l l e l i s m and a n t i g e n i c i d e n t i t y f o r the three f a c t o r s ) , and of Prigge (28) and Weinberg (29), who suggest l a c k of i d e n t i t y i n the p r o p e r t i e s . I t was P r i g g e , f o r instance, who suggested the use of the term °^ f o r the hemolytic property, and >^ f o r the l e t h a l . Some idea of the i d e n t i t y or l a c k of i d e n t i t y of the t o x i c f a c t o r s has been gained by noting what degree of consistency obtains between the respective minimal i n d i c a t i n g doses of the three t o x i c f a c t o r s , when t o x i c supernate from a given s t r a i n of 01. w e l o h i i i s prepared a number of d i f f e r e n t times and upon two or more d i f f e r e n t media. Further and equally s i g n i f i c a n t information can be derived by i n v e s t i g a t i n g the r e l a t i v e amounts of perfringens a n t i t o x i n r e q uired f o r the n e u t r a l i z a t i o n of each of these b i o l o g i c a l effects,' 45. Ipsen and Davoli (30) i n d i c a t e that the existence of m u l t i p l e t o x i n s i n perfringens f i l t r a t e i s by no means a new f i n d i n g . I n one case (Weinberg, Native l i e and Prevot, (31),) the existence of at l e a s t s i x t o x i n s (three hemotoxins and three nonhemolytic toxins) has been claimed. The former workers were able to demonstrate only three t o x i n s f o r which they claimed the f o l l o w i n g b i o l o g i c a l e f f e c t s : Table 13. Toxin Hemolysis of sheep c e l l s 1 0 L e t h a l i t y to mice Dermal r e a c t i o n U ) + Necrosis (Hemorrhagia) 0 Stewart and Clampit (26) have been able to separate the hemolytic and l e t h a l t o x i n by f i l t r a t i o n through graded c o l l o d i o n membranes. Although no work has been done i n t h i s l a b o r a t o r y w i t h the f i l t r a t i o n of 01. w e l c h i i t o x i n s through c o l l o d i o n membranes, the p o s s i b i l i t i e s f o r the use of such membranes f o r separation of d i f f e r e n t t o x i c components are worthy of some consideration. Prigge has shown that l e t h a l t o x i n may be i s o l a t e d from hemolytic by s a l t i n g out. Attempts have been made by Prigge (28) and Ipsen and D a v o l i (30) to characterize i n d i v i d u a l t o x i c f i l t r a t e s or p r e c i p i t a t e s i n respect of t h e i r ^ and £, content, by r a t i o s such as mld/mhd. Prigge contends that a "pure " t o x i n would have a r a t i o ^-1 or equal to 1. Conversely a r a t i o > 1 would i n d i c a t e the presence of o<^  t o x i n . On the b a s i s of such r a t i o s , t o x i n s have been characterized or given "antigen formulae" as The i n f l u e n c e of the medium on such r a t i o s has been noted by Stewart and Glampit (26) and Ipsen and Davoli (30). The l a t t e r found f o r example,that with the same perfringens s t r a i n (SS) on three d i f f e r e n t media,mld/mhd r a t i o s of 0.5, 10 and 100 were obtained, r e s p e c t i v e l y i n d i c a t i n g antigenic formulae ^ ,^ and (t^ c< • I n Table 14 medium c h a r a c t e r i s t i c s and r a t i o s have been t r a n s c r i b e d f o r comparison wi t h l i k e character-i s t i c s and r a t i o s obtained i n t h i s l a b o r a t o r y by growing s t r a i n s PB6H and SR12 on Reed's and glucose peptone beef i n f u s i o n media. 47. Table 14 • Meditim c h a r a c t e r i s t i c s and r a t i o s • obtained with d i f f e r e n t s t r a i n s of G l . w e l c h i i . < ^ V.f 0) , Afo meat, !<>**-> pcnkvx i n * * * •S~ 1-2.5 paj-h'eies of- /rlco.A Bis^l beef, hroth. to 7X f-t-S-S. Confer?/- (Vd/neaj-. loo /•g c.P-ff-T-Sac/ bep£- trnHi ^ 0.3> 33 -33 - •—• CPBJ- , ' , near f^rrtcl^s., hi so./ hee-f- 4^ •4-O.X 33 •33 •Reed' do-crfa peptone/ .11" M.ea4~ 75o -— — </<• 5/J./2. twjci barf--frith •0.25 si o> tneasf-. 1000 0.25 "73/« <?. / / 48. An enormously greater divergence i n mld/mhd r a t i o s due to medium v a r i a t i o n i s found i n our work, such that a given s t r a i n growing on Reed's medium produces n e a r l y ^ f r e e t o x i n , and the same s t r a i n on glucose peptone beef i n f u s i o n medium produces almost c ^ f r e e *L t o x i n . I n f a c t , with c e r t a i n experiments we have been able to say that the growth of e i t h e r SR12 or PB6H on glucose peptone beef i n f u s i o n medium produces ^ t o x i n with no detectable c ^ t o x i n whatever, as tes t e d with both sheep and r a b b i t erythrocytes. Ipsen has concluded that the present i n t e r n a t i o n a l u n i t (of a n t i t o x i n ) i s e s s e n t i a l l y a ^ u n i t . I t i s therefore a r e q u i s i t e of a t o x i n used e i t h e r f o r immunization or f o r subsequent serum assays, that i t be very high i n ^ c o n t e n t . The r e s u l t s discussed above give added weight to the s i g n i f i c a n c e of choice of medium i n t o x i n production. They a l s o i n d i c a t e that Reed's medium, w i t h i t s spectacular t o x i n y i e l d and i t s almost n e g l i g i b l e ^ production, must be most unsuitable f o r the production of toxins f o r p r a c t i c a l use. The question now a r i s e s , whether the <=< (hemolytic), the C, (mouse-lethal) and the guinea p i g dermo-necrotic f a c t o r s are a l l caused by separate, a n t i g e n i c a l l y d i s t i n c t t o x i n s . We have confirmed the r e s u l t s of previous workers showing that change of medium f o r a given s t r a i n may serve to increase the c< f a c t o r at the expense of the ^> 49. f a c t o r , or to stimulate production with a correspondingly low <yx content. We have f u r t h e r shown that i n c e r t a i n preparations, completely cx^free f a c t o r can be produced. I n a l l cases the dermo-necrotic f a c t o r remains p r a c t i c a l l y unchanged. I t i s i n d i c a t e d on t h i s b a s i s that the n e c r o t i c f a c t o r i s completely separate from the hemolytic. Conclusive proof of t h i s f a c t r e s t s i n the experiment (p /B) i n which s t r a i n SR12 was grown on Reed's broth, w i t h the usual production of cK. and he e r o t i c f a c t o r s . The hemolytic f a c t o r was completely removed by absorption with r a b b i t erythrocytes, l e a v i n g the ne c r o t i c f a c t o r unchanged, Ipsen, Smith and S o r d e l l i (25), found that the r e l a t i v e amounts of l e t h a l and n e c r o t i c substance i n three t o x i n s were equal, the LN/L+ r a t i o being about 1.20. On t h i s b a s i s they f e l t , that assumption that the same e n t i t y causes the two d i f f e r e n t e f f e c t s was quite reasonable. The r e s u l t s obtained i n t h i s i n v e s t i g a t i o n do not confirm t h i s conclusion. We have already remarked that the MM) remained almost constant i n d i f f e r i n g media i n s p i t e of great v a r i a t i o n i n c<and content, From another point of view reference to Table 9 shows that i n most t e s t s on glucose peptone beef i n f u s i o n medium, the MLD l a y between 0,10 and 0.15 cc. I n a l l cases the MND l a y between 0,05 and 0,10 cc. However one s e r i e s 50. (Sl/4/41) showed the MLD to he- considerably l a r g e r than usual ( ? 0 . 3 0 c c ) , but with the MSD remaining as i n other experiments. This d i f f e r e n c e of over t h r e e - f o l d from the normal MLD's i n our s e r i e s may of course have been due to experimental e r r o r , but t h i s i s considered u n l i k e l y in-view of the consistency of the other experiments. No v a l i d conclusion can of course be drawn from t h i s i s o l a t e d instance, but i t supports our other f i n d i n g s i n respect to v a r i a t i o n of l e t h a l f a c t o r according t o medium, wi t h n e c r o t i c f a c t o r remaining constant. I t i s therefore demonstrated that the p a r a l l e l i s m used by previous workers as an argument f o r the i d e n t i t y of l e t h a l and n e c r o t i c f a c t o r s may not- always e x i s t . 51. CONCLUSIONS: 1. CI. w e l o h i i hemolysin f o r sheep and rabbit erythrocytes i s an e n t i t y . 2. The c< (hemolytic) f a c t o r and the guinea p i g dermo-n e c r o t i c f a c t o r are two separate and d i s t i n c t substances. 3. The ,<(hemolytic) a n d ^ (mouse-lethal) f a c t o r s are not i d e n t i c a l . 4. I t i s str o n g l y suggested that the S t o x i n and the dermo-neerotic f a c t o r are not i d e n t i c a l . 5. No ^ f a c t o r , such as described by Ipsen and D a v o l i , was encountered during the course of t h i s work. 52. APPENDIX I (a) Two r a b b i t s #797 and #798 were given a se r i e s of intravenous i n o c u l a t i o n s as f o l l o w s ; the f l u i d used f o r i n j e c t i o n being supernatant ;from;centrifugated s i x t e e n hour Reed's broth cultures of PB6PI s t r a i n of CI. w e l o h i i . The in o c u l a t i o n s were given at four day I n t e r v a l s i n i n c r e a s i n g doses of 0,1 to 0.5 cc. of / l / l O t o x i n , followed by undiluted t o x i n In amounts i n c r e a s i n g from 0.3 to 2,0 cc. The a n t i t o x i n obtained i n the case of both r a b b i t s was of such strength that 0.3 cc. of 1-20 a n t i t o x i n n e u t r a l i z e d 0,5 cc. t o x i n . The strength of t h i s a n t i t o x i n c a l c u l a t e d by comparison with the known standard a n t i t o x i n was 20 u n i t s per c c , and was thus a very weak a n t i t o x i n . (b ) Two r a b b i t s #796 and #770 were given a se r i e s of intravenous i n o c u l a t i o n s of formalized supernatant from c e n t r i f u g a t e d s i x t e e n hour Reed's broth c u l t u r e s . The supernatant was toxoided by adding 0.3$ fo r m a l i n and incubating f o r 48 hours at 37°C. The i n j e c t i o n s were given at i n t e r v a l s of 5 days i n doses in c r e a s i n g from 0,1 to 1,0 cc. 53. Rabbit #770 died a f t e r f i v e i n o c u l a t i o n s . The rabbit, appeared p e r f e c t l y normal according to autopsy f i n d i n g s . The t i t r e of a n t i t o x i n In r a b b i t #796 could not be r a i s e d so that 0,5 cc. or less of 1/10 antiserum would n e u t r a l i z e 0.5 cc. t o x i n . This i s not i n agreement w i t h the f i n d i n g s of Reed (16)• The hemolytic a c t i o n of t o x i c supernatant f o r r a b b i t c e l l s i s completely destroyed by toxoiding w i t h 0.3$ f o r m a l i n . 54. APPENDIX I I . (a) Hemolytic n e u t r a l i z a t i o n t i t r e s . Standard a n t i t o x i n .05 .10 .15 .20 .25 .30 .35 .40 .45 (units) Date. PB6H s t r a i n Reed's broth J t , -II-fo 4+ 4 * 3f I t 0' 0 0 0 0 7.S-- 1 -<fl 4+ 44- 4+ 44- 5+ 1+ 0 0 4+ 4* I t I t 1* 0 0 0 0 3 - 2 - HI 4-c 4+ 4t- 4t 1+ I f 0 0 0 7-1'-f/ •4t 4f 1+ 0 4+ 4+ 4* 4 t 50$ 0 0 0 0-x 1 - X - HI 4-t- 4t 4+ 4t- 3t 0 0 0 0 n - 3 -<ft 4+ 4 t 44- 4t 4+ 5+ 1* 0 0 JM -MI 4+ 3v 34- I t 0" 0 0 0 0 l - ' r - l l 44- 44- 3+ 3+ 3+ 1+ 0 0 0 1-H-Hl 4 f 3+- 3+ I t 0 0 0 0 0 -Ll - f-HI 4+ 34- 3* 50$ 14 0 0 0 0 13-1-1.1 4-f. 4+ 3+ 0 0 •o 0 0 0 -<t( 4+ •" 4+ 4 r 50$ 0 0 0 0 0 44- 4«- 3t 5+ 3t 3t. 1+ 0 0 - i - t l 4+ 4 r 3+ 3* 34- 2t I t 0 0 SR12 s t r a i n Reed's broth SR12 st r a i n , g l u c o s e peptone beef i n f u s i o n broth 34- 50$ 1* 0 0 0 0 0 0 12358 s t r a i n vu-v» 44- 3+ 3t 50$ 50$ 50$ 1 + 0 0 Reed's broth 24->-w 4t 50$ l v 0 0 0 0 0 0 11830 s t r a i n v < x-^4+ 4f 3f 50$ 50$ 0 0 0 0 Reed's broth 55. (b) House P r o t e c t i o n Tests. A n t i t o x i n (units) .05 .1 .2 .3 .4 co n t r o l .15 cc. W-PB6H s t r a i n ^GPBI broth • Dead 0 0 0 0 0 0 Survived : 2 2 2 2 2 2 Dead 0 0 0 0 2 Survived 2 2 2 2 0 Dead 2 0 0 0 0 2 Survived 0 2 2 2 2 0 Dead 0 0 0 0 0 0 Survived 2 2 2 2 2 2 SR12 s t r a i n GPBI b r o t h ; • • o.l oc, 56-?-HI Dead 2 2 1 0 0 2 Survived 0 0 1 2 2 0 Dead 2 2 0 0 0 2 Survived 0 0 2 2 2 0 Dead 0 0 0 0 ' 0 0 Survived 2 2 2 2 2 2 Dead 2 0 0 0 0 2 Survived 0 2 2 2 2 0 SR12 s t r a i n -Reed's broth .02(W)' ^i-t-n, D e a t 2 0 0 2 .3 cc. Survived 2 2 0 0 ote - GPBI broth i s glucose peptone beef i n f u s i o n broth 56. (c) Dermo-necrotio n e u t r a l i z a t i o n teVsts. PB6H s t r a i n Reed's broth 1 - T.-HI U--L- 'HI 1-1 -HI -2J-1 -«l Toxin •loo 10mm 10mm 15mm 5mm 8mm 10mm 10mm A n t i t o x i n (units) s i .2 .5 ©4 5mm 3mm 3mm 5mm 5mm 5mm 2mm 2mm 5mm 5mm 5mm ;—- -5mm — — — PB6H s t r a i n G-BP1 broth 20mm 5mm 5mm 2mm 8mm( 5mm » -i-H-M 2.0mm( ,05oc) 8mm 8mm t -t-n 10mm. " 10mm 8mm •—••-•u-v-^ilQmm 5mm — -SR12 s t r a i n Reed's broth"'"'20mm IA-I-H 8mm( 8mm 10mm 5mm — 05cc) • — -.05 u n i t a n t i t o x i n 5mm SR12 s t r a i n GPBI 25mm 15mm 10mm - x - u i-j-v/ 8mm 4mm 5mm *( 15mm 5mm 57. BIBLIQSRAPHY (1) Pasteur, L., 1863, C.R. Acad. S c i . , P a r i s , 56, 1189 (2) B o t t i n i , 1871, Gio r . d.r, Accad. d i med. d i ,Torino, 3SX:1120. Ci t e d from Gay et a l ; Agents of Disease and Host Resistance, 1935. (3) Welch, W.H. and N u t t a l , G.H.F., 1892, Johns Hopkins Hosp. B u l l . , 3j_ 81 Cit e d from Robertson, A System of Bacteriology, 3, 1929 (4) Fraenkelj E., 1893, "Uber Gasphlegmon"» Cited from Robertson, A System of Bacteriology, 3^ 1929 (5) V e i l l o n and Zuber, 1898, Arch. Med, Exp., 10, 517 Cited-from Robertson, A System of Bacteriology, 1 3, 1929 (6) Pease, M., 1919, Proc. Soc. Exp, B i o l , and Med., 17, 30 (7) B u l l , G.G. and P r i t c h e t t , I.W., 1917, J.Exp.. Med., 36, 119 and 867 (8) Henry, H., 1923, J.Path. 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Path., 18, 475 Todd, E.W. and Hewitt, L.E., 1932, J . Path, Bact., 55, 973 Ha r t l e y , P., League of Nations Health Org., Report of Perm. Comm. on B i o l , Stand,, Lond., 1931 Ipsen, J . , Smith, M. L l e w e l l y n and S o r d e l l i , A, B u l l . Health Org., 1939, 8^ 797 Stewart, S, and Clampit, M., 1938, B u l l . Health Org., 7, 802 Glenny, A.T., Barr, M., L l e w e l l y n Jones, M., D a l l i n g , T. and Ross, Helen, 1935, J . Path. Bact., 37, 55 59 (28) Pri g g e , 1936 Z e i t s c h r . Immun. Forschg,, 89, 477 •'./ C i t e d from B u l l . Health Org. 1939, 8 (29) Weinberg, M. and Guillaumie, M., 1938, B u l l . Health Org., 7, 818 (50) Ipsen, J", and Davoli R., 1939, B u l l . Health Org., 8 , 835 (31) Weinberg, H a t i v e l l e and Prevot, 1957, sLes microbes anaerobies'• C i t e d from B u l l . Health Org. 1939, 8_ 

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