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The production of the alpha toxin of Clostridium perfringens in a chemically reproducible medium Costerton, John William Fisher 1956

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THE PRODUCTION OF THE ALPHA TOXIN OF CLOSTRIDIUM PERFRINGENS . IN A CHEMICALLY REPRODUCIBLE MEDIUM  by JOHN WILLIAM' FISHER COSTERTON A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in the Department of BACTERIOLOGY AND IMMUNOLOGY  We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF ARTS  Members of the Department of Bacteriology and Immunology.  THE UNIVERSITY OF BRITISH COLUMBIA August, 1956  ACKNOWLEDGEMENTS  I wish to express my deep a p p r e c i a t i o n to D r . D . C . B . Duff f o r h i s guidance and encouragement so f r e e l y g i v e n d u r i n g the course of t h i s r e s e a r c h . and has Dr. of  The advice of D r . J . E . B i s m a n i s , D r . J . J . Stock D r . W. E . Sheperd has proven most i n s t r u c t i v e and aided measureably i n t h i s work. I a l s o wish to thank J . J . Stock f o r h i s s u p e r v i s i o n of the l y o p h y l i z a t i o n the test s t r a i n s used h e r e i n .  Mrs. P.. C a r t e r has been extremely h e l p f u l the p r e p a r a t i o n of t h i s m a n u s c r i p t .  in  ABSTRACT Boyd, Logan and T y t e l l  (19^8)  have shown t h a t  C l o s t r i d i u m p e r f r i n g e n s BP6K w i l l grow i n a c h e m i c a l l y r e p r o d u c i b l e medium.  They recorded no  lecithinase  (<*toxin) p r o d u c t i o n under the c o n d i t i o n s  of t h e i r  T h i s t h e s i s r e p o r t s c o n d i t i o n s under which i s produced i n the above medium.  experiment.  lecithinase  Time s t u d i e s  Involving  h o u r l y sampling proved to be the most s a t i s f a c t o r y of s t u d y i n g the p r o d u c t i o n of the enzyme. sample was determined, turbidimetrically,  method  The pH of  each  the b a c t e r i a l d e n s i t y was r e c o r d e d  and the l e c i t h i n a s e  was estimated by  a m o d i f i c a t i o n of the egg y o l k suspension method. L e c i t h i n a s e was found to be produced d u r i n g the l o g a r i t h m i c phase of growth, a f t e r which i t r a p i d l y d i m i n i s h e d i n q u a n t i t y , d i s a p p e a r i n g at twelve hours growth.  approximately  The amount of the enzyme produced  i s r e l a t i v e l y s m a l l compared w i t h y i e l d s from complex organic media. lecithinase  However, the c h a r a c t e r i s t i c s  appear i d e n t i c a l w i t h those of the  produced i n complex media. lecithin,  of  the lecithinase  S i n c e the medium c o n t a i n s  no  and the enzyme i s produced d u r i n g the l o g a r i t h m i c  growth phase, constitutive  i t would appear that the enzyme i s  essentially  and e x t r a c e l l u l a r .  Phase v a r i a t i o n as i n d i c a t e d by c o l o n i a l morphology has been shown to be of c o n s i d e r a b l e lecithinase  importance i n  p r o d u c t i o n i n the c h e m i c a l l y r e p r o d u c i b l e  medium.  The o b s e r v a t i o n of a p e c u l i a r c o l o n i a l morphology  designated as a "halo" colony i s r e p o r t e d . type i s in  This morphological  seen to be c l o s e l y r e l a t e d to h i g h t o x i g e n i c  activity  G.P.B.I. In the r e p r o d u c i b l e medium a c i d p r o d u c t i o n d u r i n g  growth (pH 7*2 destruction.  to 4.8)  i s c l o s e l y r e l a t e d to enzyme  Adjustment of the pH d u r i n g growth does not  g r e a t l y enhance l e c i t h i h a s e p r o d u c t i o n but does markedly slow i t s glucose  destruction.  The s u b s t i t u t i o n of d e x t r i n f o r  i n the r e p r o d u c i b l e medium d e l a y s but does not  reduce growth and l e c i t h i n a s e p r o d u c t i o n .  The d e s t r u c t i o n  of the enzyme i s  proteinaceous  material.  slowed by the a d d i t i o n of  The c o n c e n t r a t i o n s  a l s o seen to a f f e c t  of c y s t i n e  and of i r o n are  the r a t e of d e s t r u c t i o n of  lecithinase.  /  TABLE OF CONTENTS Page I*  1  Introduction M a t e r i a l s and Methods  33  A . Media  33  B. Digestions  36  C . Removal of i r o n  36  D. Methods of c u l t i v a t i o n  37  E . Measurement of t o x i n a c t i v i t y  37  1.  L e c i t h i n a s e a c t i v i t y of * t o x i n  37  2.  H o t - c o l d h e m o l y t i c a c t i v i t y of 6 toxin  38  3# Hot h e m o l y t i c a c t i v i t y of <*. t o x i n F. Bacterial strain  39 •  39 4-0  Results A . The r e l a t i o n of d i g e s t  concentration  to <* t o x i n p r o d u c t i o n B. D i a l y s i s of the d i g e s t s C . The t o x i g e n i c a c t i v i t i e s of beef h e a r t and beef l i v e r D . The e f f e c t of d e x t r i n upon - . t o x i n production  \±1 i|2 4 4 45  E . The e f f e c t of b a c t e r i a l v a r i a t i o n upon t o x i n p r o d u c t i o n  46  F . Time s t u d i e s  5l  1. I d e n t i t y of the l e c i t h i n a s e produced i n the b a s a l medium 2.  The d e n a t u r a t i o n of <*.toxin i n c e l l - f r e e centrifugates  5l 52  - i iPage 3.  The e f f e c t o f pH adjustment upon t o x i n p r o d u c t i o n i n the b a s a l medium  52  4-. The e f f e c t o f glucose c o n c e n t r a t i o n upon < t o x i n p r o d u c t i o n  53  5. 6.  7.  The e f f e c t o f d e x t r i n upon growth and rftoxin p r o d u c t i o n  53  The e f f e c t o f v a r i o u s c o n c e n t r a t i o n of a t r y p t i c d i g e s t of beef muscle upon t o x i n p r o d u c t i o n  54-  The e f f e c t of ammonium s u l p h a t e precipitated proteins upon«toxin p r o d u c t i o n i n the b a s a l medium  54-  8.  The e f f e c t of the omission o f c e r t a i n amino a c i d s upon <<toxin p r o d u c t i o n . . . .  55  9.  The e f f e c t o f c y s t i n e upon t o x i n p r o d u c t i o n i n the b a s a l medium  55  10.  The e f f e c t of i r o n c o n c e n t r a t i o n upon « t o x i n p r o d u c t i o n i n the b a s a l medium  56  The i n f l u e n c e of b a c t e r i a l v a r i a t i o n upon ottoxin p r o d u c t i o n i n the b a s a l medium  57  11.  DISCUSSION  58  SUMMARY  67  BIBLIOGRAPHY  68  APPENDIX A  74-  1  LIST OP TABLES Page I.  Toxins produced by C l o s t r i d i u m p e r f r i n g e s  II.  E f f e c t of i r o n c o n c e n t r a t i o n upon growth and t o x i n p r o d u c t i o n  25  The e f f e c t of the components of upon o<. t o x i n p r o d u c t i o n  30  III. IV.  V. VI.  VII.  VIII.  IX.  X. XI. XII.  ..  10  G.P.B.I.  The e f f e c t of the a d d i t i o n of v a r i o u s enzymic d i g e s t s of ground beef o n * t o x i n p r o d u c t i o n i n the b a s a l medium The e f f e c t of v a r i o u s c o n c e n t r a t i o n d i g e s t upon =c t o x i n p r o d u c t i o n  1+0  of  The e f f e c t of a t r y p t i c d i g e s t and i t s d i a l y z a t e and d i a l y z a n t upon t o x i n p r o d u c t i o n i n the b a s a l medium The e f f e c t of the d i a l y z a t e and d i a l y z a n t of a p e p t i c d i g e s t upon <*. t o x i n p r o d u c t i o n i n the b a s a l medium  1+1  1+2  i|_3  The e f f e c t of p a n c r e a t i c d i g e s t s of beef h e a r t or l i v e r upon t o x i n p r o d u c t i o n i n the b a s a l medium  1+5  The e f f e c t o f the s u b s t i t u t i o n of d e x t r i n f o r glucose i n the b a s a l medium upon -t t o x i n p r o d u c t i o n  1+5  The e f f e c t of b a c t e r i a l v a r i a t i o n upon •< t o x i n p r o d u c t i o n i n G . P . 3 . I The e f f e c t of the omission of c e r t a i n amino a c i d s upon »< t o x i n p r o d u c t i o n The e f f e c t of i r o n . c o n c e n t r a t i o n upon ai t o x i n p r o d u c t i o n i n the b a s a l medium . . .  4-9 55 57  - iv  -  LIST OP ILLUSTRATIONS Page F i g u r e 1.  The a c t i o n of l e c i t h i n a s e upon lecithin;.. -  2  F i g u r e 2.  V a r i a t i o n of t o t a l c e l l y i e l d growth pH ;  F i g u r e 3»  V a r i a t i o n of <* t o x i n p r o d u c t i o n w i t h growth pH . . . " ..  22  F i g u r e l±.  Effect  26  F i g u r e 5»  The e f f e c t of a t r y p t i c d i g e s t and i t s d i a l y z a t e and d i a l y z a n t upon -«• t o x i n p r o d u c t i o n i n the b a s a l medium  F i g u r e 6.  with  of i r o n on * t o x i n p r o d u c t i o n .  «*.Toxin p r o d u c t i o n i n the medium  basal  22  I4J4Appendix :A  F i g u r e ?•  The r e l a t i o n s h i p between <* and » toxins p r o d u c t i o n i n the b a s a l medium . . . .  Appendix A  Figure 8.  The d e n a t u r a t i o n of .<. t o x i n i n free centrifugates  Appendix A  Figure 9.  The e f f e c t of c a r e f u l pH adjustment upon «t t o x i n p r o d u c t i o n  Appendix A  F i g u r e 10.  The p r o d u c t i o n of * t o x i n i n the b a s a l medium w i t h 1.0$ glucose  Appendix A  F i g u r e 11.  The p r o d u c t i o n of * t o x i n i n the b a s a l medium w i t h 0.5$ glucose . . . .  Appendix A  The p r o d u c t i o n of «*-toxin i n the b a s a l medium w i t h 0.25$ glucose . . .  Appendix A  F i g u r e 13«  The p r o d u c t i o n of <*• t o x i n i n the b a s a l medium w i t h d e x t r i n . . . . . . . . .  Appendix A  Figure  The e f f e c t of v a r i o u s c o n c e n t r a t i o n s of a t r y p t i c d i g e s t upon << t o x i n production  Appendix A  F i g u r e 12.  cell-  - v . Page Figure l £ .  F i g u r e 16.  F i g u r e 17.  The e f f e c t of v a r i o u s c o n c e n t r a t i o n s of an ammonium s u l p h a t e - p r e c i p i t a t e d p r o t e i n upon <* t o x i n p r o d u c t i o n  Appendix A  The e f f e c t of c y s t i n e c o n c e n t r a t i o n upon «t t o x i n p r o d u c t i o n i n the b a s a l medium  Appendix A  The e f f e c t of b a c t e r i a l v a r i a t i o n upon <*. t o x i n p r o d u c t i o n i n the b a s a l medium  Appendix A  1. INTRODUCTION The L e c i t h i n a s e ( * t o x i n )  of C l o s t r i d i u m P e r f r i n g e n s  The a c t i o n of the l e c i t h i n a s e s  produced by a  number of C l o s t r i d i a i s g e n e r a l l y conceded t o be of major importance i n the establishment d e s c r i b e d as gas gangrene*  and the f o r m a t i o n of  t o x i n s by C l o s t r i d i u m p e r f r i n g e n s ,  oedematiens and C l o s t r i d i u m septlcum was between 1939  19ll|.  1919 (f>0).  and  that.the  state  T h i s d i s e a s e was r e c o g n i z e d  as a c l i n i c a l e n t i t y before 191^specific  of the p a t h o l o g i c a l  Clostridium  established  I t was not however u n t i l  l i p o p r o t e i n s p l i t t i n g a b i l i t y o f the  toxin  was i n d i c a t e d by the serum c l o u d i n g r e a c t i o n observed by Nagler (68)  and known t h e r e a f t e r  as the Nagler r e a c t i o n *  F o l l o w i n g the work of N a g l e r , Macfarlane and h e r a s s o c i a t e s found that the t o x i n s  of C l o s t r i d i u m  p e r f r i n g e n s produce an opalescence i n s a l i n e of  egg y o l k *  These workers subsequently  the •<• t o x i n i s  an enzyme a c t i v e  Since l e c i t h i n i s  extracts  established  as a l e c i t h i n a s e  (i|-7).  the e m u l s i f y i n g agent which h o l d s  i n many b i o l o g i c a l aqueous s o l u t i o n s ,  that  fats  the d e s t r u c t i o n of  t h i s compound leads to a s e p a r a t i o n of f a t s and a r e s u l t a n t opalescence*  M a c f a r l a n e and Knight i n 19lp-  that the enzyme i s by I t s  specific  a c t i v a t e d by c a l c i u m i o n s ,  antiserum and by f l u o r i d e ,  o p t i m a l pH f o r i t s  (l|7)  action is  sequently been e s t a b l i s h e d  7«0 to 7*6.  showed  Inhibited  and t h a t I t has  that the l e c i t h i n a s e  the  subof  2 C l o s t r i d i u m p e r f r i n g e n s a c t s upon l e c i t h i n and to a l e s s e r extent upon sphingomyelin. unable to use c e p h a l i n ,  The enzyme i s  l y s o l e c i t h i n or  p h o s p h o r y l c h o l i n e as s u b s t r a t e s .  (1+9)  however  glycero-  (55)  The a c t i o n of t h i s enzyme upon l e c i t h i n  proceeds  w i t h the f o r m a t i o n of a d i g l y c e r i d e and p h p s p h o r y l c h o l i n e . After Porter  Figure 1  H C O - O-K, I H C O - O-K, X  I H C O ~ 7 t  (76)  W C O - O-T?, I H C O - O - K , V  >-  1  — 0-CH-CH.-N-(CH,) O OH OH Lecithin \  H,C  3  V  o H  v  Diglyceride  O I  OH-VOH  O-C  ^-CH N-(c^} OH r  3  Phosphorylcholine•  The l e c i t h i n a s e  of C l o s t r i d i u m p e r f r i n g e n s  resembles that of C l o s t r i d i u m botulinum immunologically but they d i f f e r i n that ions i n i t s  action.  haemolyticum i s perfringens  (53)  The l e c i t h i n a s e  of C l o s t r i d i u m  i d e n t i c a l w i t h that of C l o s t r i d i u m  in its  immunologically  the l a t t e r does not r e q u i r e c a l c i u m  a c t i o n (53)  and a c t i v a t i o n but  is  distinct.  The l e c i t h i n a s e  or <* t o x i n of C l o s t r i d i u m  p e r f r i n g e n s has a l s o been shown to be the  destructive  3 agent i n the " h o t - c o l d " hemolysis  of r e d b l o o d c e l l s .  (70)  L e t h a l and n e c r o t i c e f f e c t s i n experimental animals are a l s o a t t r i b u t e d to the a c t i o n of t h i s enzyme.  (70)  The  hemolysis r e f e r r e d to above has been shown to be a r e s u l t ; of the d e s t r u c t i o n of some of the of the r e d b l o o d c e l l s . rates  of hemolysis  phospholipides  I t has been e s t a b l i s h e d  of the r e d b l o o d c e l l s of  mammalian s p e c i e s p a r a l l e l the s e n s i t i v i t y red blood c e l l phospholipides lecithinase.  that  different  of the  to the a c t i o n of  isolated  the  (54-)  Gordon e t .  al.  (25)  have shown t h a t the  n e c r o t i c , h e m o l y t i c and l e c i t h i n a s e  activities  lethal,  of the <<  t o x i n are i n h i b i t e d by the a d d i t i o n of ten p e r c e n t to the system.  the  The hemolytic a c t i v i t y of  lecithin  © toxin is  also  i n h i b i t e d by l e c i t h i n but u l t r a c e n t r i f u g a t i o n at 14-7,500 g . restores  the a c t i v i t y of the e t o x i n w h i l e the  remains i n a c t i v e a f t e r i d e n t i c a l treatment, a d d i t i o n Zamecnik (lOlj-) has shown t h a t the of hog r e d b l o o d c e l l s ,  (27)  In  phospholipides  plasma and l i v e r p r o t e c t dogs and  mice from the e f f e c t s of << t o x i n .  The p h o s p h o l i p i d e s  egg y o l k and soybean o f f e r no p r o t e c t i o n . w i t h h e p a r i n prevents  <* t o x i n  the development  human s e r a exposed to the a c t i o n of The a c t i o n of l e c i t h i n a s e  ( at. t o x i n )  of  Preincubation  of opalescence i n toxin.  "in vivo".  In s t u d y i n g the e f f e c t s of i n j e c t i o n s  of the  pt t o x i n  of C l o s t r i d i u m p e r f r i n g e n s i n t o l a b o r a t o r y animals P u r r e t . a l . ( 2 0 ) have found t h a t the t o x i n causes r a p i d d e s t r u c t i o n of p l a t e l e t s  k  and r e d b l o o d c e l l s ,  as w e l l as a l t e r a t i o n s  i n plasma  p r o t e i n s and p r o l o n g a t i o n of the c o a g u l a t i o n  time.  C a p i l l a r y i n j u r y and t o x i c l i v e r damage yrere a l s o r e p o r t e d by these w o r k e r s .  Wright and Hopkins (103)  have shown  t h a t the t o x i n i s r e t a i n e d t o a l a r g e extent by the epidermal c e l l s because content.  of t h e i r r e l a t i v e l y h i g h l e c i t h i n  While P u r r e t . a l . r e g a r d e* t o x i n as the  agent of these changes,  M a c f a r l a n e and MacLennan  p o s t u l a t e d t h a t the e f f e c t  is  t h a t of a g e n e r a l  caused by the p r o d u c t s of t i s s u e d e g e n e r a t i o n . workers t h e r e f o r e conclude that e f f e c t upon the i n f e c t e d a n i m a l . there i s  specific have  toxemia These  oc t o x i n e x e r t s an i n d i r e c t T e s t s have shown that  an i n c r e a s e i n the c o n c e n t r a t i o n of r e a d i l y  extractible l i p i d e s i n affected  muscle.  As a r e s u l t of h i s t o l o g i c a l examinations  of  the  kidneys of animals which had been t r e a t e d w i t h u t o x i n , Berg and L e v i n s o n (8)  have concluded t h a t t h e r e are c o n -  s i d e r a b l e s t r u c t u r a l changes i n the g l o m e r u l i and t u b u l e s . The authors s t a t e that these changes r e s u l t e i t h e r  from  d i r e c t t o x i c a c t i o n or from c i r c u l a t o r y changes by the toxin.  A r e d u c t i o n i n the a l k a l i n e phosphatase  of the kidney was a l s o noted i n the above work.  activity Lecithinase  c, which e x i s t s i n a combined form i n guinea p i g b r a i n and kidneys,  is  seen to be r e l e a s e d  (7)  I t has been noted by Zamecnik e t . a l .  by the a c t i o n of (107)  that  toxin.  purified  oi. t o x i n does not produce the edema at the s i t e of  $ i n j e c t i o n which i s c h a r a c t e r i s t i c a l l y formed when crude filtrates  of the organism are  injected.  In s t u d i e s u s i n g e x c i s e d Le Grande  l i v e r t i s s u e Levy and  have shown t h a t the <* t o x i n of  p e r f r i n g e n s h y d r o l y z e s the l e c i t h i n s i n h i b i t s the o c t a n o a t e - o x i d i z i n g p r o t e c t i o n against the t o x i n i s  ($2),  activities  Pull  the l e c i t h i n - s p l i t t i n g a c t i v i t y of  protect p a r t i a l l y against  oxidase  of the t i s s u e and  enzyme of l i v e r .  a f f o r d e d by s p e c i f i c  o x i d i z i n g enzyme.  Clostridium  a n t i s e r a which a l s o  the i n h i b i t i o n of the  I t has a l s o been found that  a c i d phosphatase,  and d  octanoatethe  succino-  amino a c i d oxidase  of l i v e r are decreased by the a c t i o n of  (£l)  * toxin.  The l e c i t h i n of the l i v e r m i t o c h o n d r i a i s r e a d i l y a t t a c k e d by t h i s l e c i t h i n a s e .  (f?l)  I n f a t a l cases of gas gangrene, w e l l as i n guinea p i g s ,  f a t globules  and a pulmonary embolism i s human. all  are found i n the b l o o d (JUZ)  a c h a r a c t e r i s t i c f i n d i n g i n the  T h i s f i n d i n g , coupled w i t h the o b s e r v a t i o n  s t r a i n s producing l e c i t h i n a s e  f o r mice, of  (79)  <•<- t o x i n i s  that  i n h i g h l i i t r e are pathogenic  leads to the c o n c l u s i o n that the p r o d u c t i o n of h i g h Importance i n the p a t h o g e n i c i t y  Clostridium perfringens.  Kass e t .  of pathogenic  al.  (37)  that l e c i t h i n a s e  is  hyaluronidase i s  of l e s s e r s i g n i f i c a n c e .  of  have concluded  importance w h i l e  i s o l a t e d one hundred and twenty-eight sources  i n the human as  and found t h a t e i g h t y - t h r e e  These workers  s t r a i n s from random  p e r c e n t of the mouse  6  v i r u l e n t s t r a i n s produced l e c i t h i n a s e  i n synthetic  media  w h i l e o n l y f i f t y - f o u r p e r c e n t produced h y a l u r o n i d a s e . D . G . Evans (l$a» has concluded from the r e s u l t s work w i t h guinea p i g s  of  his  that s t r a i n s p r o d u c i n g only a s m a l l  amount of l e c i t h i n a s e ' ^ . n v i t r o " are unable to cause infection. H o r l i c k (35) patients  has r e p o r t e d t h a t the s e r a of  s u f f e r i n g from coronary h e a r t d i s e a s e show e a r l i e r  and h e a v i e r t u r b i d i t y than i s  seen w i t h normal s e r a when  incubated w i t h  toxin.  simple d i a g n o t i c  t e s t f o r t h i s c o n d i t i o n i f a t o x i n of  sufficiently lecithinase  T h i s f i n d i n g makes p o s s i b l e  s t a b l e c h a r a c t e r can be p r o d u c e d .  The  of C l o s t r i d i u m p e r f r i n g e n s has been used i n  the d e m y e l i n a t i o n of nerve f i b e r s failed  a  " i n v i t r o " but has  to a c t when i n j e c t e d i n t o experimental a n i m a l s .  The a c t i o n of L e c i t h i n a s e ( <* toxin)  with i t s  (65)  specific  antitoxin. Since t h i s  toxin is  which may be e s t a b l i s h e d , antitoxin is  of i n t e r e s t  an enzyme,  its  reaction with a  r e a c t i o n between enzyme and s u b s t r a t e  antitoxin is  specific  Zamecnik and  have shown that the a n t i t o x i n stops  a n t i t o x i n i s added before  of  i n a g e n e r a l c o n s i d e r a t i o n of  enzymic and immunological s p e c i f i c i t y . Lippman (105)  the k i n e t i c s  the  completely i f  the s u b s t r a t e .  However i f  added at the same time as the s u b s t r a t e  gradual deceleration i s noted.  The a n t i t o x i n i s  the the a  therefore  7  a c t i n g as a c o m p e t i t i v e  i n h i b i t o r which suggests that  a n t i g e n i c a l l y and e n z y m a t i c a l l y a c t i v e of  the enzyme molecule  In a d d i t i o n , i t  are s t e r i c a l l y  is possible  phenomenon may be a c t i v e  l o c i on the  that a s t e r i c  toxin)  While the a c t i v i t y of t h i s  surface  s i m i l a r or I d e n t i c a l . hindrancef-  i n this competitive  I n h i b i t o r s of L e c i t h i n a s e (  the  inhibition.  action. lecithinase  is  enhanced  by an o p t i m a l c o n c e n t r a t i o n of the c a l c i u m i o n , -and to a l e s s extent by the magnesium i o n , many other i n o r g a n i c are found to i n h i b i t i t s phosphate,  of«*.toxin.  Pluoro-citrate,  (1+7) Cu C l g , Sr C I 2 , Pe S0|^, A l CI3, Co C l , 2  Zn C l , (105) 2  action.  and the N i  + +  (7^)  i o n a l l i n h i b i t the  Smith and Gardner ( 8 7 ) have r e p o r t e d  some r e d u c i n g agents are a b l e to denature the Cystine,  ions  thioglycolic  action  that  toxin.  a c i d , hydrogen s u l p h i d e and sodium  b i s u l p h i t e were found to i n a c t i v a t e  the t o x i n w h i l e  a c i d and gaseous hydrogen over p l a t i n u m b l a c k were  ascorbic inactive  In t h i s r e s p e c t . In a d d i t i o n to the i n o r g a n i c i n h i b i t o r s  detailed  above Wagner ( 9 7 ) has r e p o r t e d an organic i n h i b i t o r of lecithinase  present  i n human s e r a .  This f a c t o r i s  antibody nor l e c i t h i n and i s found to be heat  neither  labile.  Heat d e n a t u r a t l o n of L e c i t h i n a s e ( <^ t o x i n ) Guillaumie et.  al.  ( 2 9 ) have r e p o r t e d an unusual  8 behaviour of the l e c i t h i n a s e  of C l o s t r i d i u m p e r f r i n g e n s  w i t h r e g a r d to heat d e n a t u r a t i o n . 6.8  to 7.6  of the  W i t h i n the pH range  h e a t i n g to 70°C.for f i v e minutes d e s t r o y s  all  <* t o x i n , © t o x i n and g e l a t i n l i q u i f y i n g enzyme.  However, subsequent restores  r e h e a t i n g to 100o<e.for f i v e  some of the l e c i t h i n a s e  the g e l a t i n l i q u i f y i n g a b i l i t y .  minutes  a c t i v i t y and some of The a c t i v i t y of 0 t o x i n  i s not r e s t o r e d by t h i s r e h e a t i n g .  In another p u b l i c a t i o n  (31)  t h a t the  G u i l l a u m i e et*  a l . have s t a t e d  << t o x i n of  C l o s t r i d i u m p e r f r i n g e n s i s denatured i n twenty minutes at 60°(C.or i n f i v e minutes at 70°C. hour at 100°c however, toxin.  R e t e n t i o n f o r one  f a i l e d to w h o l l y i n a c t i v a t e  Smith and Gardner (86)  have supported the  of G u i l l a u m i e and h i s a s s o c i a t e s by r e p e a t i n g experiments.  Mason (58)  has r e p o r t e d t h a t  the findings  their toxin w i l l  p a r t i a l l y w i t h s t a n d h e a t i n g to 120°<C.for one h o u r . Measurement of L e c i t h i n a s e ( <* toxin) The i n i t i a l measurement of  activity. t o x i n a c t i v i t y was  by Nagler who was able to compare the opalescence caused by v a r i o u s samples when incubated w i t h a constant of p o o l e d serum. (68) a c t i o n of  A f t e r M a c f a r l a n e ' s d i s c o v e r y of  << t o x i n on a s a l i n e  Heyningen (92)  measurement of l e c i t h i n a s e  the  e x t r a c t of egg y o l k Van  d e v i s e d a q u a n t i t a t i v e method f o r  sample to be t e s t e d i s  volume  activity.  the  In t h i s method the  incubated w i t h a s a l i n e  egg y o l k  9  suspension resultant  i n a d i l u e n t c o n t a i n i n g c a l c i u m and the opalescence i s a s s e s s e d .  McClung and Toabe, (56)  The s o l i d medium of  which c o n t a i n s  egg y o l k  suspension,  has been used i n the d e t e r m i n a t i o n of l e c i t h i n a s e  activity.(36)  However attempts to use t h i s medium i n q u a n t i t a t i v e d u r i n g t h i s work have been  studies  unsuccessful. 1  The phosphate group exposed as the r e s u l t s p l i t t i n g off  the d i g l y c e r i d e p o r t i o n of l e c i t h i n  of (63)  may be measured manometrieally s i n c e the r e a c t i o n of phosphate w i t h sodium b i c a r b o n a t e produces carbon dioxide.(105) The exposed phosphate group may a l s o be d e t e c t e d by other chemical means.  (106)  The p u r i f i c a t i o n of l e c i t h i n a s e I t has been shown that  (  toxin)  •< t o x i n w i l l pass through  a n e u t r a l f i l t e r w i t h a pore s i z e of 55 mu but not a s i m i l a r f i l t e r w i t h a pore s i z e of 5 l mu. (59) thus apparent that the  «<. t o x i n molecule i s  s u p p o r t i n g more r e l i a b l e evidence, that i t  through It  is  l a r g e , thereby is  a protein.  Methods of p u r i f i c a t i o n are s e l e c t e d on the b a s i s of  the  p r o b a b l e nature and s i z e of the d e s i r e d p r o d u c t . In 19i|2 Van Heyningen (93)  partially purified  t o x i n by a b s o r p t i o n on t r i c a l c i u m phosphate.  Anderson  achieved a b e t t e r p u r i f i c a t i o n u s i n g aluminum hydroxide and P i l l e m e r and Roth (75)  achieved almost  p u r i f i c a t i o n u s i n g methanol f r a c t i o n a t i o n .  complete The above  the (73)  10 p u r i f i c a t i o n showed c o n c l u s i v e l y hemolytic and l e c i t h i n a s e only one  that  activities  the l e t h a l ,  necrotic,  are the f u n c t i o n s  of  enzyme.  OTHER TOXINS PRODUCED BY CLOSTRIDIUM PERFRINGENS Of the s i x r e c o g n i z e d types of  Clostridium  p e r f r i n g e n s only one i s r e c o g n i z e d as a human pathogen while  the remaining f i v e  infections  are g e n e r a l l y i n c r i m i n a t e d i n  and I n t o x i c a t i o n s  of domestic  i s found i n human gangrenous for  infections  animals. and i s  some cases of food p o i s o n i n g i n the human.  Type A  responsible This  may be i d e n t i f i e d by c e r t a i n c o l o n i a l c h a r a c t e r i s t i c s by the heat r e s i s t a n c e  of i t s  a comparative study of the following the  table,  spores  (3h)  and  as w e l l as by  toxins which i t p r o d u c e s .  a f t e r Oakley and Warrack,  t o x i n p r o d u c t i o n of the s i x types of  type  The  (70) summarizes  Clostridium  perfringens. Table 1 Toxin  Activity  B a c t e r i a l type A  p  & £  e  Lethal Lethal Lethal Lethal Lethal Lethal  necrotizing hemolytic lecithinase necrotizing hemolytic necrotizing  B +  —  +++  —  +  —  -r f+  —  c +  D.CE +  -  Hemolytic Lethal  K  Collagenase  k  Proteinase  M  H ya luronidase . Desoxyri bonuclease  gelatinase gelatinase  F +  - + + - ++ - - - -  +  i  V  +++  ++  4  •t-  +  +  -  -  -  -  -  ++  —  +  -  + +  -  ++  -  +  - -  +•  +  +  11 Some degraded s t r a i n s type A may produce l i t t l e >\  toxin;  are always found  ot t o x i n ;  or type C may f a i l  and  type B may y i e l d no  to produce /B t o x i n .  £ t o x i n - T h i s t o x i n i s produced as a p r o t o x i n which i s a c t i v a t e d by such p r o t e o l y t i c  enzymes as  trypsin.  of type D organisms  (82)  lose their  Thus f i l t r a t e s  and Q t o x i c a c t i v i t i e s  when  exposed to t r y p s i n , w h i l e t h e i r e p s i l o n a c t i v i t y i s c o n s i d e r a b l y enhanced.  toxin  This  toxin  has been p a r t i a l l y p u r i f i e d by S a i s s a c and Raynard (82)  by means of e x t r a c t i o n w i t h  sodium c i t r a t e . Q t o x i n - Theta t o x i n causes the well-known "hot of sheep r e d b l o o d c e l l s .  This toxin i s  a c t i v a t e d by o x i d a t i o n (91) months of storage  hemolysis"  but a f t e r  the a c t i v i t y of the  several toxin  may be r e s t o r e d by the a d d i t i o n of 0.1 p e r c e n t c y s t e i n e h y d r o c h l o r i d e (30) a d d i t i o n of c a l c i u m c h l o r i d e . ( 3 2 ) is  to  0.2  or by the Theta t o x i n  i n a c t i v a t e d by t r y p s i n .  An i n v e s t i g a t i o n by Todd (91) this  in-  has shown t h a t  oxygen l a b i l e hemolysin i s n e u t r a l i z e d by a n t i s e r a  to s t r e p t o l y s i n 0 and t h a t the s p e c i f i c toxin neutralizes Streptococci;  s t r e p t o l y s i n 0.  antibody to  The Pneumococci;  theta the  C l o s t r i d i u m t e t a n i and C l o s t r i d i u m  p e r f r i n g e n s a l l produce oxygen l a b i l e hemolysins which can  12 be r e a c t i v a t e d by r e d u c t i o n . related /  These hemolysins  are  closely  serologically.  t o x i n - I o t a t o x i n resembles is  e p s i l o n t o x i n i n that  it  a c t i v a t e d by t r y p s i n . ( 8 l )  K t o x i n - In 19!?1  Guillaumie et.  al.  (33)  reported a d i s t i n c t  c o l l a g e n a s e or kappa t o x i n In the f i l t r a t e s  of  C l o s t r i d i u m h i s t o l y t l c u m and of C l o s t r i d i u m perfringens. by t h i s  The breakdown of c o l l a g e n  enzyme i s prevented by the' a d d i t i o n of  a n t i s e r a prepared a g a i n s t of  pellets  e i t h e r organism.  the crude  filtrates  G u i l l a u m i e has r e p o r t e d  some l o c a l i z e d r e s o r p t i o n i n a r t i f i c i a l animal s c l e r o s e s through the use of kappa t o x i n . X t o x i n - The lambda t o x i n of C l o s t r i d i u m p e r f r i n g e n s a t t a c k s h i d e , powder, s e r i c i n , g e l a t i n ,  casein  and hemoglobin but w i l l not act upon n a t i v e collagen.  (9)  In a d d i t i o n to the c l a s s i c a l t h i s organism produces a phagocytosis  toxins  listed  above  inhibiting factor  d i s c o v e r e d r e c e n t l y by Gsratfcey, Merchant and Bohr.  (22)  THE PRODUCTION OP LECITHINASE ( <* TOXIN)IN VITRO In view of the abvious importance of  antilecithinase  a c t i v i t y i n the immunological defense a g a i n s t gas  gangrene,  there have been numerous attempts to p r o v i d e a maximum  13 c o n c e n t r a t i o n of oL t o x i n i n p r e p a r a t i o n s to be used as toxoids.  I n i t i a l l y t h i s work was concerned o n l y w i t h  complex and r e l a t i v e l y undefined media, but i n r e c e n t years an i n c r e a s i n g number of attempts have been made to develop a more d e f i n e d medium s u i t a b l e f o r «x t o x i n production. THE EFFECT OF .THE MEDIUM ON LECITHINASE ( oi TOXIN) PRODUCTION The p r o d u c t i o n of  <* t o x i n " i n v i t r o "  is  affected  by the n a t u r e of the medium, the p e c u l i a r  kinetics  of each i n d i v i d u a l s t r a i n of the organism, and  by  such f a c t o r s  influence  as pH and temperature which are known to  a l l biological  systems.  T o x i n P r o d u c t i o n i n Complex Media A peptone medium c o n t a i n i n g 2 to 5 p e r c e n t normal horse serum was used by Robertson and Keppie (79) produce 100  mouse m . l . d . of * t o x i n p e r m l .  Macfarlane and  Knight (1+7) used a medium c o n t a i n i n g e x t r a c t s muscle to produce 200 Hendee (1)  mouse m . l . d . per m l .  to  of horse  Adams and  employed a medium c o n t a i n i n g tryptone and a  p a n c r e a t i c d i g e s t of c a s e i n to produce 1+00 mouse m . l . d . of  the t o x i n p e r m l .  produce up to 1000  And i n 19l\$ Logan (\+%)  mouse m . l . d . per m l . i n a medium  c o n t a i n i n g the f o l l o w i n g  components.  Casein hydrolysate Fat  was able  solvent  e x t r a c t e d beef  heart  to  i4 D e x t r i n ( a d j u s t e d to o p t i m a l  concentration)  L Cystine L Tryptophane Calcium D pantothenate Pimelic acid Nicotinic  acid  Pyridoxine Thiamine Riboflavine Iron (adjusted  to o p t i m a l  concentration)  T h i s and other s i m i l a r media have s i n c e used i n the commercial p r o d u c t i o n of Mason (58) toxins  of both  type A are produced i n  i n f u s i o n b r o t h but that o n l y  i s produced i n n u t r i e n t b r o t h . good y i e l d s  toxin.  has s t a t e d t h a t both the <>l and the &  of C l o s t r i d i u m p e r f r i n g e n s  glucose peptone beef  and S  a v a r i e t y of p r o t e o l y s a t e s ,  Malgras (57)  toxins  on media c o n t a i n i n g  w h i l e R u s s i a n workers  glucose to be the most s u i t a b l e f o r  (72)  percent  <* t o x i n p r o d u c t i o n .  In r e c e n t work Gooder and h i s c o l l e a g u e s (25) a medium c o n t a i n i n g a constant  & toxin  has r e p o r t e d  have found a meat peptone medium c o n t a i n i n g 0.5  have found  amount of a p a n c r e a t i c  d i g e s t of beef h e a r t to be s u i t a b l e with  been  f o r s t u d i e s concerned  toxin production. It is  of t e c h n i c a l importance to note t h a t ,  the media r e f e r r e d to above are g e n e r a l l y c o n s i d e r e d  while to  15  p r o v i d e anaerobic c o n d i t i o n s culture vessels,  this  suitable  organism i s capable of p r o l i f e r a t i o n  i n simple media w i t h l i t t l e Poss (19)  f o r growth, i n  provision for  anaerobiosis.  has r e p o r t e d that C l o s t r i d i u m p e r f r i n g e n s  grow r e a d i l y i n a medium c o n t a i n i n g c a s e i n , b l o o d and g l u c o s e without any treatment c o n d i t i o n s f o r growth.  yeast  to assure  will  extract, anaerobic  During t h i s work i t was noted  i n c e r t a i n culture vessels a synthetic only amino a c i d s , v i t a m i n s  that  medium c o n t a i n i n g  and s a l t s supported a l u x u r i a n t  growth of the organism without p r e l i m i n a r y b o i l i n g . T o x i n P r o d u c t i o n i n Less Complex Media The medium of Reed and O r r (78) y i e l d of  had g i v e n some  t o x i n but has not produced t o x i n i n as h i g h a  c o n c e n t r a t i o n as have the more complex media. contains  sodium t h i o g l y c o l l a t e ,  dextrose  has been r e p o r t e d t h a t h i g h e r y i e l d s obtained i f  and g e l a t i n .  of the  t o x i n are  (89)  have r e p o r t e d y i e l d s  * t o x i n h i g h e r than those obtained i n g l u c o s e  beef  It  the g e l a t i n used has not been h i g h l y p u r i f i e d .  Tamura, Boyd, Logan and T y t e l l of  T h i s medium  i n f u s i o n b r o t h u s i n g a medium c o n t a i n i n g the  components A c i d h y d r o l y s a t e of C a s e i n Buffer Mg SOij. L Tryptophane Pantothenic acid'  peptone following  16  Pimelic acid Nicotinic acid Riboflavins I r o n ( a d j u s t e d to optimal c o n c e n t r a t i o n ) A d j u s t e d to pH 7.9 The s e m i - s y n t h e t i c  medium of T a y l o r and Stewart  has been r e p o r t e d to y i e l d no the f o l l o w i n g  toxin.  (90)  T h i s medium c o n t a i n s  substances:-  Casein Peptone Buffer I r o n ammonium c i t r a t e Glucose In view of the work c a r r i e d out i n t h i s l a b o r a t o r y , it  Is u n f o r t u n a t e that these workers d i d not r e p o r t  the  l e n g t h of time which had elapsed between I n o c u l a t i o n and the t e s t i n g  of samples f o r t h e i r p r o d u c t i o n of oC t o x i n .  Growth i n a C h e m i c a l l y R e p r o d u c i b l e Medium In 1948  Boyd, Logan, and T y t e l l  (11)  developed a  c h e m i c a l l y r e p r o d u c i b l e medium f o r the growth of C l o s t r i d i u m p e rf r i n g e s  T h i s medium c o n t a i n s n i n e t e e n amino a c i d s ,  seven accessory growth f a c t o r s , glucose  as the energy s o u r c e .  four s a l t s ,  b u f f e r and  These workers r e p o r t e d that  no «i. t o x i n was produced i n t h i s medium under the of t h e i r experiment.  conditions  While the development of a c h e m i c a l l y  17 r e p r o d u c i b l e medium i s  always  to be c o n s i d e r e d a m i l e s t o n e  i n the e l u c i d a t i o n of m i c r o b i a l metabolism, fortunate  i t was u n -  t h a t the a l l - i m p o r t a n t * t o x i n was r e p o r t e d  absent i n f i l t r a t e s  from growth i n t h i s medium.  This  would suggest that the medium, although capable of s u p p o r t i n g c e l l p r o l i f e r a t i o n , was not complete sense that the organism f a i l e d to f u l f i l potentialities.  Under the  to be r e p o r t e d here the medium i s  it  full  As w i l l be seen l a t e r however,  proved a mistaken I n t e r p r e t a t i o n .  allowing  its  toxin production.  metabolic  this  conditions  i n f a c t capable of  In t h i s manner,  can be compared In s i g n i f i c a n c e  of M u e l l e r , ajg&v'Dfeti R v - i (69)  i n the  therefore,  w i t h the c o n t r i b u t i o n  i n developing a r e p r o d u c i b l e  medium f o r d i p h t h e r i a t o x i n p r o d u c t i o n . The K i n e t i c s of Toxin P r o d u c t i o n In any c o n s i d e r a t i o n of the p r o d u c t i o n of << t o x i n by C l o s t r i d i u m p e r f r i n g e n s against  denaturation.  p r o d u c t i o n must be balanced  F a c t o r s which seem to a;ffect p r o -  d u c t i o n may indeed i n f l u e n c e relatively labile  the d e n a t u r a t i o n of  this  toxin.  The Formation of L e c i t h i n a s e ( «>c toxin) Alpha t o x i n i s  secreted  (94)  i n t o the medium  d u r i n g the l o g a r i t h m i c growth phase a f t e r a c e r t a i n d e l a y which i s dependent upon the d e n s i t y  of the inoculum u s e d .  T h i s delay does not i n d i c a t e an i n t r a c e l l u l a r r e t e n t i o n  (10)  18 but merely r e f l e c t s  the k i n e t i c s  P a s i c h n i k and Chernishova (72)  of <x t o x i n p r o d u c t i o n .  (77)  have concluded t h a t i n d i v i d u a l  s t r a i n s of the organism e x h i b i t I n d i v i d u a l p r o p e r t i e s w i t h r e g a r d to the d u r a t i o n and I n t e n s i t y  of t o x i n p r o d u c t i o n .  F i g u r e 1 i s based upon the work of Gale van Heyningen (21)  who found that  <*• t o x i n p r o d u c t i o n i n a complex medium  occurs only d u r i n g the l o g a r i t h m i c growth phase. i n i t i a l delay i n  The  •* t o x i n p r o d u c t i o n which i s evident  in  f i g u r e 1 s u b s t a n t i a t e s the statement of Raynaud and Bizzini is  (10)  and has been r e p e a t e d i n our e x p e r i e n c e .  obvious from a c o n s i d e r a t i o n of these and of  experiments  that  It  later  «* t o x i n p r o d u c t i o n takes p l a c e d u r i n g the  l o g a r i t h m i c growth phase and that p r o d u c t i o n ceases near the beginning of the s t a t i o n a r y  phase.  The D e n a t u r a t i o n of L e c i t h i n a s e ( •< t o x i n ) I t has been r e c o g n i z e d that the C l o s t r i d i u m perfringens i s unstable of that organism.  t o x i n of  i n culture  filtrates  Kreguer (i|.0) has r e p o r t e d that «L t o x i n  i n b o u i l l o n d e c l i n e s more q u i c k l y on s t a n d i n g than does the  t o x i n i n p r e p a r a t i o n s c o n t a i n i n g more meat  D a l l i n g and Ross (13)  have supported the r e s u l t s  solids. quoted  above and have concluded that f i n a l t o x i n p r o d u c t i o n i s i n f l u e n c e d by the p r o p o r t i o n of meat s o l i d s  i n the medium.  F i l t r a t e s of the type D organisms c o n t a i n <* and 6  toxins  a f t e r a few hours i n c u b a t i o n , but i t i s found t h a t  the  19 toxin has disappeared when these cultures are tested at five days, while the  toxin remains.  Thus while the  lecithinase of Clostridium botulinum can he stored i n definitely the corresponding product of Clostridium perfringens is readily denatured upon standing.  (63)  It has "been shown that the lecithinase of Clostridium perfringens, is more easily denatured "by trypsin (81) papain and pepsin (88) than are the toxins of Clostridium tetani and of Corynebacterium diphtheriae. In view of the extensive battery of proteolytic enzymes known to be present in supernatants of Clostridium perfringens i t is not surprising that the to denaturation.  toxin is subject  Gonzales (23) has reported that the  proteolytic enzymes of this organism are most active at 50°C and that they are inactivated at 63°C The work of Vyshepan and Krasnova (96) constitutes a most significant development in the consideration of the denaturation of  toxin.  These workers have isolated an en-  zyme from filtrates and centrifugates of Clostridium perfringens which denatures  toxin by splitting the protein  moiety of the molecule, thus rendering i t inactive as a lecithinase.  While the optimal pH for its action is 6.0 to  7.0 the enzyme is irreversibly inactivated at a pH below 5.5. Cysteine is required as a co-factor by this lecithinase denaturing enzyme. Besides attacking the protein of  toxin the  20 above enzyme i s found  to a c t upon c a s e i n and g e l a t i n  but not upon egg albumin.  Therefore,  the a d d i t i o n o f  c a s e i n , g e l a t i n o r meat p r o t e i n prevents of  t o x i n by p r o v i d i n g an a l t e r n a t e s u b s t r a t e f o r the  enzyme. and that  the breakdown  The a c t i v i t y o f t h i s enzyme i s slowed by g l y c e r i n  t h i s f a c t e x p l a i n s the long s t a n d i n g o b s e r v a t i o n  (96)  t o x i n r e t a i n s i t s a c t i v i t y w e l l when s t o r e d i n  t w e n t y - f i v e percent  glycerin.  These authors denaturing  state that t h e i r  lecithinase  enzyme resembles the anaerobase r e p o r t e d by  Machman i n 1937  i n i t s optimal pH, i t s a c t i v a t i o n by  c y s t e i n e , and i n i t s s u b s t r a t e s p e c i f i c i t y .  However  u n l i k e anaerobase t h i s enzyme does not r e q u i r e codehydrases 1 and 2 as coenzymes. The the nature  e x i s t e n c e o f such an enzyme Is suggested by  o f the d e n a t u r a t i o n process and by I t s r e l a t i o n  to the p r o t e i n content production. questioned,  of the medium used i n t o x i n  While the p u r i t y o f the above enzyme may be the e x i s t e n c e o f some p r o t e o l y t i c agent a c t i v e  In the d e n a t u r a t i o n of the «*. t o x i n o f C l o s t r i d i u m p e r f r i n g a a s i s s t r o n g l y suggested  by the evidence  at hand.  FACTORS INFLUENCING LECITHINASE ( «* TOXIN) PRODUCTION Every b i o l o g i c a l system i s i n f l u e n c e d by such environmental  f a c t o r s as pH, temperature and c o n c e n t r a t i o n  of m i n e r a l s .  Since each o f these f a c t o r s may i n f l u e n c e  t o x i n p r o d u c t i o n , or d e n a t u r a t i o n , or both o f these  21  p r o c e s s e s , the system assumes a d d i t i o n a l  complexities.  In a d d i t i o n to the environmental f a c t o r s  c e r t a i n compounds  have been r e p o r t e d to s t i m u l a t e of the  or i n h i b i t e the p r o d u c t i o n  toxin.  Temperature I n c u b a t i o n at 37°C has g e n e r a l l y been used i n the p r o d u c t i o n of  «•*• t o x i n (21)  grows w e l l at t h i s temperature.  (80)  since  the organism  Boyd, Logan and T y t e l l  (11)  have r e p o r t e d that the b e s t growth i n t h e i r b a s a l medium is  a t t a i n a b l e at 45°C.  However, s i n c e C u r r i e (12)  has  shown that the same medium supports growth as w e l l at 37°C as at the h i g h e r temperature t h i s work was c a r r i e d out u s i n g i n c u b a t i o n at 37°C as standard p r o c e d u r e .  The  e f f e c t of I n c u b a t i o n at l±$°G upon p r o d u c t i o n and d e n a t u r a t i o n of the t o x i n has not been s t u d i e d . Hydrogen Ion C o n c e n t r a t i o n Although I n i t i a l pH values were g i v e n i n r e p o r t s of e a r l y t o x i n p r o d u c t i o n experiments,  remarkably l i t t l e  a t t e n t i o n was p a i d to the pH a t t a i n e d d u r i n g the growth of the organism.  The complex media used were u s u a l l y  to pH 7.6  or 7.9  (78)  (89)  before i n o c u l a t i o n .  Boyd, Tamura, Logan and T y t e l l  (89)  adjusted  However,  s t a t e t h a t the  success  of t h e i r peptone f r e e medium may be a t t r i b u t e d to the  fact  t h a t the pH of the medium d i d not f a l l below 6.0 d u r i n g growth.  22 A study of the e f f e c t of pH growth and t o x i n p r o d u c t i o n was c a r r i e d out by Gale" and van Heyningen i n 19lj2.  Figure 2 represents  the e f f e c t of pH s t a b i l i z -  a t i o n at values between 5 « 5 and 8.5 weight  (21)  upon the f i n a l dry  of c e l l s ' p e r m l . of the medium. Figure 2  Gale and van Heyningen  (21)  V a r i a t i o n of t o t a l c e l l y i e l d w i t h growth p H .  -  4  i  E  3-  V-  •o  |-  E  pH d u r i n g growth ° w i t h 0«ij.5$ glucose. • without g l u c o s e . F i g u r e 3 r e p r e s e n t s the e f f e c t of pH s t a b i l i z a t i o n a t v a r i o u s values between pH 5-5 and 8.0 upon the toxin produced per c e l l . Figure 3 V a r i a t i o n of  Gale and van Heyningen  t o x i n p r o d u c t i o n w i t h growth p H .  2CM  6.0  70  pH d u r i n g growth 0  w i t h 0.4-5$ glucose • without glucose  8.0  (21)  23 Nature and C o n e e n t r a t i o n of Carbohydrate Adams and Hendee (1)  r e p o r t e d i n 1945  were able to produce 50 to 100 p e r c e n t more d e x t r i n was s u b s t i t u t e d Vyshepan e t .  al.  (95)  f o r g l u c o s e i n t h e i r medium.  have found that g l u c o s e i s  <*• t o x i n p r o d u c t i o n i s  Since  organism the p r o d u c t i o n  the t o x i n begins and ends more q u i c k l y i f glucose  present  as the energy  Is  source.  The s u b s t i t u t i o n of  and  fermented  r e l a t e d to the l o g a r i t h m i c  growth phase i n c u l t u r e s of t h i s  of  they  t o x i n when  more q u i c k l y than e i t h e r d e x t r i n or g l y c e r o l .  of  that  the water s o l u b l e p o r t i o n  d e x t r i n f o r glucose i n the b a s a l medium of Boyd, Logan T y t e l l f a i l e d to promote the p r o d u c t i o n of  *• t o x i n .  (12)  With r e g a r d to the c o n c e n t r a t i o n of the c a r b o hydrate energy source M e i s e l and A l b r y c h t (59)  have  stated  t h a t the c o n c e n t r a t i o n of g l u c o s e or d e x t r i n has no  effect  upon the p r o d u c t i o n of by  toxin.  This f i n d i n g i s  the work of Gale and van Heyningen (21),  contradicted  and s i n c e  the  a v a i l a b i l i t y of fermentable carbohydrate a f f e c t s the pH of  the c u l t u r e some e f f e c t of carbohydrate c o n c e n t r a t i o n  would be  expected. NATURE AND CONCENTRATION OF MINERAL IONS The nature and c o n c e n t r a t i o n of m i n e r a l s has  been shown to a f f e c t  both growth and t o x i n p r o d u c t i o n i n  Clostridium perfringens  2k  E f f e c t of m i n e r a l s upon growth Shankar and B a r d ,  (83)  and Webb, (98)  r e p o r t e d that c a l c i u m , magnesium,  iron,  have  sodium and  potassium are r e q u i r e d to support the growth of  the  organism w h i l e c o b a l t i s r e q u i r e d by some s t r a i n s . Growth on magnesium or potassium d e f i c i e n t to the f o r m a t i o n of filamentous deficiency  results  media l e a d s  c e l l s while calcium  i n the f o r m a t i o n of c e l l u l a r  These authors a l s o r e p o r t that copper i s that  z i n c and manganese  (8k)  aggregates.  i n h i b i t o r y and  are not r e q u i r e d by the  organisms.  A l d o l a s e has been i s o l a t e d from C l o s t r i d i u m p e r f r i n g e n s b y Bard and Gunsalus ( £ ) and i t has shown that t h i s factor.  been  enzyme i s dependent upon i r o n as a c o -  I t has been found (89)  t h a t 1.11  mg. of phos-  phorus per m l . o f medium i n h i b i t s the growth of  the  organism. E f f e c t of I r o n upon t o x i n p r o d u c t i o n Ajl  et.  a l . (3)  by the S t r e p t o c o c c i , diphtheriae, Is a f f e c t e d  have s t a t e d that t o x i n p r o d u c t i o n  the S h i g e l l a , Corynebacterium  Clostridium tetani  and C l o s t r i d i u m p e r f r i n g e n s  by the c o n c e n t r a t i o n of i r o n i n the medium.  These authors s t a t e that i r o n c o n c e n t r a t i o n a f f e c t s the  both  t o x i n p r o d u c t i o n and the metabolic behaviour o f  C l o s t r i d i u m perfringgns but that the seem to be  connected.  two e f f e c t s do not  25 Pappenheimer and Shaskan (71)  have shown t h a t a  c o n c e n t r a t i o n of i r o n o p t i m a l f o r growth l e a d s to a t y p i c a l f e r m e n t a t i o n of g l u c o s e to a c e t i c carbon d i o x i d e and hydrogen.  and b u t y r i c a c i d s ,  In c o n t r a s t ,  establishes a glucose metabolism the )  which i s  lactic  acid.  c o n c e n t r a t i o n of 0.73 medium r e p r e s e n t s  an i r o n  deficiency  c h i e f p r o d u c t of  These authors s t a t e that an i r o n mg« p e r m l . of g e l a t i n  hydrolysate  the o p t i m a l l e v e l f o r growth and f o r  «*• t o x i n p r o d u c t i o n w h i l e y i e l d i n g a minimum of  lactic  acid. I t has been r e p o r t e d (6)  that the a d d i t i o n of  i r o n to complex media i n c o n c e n t r a t i o n s per m l . s t i m u l a t e s workers (89)  up to 0-1|_ mg.  t o x i n p r o d u c t i o n . • However other  have found 0.2  an optimal c o n c e n t r a t i o n .  to 0.25  mg. per m l . to be  K r i t s k a y a (1+1)  has  studied  the e f f e c t s of i r o n c o n c e n t r a t i o n upon the growth and «<. t o x i n p r o d u c t i o n of C l o s t r i d i u m p e r f r i n g e s . following table  i s d e r i v e d from h i s  The  results:-  Table 2 Iron Concentration  •333 •166 .083 .016  mg/ml. mg/ml. mg/ml. mg/ml.  Effect No growth. Good growth. Optimal t o x i n p r o d u c t i o n . Good growth. L e s s , t o x i n p r o d u c t i o n . Good growth. No t o x i n p r o d u c t i o n .  F i g u r e l+, a f t e r Rogers and K n i g h t ,  (80)  illustrates  the  optimal c o n c e n t r a t i o n of i r o n f o r « t o x i n p r o d u c t i o n i n a beef h e a r t medium.  26 F i g u r e Ij. E f f e c t of I r o n on «. T o x i n  Production.  150-  z bacter  110-  ^» (0  70-  c  •  i  0.1  0.2  i  0.3  i  0-4  I r o n i n mg./ml. of medium. In c o n t r a s t w i t h the above f i n d i n g s Adams and Hendee (1)  have s t a t e d t h a t when c o n c e n t r a t i o n s of p r o t e i n ,  carbohydrate  and b u f f e r are such as to ensure good growth  the system becomes i n s e n s i t i v e to s t i m u l a t i o n by I n h i b i t i o n of  *  iron.  t o x i n p r o d u c t i o n by i r o n d e f i c i e n c y i s  however r e c o g n i z e d by these  authors.  E f f e c t of other m i n e r a l s upon t o x i n p r o d u c t i o n . Gooder (2l\.) has r e p o r t e d t h a t the use of c a l c i u m phosphate to remove i r o n from c e r t a i n media has r e s u l t e d In the concurrent removal of z i n c to such an extent as to cause a decrease found  i n toxin production.  C u r r i e (12)  has  t h a t the a d d i t i o n of c a l c i u m or magnesium to the  b a s a l medium of Boyd, Logan and T y t e l l f a i l e d to promote oc t o x i n p r o d u c t i o n under the c o n d i t i o n s of h i s experiment.  27 CHEMICALLY DEFINED COMPOUNDS INFLUENCING TOXIN PRODUCTION Using e i g h t  s t r a i n s of C l o s t r i d i u m p e r f r i n g e n s  grown i n beef i n f u s i o n b r o t h at pH 7«5 M e i s e l e t . a l . ( 6 2 ) have found that one e i g h t h of a b a c t e r i o s t a t i c of p e n i c i l l i n s t i m u l a t e s  the p r o d u c t i o n of  e f f e c t disappears at pH 7 « 0 . by Miss Kron (i+2)  «t t o x i n .  "This  A v a l i d a t i o n of t h i s work  u s i n g the b a s a l medium of Boyd, Logan  and T y t e l l has shown that a s l i g h t production i s  concentration  increase i n toxin  indeed seen under these c i r c u m s t a n c e s .  M e i s e l found that the same e f f e c t was not obtained w i t h procaine,  s e v e r a l sulphonamides,  s a l i c y l i c a c i d (61)  (60)  and p - amino  and that p - amine benzoic  acid  reduced the t o t a l p r o d u c t i o n of «* t o x i n . However Kato and I l l e n y : benzoic a c i d i n c r e a s e s  the  (38)  s t a t e t h a t p-amino  t o x i n produced by C l o s t r i d i u m  p e r f r i n g e n s i f added at the b e g i n n i n g of the growth p e r i o d . P-amino benzoic a c i d , i n 0,0$ increases  percent  concentration,  t o x i n p r o d u c t i o n by C l o s t r i d i u m t e t a n i  without a f f e c t i n g  (ij-3)  the growth of the organism.  The a d d i t i o n of c e r t a i n amino a c i d s to a medium c o n t a i n i n g p a n c r e a t i c d i g e s t of beef h e a r t has been found to a f f e c t  «c t o x i n p r o d u c t i o n . (25)  The a d d i t i o n of  valine  to the medium decreases °i t o x i n p r o d u c t i o n by 25 p e r c e n t w h i l e the a d d i t i o n of c y s t e i n e b r i n g s about a 37 p e r c e n t decrease  i n the t o x i n produced.  The a d d i t i o n of  completely i n h i b i t s t o x i n p r o d u c t i o n without  cystine  affecting  28 the growth of the organism. however,  Vyshepan (95>)  that cysteine increases  but r e s u l t s  contends  «* t o x i n p r o d u c t i o n  In the p r o d u c t i o n o f a t o x i n more  easily  denatured than that produced i n normal c u l t u r e s .  A  c o n s i d e r a t i o n of the phase of growth of the c u l t u r e s the time of  «* t o x i n d e t e r m i n a t i o n would p o s s i b l y  to r e c o n c i l e these seemingly  at  serve  opposed r e p o r t s .  In view of more r e c e n t l y a c q u i r e d knowledge, the phase of growth i n which a c u l t u r e e x i s t s at time of sampling i s  of great  importance i n the  p r e t a t i o n of s t u d i e s concerned w i t h  inter-  * toxin production.  The phase of growth i n which a c u l t u r e e x i s t s i s p a r t i c u l a r Importance when c y s t e i n e i s p r e s e n t this  <>L t o x i n .  of  since  amino a c i d has been d e s c r i b e d as a c o f a c t o r  an enzyme which a c t s to denature  the  for  (96)  COMPLEX AND UNDEFINED FACTORS INFLUENCING TOXIN PRODUCTION Although i t has been e s t a b l i s h e d  t h a t some  b a c t e r i a l t o x i n s may be formed i n a c h e m i c a l l y medium (66) factors  there are other i n s t a n c e s i n which u n r e s o l v e d  are r e q u i r e d f o r t o x i g e n e s i s .  M u e l l e r and M i l l e r  have shown that the a c i d i c , b a s i c and n e u t r a l of  defined  fractions  a t r y p t i c d i g e s t of c a s e i n are a l l r e q u i r e d i n the  p r o d u c t i o n of t o x i n by C l o s t r i d i u m t e t a n i .  Each  toxigenic  f a c t o r was destroyed by prolonged a c i d h y d r o l y s i s , i n d i c a t i n g that these f a c t o r s may be p a r t i a l l y p e p t i d e in  nature.  (67)  29  In 191+6 Rogers and Knight (80)  undertook an  i n v e s t i g a t i o n to determine the nature of c e r t a i n f a c t o r s i n a p r o t e i n f r e e e x t r a c t of horse muscle which s t i m u l a t e <* t o x i n p r o d u c t i o n by C l o s t r i d i u m p e r f r i n g e s . substance used as a s t a r t i n g m a t e r i a l i n t h i s  The  investigation  was prepared by e x t r a c t i n g horse muscle w i t h a s o l u t i o n c o n t a i n i n g sodium s u l p h a t e .  The sodium sulphate p r e -  c i p i t a t e s the p r o t e i n s p r e s e n t i n the e x t r a c t w h i l e p r e s e r v i n g many of the heat l a b i l e components of animal t i s s u e . of  the  A c c o r d i n g to these workers the p r o p e r t i e s  the p a r t i a l l y p u r i f i e d t o x i g e n i c f a c t o r c l o s e l y  those of an amino-hexose. small molecular s i z e ,  resemble  The f a c t o r i s of r e l a t i v e l y  i s dependent upon the presence of  primary amine groups, and i s readily altered i n a l k a l i .  s t a b l e i n a c i d but very A c c o r d i n g l y glucosamine,  N - a c e t y l glucosamine and chrondrosamine were t e s t e d f o r toxigenic  activity.  A c o n c e n t r a t i o n of glucosamine judged  to be e q u i v a l e n t to the c o n c e n t r a t i o n of the  toxigenic  f a c t o r i n the muscle e x t r a c t caused an i n c r e a s e i n << t o x i n production.  The i n c r e a s e was, however,  only approximately  30 p e r c e n t of t h a t caused by an e q u i v a l e n t amount of muscle e x t r a c t .  N - a c e t y l glucosamine had a corresponding  e f f e c t but chondrosamine was t o t a l l y i n a c t i v e .  ATTEMPTS TO PRODUCE «< TOXIN IN_THE BASAL MEDIUM OP BOYD, LOGAN AND TYTELL. In 1950 C u r r i e (12)  e s t a b l i s h e d t h a t , under the  c o n d i t i o n s of h i s experiment, no l e t h a l or l e c i t h i n a s e  30 a c t i v i t y c o u l d be a t t r i b u t e d to the supernatant of C l o s t r i d i u m p e r f r i n g e r s grown i n the b a s a l medium of Boyd, Logan and T y t e l l .  T h i s medium w i l l  henceforth  be r e f e r r e d to simply as the b a s a l medium.  In a d d i t i o n  to h i s  and h i s  adjustment of m i n e r a l c o n c e n t r a t i o n s  s u b s t i t u t i o n of d e x t r i n f o r g l u e o s e , C u r r i e added y e a s t extract,  choline,  c h o l i n e c h l o r i d e , sodium glycerophosphate,  n i c o t i n i c a c i d and glucosamine t o  the b a s a l medium without  a c h i e v i n g •<. t o x i n p r o d u c t i o n . An i n v e s t i g a t i o n was then undertaken to the nature of the t o x i g e n i c beef  factors  i n glucose  determine  peptone  i n f u s i o n ( G . P . B . I . ) and t o apply t h i s f a c t o r to  p r o d u c t i o n of  ot. t o x i n i n the b a s a l medium. G . P . B . I .  c o n t a i n s beef  i n f u s i o n , glucose,  peptone,  sodium c h l o r i d e and b u f f e r , w h i l e beef (B.I.B.)  is  attempt by C u r r i e (12) G.P.B.I.  meat p a r t i c l e s ,  infusion broth  composed of the above compounds without  and meat p a r t i c l e s .  contains  the  peptone  The f o l l o w i n g t a b l e I l l u s t r a t e s  an  to determine which component of  the t o x i g e n i c  factor.  Table 3 The e f f e c t of the components  of G . P . B . I . upon  toxin  production. Medium Beef i n f u s i o n (without b u f f e r ) B . I . B . (without glucose) B . I . B . w i t h 0.5$ g l u c o s e B . I . B . w i t h 1.0$ g l u c o s e B . I . B . w i t h 2.0$ g l u c o s e B . I . B . p l u s 1.0$ peptone  qC t o x i n  activity  0 0 0 0 0 1/96  31 B . I . B . p l u s 1.0$ tryptone B . I . B . p l u s 1,0% proteose peptone G . P . B . I . supernatant G.P.B.I. B a s a l medium p l u s 2% B . I . B . B a s a l medium p l u s \$> B . I . B . Prom the r e s u l t s  of t h i s  0 0 experiment i t  was  concluded that t r y p t o n e , proteose peptone and meat c o n t a i n the t o x i g e n i c  factor.  To e l i m i n a t e the  particles  possibility  that the p a r t i c u l a t e form of the meat might e f f e c t  <*. t o x i n  production,  G.P.B.I.  sand and g l a s s beads were added to the  supernatent. of  the  The a d d i t i o n s d i d not enhance the p r o d u c t i o n  toxin. Soxhlet  toxigenic  e x t r a c t i o n w i t h ether d i d not reduce  a c t i v i t y of the d r i e d meat p a r t i c l e s .  a l k a l i n e h y d r o l y s i s of the e x t r a c t e d meat  the  A c i d or  particles  d e s t r o y e d t h e i r a b i l i t y to promote oc t o x i n p r o d u c t i o n b u t J  a t r y p t i c d i g e s t r e t a i n e d a l l of the t o x i g e n i c of  the o r i g i n a l p a r t i c l e s .  The t o x i g e n i c  t r y p t i c d i g e s t i s d i a l y z a b l e and i s  The e x i s t e n c e of  f a c t o r had p r e v i o u s l y been r e p o r t e d  by Adams, Hendee and Pappenhe&mer (2) Knight.  f a c t o r i n the  t h e r e f o r e presumed to  be of r e l a t i v e l y s m a l l m o l e c u l a r s i z e . a dialyzable toxigenic  activity  and by Rogers and  (80) C u r r i e found that the t o x i g e n i c  m a t e r i a l was  l a r g e l y absorbed upon P u l l e r ' s e a r t h a t a c i d pH v a l u e s . T h i s absorbed m a t e r i a l was then t e s t e d to determine nature,  and spectrophotometric  its  and chromatographic analyses  32 of the absorbed and unabsorbed samples were undertaken. Tests upon the absorbed material showed only that tryptophane was present and that pentoses and reducing sugars were absent.  The chromatographic and spectrophotometric  analyses showed no difference between the absorbed and unabsorbed samples. BACTERIAL VARIATIONS INFLUENCING LECITHINASE fcTOXEN) PRODUCTION Work conducted in this department has shown that toxin production by Clostridium botulinum (14.) is influenced to a very large extent by the strain of the organism used. Our studies have indicated a similar variation in the  toxin  produced by different variants of Clostridium perfringens. In a recent paper Lominski et a l . (46) have reported that the coagulase produced by "R" variants of Micrococcus pyogenes variety aureus is denatured by products of the "S" variant of the same organism. In the production of  toxin by Clostridium  perfringens some workers have favored the use of animal passage for the selection and maintenance of toxigenic strains while others have insisted that animal passage is unnecessary.  However strains which exhibit pathogenicity  for man or for experimental animals are generally found to be good<*.toxin producers.  (37) (79) Gooder (24.) does  not consider animal passage necessary and Taylor and Stewart (90) state that daily transfers in a peptone . medium for one hundred and five days did not decrease  33  the t o x i g e n i c i t y  of t h e i r s t r a i n .  Keppie and Robertson  have r e p o r t e d that a wide capsule i s  generally  (39)  associated  w i t h low ot t o x i n p r o d u c t i o n . Logan e t . always i n c r e a s e s strains.  al.  the  have found that pigeon passage  ottoxin p r o d u c t i o n of l e s s  However W i l d f u h r (99)  toxigenic  has found t h a t ,  s t r a i n s p r o d u c i n g minimal amounts of  while  * t o x i n can be r a i s e d  to good t o x i n p r o d u c t i o n by animal passage, t h i s f a i l e d to promote strains.  technique  «* t o x i n p r o d u c t i o n by n o n t o x i g e n i c  The best r e s u l t s were o b t a i n e d when c u l t i v a t i o n  i n a b r a i n medium was a l t e r n a t e d w i t h p i g e o n p a s s a g e . W i l d f u h r (100)  concludes  that  * toxin production i s  dependent upon the medium, the age and q u a l i t y of p r e cultures,  and the b a c t e r i a l s t r a i n employed.  MATERIALS AND METHODS Media Glucose peptone beef the maintenance of stock  i n f u s i o n b r o t h was used f o r  cultures.  A medium suggested by E l l n e r (jj#)) f o r  the  I n d u c t i o n of s p o r u l a t i o n i n C l o s t r i d i u m p e r f r i n g e s s was used f o r the p r e s e r v a t i o n of the s t r a i n s used i n t h i s work. These s t r a i n s were a l s o l y o p h i l i z e d under the of D r . J . J . S t o c k .  supervision  3kThe b a s a l medium o f Boyd, Logan and T y t e l l (11) was  used  throughout  following  t h i s work.  T h i s medium c o n t a i n s the  substances:-  Glucose Ascorbic acid DL- A l a n i n e D - Arginine # DL - A s p a r t i c a c i d L - Cystine # Glycine L - Glutamic a c i d # L - Histidine # Hydroxy L - p r o l i n e DL - I s o l e u c i n e # L - Levcine # L - Lysine # DL - Methionine # L - Proline DL - P h e n y l a l a n i n e # DL - S e r i n e # DL - Threonine #  2.0gm. 50.0mg. 100.0 " £0.0 " 100.0 " \ 20.0 " 100.0 "  50.0mg. L-Tryptophan # 50.0 * L-Tyrosine # 75.0 » DL-Valine # 2.£ " Uracil A denine Sulphate 34 " Ca D-pantothenate 100.0 gamma Pynidoxamine dihydro100.0 " chloride 200.0 " Riboflavine Biotir 1.0 " Mg S0[,.7H20 4-0. Omg. Pe S01T.7H20 2.0 * Mn SOlI. I4il20 2.0 " Na C I 2.0 "  150.0 50.0 25.0 50.0 75*0 100.0 5*0.0 *» 25.0 K 2 HPOh 50.0 K H2 P % 150.0 D i s t i l l e d Water 50.0 11  1.66gm. 0.32 " 200 c c  # E s s e n t i a l f o r growth. C y s t i n e i s d i s s o l v e d s e p a r a t e l y w i t h the a d d i t i o n o f HC1, and added t o the medium a f t e r the other c o n s t i t u e n t s have been d i s s o l v e d . ~Stock s o l u t i o n s o f the accessory growth f a c t o r s and m i n e r a l s are prepared as o u t l i n e d below. 50mg. o f u r a c i l and 87 mg. o f adenine are d i s s o l v e d i n 50 cc o f 0.2N. HC1.  sulphate  1.75cc o f t h i s  solution  are added p e r 100 cc o f medium. 10 gm. o f Mg S0|^. 7 H 0 , 0.5gm o f PeSO^. 7 H 0 , 2  2  0.5gm o f NaCl, and 0.5gm o f Mn S 0 ^ . I4H2O a r e d i s s o l v e d , l.Occ o f syrupy HNO3 i s added, and mixture made t o lOOcc w i t h water. of medium.  0.5cc o f t h i s s o l u t i o n i s r e q u i r e d p e r lOOcc  35  10 mg. of r i b o f l a v i n and 20 mg. o f c a l c i u m D pantothenate water.  are d i s s o l v e d  and made up t o lOOcc w i t h  0.5cc o f t h i s s o l u t i o n i s r e q u i r e d p e r lOOcc  of medium* 20 mg. o f pyridoxamine d i h y d r o c h l o r i d e are dissolved  and made up to lOOcc w i t h water.  i s kept i n a dark b o t t l e at \\.°C, is  This  solution  0 « 2 5 c c of t h i s  solution  added p e r lOOcc o f medium. A v i a l o f b i o t i n 25 gamma i s made up t o 25cc  w i t h d i s t i l l e d water.  0.5cc o f t h i s s o l u t i o n i s r e q u i r e d  per lOOcc o f medium. The phosphate b u f f e r i s prepared by d i s s o l v i n g  52 gm. o f K HPO^. 3H 0 and 10 gm. o f KH P0^ i n 250cc 2  of water.  2  2  I|..0cc o f t h i s b u f f e r are added p e r lOOcc o f  medium. The Amino a c i d s and a s c o r b i c a c i d are d i s s o l v e d by b o i l i n g .  The medium i s then c o o l e d and the accessory  growth f a c t o r s , is  cystine  and s a l t s are added.  The medium  a d j u s t e d t o pH 7 « l 5 and the phosphate b u f f e r i s added  and the medium s t e r i l i z e d .  T h i s medium may be s t o r e d  under r e f r i g e r a t i o n f o r at l e a s t two weeks w i t h results.  P r i o r to i n o c u l a t i o n the glucose i s added from  a 50 p e r c e n t s t o c k s o l u t i o n . before  satisfactory  The medium may be b o i l e d  i n o c u l a t i o n to ensure a n a e r o b i o s i s  found t h a t t h i s procedure i s not s t r i c t l y  but i t has been necessary.  36 The medium of McClung and Toabe (£6) was used i n an attempt  to measure * t o x i n p r o d u c t i o n on a s o l i d  medium. DIGESTIONS Pancreatin.  21.6 mg. of p a n c r e a t i n were used  p e r gram of m a t e r i a l to be d i g e s t e d . were maintained under t o l u e n e , mittent  The p r e p a r a t i o n s  at pH8.5, w i t h  inter-  stirring. Trypsin.  100 mg. of t r y p s i n were added p e r  gram of d r i e d weight of the m a t e r i a l to be  digested.  T h i s d i g e s t i o n was c a r r i e d out under toluene w h i l e pH was maintained at 2.0 by p e r i o d i c  the  adjustment.  REMOVAL OP IRON The aluminum oxide method of i r o n removal recommended by D o n a l d , Passey and Swaby (!§•)) was used i n t h i s work.  F i v e grams of chromatographic grade  aluminum oxide were added to one l i t r e of the  basal  medium at pH 7«5 and the f l a s k was shaken and then autoclaved at 10 l b s . / s q . i n . removal from the autoclave wet  the i n s i d e  f o r 20 minutes.  the contents were s w i r l e d  of the f l a s k .  A f t e r standing  the medium was sucked through an a c i d t r e a t e d glass f i l t e r flask.  After  connected by g l a s s j o i n t s  its to  overnight sintered  to a pyrex  filter  The f i l t r a t e was then r e a d j u s t e d to pH 7«15> and  sterilized.  37  METHODS OF CULTIVATION Daily transfers  of the t e s t s t r a i n s  medium were continued throughout that i f the c u l t u r e s  t h i s work.  i n the  basal  I t was found  i n the b a s a l medium were r e f r i g e r a t e d  at a p o i n t e a r l y i n t h e i r l o g a r i t h m i c growth phase they remained a c t i v e inoculation.  as i n o c u l a f o r as l o n g as three weeks  If, however,  after  i n c u b a t i o n of the c e l l s was  continued f o r twenty-four hours i n the b a s a l medium the c u l t u r e s were i n a c t i v e  as i n o c u l a and were presumed  to  be dead. F o r purposes of t i m e - s t u d i e s the organisms were grown i n l a r g e b r o t h tubes or i n 2 £ 0 ml graduated c y l i n d e r s . D u r i n g these s t u d i e s h o u r l y samples were removed. was measured t u r b i d i m e t r i c a l l y and the -pH of each  Growth sample  was determined by the use of a Beckman model G pH meter. The  adjustment  o f pH was accomplished by a d d i t i o n of  1.0N NaOH or 1.0N HC1 to the  cultures.  MEASUREMENT OF TOXIC ACTIVITY - A l p h a t o x i n I s measured as a l e c i t h i n a s e  by a  m o d i f i c a t i o n of the method of van Heyningen, or as a "hot-cold" hemolysin. L e c i t h i n a s e a c t i v i t y of  <* t o x i n .  Samples to be t e s t e d were c e n t r i f u g e d at 3000 r . p . m . for  one hour and the supernatant was removed and a d j u s t e d  38 to pH 6.5.  I t has been found d u r i n g t h i s work t h a t  measurement of l e c i t h i n a s e  the  a c t i v i t y i s made d i f f i c u l t  at  pH 7«0 by a f i n e white p r e c i p i t a t e o c c u r r i n g i n the medium at that p H . One m l . of the supernatant was then d i l u t e d s e r i a l l y i n a saspending f l u i d c o n t a i n i n g 0.89 gm. of c a l c i u m a c e t a t e i n one l i t r e of d i s t i l l e d water.  Two  m l . of the egg y o l k suspension of van Heyningen (92) were then added to each tube and the d i l u t i o n s p l a c e d i n a 37°G water bath f o r 15 minutes.  A f t e r a 21). hours p e r i o d  at I}.e the tubes were examined v i s u a l l y f o r t u r b i d i t y due 0  to l e c i t h i n a s e  a c t i o n and the h i g h e s t  a c t i v i t y was r e p o r t e d as the  d i l u t i o n showing  titre.  " H o t - c o l d " hemolytic a c t i v i t y of  toxin.  F o r the purpose of measuring " h o t - c o l d " hemolysis  due to the a c t i o n of  «* t o x i n the  supernatant  was p r e p a r e d as above and adjusted to pH 7«0.  Serial  d i l u t i o n s i n one m l . amount were prepared i n the c a l c i u m a c e t a t e suspending f l u i d .  The presence  the a c t i o n of both °t and Q t o x i n s ,  of c a l c i u m allows  so t h a t the " h o t - c o l d "  hemolysis may be masked by the "hot" hemolysis due t o  the  action of & t o x i n . Two m i l l i l i t r e s of a two percent suspension sheep's r e d b l o o d c e l l s was added to each of the  of  above  d i l u t i o n s which were then incubated a t 37°c f o r 15 minutes,  39  then r e f r i g e r a t e d f o r twenty-four h o u r s . hemolysis  The degree of  a t the end of both the I n c u b a t i o n and r e f r i g -  e r a t i o n p e r i o d s was r e c o r d e d . "Hot" hemolysis  a c t i v i t y of  6 toxin.  To measure the "hot" hemolysis of  due t o the  action  & t o x i n the supernatant i s p r e p a r e d as o u t l i n e d above  and a d j u s t e d to pH 7*0.  S e r i a l d i l u t i o n s were made i n a  phosphate b u f f e r c o n t a i n i n g 73• 6 gms of Na2HP0J^ and 27.1  gms of KR^POj^ per l i t r e o f w a t e r .  suspension  A two percent  of sheep's b l o o d was t h e n added and the  d i l u t i o n s were incubated f o r one hour at 3 7 ° c no c a l c i u m i s •*»  added to t h i s  Since  system there i s no a c t i o n of  toxin. The sheep's b l o o d used i n these s t u d i e s was  used When f r e s h or was p r e s e r v e d i n A l s e v e r ' s made up as  solution  followst Dextrose Na CI Trisodium c i t r a t e C i t r i c acid  - 2.05% - 0*k2% - 0.80$ - 0.055$  BACTERIAL STRAIN The BP6K s t r a i n and the "W" s t r a i n of C l o s t r i d i u m perfringefiBwere used throughout t h i s work.  The "W" s t r a i n  was obtained from the Connaught L a b o r a t o r i e s i n T o r o n t o .  RESULTS Since enzymic d i g e s t s of animal t i s s u e had p r e v i o u s l y been shown to be good s u b s t r a t e s f o r * t o x i n p r o d u c t i o n by C l o s t r i d i u m p e r f r i n g e n s  a s e r i e s of  such  d i g e s t s which had been p r e p a r e d by M r . C u r r i e and by D r . Duff were added to the b a s a l medium. was measured at s i x t e e n h o u r s .  F o r each p r e p a r a t i o n  a c a l c u l a t i o n was made to determine equivalent beef.  Toxin production  the volume of  digest  to one gram dry weight of the o r i g i n a l ground  T h i s u n i t w i l l be r e f e r r e d to as  the gram e q u i v a l e n t  or G . E . Table k The e f f e c t of the a d d i t i o n of v a r i o u s enzymic d i g e s t s of ground beef on * t o x i n product i n the b a s a l medium. Digest ("3) P e p t i c d i g e s t ( C u r r i e ) (k) T r y p t i c d i g e s t (Costerton) (5) T r y p t i c d i g e s t (Duff) (6) T r y p t i c d i g e s t ( C u r r i e ) (7) P e p t i c and T r y p t i c d i g e s t (Duff) (8) P e p t i c and T r y p t i c d i g e s t ( C u r r i e ) (9) P a n c r e a t i n d i g e s t (Duff) (10) P a n c r e a t i n d i g e s t ( C u r r i e ) ik)^Papain d i g e s t (Duff) (12)Papain d i g e s t ( C u r r i e )  G.E./ml.  * Toxin *  l/6 l/6 l/6 l/6 l/6 l/6 l/6 l/6 l/6 l/6  1:96 1:96 1:96 1:96 1:96 1:96 1:96 1:96 1:96 1:192  * The l e c i t h i n a s e a c t i v i t y of •*• t o x i n i s expressed h i g h e s t d i l u t i o n i n which t u r b i d i t y i s e v i d e n t . As w i l l be seen from Table Ij. the method of employed has l i t t l e the r e s u l t a n t  e f f e c t upon the t o x i g e n i c  digest.  as  the  digestion  capacity  of  41  THE RELATION OP DIGEST CONCENTRATION TO * TOXIN PRODUCTION Various concentrations  of enzymic d i g e s t were  added to the b a s a l medium i n order to e s t a b l i s h a minimum concentration f o r toxigenic  activity.  Table f> shows the  e f f e c t upon t o x i n p r o d u c t i o n i n the b a s a l medium of a d d i t i o n of the f o l l o w i n g c o n c e n t r a t i o n s  the  of the p a n c r e a t i c  d i g e s t of ground beef prepared by D r . D u f f . Table $ The e f f e c t of v a r i o u s c o n c e n t r a t i o n s production.  of d i g e s t upon <* t o x i n  G.E./ml.  c< t o x i n  1/2  (growth i n h i b i t e d )  1:96 1:96 1:96 l:2i+  l/k  1/8 1/16 1/32  nil nil  1/64  Basal Control  S i m i l a r r e s u l t s were obtained u s i n g a t r y p t i c d i g e s t except t h a t i n h i b i t i o n of growth was not seen i n c o n c e n t r a t i o n s below 2 G . E . t h a t the i n h i b i t o r y f a c t o r i s Since i n this  of  * t o x i n was measured at  g i v e no i n d i c a t i o n of the  «*- t o x i n p r o d u c t i o n and d e n a t u r a t i o n .  a time study i l l u s t r a t e d i n f i g u r e l 4  tabulated  indicate  dialyzable.  experiment  s i x t e e n hours the r e s u l t s p i c t u r e of  Further studies  above.  overall  The r e s u l t s  augment those  k2 DIALYSIS OP THE DIGESTS The e x i s t e n c e of a d i a l i z a b l e t o x i g e n i c had been r e p o r t e d by C u r r i e ; Papenhiemer: (2)  (12)  factor  by Adams, Hendee and  and by Rogers and K n i g h t ,  (80)  In view  of these r e p o r t s two t r y p t i c d i g e s t s and one p e p t i c were d i a l y z e d i n 20 m l . amount a g a i n s t water a t ;J+° »  distilled  A c u l t u r e i n G . P . B . I . was used as a t o x i n -  C  p r o d u c t i o n c o n t r o l to guard a g a i n s t variation.  1000 m l . of  digest  the e f f e c t s of b a c t e r i a l  F o r the purpose of e s t a b l i s h i n g  term f o r the m a t e r i a l l e f t  a  suitable  i n the sac a f t e r d i a l y s i s  m a t e r i a l w i l l be r e f e r r e d t o as the  this  dialyzant.  Table 6 The e f f e c t of a t r y p t i c d i g e s t and i t s d i a l y z a t e and d i a l y z a n t upon t o x i n p r o d u c t i o n i n the b a s a l medium. G.E./ml.  •  Complete Digest no growth • s l i g h t growth 1:96 1:96  1/2 1/16 1/32  iM  Basal Control G . P . B . I . Control  It  nil 1:192  From these f i g u r e s i n h i b i t o r y substance p r e s e n t The t o x i g e n i c  it  is  •c T o x i n Dialyzant  DIalysate  1:96 1:96 1:96 1:1+8 1:21+ 1:6 nil 1:192  no growth no growth 1:96 1:1+8 nil nil nil 1:192  at T « J t r >  seen t h a t a growth  i n the d i g e s t i s  c a p a c i t y of the d i a l y z a t e  is  dialyzable.  found t o be much  l e s s than t h a t of the d i a l y z a n t which r e t a i n s most of a c t i v i t y of the  digest.  the  43 I n a d d i t i o n to the above experiment activities  of the d i a l y z a t e  were determined.  the  toxigenic  and d i a l y z a n t of a p e p t i c  A p o o l e d inoculum was used i n  experiment to c o u n t e r a c t the i n f l u e n c e  digest  this  of b a c t e r i a l v a r i a t i o n  i n the t e s t s t r a i n . Table 7 The e f f e c t of the d i a l y z a t e and d i a l y z a n t of a p e p t i c upon * t o x i n p r o d u c t i o n i n the b a s a l medium. XJ.E./ml.  * Toxin Dialyzant  1/2 1/4 1/8 1/16 1/32 1/64  1:6 1:6  nil nil nil nil nil  nil nil nil  The experiment demonstrates  i n this  Dialyzate  1:1+8 1:48 1:48 1:12  Basal Control  significant  that there i s  no  c o n c e n t r a t i o n of a d i a l y z a b l e t o x i g e n i c  factor  digest. Next,  the e f f e c t  a time study was undertaken to  of a t r y p t i c d i g e s t and I t s  «<• t o x i n .  determine  d i a l y z a t e and  d i a l y z a n t upon both the p r o d u c t i o n and the of  digest  denaturation  Figure 5 The e f f e c t of a t r y p t i c d i g e s t and i t s d i a l y z a t e and d i a l y z a n t upon * t o x i n p r o d u c t i o n i n the b a s a l medium. 1.72-  3SDUI  |:48-  •  » .  a  /  Leci  JZ  |:241:12-  4  10  18  Hours  Time * d i a l y z a t e 1 G.E. p e r ml. o d i a l y z a n t 1 G.E. p e r ml. X digest 1 G.E. p e r ml. Figure 5 i l l u s t r a t e s  that one or more f a c t o r s a r e  p r e s e n t i n the d i g e s t which e i t h e r p r o t e c t the <* t o x i n from d e n a t u r a t i o n o r enhance i t s p r o d u c t i o n or both.  This  a c t i v i t y i s p r e s e n t i n the d i a l y z a n t t o a g r e a t e r extent than i n the d i a l y z a t e . THE TOXIGENIC ACTIVITY OF BEEF HEART AND BEEF LIVER T h i s study was undertaken t o compare the t o x i g e n i c p r o p e r t i e s of beef h e a r t and l i v e r w i t h the beef muscle used i n the above experiments.  G.P.B.I. medium was made  up u s i n g beef h e a r t and beef l i v e r i n the p l a c e o f ground muscle.  The p r o d u c t i o n o f  t o x i n i n both o f these media  was one-half of the t i t r e produced i n the c o n v e n t i o n a l  G.P.B.I.  P o r t i o n s of ground h e a r t and l i v e r were allowed to i n f u s e w i t h tap water o v e r n i g h t a t i|_°c •>  These  Infused  p r e p a r a t i o n s and corresponding u n i n f u s e d samples were then digested with pancreatine Table 8 The e f f e c t of p a n c r e a t i c d i g e s t s of beef h e a r t and l i v e r upon «* t o x i n p r o d u c t i o n i n the b a s a l medium. Digest Pancreatic Pancreatic Pancreatic Pancreatic Pancreatic  digest digest digest digest digest  <* T o x i n  of of of of of  Infused beef h e a r t u n i n f u s e d beef h e a r t i n f u s e d beef l i v e r u n i n f u s e d beef l i v e r i n f u s e d beef muscle  Table 8 i l l u s t r a t e s beef h e a r t or beef  that p a n c r e a t i c digests of  l i v e r are approximately e q u i v a l e n t  those of beef muscle i n t o x i g e n i c THE EFFECT OF DEXTRIN UPON Adams and Hendee (1) of  1:96 1:96 1:96 1:192 1:96  to  activity.  TOXIN PRODUCTION had found t h a t the  substitution  d e x t r i n f o r g l u c o s e i n t h e i r complex medium had i n c r e a s e d t o x i n p r o d u c t i o n as much as 100 p e r c e n t .  Accordingly  the e f f e c t of d e x t r i n upon «>ctoxin p r o d u c t i o n i n the b a s a l medium and i n the b a s a l medium w i t h a d i g e s t of beef was  muscle  investigated. Table 9  The e f f e c t of the s u b s t i t u t i o n of d e x t r i n f o r g l u c o s e i n the b a s a l medium upon e^-toxin p r o d u c t i o n . Glucose  Dextrin  1.2$  1.2$ 1.2$ -  1.2$ 1.2$  Tryptic Digest  1/12  G.E.  of Beef Muscle  ©cToxin nil  1:48 nil  1:48  (Table 8 continued) Glucose  Dextrin  1.2$  1.2$ 1.2$  1+6 T r y p t i c Digest  1/12 1/12  of Beef Muscle  Toxin  1:192  G.E. G.E.  1:1+8  The above work, which was repeated w i t h i d e n t i c a l results,  shows t h a t i n the absence of glucose, d e x t r i n serves  to i n c r e a s e  <*• t o x i n p r o d u c t i o n as measured at 16 h o u r s .  Time s t u d i e s undertaken l a t e r i n t h i s work have f u r t h e r e x p l a i n e d the e f f e c t See f i g u r e  of d e x t r i n upon <*toxin p r o d u c t i o n .  13.  A s e r i e s of c u l t u r e s  i n which d e x t r i n had been  s u b s t i t u t e d f o r g l u c o s e showed t h a t growth of the  organisms  became much f a s t e r  strongly  a f t e r the  suggested the development became more r a p i d , disappeared.  It  first culture.  This  of an induced enzyme.  c< t o x i n ,  As growth  as measured at 16 h o u r s ,  then became evident  that the e f f e c t  d e x t r i n was that of a l e s s a v a i l a b l e energy s o u r c e , presence  of the  of which slows the e n t i r e c e l l u l a r metabolism.  In t h i s manner the peak of t o x i n p r o d u c t i o n may c o i n c i d e more c l o s e l y w i t h the time at which the t o x i n i s  measured.  THE EFFECT OF BACTERIAL VARIATION ON <* TOXIN PRODUCTION I t was found d u r i n g t h i s work that c o n t r o l s guard a g a i n s t m i s i n t e r p r e t a t i o n due to t o x i g e n i c  to  variations  i n the t e s t organism had c o n t i n u a l l y to be i n c l u d e d i n s t u d i e s of  << t o x i n p r o d u c t i o n .  No c h a r a c t e r i s t i c  pattern  was f o l l o w e d i n the v a r i a t i o n s i n t o x i n p r o d u c t i o n by the s t r a i n s used i n t h i s l a b o r a t o r y .  While the l e c i t h i n a s e  t i t r e of one s t r a i n i n  G . P . B . I . had d e c l i n e d from 1:96  to n i l a f t e r 36  transfers  i n the b a s a l medium, another s t r a i n had i n c r e a s e d toxigenic  a c t i v i t y after  in  the same number of p a r a l l e l  transfers. Upon p l a t i n g a l l the s t r a i n s of C l o s t r i d i u m perfringensat  our d i s p o s a l ,  colonies  of three  distinct  m o r p h o l o g i c a l types c o u l d e a s i l y be d i s t i n g u i s h e d by the use of a colony microscope w i t h an i n d i r e c t l i g h t These c o l o n i e s very c l o s e l y  source.  resembled those of C l o s t r i d i u m botulinum (Q^i$  and the terminology c o i n e d f o r that  organism  w i l l be used h e r e . Opaque - T r a n s l u c e n t ( O T ) .  These c o l o n i e s  opaque and t r a n s l u c e n t  appear  i n an a l t e r n a t i n g  mosaic p a t t e r n which changes i n shape and i n p o s i t i o n as the p l a t e effect  is  rotated.  i s caused by an alignment of  This the  c e l l s on t h e i r l o n g e r a x i s by attachment ( 8 5 ) end to end to form a " f i n g e r p r i n t " p a t t e r n . Translucent ( T ) .  These c o l o n i e s  are e n t i r e l y t r a n s - -  l u c e n t and are seen to be f l a t t e r OT c o l o n i e s  although not as f l a t  as the T c o l o n i e s  than the and s p r e a d i n g  of C l o s t r i d i u m b o t u l i n u m .  The c e l l s i n these c o l o n i e s  are not arranged  i n any p a t t e r n but are randomly d i s t r i b u t e d . Opaque ( 0 ) .  These c o l o n i e s  are opaque and o f t e n  s l i g h t l y y e l l o w i n the l i g h t used f o r these  1+8 observations.  A f a i n t mosaic  pattern  which i n c r e a s e s i n d e f i n i t i o n near the edges of the colony i s  always seen i n 0  c o l o n i e s d e r i v e d from an OT s t r a i n .  While  the only spores seen are i n c o l o n i e s of type they do not c o n s i s t e n t l y and are o f t e n  this  c o n t a i n spores  non-sporulating.  While the c o l o n i a l morphology of some s t r a i n s  has  been seen to remain s t a b l e throughout more than one hundred transfers  i n the b a s a l medium other s t r a i n s have  from pure T to pure OT c u l t u r e s  i n four  changed  transfers.  C u l t u r e s of these three m o r p h o l o g i c a l types of the organism In G . P . B . I . were i d e n t i c a l i n t h e i r and o v e r a l l b i o c h e m i c a l b e h a v i o r . sacchrolytic differentiation  appearance  Thus the p r o t e o l y t i c  and  seen i n C l o s t r i d i u m botullnum  i s not apparent i n t h i s organism. these three types was p o s s i b l e  No d i f f e r e n t i a t i o n  by the degree of  of  hemolysis  on b l o o d agar p l a t e s . During these s t u d i e s a p e c u l i a r c o l o n i a l here r e f e r r e d to as a "halo" c o l o n y , was seen.  type,  intermittently  The c e n t r a l p o r t i o n of such c o l o n i e s c o n s i s t e d  a normal T or OT growth,  s h a r p l y d e l i n e a t e d by a  which separated i t from an outer f r i n g e of growth, of the opposite m o r p h o l o g i c a l t y p e . were not s t a b l e and would r e v e r t transfer,  of  depression usually  The "halo" c o l o n i e s  to normal morphology upon  although some s t r a i n s were developed which showed  an e x c e p t i o n a l l y h i g h i n c i d e n c e o f t h i s c o l o n i a l morphology. Table 10 i l l u s t r a t e s  the f i n d i n g that these c o l o n i e s are  i n d i c a t i v e of a greatly increased toxigenic G.P.B.I.  T h i s t a b l e does not represent  activity in  the r e s u l t  o f the  mixing o f phases but only r e p o r t s the approximate p r o p o r t i o n of v a r i o u s c o l o n i a l morphologies seen when each t e s t  culture  was p l a t e d . T a b l e 10 The e f f e c t  of v a r i a t i o n upon  toxin production i n G . P . B . I .  C o l o n i a l Morphology  <* T o x i n  (1) £ 0 $ "Halo* c o l o n i e s . [|.0$ C o l o n i e s resembling "halo" c o l o n i e s but without the d e l i n e a t i n g d e p r e s s i o n . 10$ 0T c o l o n i e s . (2) 30$ "Halo" c o l o n i e s . 50$ E x c e p t i o n a l l y opaque 0T c o l o n i e s . . 20$ T c o l o n i e s . ( £ ) 100$ "Halo" c o l o n i e s . (Ij.) 30% "Halo" c o l o n i e s . 50$ E x c e p t i o n a l l y opaque 0T c o l o n i e s . 20$ T c o l o n i e s . (5) 1+0$ "Halo" c o l o n i e s . 30$ Opaque 0T c o l o n i e s . 30$ T c o l o n i e s . (6) 20$ "Halo" c o l o n i e s . 50$ Opaque 0T c o l o n i e s 30$ T c o l o n i e s (7) 100$ P l a t T c o l o n i e s w i t h an evenly d i s t r i b u t e d opaque tendency. (8) 100$ C o l o n i e s resembling the "halo" colony but w i t h o n l y a few shallow delineating depressions. (9) 80$ C o l o n i e s resembling the "halo" colony but w i t h only a few shallow d e l i n e a t i n g depressions. - 20$ 0T c o l o n i e s w i t h a w e l l - d e f i n e d coarse mosaic p a t t e r n . (10) 1+0$ E x c e p t i o n a l l y w e l l - d e f i n e d "halo" colonies. 1+0$ T c o l o n i e s . 20$ Opaque 0T c o l o n i e s . 1  S i n c e the h i g h e s t l e c i t h i n a s e  titre  1:1536  1:768 1:1536 1:3076 1:1536 1:1536  1:192 1:3072 1:3072  1:1536  i n G.P.B.I.  50 p r e v i o u s l y seen d u r i n g t h i s work was results  indicate that the factors  1:381+  responsible f o r  p r o d u c t i o n of the "halo" colony g r e a t l y production.  exceptional the  stimulate^toxin study was  the  same medium as that p r e v i o u s l y used i n t h i s p r o j e c t .  It  is  The G . P . B . I .  these  used d u r i n g t h i s  seen t h a t the only c u l t u r e p r o d u c i n g «*. t o x i n i n the  titre  that had come to be accepted i n G . P . B . I .  which d i d not g i v e r i s e to "halo"  colonies.  Subsequently the T and OT phases of s t r a i n s were p u r i f i e d and t h e i r t o x i g e n i c i n v a r i o u s media. of  was one  several  abilities  Time s t u d i e s showed t h a t  tested  the T phase  the organism produces no «<. t o x i n i n the b a s a l medium  but i s  capable of normal t o x i n p r o d u c t i o n i n the  medium w i t h added enzymic d i g e s t of beef muscle. other hand the w e l l - d e f i n e d  OT c u l t u r e s  basal On the  t e s t e d produced  t o x i n i n the b a s a l medium and i n the b a s a l medium p l u s digest.  However, one OT c u l t u r e which grew i n  colonies  which were somewhat opaque and i n which the mosaic p a t t e r n was f a i n t l y o u t l i n e d produced no t o x i n i n the b a s a l medium. No capsules  c o u l d be seen i n any of these c u l t u r e s .  In view of the r e s u l t s  i l l u s t r a t e d i n table  the p u r i f i e d phases were mixed and t e s t e d f o r production.  10  eC t o x i n  T h i s mixture of c u l t u r e s produced no  s t i m u l a t i o n of t o x i n p r o d u c t i o n i n the b a s a l medium w i t h enzymic d i g e s t or i n G . P . B . I .  The e f f e c t of mixing the  T and OT phases of t h i s organism i n the b a s a l medium was determined by means of a time study i l l u s t r a t e d by f i g u r e  17*  51 TIME STUDIES  All  the r e s u l t s  d e t a i l e d to t h i s p o i n t have  been obtained u s i n g the techique of C u r r i e and of many p r e v i o u s workers In t h i s f i e l d who measured production after  s i x t e e n hours of i n c u b a t i o n .  s e c t i o n s d e a l w i t h experiments  u s u a l l y of one h o u r ,  d u r i n g the growth of the organisms. are r e f e r r e d t o h e r e i n as time Figure 6 represents  The f o l l o w i n g  i n which t o x i n p r o d u c t i o n  was measured at s t a t e d i n t e r v a l s ,  that  toxin .  These  experiments  studies.  the experiment which showed  t o x i n i s produced i n the b a s a l medium.  experiment was repeated as a c o n t r o l i n each  This -'toxin  p r o d u c t i o n experiment undertaken d u r i n g t h i s work. I d e n t i t y of the l e c i t h i n a s e  produced i n the b a s a l medium.  When 0.2 u n i t s / m l . of a n t i t o x i n was added to t o x i n measuring system no l e c i t h i n a s e demonstrated.  The " h o t - c o l d " hemolysis  activity  the  could.be  a c t i v i t y of  the  c u l t u r e i n the b a s a l medium was seen to p a r a l l e l the lecithinase  activity.  Centrifugates  of the c u l t u r e i n the b a s a l medium  were i n j e c t e d i n t o mice and i t was found that  a  lecithinase  t i t r e of 1 s2if. corresponded to 5 LD/50 of the t o x i n p e r m l . I t was determined t h a t 0.3 u n i t s of the s p e c i f i c  antitoxin  p r o v i d e d p r o t e c t i o n f o r a mouse I n j e c t e d w i t h 5 LD/50 of the t o x i n .  T h e r e f o r e 0.060- u n i t s o f a n t i t o x i n  protect  52 against  one LD/50 of  ««-toxin.  Since i t had thus been shown that a l e c i t h i n s p l i t t i n g , h e m o l y t i c and l e t h a l f a c t o r i s present i n the supernatants o f c u l t u r e s i n the b a s a l medium and that f a c t o r i s n e u t r a l i z e d by the s p e c i f i c concluded that  «t t o x i n i s  The Denaturaticn of  a n t i t o x i n I t was  indeed produced i n the b a s a l medium.  * Toxin i n c e l l - f r e e  F i g u r e 8 shows the e f f e c t pH upon the d e n a t u r a t i o n of centrifugate  Centrifugates  of temperature and of  « < t o x i n i n the  cell-free  of a c u l t u r e at i t s maximum t o x i n p r o d u c t i o n  in  the b a s a l medium.  of  a pH value above 7«0 a c t to decrease  denaturation.  R e f r i g e r a t i o n and the maintenance the r a t e of  toxin  The i n c u b a t i o n of d u p l i c a t e c u l t u r e s i n a  M a c k i n t o s h - F i l d e s j a r and under a e r o b i c c o n d i t i o n s that a n a e r o b i o s i s its  this  showed  a f f e c t s n e i t h e r t o x i n p r o d u c t i o n nor  denaturation.  The E f f e c t of pH adjustment upon ^ t o x i n p r o d u c t i o n i n the b a s a l medium. During t h i s  experiment the pH of the c u l t u r e was  measured at f i f t e e n minute i n t e r v a l s and the r e q u i r e d adjustment made by a d d i t i o n o f 1.0N  NaOH. F i g u r e 9 shows  t h a t the d e n a t u r a t i o n of  r e t a r d e d by the main-  * toxin is  tenance of the pH at a value above 7.0.  During a l l pH  adjustment s t u d i e s i t was observed t h a t the o p t i c a l of  density  the c u l t u r e s d e c l i n e s f o l l o w i n g the l o g a r i t h m i c growth  53 phase.  Often t h i s decrease i n o p t i c a l d e n s i t y  abrupt than t h a t shown i n f i g u r e  i s more  9.  The E f f e c t of Glucose C o n c e n t r a t i o n upon * t o x i n P r o d u c t i o n . The c o n c e n t r a t i o n of glucose a f f e c t s p r o d u c t i o n i n the b a s a l medium i n t h a t i t  toxin  determines  b o t h the amount of growth and the pH r e s u l t a n t from that growth.  F i g u r e 10 shows the c h a r a c t e r i s t i c p r o d u c t i o n and  r a p i d d e n a t u r a t i o n of figures  << t o x i n i n the b a s a l medium.  However,  11 and 12 show the slower t o x i n d e n a t u r a t i o n and  the d e c l i n e i n o p t i c a l d e n s i t y p r e v i o u s l y seen i n pH adjustment  studies.  The E f f e c t of D e x t r i n upon Growth and  < = < • toxin Production.  In the experiment r e p r e s e n t e d i n f i g u r e 13 values f o r the  t o x i n t i t r e of samples  the  taken before  ten  hours are omitted because i t has s i n c e been found that a f a l s e opalescence develops 6.5«  In subsequent  i f the pH of the sample i s  over  s t u d i e s pH adjustments were made and  the m a j o r i t y of the e a r l i e r experiments were r e p e a t e d . It i s  shown i n t h i s  and the r e s u l t a n t  decline  experiment t h a t the growth  i n pH are slowed by the  sub-  s t i t u t i o n of d e x t r i n f o r glucose i n the b a s a l medium. When the pH of a c u l t u r e i n the b a s a l medium which c o n t a i n s d e x t r i n i s maintained at a value above 7«0 a decrease i n the r a t e of figure 9  * t o x i n d e n a t u r a t i o n s i m i l a r to that seen i n  results.  5k The E f f e c t of V a r i o u s C o n c e n t r a t i o n s of a T r y p t i c  Digest  of Beef Muscle upon •*Toxin P r o d u c t i o n . F i g u r e lk and i t s of  Illustrates  the p r o d u c t i o n of  d e n a t u r a t i o n In the presence  a t r y p t i c d i g e s t of beef muscle.  d i g e s t g r e a t e r than l / l 6  G.E./ml.  ot t o x i n  of v a r y i n g c o n c e n t r a t i o n s C o n c e n t r a t i o n s of  had the same e f f e c t  t h a t c o n c e n t r a t i o n on the d e n a t u r a t i o n of the t o x i n . maintenance of the pH of these c u l t u r e s at a l e v e l 7.0  does not a f f e c t  its  r a t e of d e n a t u r a t i o n .  the as The  above  t o x i n p r o d u c t i o n but f u r t h e r decreases  The E f f e c t of Ammonium S u l p h a t e - P r e c i p i t a t e d P r o t e l m  upon  * t o x i n P r o d u c t i o n i n the B a s a l Medium. The p r o t e i n s remaining from a t r y p t i c d i g e s t of beef muscle were p r e c i p i t a t e d by s a t u r a t i o n w i t h ammonium sulphate.  The p r e c i p i t a t e d p r o t e i n and the ammonium  sulphate - s a t u r a t e d supernatant were then t e s t e d f o r t h e i r e f f e c t upon < t o x i n p r o d u c t i o n i n the b a s a l medium. An ammonium sulphate - s a t u r a t e d s o l u t i o n of the had  supernatant  to be used s i n c e d i a l y s i s would have removed the  d i a l y z a b l e components  of the d i g e s t .  3.6  G.E./ml.  of  this  supernatant allowed good growth of the organisms but d i d not enhance t o x i n p r o d u c t i o n n o r p r o t e c t The e f f e c t p r o t e i n s upon  the t o x i n formed.  of the i n c l u s i o n of the p r e c i p i t a t e d  toxin production is  i l l u s t r a t e d in figure  While the gram equivalence  values quoted i n t h i s  figure  are not s t r i c t l y a c c u r a t e ,  due to the d i f f i c u l t y of  15.  r e c o v e r i n g a l l of the v i s c o u s p r o t e i n p r e c i p i t a t e , seen t h a t t h i s f r a c t i o n h o l d s most of the a c t i o n of the d i g e s t ,  effect  is  toxin-protective  A marked s i m i l a r i t y between the  a c t i v i t y o f l/2f> G . E . / m l . adjustment  it  of the p r o t e i n and the pH  seen i n f i g u r e 9 i s  immediately  obvious.  The E f f e c t of the Omission of C e r t a i n Amino A c i d s upon <*• t o x i n P r o d u c t i o n . The f o l l o w i n g study was undertaken to  determine  the e f f e c t of the omission of those amino a c i d s  not  c o n s i d e r e d by Boyd, Logan and T y t e l l  necessary  for  growth upon  (11)  to be  toxin production. Table  11  The e f f e c t of the omission of c e r t a i n amino a c i d s upon ^ toxin production. Amino a c i d s omitted  Growth  Control Normal DL-aline, glycine. Nil DL-aspartic acid. Normal L-proline, hydroxy-L-proline. Normal Glycine, DL-alanine, DL-aspartic acid, L-proline, hydroxy-L-proline. N i l Thus i t  is  <* T o x i n P r o d u c t i o n Normal Normal Normal  seen t h a t there are no amino a c i d s  i n the b a s a l medium which are r e q u i r e d f o r  << t o x i n p r o d u c t i o n  but not f o r the growth of the organism. The E f f e c t of C y s t i n e upon « t o x i n P r o d u c t i o n i n the B a s a l Medium. The c o n c e n t r a t i o n of c y s t i n e  i n the b a s a l medium  56 has been shown to a f f e c t shows the e f f e c t  «< t o x i n p r o d u c t i o n .  F i g u r e 16  of the normal 10 mg./lOO m l . c o n c e n t r a t i o n  used i n the b a s a l medium.  Higher c o n c e n t r a t i o n s of  cystine  y i e l d l e s s <<• t o x i n , w h i l e 6 mg./lOO m l . appears to be o p t i m a l w i t h r e g a r d to t o x i n p r o d u c t i o n .  At l e v e l s below  5 mg/100 m l . the p r o d u c t i o n of t o x i n i s decreased but denaturation i s  c o n s i d e r a b l y slower at these lower  c o n c e n t r a t i o n s so t h a t the peak of t o x i n p r o d u c t i o n i s lower but of longer d u r a t i o n . A r e p e t i t i o n of the above experiment w i t h the maintenance of a c u l t u r a l pH above 7»0 showed no i n c r e a s e i n t o t a l t o x i n p r o d u c t i o n a t the o p t i m a l c y s t i n e  level.  However at h i g h e r c y s t i n e c o n c e n t r a t i o n s pH adjustment served to i n c r e a s e t o t a l <*• t o x i n p r o d u c t i o n as w e l l as decrease  the r a t e of i t s  to  denaturation.  The E f f e c t of I r o n C o n c e n t r a t i o n upon <<Toxin P r o d u c t i o n i n the B a s a l Medium. F o r the purpose of t h i s study i r o n was removed from the b a s a l medium by a b s o r p t i o n on aluminum o x i d e . I t was e s t a b l i s h e d t h a t the second t r a n s f e r of the organism on t h i s absorbed medium f a i l e d to grow.  Therefore,  inoculum used i n t h i s work c o n s i s t e d of the f i r s t  the  transfer  of the t e s t s t r a i n i n the absorbed medium to minimize a " c a r r y - o v e r " of the element under these c o n d i t i o n s . Table 12 i l l u s t r a t e s  the e f f e c t  of a d d i t i o n s of  i r o n to the absorbed medium upon growth and < t o x i n production.  57 Table 12 The e f f e c t o f i r o n c o n c e n t r a t i o n i n the b a s a l medium. Iron Addition  upon <* t o x i n p r o d u c t i o n  Growth  0 mg/ml 0.012 mg/ml 0.02k mg/ml O.Olj.8 mg/ml O.llm. mg/ml 0.3oq. mg/ml  none slight good good good good  0.860 mg/ml 1.730 mg/ml  good slight  «<* T o x i n P r o d u c t i o n  normal normal. normal normal w i t h more r a p i d d e n a t u r a t i o n than i s u s u a l none  Thus i t i s seen that the o p t i m a l c o n c e n t r a t i o n o f i r o n f o r << t o x i n p r o d u c t i o n i s 0.02l|. mg/ml. t o 0.lijJ+ mg/ml. and that lower and extremely h i g h c o n c e n t r a t i o n s o f i r o n i n h i b i t growth. upon d e n a t u r a t i o n  An i n f l u e n c e  iron  concentration  i s suggested and i t i s t h e r e f o r e  t h a t the pH of those c u l t u r e s and low t o t a l  of higher  showing r a p i d  noted  denaturation  t o x i n p r o d u c t i o n were n o t abnormally low.  The I n f l u e n c e o f B a c t e r i a l V a r i a t i o n upon * T o x i n P r o d u c t i o n i n the B a s a l Medium. T h i s study was undertaken to determine  the e f f e c t  on «c t o x i n p r o d u c t i o n i n the b a s a l medium of mixing the T and OT phases o f the organism.  F i g u r e 17 shows t h a t the  mixed phases produced approximately produced by the OT c u l t u r e .  one-half  of the t o x i n  58  DISCUSSION I t has been shown that C l o s t r i d i u m p e r f r i n g e n s produces a l e c i t h i n a s e Logan and T y t e l l .  i n the b a s a l medium of Boyd,  This l e c i t h i n a s e  i s r a p i d l y denatured  as growth proceeds and d e n a t u r a t i o n i s complete b e f o r e c o n v e n t i o n a l time f o r the measurement of For t h i s r e a s o n the measurement of  the  <* t o x i n a c t i v i t y .  «< t o x i n a f t e r  hours i n the b a s a l medium had always y i e l d e d  sixteen  negative  results. An i n v e s t i g a t i o n has shown t h a t the of the l e c i t h i n a s e  activity  i s p a r a l l e l e d by l e t h a l i t y and by the  a b i l i t y of the supernatant to induce " h o t - c o l d " hemolysis i n sheep's e r y t h r o c y t e s . activities  is  S i n c e each of the  i n h i b i t e d by the presence  above-mentioned  of the  specific  antiserum to C l o s t r i d i u m p e r f r i n g e n s we have concluded t h a t the agent of these a c t i v i t i e s The f o r m a t i o n of  i s a c t u a l l y <* t o x i n .  << t o x i n Is d i r e c t l y r e l a t e d  to  the l o g a r i t h m i c growth phase and i t has been shown that the maximum t i t r e of the t o x i n i s p r e s e n t d u r i n g the most a c t i v e growth. a slight  At the end of the l o g a r i t h m i c growth phase  i n c r e a s e i n the pH of the b a s a l medium i s  i n v a r i a b l y seen.  A s i m i l a r i n c r e a s e i s r e p o r t e d by Gros  and ; Macheboeuf,(28)  as a r e s u l t of the l i b e r a t i o n of  59 ammonia d u r i n g the p r e l i m i n a r y a u t o l y s i s Clostridium  of the c e l l s of  sporogenes.  The omission of L p r o l i n e , h y d r o x y - L - p r o l i n e or D L - a s p a r t i c a c i d from the b a s a l medium does not the  affect  •< t o x i n p r o d u c t i o n or the growth of the organism*  Since Woods and Trim (101)  have found that these amino  a c i d s are p r e s e n t In the p r o t e i n s of the organism i t must be assumed that they are formed by s y n t h e s i s or by t r a n s amination. Prom v a r i o u s experiments r e p o r t e d above,  it  obvious that «c t o x i n i s v e r y r e a d i l y denatured i n the natants  of c u l t u r e s from the b a s a l medium.  enzymes the  super-  Since l i k e most  of the C l o s t r i d i a these organisms- produce h i g h of p r o t e o l y t i c  is  concentrations  t o x i n , which i s known to be  e a s i l y denatured by p r o t e o l y t i c agents, expected to escape d e s t r u c t i o n .  (81)  The presence  cannot be of the  cells  i s not r e q u i r e d f o r the r a p i d d e n a t u r a t i o n of •<. t o x i n i f sufficient  growth of the organism i n the b a s a l medium has  been a l l o w e d .  Gonzalez (23)  has s t a t e d  that  temperatures  above 63°c or the a d d i t i o n of a 1:88,000 c o n c e n t r a t i o n of mercuric c h l o r i d e serve to i n a c t i v a t e enzymes of t h i s  the  proteolytic  organism.  Theta t o x i n i s produced i n the b a s a l medium i n amounts comparable w i t h those produced i n complex media. T h i s t o x i n i s a l s o formed d u r i n g the l o g a r i t h m i c growth phase and i s  subject to a d e n a t u r a t i o n somewhat l e s s r a p i d  60  than t h a t of the  <* t o x i n ,  has a l s o been shown t h a t the common p r o t e a l y t i c to t h a t of ft t o x i n i s  i n the b a s a l medium.  Since  it  % t o x i n i s r e a d i l y denatured by  enzymes (81) to be  a denaturation s i m i l a r  expected.  The apparent s t i m u l a t i o n of ^ t o x i n p r o d u c t i o n by the s u b s t i t u t i o n of d e x t r i n f o r glucose i n the b a s a l medium, which has been r e p o r t e d by many e a r l i e r workers,  is  e x p l a i n e d by the time study i l l u s t r a t e d i n f i g u r e 13* e f f e c t of d e x t r i n i s  The  to delay the growth of the organism  w h i l e an adaptive enzyme i s formed to break down t h i s r i n g - s h a p e d p o l y s a c c h a r i d e i n t o energy y i e l d i n g g l u c o s e units. The delay caused by d e x t r i n allowed the maximum t o x i n p r o d u c t i o n p e r i o d to c o i n c i d e more c l o s e l y w i t h time at which effect  «< t o x i n was commonly measured.  the  Thus the  of d e x t r i n i s not an a c t u a l s t i m u l a t i o n of * t o x i n  production. however,  The t o x i n p r o d u c t i o n i n these c u l t u r e s  not i d e n t i c a l w i t h t h a t i n glucose c u l t u r e s  t h a t •<toxin i s  evident  at a lower o p t i c a l d e n s i t y .  e a r l i e r t o x i n p r o d u c t i o n may r e s u l t from the  is, in This  toxigenic  a c t i v i t y of c e l l s c a r r y i n g on a " r e s t i n g " metabolism b e f o r e c e l l u l a r r e p r o d u c t i o n i s made p o s s i b l e . The .c t o x i n p r e s e n t thus r e p r e s e n t s  the r e s u l t  and d e n a t u r a t i o n . may r e s u l t  at any time i n any medium of a balance between p r o d u c t i o n  Since an i n c r e a s e  i n toxin production  i n the same maximum t o x i n l e v e l as a decrease  61 I n the r a t e of d e n a t u r a t i o n would produce, i t difficult  to d i f f e r e n t i a t e  a lower d e c l i n e i n the  often  between stimulation of p r o d u c t i o n  and i n h i b i t i o n of d e n a t u r a t i o n . the r a t e of d e n a t u r a t i o n i s  is  However, a decrease  in  o f t e n obvious as a r e s u l t  of  <*. t o x i n l e v e l a f t e r maximum p r o d u c t i o n  has been r e a l i z e d . The f a c t o r s found i n these experiments, the d e n a t u r a t i o n of  to  affect  <<• t o x i n had a l l been p r e v i o u s l y  d e s c r i b e d as i n c r e a s i n g the p r o d u c t i o n of the t o x i n i n complex media.  The d e n a t u r a t i o n of «* t o x i n i s  by the pH of the medium, by the presence material,  and by the c o n c e n t r a t i o n s  of  affected  proteinaceous  of g l u c o s e ,  cystine  and i r o n . The pH of the medium has been shown to the d e n a t u r a t i o n of  t o x i n to a great e x t e n t .  maintenance of the c u l t u r a l pH at values  affect The  above 7«0 slows  d e n a t u r a t i o n to a c o n s i d e r a b l e degree but does not stop i t .  entirely  T h i s pH c o i n c i d e s w i t h the upper l i m i t of  the  o p t i m a l pH range of the L e c i t h i n a s e - d e s t r o y i n g enzyme r e p o r t e d by Vyshepar and Krasnova.(95)  However,  workers gave the lower l i m i t of the a c t i v i t y of enzyme as pH 6.0.  these their  I t has been shown that <*toxin i s  r a p i d l y denatured at pH 5.5>» a value at which the enzyme was irareversibly i n a c t i v a t e d . possible  It  is,  above  however,  that Vyshepan and Krasnova have p u r i f i e d  only  one of a number of enzymes capable of denaturing the  toxin.  62 The c o n c e n t r a t i o n of glucose In the medium a f f e c t s <x-toxin p r o d u c t i o n i n t h a t i t resultant  c u l t u r a l pH.  a f f e c t s growth and the  Studies  of the e f f e c t  of g l u c o s e  upon t o x i n p r o d u c t i o n have shown t h a t the d e n a t u r a t i o n of the t o x i n i s more r a p i d at pH I t was seen i n these s t u d i e s , pH was a d j u s t e d , below 6.0  than at pH 6.0  or  6.5.  as i n those i n which the  t h a t f a i l u r e to produce a c u l t u r a l pH  d u r i n g growth i n the b a s a l medium r e s u l t s  d e c l i n e i n o p t i c a l d e n s i t y f o l l o w i n g the  in a  logarithmic  growth phase. S t u d i e s undertaken e a r l y i n t h i s work showed  that  the a d d i t i o n of enzymic d i g e s t s of beef muscle to  the  b a s a l medium r e s u l t e d i n the presence  after  s i x t e e n hours i n c u b a t i o n .  of  <* t o x i n  I t was shown that the  enzyme used i n the d i g e s t i o n was of l i t t l e that d i g e s t s of beef h e a r t , equal i n t h e i r a c t i v i t y .  specific  importance and  l i v e r and muscle were approximately  Later, work e s t a b l i s h e d  that  <* t o x i n  i s p r o t e c t e d from d e n a t u r a t i o n i n p r o p o r t i o n to the c o n c e n t r a t i o n of the d i g e s t added to the medium. I t has been shown that the t o x i n p r o t e c t i v e present  factor  i n the d i g e s t s i s d i a l y z a b l e to o n l y a s m a l l e x t e n t .  A thoroughly d i g e s t e d p r e p a r a t i o n must be presumed to c o n t a i n a h i g h c o n c e n t r a t i o n of s m a l l p e p t i d e s which would be d i a l y z a b l e . and p e p t i d e s  P r e c i p i t a t i o n of the remaining p r o t e i n s  of a t r y p t i c d i g e s t by s a t u r a t i o n w i t h  ammonium sulphate l e a v e s the supernatant d e v o i d of protective  a c t i v i t y while  the p r o t e i n s and l a r g e r  toxinpeptides  63 r e t a i n most of the a c t i v i t y of the d i g e s t The above f i n d i n g s , observations  itself.  together with previous  that p r o t e i n concentration i s  of importance  i n t o x i g e n e s i s i n complex media, l e a d t o the that the p r o t e i n s toxin-protective is,  therefore,  alternate  influence.  protective  In  production i s  The most obvious  a  possibility  and peptides act  f o r the enzymes which are  * toxin denaturation.  effect  similar effect  of the d i g e s t s exert  t h a t these p r o t e i n s  substrates  to be a c t i v e  further  and p e p t i d e s  conclusion  I f the  as  believed toxin-  of the d i g e s t s i s coupled w i t h the  of pH adjustment,  no i n c r e a s e i n  toxin  seen but the r a t e of d e n a t u r a t i o n  is  decreased. Prom the s t u d i e s concerning the e f f e c t  of  the  c o n c e n t r a t i o n of c y s t i n e upon << t o x i n p r o d u c t i o n i t seen that t h i s  amino a c i d i s r e q u i r e d f o r growth and  t o x i n p r o d u c t i o n by C l o s t r i d i u m p e r f r i n g e n s /lOO ml. i s w h i l e 2.5  was  o p t i m a l f o r both growth and  6mg.  cystine  t o x i n formation  mg./lOO m l . allows good growth but reduces  amount of t o x i n produced.  However, at the lower  c o n c e n t r a t i o n d e n a t u r a t i o n i s markedly slowed. concentrations  of c y s t i n e  the  cystine At h i g h e r  d e n a t u r a t i o n becomes i n c r e a s i n g l y  r a p i d u n t i l at 20 mg./lOO m l . no < t o x i n p r o d u c t i o n can be  detected. The e f f e c t of c y s t i n e upon  is  of c o n s i d e r a b l e i n t e r e s t  toxin denaturation  because of the  description  6l+ of c y s t e i n e as a c o f a c t o r of the enzyme i s o l a t e d by Vyshepan and Krasnova.(95)  C y s t i n e i s very  easily  reduced to two c y s t e i n e molecules which, a c c o r d i n g to the above a u t h o r s ,  activate  i n ot t o x i n d e n a t u r a t i o n . is  often associated  easily  enzyme  The c y s t i n e - c y s t e i n e  o x i d i z e d s u l p h y d r y l group of c y s t e i n e  There i s  system  an i n d i c a t i o n that h i g h  protects  concentrations  the d e n a t u r a t i o n o f o t t o x i n .  an i r o n c o n c e n t r a t i o n of  0.381+ m g . / m l .  mg/ml. no <* t o x i n c o u l d be d e t e c t e d .  It  aid at is  of i r o n c o n c e n t r a t i o n upon the  metabolism may be r e f l e c t e d  At  more r a p i d de-  seen than at lower c o n c e n t r a t i o n s ,  that the i n f l u e n c e  the  enzymes from i n a c t i v a t i o n by o x i d a t i o n .  of i r o n may a c c e l e r a t e  naturation is  active  w i t h p r o t e o l y t i c systems because  s i m i l a r groups on.the  0.860  the p r o t e o l y t i c  possible general  i n changes i n t o x i n p r o d u c t i o n  a n d / o r d e n a t u r a t i o n but no d i r e c t mechanism can be suggested at t h i s t i m e . not a f f e c t e d this  The c u l t u r a l pH d u r i n g growth was  by the range of i r o n c o n c e n t r a t i o n s used i n  study. The o p t i m a l c o n c e n t r a t i o n of i r o n f o r growth  from  0.021+ mg/ml.  to  0.860  mg/ml. w h i l e the o p t i m a l i r o n  c o n c e n t r a t i o n f o r << t o x i n p r o d u c t i o n The i n f l u e n c e of is,  is  Is 0.021+ mg/ml.  "  of i r o n c o n c e n t r a t i o n upon the p r o d u c t i o n  < t o x i n by C l o s t r i d i u m perfringens i n the b a s a l medium however,  not as pronounced as i n the case of  Corynebacterium d i p h t h e r l a e .  65 Although s t u d i e s i n the b a s a l medium have shown that  toxin is  a concomitant of normal growth  amounts of the t o x i n much g r e a t e r than those obtained here have been r e p o r t e d i n complex undefined organic media.(1)  It i s ,  therefore,  possible  that other and  at p r e s e n t unknown genuinely t o x i g e n i c which are capable of s t i m u l a t i n g  f a c t o r s may e x i s t  <* t o x i n p r o d u c t i o n .  P a r t of the apparent enhancement might be a t t r i b u t e d  to  the i n c r e a s e i n b a c t e r i a l numbers due to o p t i m a l n u t r i t i o n . One i s ,  however,  i n c l i n e d to doubt t h i s  interpretation  on the b a s i s of a comparison of dry weights Miller  (61+) has shown t h a t a p e p t i d a s e  h i s t i d i n e peptides  attained.  capable of  splitting  to form c e r t a i n as yet unknown products  i s r e q u i r e d f o r t o x i g e n e s i s i n C l o s t r i d i u m , it eta'nj.. •'. The T - O T n  n  phase v a r i a t i o n o r i g i n a l l y seen to  occur i n C l o s t r i d i u m botullnum has a l s o been noted i n Clostridium perfringens  T h i s phase v a r i a t i o n  affects  <»<- t o x i n p r o d u c t i o n i n the b a s a l medium but not In more complex media.  These f i n d i n g s  suggest t h a t the OT phase  has the a b i l i t y to produce «*. t o x i n from the amino a c i d s present  i n the b a s a l medium w h i l e the T phase r e q u i r e s a  more complex p r e c u r s o r f o r t o x i g e n e s i s .  The p o s s i b i l i t y  t h a t the T phase i s a more p r o l i f i c producer of an d e n a t u r i n g f a c t o r has been d i s c o u n t e d s i n c e  the mixture  of the two phases f a i l e d to show any unusual t o x i n denaturation.  toxin-  66 The occurence of the "halo" c o l o n i e s  seen d u r i n g  t h i s work was shown to be r e l a t e d i n some manner to a g r e a t l y i n c r e a s e d p r o d u c t i o n of <* t o x i n i n G . P . B . I . c i r c u l a r depressions  The  i n these c o l o n i e s are p r o b a b l y not  caused by the l i b e r a t i o n of a bacteriophage s i n c e e x i s t e n c e of a u n i f o r m " r i n g " of c e l l s bacteriophage seems improbable.  sensitive  the to  The e x i s t e n c e of a "halo"  of one phase around a colony of another cannot be accounted f o r by the c o l o n i a l p a t t e r n s u s u a l l y r e s u l t a n t from a mutation.  Andervont and Simon (If) have shown t h a t  vegetative  cells  of c e r t a i n B a c i l l u s s p e c i e s are l y s e d  when grown i n c o n t a c t w i t h spore b e a r i n g c e l l s same s p e c i e s .  of  the  Some i n t r a s p e c i f i c antagonism may c o n t r i b u t e  to t h i s phenomenon which, however, unexplained.  the  remains  essentially  67 SUMMARY  1.  A lecithinase is produced and subsequently-  destroyed during the growth of Clostridium perfringens in the chemically reproducible medium of Boyd, Logan and Tytell.  The lecithinase possesses the biological and  immunological properties of «*• toxin. 2.  Lecithinase production is markedly affected by  bacterial variations which are reflected in colonial morphology. 3«  The apparent stimulation of lecithinase production  by the substitution of dextrin for glucose is explained on the basis of delayed growth and enzyme production. ij..  The rate of lecithinase destruction is slowed  by pH adjustment, addition of proteinaceous material, and by the adjustment of the concentrations of cystine and of iron.  6a  BIBLIOGRAPHY 1. 2.  Adams, M. H. and Hendee, E . D., J . Immunol., jjl, 249, 1945. Adams, M. H . , Hendee, E. D. and Pappenheimer, A. M., J . Immunol., ,47, 503, 1941-  3.  A j l . S. Bact. Rev., 12, 263, 1955.  4.  Andervont, H. J . and Simon, C. E . , Am. J . Hyg., 386, 1924.  5.  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D. , 40+  Hours  -1:48  FIGURE II Enz y me 0.5%  GLUCOSE -I-.4  O.D. 40-  pH  7.0-  30-  -^PlH  /  \  v Optical  Density -l: 2 4  6.0-  20-  o 5.0-  10-  \ o  -1:12  Lecithinase  -I: 6  i  2  T 4  1  1  6  8  Hours  lb  12  8  FIGURE 0.25%  GLUCOSE  O.D. 404  Hours  12  Enzyme |_i: 8 4  FIGURE  Hours  13  Hours  FIGURE AMMONIUM  15  SULPHATE - PRECIPITATED  Hours  PROTEIN  FIGURE THE  EFFECT OF CYSTINE  ON <X TOXIN  Hours  16 PRODUCTION  FIGURE THE  17  INFLUENCE OF BACTERIAL VARIATION  ON C< TOXIN  PRODUCTION Enzyme  O.D. Optical Density  5 OH  1. "0 T" Phase 30-J  2. Mixed"T"and "Ot" Phases 3. "T Phase H  20H  10-  &  i  r  h — t — ?  Hours  

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