UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A contribution toward the development of chemically reproducible media for the production of CLOSTRIDIUM… Currie, John Fisher 1951

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1951_A8 C8 C6.pdf [ 3.51MB ]
Metadata
JSON: 831-1.0106583.json
JSON-LD: 831-1.0106583-ld.json
RDF/XML (Pretty): 831-1.0106583-rdf.xml
RDF/JSON: 831-1.0106583-rdf.json
Turtle: 831-1.0106583-turtle.txt
N-Triples: 831-1.0106583-rdf-ntriples.txt
Original Record: 831-1.0106583-source.json
Full Text
831-1.0106583-fulltext.txt
Citation
831-1.0106583.ris

Full Text

A CONTRIBUTION TOWARD THE DEVELOPMENT OF CHEMICALLY REPRODUCIBLE MEDIA FOR THE PRODUCTION OF CLOSTRIDIUM PERFRINGENS TOXINS by John Fisher Currie A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of Bacteriology and Preventive Medicine ¥e accept this thesis as conforming ifco the standard required from candidates for the degree of Master of Arts. Members of the Department of Bacteriology and Preventive Medicine. THE UNIVERSITY OF BRITISH COLUMBIA Ap r i l , 1951 ABSTRACT. A method for the production of a hot hemolysin by CI. perfringens in a chemically defined medium i s described. A factor essential for the production of lecithinase ( alpha toxin ) has been par t i a l l y purified from dried meat. I t i s suggested that this toxigenic factor i s a protein derivative. T h i s work was aided by grants from the National Research Cou n c i l . Acknowledgement i s also made: To Dr. D.C.B. Duff f o r h i s advice and encouragement throughout the course of t h i s i n v e s t i g a t i o n . To the Departments of Chemistry, Biochemistry, and Dairying whose co-operation increased the scope of t h i s i n v e s t i g a t i o n . To Mr. R.H. Currie f o r the preparation of the f i g u r e s . To my wife who has been a constant source of i n s p i r a t i o n and encouragement. Without her help, t h i s work would not have been p o s s i b l e . TABLE OF CONTENTS. Introduction. page. 1. Experimental Work. I . Standard Procedures. 1. Organism. 6. 2. Turbidimetric Estimation of Growth. 7. 3. Determination of Toxin T i t e r s . a. Preparation of 'Toxic Supernatants. 9. b. Hot Hemolysin 10. c. Hot-Cold Hemolysin 10. d. Lecithinase A c t i v i t y . 11. I I . Growth i n a Chemically Defined Medium. 12. I I I . Toxin Production i n the Basal Medium 1. E f f e c t of Temperature. 17. 2. Adsorption of Theta Toxin. 19. 3. L e t h a l A c t i o n . 21. 4. Summary of C h a r a c t e r i s t i c s of Toxin i n Basal Medium 22. IV. The E f f e c t of Calcium,, Magnesium, and Iron on Growth, and on Toxin production i n the Basal Medium . 23. V The E f f e c t of Toxigenic Organic Substances which Stimulate Lecithinase Production. 28. VI. Complex Factors Stimulating Lecithinase Production. 1. Toxigenic A c t i v i t y of Dextrin. 31. 2. Toxigenic A c t i v i t y of Adenosine Triphosphate. 32. 3. The E f f e c t of Yeast Extract on Growth and Toxin Production. 33. 4. Lecithinase Production i n G.P.B.I. page. 34« 5. E f f e c t of Tryptone and Proteose Peptone, i n the Basal Medium. 37. 6. E f f e c t of P a r t i c u l a t e M a t e r i a l . 39-7. Soxhlet E x t r a c t i o n . 41 • 8. Hydrolysis of Extracted meat. 4-2. 9. D i a l y s i s of the T r y p t i c Hydrolysate. 46. 10. Adsorption of the Toxigenic Factor. 4-7. 11. Chemical Tests to Determine the Nature of the Adsorbed Substances. 49. Discussion. 51. Conclusions. 59. Appendix. A. Source of CI. perfringens Cultures. 60. B. Medium of Boyd, Logan and T y t e l l . 61. C. U t i l i z a t i o n of Carbon. 62. Bibliography. 66. LIST OF FIGURES. Figure. 1. Hydrolysis of L e c i t h i n by CI. perfringens L e c i t h i n a s e . 3. Figure. 2. The Relationship of B a c t e r i a l Dry Weight to T u r b i d i t y Readings. 8. Figure. 3. Size of Inoculum. 14-. Figure. 4« Modified Growth Curve. 16. LIST OF TABLES. Table. 1. E f f e c t of carryover on toxin production. page. IS. Table. 11. Adsorption of Toxins from Reed's Medium. 20. Table. 111. Adsorption of Toxin from the Basal Medium. 20. Table. IV. L e t h a l A c t i v i t y of Toxin Produced' i n the Basal Medium. 21. Table. V. Varying Concentrations of Inorganic Ions. 25. Table. VI. E f f e c t of Toxigenic Organic Substances. 29. Table. V l l . A d dition of Yeast Extract' to Basal Medium. - 33. Table. IX. Toxin Production i n G.P.B.I. F r a c t i o n s . 36. Table. X. Tryptone and Proteose peptone i n the Basal Medium. 38. Table. XI. E f f e c t of P a r t i c u l a t e M a t e r i a l . 40. Table. X l l . E f f e c t of Al c o h o l and Ether E x t r a c t i o n . 42. Table. X l l l . E f f e c t of Hydrolysis. 45. Table. XIV. D i a l y s i s of T r y p t i c Hydrolysate. 47. Table. XV. Adsorption of Toxigenic Factor. 48. Table. XVI. End Products of G l y c o l y s i s . 63. 1. INTRODUCTION. Classical emphasis i n the study of bacterial metabolism has, to a large extent, been concerned with non pathogenic organisms, principally of the l a c t i c acid group (1 ). While there are increasing numbers of reports concerning the metabolism of certain of the pathogens, as yet no one has been able to trace the actual metabolic pathways involved i n , for example,the production of any bacterial toxin. Such knowledge i s of great theoretical interest and furthermore, once such information i s obtained, numerous practical applications w i l l undoubtedly follow. Clostridium perfringens ( welchii ) Type A. forms a most suitable organism for a study of metabolism i n relation to toxin production. In a meat medium, such as glucose-peptone- beef- infusion, i t produces predominately i t s alpha toxin; i n a medium with gelatin as a source of protein, i t produces largely theta toxin; while in certain chemically reproducible media, l i t t l e or no toxin i s formed (2 ). Thus with varying types or quantities of metabolites, this organism produces one or the other, or at least, very different ratios of these two toxins. This information leads one to postulate varying metabolic pathways, dependent upon differences i n the i n i t i a l available constituents, and leading i n turn to different end products, among which one or the other toxin may be included. Even in regard to the toxins themselves, CI. perfringens i s of special interest because several of i t s end- products, including a toxin (alpha ) have clearly been shown to be enzymes active upon definable substrates. Invasive power of the organism i s , at least in part, due to an enzyme, hyaluronidase, which hydrolyzes hyaluronic acid, the polysaccharide portion of the viscous 2. mueoprotein present between muscle tissue cells. A second enzyme, collagenase, is considered to act partially as an invasin and partially as a toxin (3 )• This enzyme attacks the collagen of the muscle fibers. By hydrolyzing the collagen of the sarcolemma, the fiber bundles disintegrate causing what appears to be the pulping of tissue. In terms of pathogenicity, the most important enzyme is generally regarded to be lecithinase. One study has shown that virulence to mice depends upon lecithinase and not hyaluronidase (4 )• It is generally stated that lecithinase and the hot- cold hemolysin are two activities of one substance, alpha toxin ( 5 ), although some of the earlier work in this laboratory ( 6,7 ) suggested that they might be two separate factors. The mechanism of lecithinase activity will be more fully discussed below. Theta toxin, a hot hemolysin, has not yet been shown to be an enzyme. That is, its specific substrate has not yet been demonstrated. To assume that this toxin is also an enzyme is at the moment premature. Grabar ( 8 ) has stated dogmatically that all toxins are enzymes. While there is no experimental proof that this is the case, such a hypothesis, as Gale points out (9) , fits in with what little is known about the nature and properties of bacterial toxins. If a toxin is an enzyme whose specific substrate is a known chemical entity, the mechanism of toxic activity can be determined in part by analyzing the products of the enzymatic cleavage of the substrate. As always, however, in-vivo action need not parallel in vitro action. 3 In any study of the toxins produced by an organism when grown upon various types of media, an understanding of the mode of action of the toxins i n question i s essential for a f u l l appreciation of the problem. I t i s therefore relevant b r i e f l y to discuss the mechanism of lecithinase ac t i v i t y . Macfarlane and Knight (5) have shown that the lecithinase of CI. perfriengens hydrolyzes le c i t h i n to a diglyceride and phosphocoline as shown in figure 1. It i s of interest to note that these workers were the f i r s t to show the identity of the fatty acids present i n l e c i t h i n . Figure 1. Hydrolysis of Lecithin by CI. perfringens Lecithinase. CH20C0R« CHoOCOR' 1 CHOCOR" CHOCOR" | CH 20-P4J— 0C2H^N(CH3)3 CH20H + © © H20 0-P. OCgH^CH^-i-H ( R' and R" are fatty acid residues ) The action by which lecithinase causes hemolysis i s not f u l l y understood. Lecithin i s an essential component of the membrane of c e l l walls and Gale (9) has postulated that i f the l e c i t h i n in the red blood c e l l wall i s hydrolyzed, the c e l l wall disrupts and hemoglobin i s released. Very recently, Macfarlane (10) has shown experimentally that before hemolysis there i s always a decomposition u. of some of the erythrocyte phospholipid, as measured by the release of a c i d soluble phosphorous. Thi s enzymatic decomposition of l e c i t h i n i s then followed by hemolysis. Th i s explains at l e a s t i n part why alpha hemolysis i s a hot-cold e f f e c t because, as suggested by Macfarlane, the reaction i s not only a two stage one, but a l s o depends upon d i f f e r e n t p h y s i c a l conditions f o r the performance of each stage. Investigation of l e c i t h i n a s e a c t i v i t y on other types of c e l l s (12,13) suggests that t h i s enzyme can i n some way i n h i b i t s u c c i n i c oxidase, s u c c i n i c dehydrog:enase and cytochrome oxidase. Zamecnik, Brewster and Lipmann (14) have devised an ingenous method of measuring l e c i t h i n a s e a c t i v i t y manometrically. T h i s method i s based upon the f a c t that phosphocholine, with a pK 2 of 5.2 w i l l release carbon dioxide from a carbonate b u f f e r . Thus, the volume of carbon dioxide released w i l l be a function of the l e c i t h i n a s e a c t i v i t y . I f large amounts of substrate can be prepared i n a stable state, such a procedure i s preferable to the l e c i t h o v i t e l l i n e ' r e a c t i o n of van Heyningen (15), i n that the substrate w i l l not be subject to b i o l o g i c a l v a r i a t i o n . Thus, much i s known about the mechanism of t h i s enzyme's a c t i v i t y . I f the mechanism by which the b a c t e r i a l c e l l can synthesize l e c i t h i n a s e can be demonstrated, i t may be possible, from a knowledge of the enzyme's formation and a c t i o n , to modify present day concepts of toxins, i n view of f a c t s rather than theories. Several workers (2, 16, 17, 18) have reported growth of. G l . perfringens i n semi- synthetic medium. 5. Boyd, Logan and T y t e l l (19) f i n a l l y reported a chemically reproducible medium which allowed good growth but, under the conditions of t h e i r experiment, no toxin production. The production of alpha t o x i n has been investigated i n complex media and information i s a v a i l a b l e as t o such f a c t o r s as optimal time of incubation ( 7 ) optimal pH ( 20 ) and optimal concentration of i r o n ( 21, 22 ) . The majority of reports on alpha t o x i n production, however, concern the addi t i o n of some toxigenic f a c t o r s to a complex medium which thereby increases the production of l e c i t h i n a s e ( 16, 17, 23, 24, 25, 26 ) . Such papers are numerous, and where relevant, they w i l l be discussed i n the t e x t . As yet, no one has reported e i t h e r l e c i t h i n a s e . ( alpha t o x i n ) or theta toxin i n a chemically defined medium. I f one i s to hope f i n a l l y to trace the metabolic pathways involved i n toxin production, the constituents of the s t a r t i n g material must be f u l l y known; hence much of t h i s i n v e s t i g a t i o n has constituted an attempttto produce l e c i t h i n a s e i n a medium of chemically s p e c i f i e d composition. 6. EXPERIMENTAL WORK. 1. Standard Procedures. The procedures described here are those of a general nature which were employed throughout the i n v e s t i g a t i o n . S p e c i a l methods used i n some of the work w i l l be described i n t h e i r s p e c i f i c section. 1. Organism. Stock cultures of the s t r a i n s of CI. perfringens used ( see appendix A ) were preserved i n a l y o p h i l i z e d state by means of the following procedure. Single colony i s o l a t i o n s were c a r r i e d out on blood agar plates and t y p i c a l organisms cultured i n a f l u i d medium. The c e l l s were then separated, by c e n t r i f u g a t i o n and resuspended i n a small volume of s t e r i l e skim milk. This suspension was frozen r a p i d l y i n a f r e e z i n g mixture of seventy percent e t h y l a l c o h o l and dry i c e ( s o l i d carbon dioxide ) and then d r i e d from the frozen state by evacuation. A l l d r i e d stocks revived so f a r have been f u l l y v i a b l e giving immediate lu x u r i a n t growth. The d i s s o c i a t i o n of CI. perfringens has been the object of many in v e s t i g a t i o n s ( 27, 28, 29, 30 ) . I t has been shown that the organism w i l l d i s s o c i a t e r a p i d l y when c a r r i e d i n a r t i f i c a l media f o r any length of time. By l i m i t i n g the number of subcultures from each revived culture the p o s s i b i l i t y of v a r i a t i o n of the organism was reduced t o a minimum. I In view of the s i m p l i c i t y , e f f i c i e n c y and r a p i d l y of t h i s method of preparing d r i e d cultures, a large supply of various s t r a i n s of Type A CI. perfringens has been prepared f o r use as departmental stocks. 7. An i n t e r e s t i n g s i d e l i g h t noted was that a smear of the d r i e d organisms showed them to be strongly Gram p o s i t i v e , t y p i c a l , vegetative c e l l s with no v i s i b l e spores. 2. Turbidimetric Estimation of growth. The majority of present day i n v e s t i g a t i o n s estimate the amount of b a c t e r i a l growth by reference to the p h y s i c a l measurement of the amount of l i g h t which w i l l pass through a given depth of f l u i d medium, and c o r r e l a t e t h i s measurement to b a c t e r i a l dry weight or t o t a l b a c t e r i a l nitrogen. The method employed, i n the portion of t h i s i n v e s t i g a t i o n reported here, was to standardize c e l l suspensions ( i n p h y s i o l o g i c a l s a l i n e ) of kno?m opacity t o t h e i r dry weight. By using washed c e l l s and by taking the weight of an equal volume of d r i e d s a l i n e as a blank, the a c t u a l dry weight of bacteria was determined. Dry weight was determined by drying at 65 degrees C. u n t i l three consecutive weighings gave r e s u l t s accurate to 0.1 mgm; the maximum s e n s i t i v i t y of the balance used. The r e s u l t s are shorn i n f i g u r e 11. The s t r a i g h t l i n e r e l a t i o n s h i p obtained shows the r e l i a b i l i t y of opacity readings. The instrument used i n t h i s laboratory i s an EEL colorimeter f i t t e d with a n e u t r a l f i l t e r so that i t w i l l record t u r b i d i t y (opacity) of b a c t e r i a l suspensions. The great advantage of t h i s instrument i s that i t has tube adapters so that t e s t tubes containing one, four, or eight m i l l i l i t e r s may be used e i t h e r f o r c o l o r i m e t r i c or t u r b i d i m e t r i c determinations. Through the use of one of these adapters, i t was possible to continue use of the micro technique previously described with a consequent s i g n i f i c a n t saving i n the amount of media •8 9 required f o r t h i s work ( 31 )• Since i t was im p r a c t i c a l to t r a n s f e r each culture to one of the matched tubes provided with the colorimeter, t e s t tubes of s i m i l a r diameter used f o r growing the organism, were standardized. In the case of the f i v e mm by one hundred mm t e s t tubes, one ml amounts of water were placed i n each tube. A f t e r determining the t u r b i d i t y reading, the tubes were rotated i n the machine so as to detect any flaws i n the consistency of the g l a s s . T h i s procedure was repeated by s e t t i n g the instrument at three a r b i t r a r y readings. Any tubes g i v i n g a v a r i a t i o n of more than one d i v i s i o n were discarded By r e f e r r i n g to f i g u r e 11, i t can be seen that the experimental e r r o r of the method i s , therefore, 0.1 mgm. dry weight. It. should a l s o be noted that the sing l e point not f a l l i n g on the curve i s within t h i s l i m i t of e r r o r . Other s i z e s of t e s t tubes were standardized i n a s i m i l a r manner. However, i n most cases i t was necessary to mark the tubes with a diamond p e n c i l so that they should be inse r t e d i n t o the colorimeter i n the same p o s i t i o n each time. 3. Determination of toxin t i t e r s ,  a. Preparation of t o x i c supernatants. Both l e c i t h i n a s e and theta toxin are very l a b i l e substances. As e a r l y as 1927, Reed and Orr ( 24. ) showed that exposure of t o x i n to a i r f o r 24 hours reduced i t s hemolytic a c t i v i t y by f i f t y per cent. Theta t o x i n i s a c t i v e only i n a reduced state and i t has been suggested by Gale ( 9 ) that oxidation of s u l f h y d r y l groups ( i e -SH to -SS- ) i s responsible f o r the i n a c t i v a t i o n . As yet, the reason f o r the l a b i l i t y of l e c i t h i n a s e i s not understood. 10. Previous work i n this laboratory ( 33 ) has shown that both of these toxins are pa r t i a l l y adsorbed on Seitz f i l t e r pads. Thus the classical methods of preparing a sterile toxic f i l t r a t e cannot be used for c r i t i c a l work. A suitable method for obtaining a c e l l free toxin i s to centrifugate the culture at 3000 RPM for one hour followed by immediate removal of the toxic supernatant from the c e l l s . Unless the toxin i s removed at once, the metabolic processes of the organism continue and the gas so produced tends to break up the " c e l l clot " at the bottom of the tube. I f the cultures are centrifugated at lower speeds or for shorter periods of time, inconsistent toxin t i t e r s are obtained. Using the method described, t i t e r s are as constant as i s biologically possible. b. Hot Hemolysin. The diluent for theta toxin titrations was a phosphate buffer composed of M/5 Na2HP0^ and M/5 KH 2 P0^ at pH 6.5. This buffer inhibits hot- cold hemolysin by removing any calcium ions as insoluble calcium phosphate. The indicator was 2 per cent sheep's red blood cells and the incubation was one hour at 37 degrees C. c. Hot- Cold Hemolysin. The diluent used was isotonic sodium chloride to which was added calcium acetate to a 0 .005M concentration. The indicator was sheep's red blood cells and the incubation was one hour at 37 degrees C and overnight at four degrees C. I t should be noted that any hemolysis after the preliminary hot incubation i s due to theta toxin which i s not inhibited. 11. d. Lecithinase A c t i v i t y . Lecithinase was a l s o d i l u t e d i n calcium s a l i n e as above, however, the i n d i c a t o r was an egg yolk suspension prepared by the method of van Heyningen ( 15 ) . The incubation was f i f t e e n minutes at 37 degrees C- and overnight at U degrees G*. To one f a m i l i a r with hemolytic t i t r a t i o n s , the l e c i t h i n a s e t i t e r s may appear to be very low. T h i s i s however a f a l s e assumption as a l e c i t h i n a s e t i t e r of 1/96 corresponds to a hot- cold hemolysin of 1/2560. 12. 11. Growth i n a Chemically Defined Medium . The f i r s t h a l f of t h i s i n v e s t i g a t i o n comprised an attempt to i d e n t i f y a growth f a c t o r i n a r i b o f l a v i n - f r e e , pyridoxine- free F u l l e r ' s earth eluate of yeast extract. This unknown f a c t o r , when added to the t h i r t e e n amino a c i d medium of Duff ( 2 ) allowed f a i r l y good growth of a l l s t r a i n s then a v a i l a b l e . However, the eventual success, described below, of another and f u l l y reproducible medium caused the abandonment of t h i s part of the i n v e s t i g a t i o n , since the chief purpose of the search had been the attainment of a reproducible medium f o r use i n studies on t o x i n production. The f i r s t report of a chemically reproducible medium f o r CI. perfringens i s that of Boyd, Logan and T y t e l l ( 19 ) . The materials a v a i l a b l e i n t h i s laboratory necessitated the s u b s t i t u t i o n of pyridoxin f o r pyridoxamihe i n the formula. T h i s medium however would not support the growth of any of our stock s t r a i n s . Some pyridoxamine was obtained and a f r e s h batch of medium was prepared. Again no growth was obtained. A t h i r d batch of medium was prepared which y i e l d e d s i m i l a r r e s u l t s . In view of the f a c t that many of the materials a v a i l a b l e were undoubtedly old ( although they were not marked as to the date they were f i r s t opened ) and that a culture of BP6K ( used by Boyd et a l ) was not a v a i l a b l e , t h i s medium was temporarily discarded. With the a r r i v a l of a new stock of growth f a c t o r s and a culture of s t r a i n BP6K, another attempt was made to grow CI. perfringens i n the medium of Boyd, Logan and T y t e l l . The medium was prepared according to the o r i g i n a l d i r e c t i o n s ( see Appendix B ) and tubed i n 8 ml amounts. St r a i n s PB6H and BP6K were subcultured from GPBI to a casein hydrolysate-13. yeast extract medium, washed and inoculated into previously boiled and cooled medium. A l l cultures were incubated at 45 degrees C. Good growth was obtained with both strains for 4 generations. This indicated that previous failures to obtain growth must have been due to old ingredients. The importance of marking the date of the f i r s t opening of any chemical i s a practice too often overlooked by many workers. The fourth tube generation was subcultured into micro tubes containing 1 ml. media ( 31 ) and carried for two generations with no diminution of growth. Therefore i t was decided to use micro tubes wherever possible. The inoculum used above was a r b i t r a r i l y chosen as 15 per cent of the volume of medium as this was found to be optimal for Duff's medium. The next step was to determine the optimal inoculum InJJthe medium of Boyd et a l ( hereafter referred to as the basal medium ). Washed cells were inoculated into 1 ml. amounts of the basal medium from a pasteur pipette. The inoculum was from 1 to 7 drops ( each drop approximately 0.05ml. ) and the cultures carried for four generations." The results of 21 hour growth in the fourth tube generation are shown in figure 111. Similar curves were obtained for the other three generations and for incubation periods of 48 and 72 hours. A 2 dibp quantity was chosen as the standard inoculum for future work. # The term " generation " refers to tube generation whenever used. GROWTH (mgms per ml) . oO 0.5 1.0 1.5 2.0 •7T 15. It should be pointed out that this inoculum i s 10 per cent which i s the maximum desirable. It should be noted that figure 111 shows an inherent fallacy i n the idea that a very large inoculum i s desirable although this decrease in growth may be due entirely to the carry over of large amounts of acidified medium in later generations. Since another series of experiments had shorn that growth at 37 degrees C.was equivalent to growth at 45 degrees a very crude approximation of the growth curve was obtained. The object of this test was to determine whether or not two generations per day could be used as such a procedure would greatly increase the volume of work possible. For this, f i f t h generation organisms were used and the growth determined at 4 , 5 , 6 , 7 , 8 , and 25 hours. The results are presented i n figure I V . On the basis of these findings, the standard procedure was to transfer cultures both i n the morning and in the evening. This means that there i s a variation in the incubation period of odd and even generations. Thus, in comparing results of different series of tests, this time variation must be taken into consideration. However, so far as growth alone i s concerned, both cultures w i l l show l i t t l e i f any variation in the amount of growth ( See figure IV ) Thus a procedure has been perfected for obtaining satisfactory growth of CI. perfringens BP6K at both 45 and 37 degrees C, in a chemically defined medium; hereafter called the basal medium. GROWTH (mgms per ml ) ro o ro •9T 17. 111. Toxin Production i n the Basal Medium.  1. E f f e c t of Temperature. Boyd, Logan and T y t e l l ( 19 ) i n t h e i r o r i g i n a l work reported that, under the conditions of t h e i r experiment, there was no formation of oc t o x i n ( l e c i t h i n a s e ) or any other known toxins or e^nzymes ( hyaluronidase, @ t o x i n , or gelatinase ) i n t h e i r medium. These workers claimed that the optimal temperature f o r growth was 45 degrees C. thus confirming the work of Chapman ( 34 )» However, r e s u l t s i n t h i s laboratory have shown that j u s t as good growth occurred, over a twenty four hour period, at 37 degrees as at the' higher temperature. In view of the f a c t that t o x i n synthesis undoubtedly depends upon the functioning of at l e a s t a few enzyme systems, and that enzymes often e x i b i t a rather narrow temperature optimum, i t was decided to t e s t the basal medium f o r toxi n production a f t e r growth a t 37 degrees . The f i r s t method of t i t r a t i o n was that of hot- cold hemolysis. T h i s would show, i n a si n g l e t e s t , e i t h e r alpha or theta t o x i n . T r i p l i c a t e cultures over f i v e generations were t i t r a t e d together with a c o n t r o l of s t e r i l e media. In a l l but the control hot hemolysis occurred with an average t i t e r of 1/4096. There was no decline i n t i t e r over the f i v e generation period. Thus, a hot hemolysin was found which may or may not be masking a lower t i t e r of hot- cold hemolysin. The experiment which follows gave r e s u l t s which i l l u s t r a t e a very important p r a c t i c a l point i n any study of b a c t e r i a l 13. physiology. A t i t r a t i o n was set up f o r hot- cold hemolysis, hot hemolysis, and l e c i t h i n a s e a c t i v i t y over a four generation period. Each generation was grown and t i t r a t e d i n t r i p l i c a t e and the r e s u l t s i n Table 1 represent modal oay.erages. Table 1 . E f f e c t of Carry over on Toxin Production. Generation Growth mgms/ml Hemolytic T i t e r Iftelr ; H 6 t T:,:Ur Incubation Hemolytic T i t e r a f t e r R e f r i g e r a t i o n L ecithinase A c t i v i t y 1 . 1.50 V 5 1 2 0 1/5120 1/192 2 . 1.40 1/5120 1/5120 1/96 3 . 1.56 1/5120 1/5120 1/48 # 0 4. 1.30 1/5120 1/5120 # Incubated one hour with readings every 15 minutes. The hemolytic t i t e r s a f t e r r e f r i g e r a t i o n simply i n d i c a t e s that hot- cold hemolytic t i t e r s greater than 1/5120 d i d not occur. Concentrations of hot- cold hemolysin l e s s than 1/5120 cannot be determined because of the masking e f f e c t of the hot hemolysin. Repetition of t h i s experiment showed that the l e c i t h i n a s e a c t i v i t y present i n the f i r s t three generations was due to carry over of toxigenic materials probably through i n s u f f i c i e n t washing. I t cannot be overemphasized that during the e a r l y part of an i n v e s t i g a t i o n , when adequate controls with previously characterized media are not possible, great care must be employed i n assessing any r e s u l t s . Thus i t was found that the basal medium produced at l e a s t one type of hemolysin. 19. 2. Adsorption of Theta Toxin. van Heyningen ( 35 ) found that theta- toxin could be adsorbed out of a mixture of alpha and theta toxins by exposure of the mixture to packed sheep's erythrocytes for five minutes at zero degrees C. Excessive exposure will further result in adsorption of the alpha toxin. This method was previously used in this laboratory by Morton ( 6 ) However, his work did not include any determination of alpha toxin. After adsorption, it is necessary to remove the red blood cells by centrifiugation. In view of the importance of the time factor in adsorbing all the theta toxin without alpha toxin, it was decided to try a few preliminary runs to perfect the technique. If Reed's medium is inoculated directly from a glucose beef infusion medium, there is sufficient carry over of toxigenic factors to enable the organisms to produce both theta toxin and lecithinase for at least two, and often three or more, generations. Such a procedure was used to produce a mixture of alpha and theta toxins. The polyvalent toxin was adsorbed with packed sheep's erythrocytes at zero degrees C. for periods of 2,3,4, and 5 minutes. The cells were centrifuged out rapidly ( about three minutes ) and the adsorbed toxin removed as quickly as possible and titrated for hot and hot- cold hemolysin and lecithinase activity. The results are shown in Table 11. It should be noted that five minute adsorption plus centrifugation resulted in some hemolysis of the cells used for adsorption. This tended to mask the hemolytic titrations and made them difficult to read. 20. Table 11. Adsorption of Toxins from Reed's Medium. Time of Adsorption. Hemolytic T i t e r a f t e r Hot Incubation Hemolytic T i t e r a f t e r R e f r i g e r a t i o n L ecithinase A c t i v i t y Non Adsorbed. 1/2560 1/2560 1/96 2 minutes. 0 1/1280 1/96 3 minutes. 0 1/1280 1/96 4 minutes. 1/40 1/640 1/96 5 minutes. 0 1/1280 1/48 The modification of the o r i g i n a l method ( i . e . not using a r e f r i g e r a t e d centrifuge ) appears to have reduced the optimal " adsorption " time from 5 minutes to two minutes. However, even a t two minutes, the hot- cold hemolysis was reduced while at f i v e minutes the l e c i t h i n a s e a c t i v i t y was reduced. Toxin produced i n the basal medium was therefore adsorbed f o r periods of two and four minutes. The r e s u l t s are shown i n Table 111 Table 111 Adsorption of Toxin from the Basal Medium. Adsorption Time Hemolytic T i t e r a f t e r Hot Incubation Hemolytic T i t e r a f t e r R e f r i g e r a t i o n L ecithinase A c t i v i t y Non Adsorbed. 1/5120 1/5120 0 2 minutes 1/160 1/160 0 4 minutes . 3/160 1/160 0 21. While these r e s u l t s do not show complete adsorption of theta toxin, i t would c e r t a i n l y seem l o g i c a l to suppose that the hot l y s i s , which occurred a f t e r primary incubation, was due to theta t o x i n . This i s furth e r borne out by the f a c t that the toxin had no l e c i t h i n a s e a c t i v i t y . 3. L e t h a l E f f e c t . A portion of the toxin which was used i n the adsorption experiments above, was inoculated i n t r a p e r i t o n e a l l y i n t o mice i n varying quantites. The r e s u l t s are shown i n Table IV. Table IV Le t h a l A c t i v i t y of Toxin Produced i n Basal Medium Toxin Injected. T o t a l Number of Mice. Died 19 hours. Survived f o r at l e a s t 5 days. 1.0ml. 6 1 5 0.75ml. 6 0 6 0.50ml. 6 0 6 0.25ml. 3 1 2 22. 4. Summary of C h a r a c t e r i s t i c s of. Toxin i n Basal Medium. Some of the main c h a r a c t e r i s t i c s of t h i s toxin produced i n the basal medium have now been determined and i t possesses the following properties: 1) A hot hemolysin which i s not increased by hot- cold incubation and which can be almost e n t i r e l y removed by adsorption with sheep's erythrocytes. 2) Not l e t h a l to mice i n amounts up to 0.1953 M.H.D's ( 1.0 ml.) 3) No l e c i t h i n a s e a c t i v i t y . On the basis of these properties, t h i s toxin has been c l a s s i f i e d as theta toxin;. Lowering of the incubation temperature from 45 to 37 degrees therefore enables CI. perfringens to produce theta t o x i n i n the medium of Boyd, Logan and T y t e l l . 23. IV. The E f f e c t of Calcium, Magnesium, and Iron on Growth,  and on Toxin i n Production i n the Basal Medium. The r o l e of inorganic i r o n i n metabolism i s f a r from understood. Some enzymes are metalloproteinsj Cytochrome oxidase, f o r example, contains e i t h e r copper or i r o n . At l e a s t one vitamin, Bm contains cobalt. Phosphorous has an e s s e n t i a l r o l e i n ftk)  1—> ' g l y c o l y s i s . Inorganic ions may also be i n h i b i t o r s or a c t i v a t o r s of enzymes or enzyme systems. van Heyningen ( 15 ) MacFarlane and Knight ( 36 ) and Zamecnik ( 14- ) have showed that both calcium and magnesium a c t i v a t e l e c i t h i n a s e a c t i v i t y . In the case of calcium, the optimal concentration i s 0.005M, increasing the concentration to 0.1M i s i n h i b i t o r y . In the case of magnesium, 0.0001M i s stimlatory, while 0.001M i s i n h i b i t o r y . The a c t i v a t i o n of the l e c i t h i n a s e enzyme need not, of course, have any r e l a t i o n s h i p to the stimulation of i t s formation. However, i n view of the intimate connection of these two ions with the deamination and dephosphorylation of adenosine triphosphate (37 ), the transport system f o r high energy phosphate bonds, they might w e l l stimulate the metabolic processes i n general through a stimulation of g l y c o l y s i s . Such reasoning was based on the hypothesis, so f a r unsupported by experimental evidence,of Adams Hendee and Pappenheimer ( 26 ) that l e c i t h i n a s e production might be solely a function of the amount of growth. 24-The role of iron in toxin production by Clostridium 'tetani  p and Corynebacterium di^htheriae i s well known ( 38, 39 ). Pappenheimer and Shaskan ( 21, 22 ) f i r s t showed that iron affected the production of lecithinase by CI. perfringens .. They further showed that as the iron content of the medium was decreased, the l a c t i c acid formation approaches two moles per mjofe of glucose fermented. These workers found that the iron concentration for maximum lecithinase ac t i v i t y , maximum growth and minimum l a c t i c acid coincided. On these results they postulated the following metabolic pathways: Glucose intermediate Iron ^ 2 lactate (pyruvate? ) deficiency Iron containing enzyme acetate, butyrate and carbon dioxide. Bard and Gunscilus ( 40 ) have reported evidence for the existence of a metallo-aldolase i n CI. perfringens and have indicated, through the use of a c e l l free enzyme preparation, that this i s a specific enzymatic site of action for iron. Ho?;ever, they do not rule out the possibility that some other iron containing enzyme such as that postulated by Pappenheimer and Shaskan may exist. The fact that iron i s essential for growth of CI. perfringens and that i t s role i s that of a metallo-aldolase which catalyzes the conversion of fructose-1,6- diphosphate to 3- glyceraldehyde phosphate also suggests the existence of an Imbden-Meyerhof system in this organism. I f such i s the case, iron i s essential for growth in that i t i s essential for glycolysis, and only i n this role i s i t essential for lecithinase production. I t should be pointed out that Pappenheimer and Shaskan ( 22 ) did actually show - 25. that i n a medium which produced no toxin, the i r o n concentration influenced only growth and l a c t a t e formation. Since calcium, magnesium and i r o n are so c l o s e l y associated with the e a r l y reactions i n the breakdown of glucose, i t was decided to t e s t the e f f e c t of t h e i r concentration on growth and toxin production i n the basal medium. The r e s u l t s are shown i n Table V. The a d d i t i o n of calcium acetate to the basal medium resu l t e d i n the precipitation of some of the phosphate ( from the buffer ) . For t h i s reason,the f i n a l medium i n t h i s table contains no buffer. The normal concentrations i n the basal medium are: -5 -5 90X10 M Mg S0^. 7H 20, 4X10 M Fe SO^. 7H 20, and Ca^ as Calcium Pantothenate. Table V Varying Concentrations of Inorganic Ions. M o d i f i c a t i o n i n Basal Medium Growth Hot Hemolytic T i t e r Lecithinase A c t i v i t y Compound Concentration Control Usual 1.40 1/5120 0 MgSo,4.7H20 N i l 0 0 0 22.5X10~5M 1.4Q 1/20480 0 -5 45X10 M 1.32 1/5120 0 67.5X10"5M 1.32 I / I O 2 4 O 0 Fe S0^.7H20 N i l 0.95 1/5120 0 1X10" 5 1.22 1/5120 0 2X10~5M 1.56 1/1280 .0 -5 3X10 1.50 1/10240 0 ( C H 3 C 0 0 ) 2 C a -3 5X10^ M 1.05 1/160 0 (CH 3 C 0 0 ) 2 C a 5X10"* M \ 0 0 0 Buffer N i l J 26. The a d d i t i o n of calcium has apparently i n h i b i t e d the formation of theta t o x i n . Since theta toxin i s normally produced i n complex media which contain calcium, one would not expect calcium ion i t s e l f to be i n h i b i t o r y to theta t o x i n production. The i n h i b i t o r y a c t i o n i s , therefore, more l i k e l y a t t r i b u t a b l e to a reduction, by p r e c i p i t a t i o n , of the phosphate added as buffer, with consequent reduction i n growth. Lat e r work has shown that a 25 per cent reduction i n the phosphate concentration of the basal medium reduces the hot hemolysin to 1 to 320 while reductions of 50 per cent or more completely i n h i b i t theta t o x i n production. I t i s i n t e r e s t i n g to note that there i s no diminution of growth u n t i l the buffer concentration i s l e s s than 50 per cent of that i n the basal medium. Magnesium, as might be expected, proved e s s e n t i a l f o r growth. However, as the concentrations tested resulted i n growth within the experimental e r r o r of the method no f u r t h e r conclusion can be drawn. I t might appear that reducing the concentration of magnesium increases the theta t o x i n production. However, the f a c t that a d i r e c t r e l a t i o n s h i p between concentration and toxin production was not obtained does not j u s t i f y such an assumption. I t has been shown by Bard and Gunsulus ( 4-0 ) that i r o n i s necessary f o r normal growth of CI. perfringens. I t w i l l be noted that growth and theta t o x i n production occurred without the addition of ferrous s u l f a t e to the basal medium. I t was, however, not e n t i r e l y absent, as was found by inspecting the a n a l y t i c a l l a b e l s of the other chemicals used to form the basal medium. In several cases, traces of i r o n were reported. The i r o n requirements f o r optimal theta t o x i n production, however, i s here shown to be 27. g r e a t e r t h a n t h e t r a c e s o r i g i n a l l y p r e s e n t , s i n c e t h e g r e a t e s t t i t e r o f t o x i n o c c u r r e d a t a n added c o n c e n t r a t i o n o f 3X10 M o l a r F e ^ . T B ^ O . I t i s f u r t h e r shown t h a t m o d i f i c a t i o n o f t h e r e s p e c t i v e magnes ium, i r o n a n d c a l c i u m c o n t e n t o f t h e b a s a l medium d o e s n o t r e s u l t i n t h e p r o d u c t i o n o f d e t e c t a b l e a l p h a t o x i n , measured a s l e c i t h i n a s e . W h i l e o t h e r i o n s m i g h t p o s s i b l y have been t e s t e d i n t h i s r e s p e c t , i t was f e l t t h a t f u r t h e r w o r k o f t h i s n a t u r e w o u l d p r o v e u n p r o f i t a b l e , a n d t h a t a t t e n t i o n s h o u l d be d i r e c t e d s p e c i f i c a l l y t o t h e a l p h a - t o x i g e n i c f a c t o r , w h a t e v e r i t s n a t u r e , w h i c h i s known t o e x i s t i n c o m p l e x o r g a n i c m e d i a . 28. V. The E f f e c t of Toxigenic Organic Substances which  Stimulate Lecithinase Production, An a r b i t r a r y d i v i s i o n has been made of the material to be included i n Sections V and VI so that the former section w i l l include only those substances that are chemical e n t i t i e s and whose structure i s known. Tamura, T y t e l l , Boyd and Logan ( 16, 17 ) found that, i n a casein hydrolysate medium, glucosamine, n i c o t i n i c a c i d , and r i b o f l a v i n stimulated toxi n production. However, i n t h e i r o r i g i n a l work, one cannot determine whether they measured hot or hot-cold hemolysis or both. Adams, Hendee and Pappenheimer ( 26 ) found that both l e c i t h i n and glycerophosphorylcholine exerted a s l i g h t e f f e c t on the production of l e c i t h i n a s e . Previous work i n t h i s laboratory ( 6, 41 ) gave c o n f l i c t i n g evidence as to whether or not l e c i t h i n a s e was an adaptive enzyme. Thi s discrepancy may well be due to the lack of other e s s e n t i a l toxigenic factors i n the f i r s t case, or the presence of other f a c t o r s i n the egg yolk suspension i n the l a t t e r . Adams et a l ( 26 ) p u r i f i e d l e c i t h i n from egg yolk and do not report any data as t o the p u r i t y of t h e i r product, hence t h e i r r e s u l t s also may be due to " contaminating " f a c t o r s . Glycerophosphocholine i s not a v a i l a b l e commercially. However, both choline chloride and sodium glycerophosphate were tested f o r toxigenic properties both separately and together. Some of the t h e o r e t i c a l f a l l a c i e s i n expecting these two components to function i n a manner s i m i l a r to the i n t a c t molecule w i l l be discussed i n a l a t e r section of t h i s report. 29. The concentration of r i b o f l a v i n i n the basal medium was the same as that found to be optimal f o r toxin production. The concentrations of n i c o t i n i c a c i d and glucosamine used were those of the o r i g i n a l papers. No data are a v a i l a b l e as to the optimal choline concentx-ation f o r t o x i n production of any of the C l o s t r i d i a . The amount used was a r b i t r a r i l y chosen as that found most sui t a b l e f o r pneumococcal growth ( 4 2 ) . The concentration of sodium glycerophosphocholine was calculated on the basis of a mole to mole r a t i o with the choline ch l o r i d e . The r e s u l t s of these experiments, shown i n Table VI, are a modal average of s i x t e s t s . Table VI. E f f e c t of Toxigenic Organic Substances Additions to basal mediunr Growth mgms/ml Hot Hemolytic T i t e r Lecithinase A c t i v i t y Control 1.80 1/1280 0 l$glucosamine-l$glucose I . 8 4 1/640 0 l$glucosamine-0.l^glucose 0.11 - -l$glucosamine-Oglucose 0.00 - -glucosamine-nicotinic a c i d 1.72 1/2560 I 0 0.0001$ n i c o t i n i c a c i d 1.50 1/2560 \^ 0 O.Ol^sodium glycerophosphate 1.10 • - V 0 0.0025$ choline chloride 1.54 - \ 0 cholina+glycerophosphate 1.47 - 0 '• / # Unless otherwise noted, glucose concentration was one per cent. 30. As can be r e a d i l y seen, none of these substances a re , i n themselves, capable of s t imulat ing the product ion of l e c i t h i n a s e . One would hes i ta te to assess the in f luence of glucosamine and n i c o t i n i c a c i d on theta t o x i n product ion as the three t e s t t i t e r s determined d i f f e r from the c o n t r o l by only one tube i n the t i t r a t i o n s e r i e s . None of the mater ia l s te s ted increases the growth of the organism over that obtained i n the basa l medium. Chol ine and glycerophosphate s i g n i f i c a n t l y depress growth e i t h e r s i n g l y or together . N i c o t i n i c a c i d a l s o depresses growth'but,whan usad i n . . . / combination wi th glucosamine, itopermitsunormal! growth. In the t e s t media when the glucose concentrat ion was depressed, one must pos tu la te that BP6K does not possess the enzyme systems necessary to deaminate or transaminate glucosamine. No information has been found as to what other substrates would be required f o r the t r a n s f e r of these amino groups. 31. VI. Complex Factors Stimulating Lecithinase Production. ( Two substances, adenosine triphosphate and d e x t r i n havejbeen included i n t h i s section because, although t h e i r structure i s known, there was some doubt as to the p u r i t y of the material employed. ) 1. Toxigenic a c t i v i t y of Dextrin. Adams, Hendee and Pappenheimer ( 26 ) found that the water in s o l u b l e f r a c t i o n of d e x t r i n ( Merck NFV ) would increase the production of l e c i t h i n a s e i n a complex medium. However, i n the presence of any other carbohydrate, t h i s stimulatory action d i d not occur. In e a r l i e r work, using Duff's semi-synthetic medium ( 2 ), i t was found that when dex t r i n was added, i n place of glucose as the carbohydrate source, no growth occurred. T h i s experiment was repeated i n t r i p l i c a t e on four d i f f e r e n t occassions and each time the same negative r e s u l t s were obtained. Merck NFV d e x t r i n was not a v a i l a b l e f o r t e s t i n g i n the basal medium and i t was necessary to substitute Eastman b a c t e r i o l o g i c a l d e x t r i n . While t h i s material has a white l a b e l i n d i c a t i n g the highest degree of p u r i t y , no a n a l y s i s of other chemicals present i s given. In attempting to i s o l a t e the water in s o l u b l e f r a c t i o n by the procedure of Adams et a l , i t was found that i n place of a two phase mixture, only a water insol u b l e f r a c t i o n was formed, with a l l the water being adsorbed by the d e x t r i n . On the basis of these f i n d i n g s , i t was necessary to assume that the water soluble .material had previously been removed from t h i s Eastman b a c t e r i o l o g i c a l d e x t r i n . Since t h i s assumption was not proved, the r e s u l t s obtained do not n e c e s s a r i l y contradict those of the previous workers. 3 2 . I n a s i n g l e ( t r i p l i c a t e ) e x p e r i m e n t i t was f o u n d t h a t t h e s u b s t i t u t i o n o f d e x t r i n f o r g l u c o s e i n t h e b a s a l medium r e s u l t e d i n t h e same amount o f g r o w t h , w i t h n o e v i d e n c e o f l e c i t h i n a s e a c t i v i t y . 2. T o x i g e n i c a c t i v i t y o f A d e n o s i n e t r i p h o s p h a t e ( ATP ) T h e A T P , w h i c h was s u p p l i e d t h r o u g h t h e k i n d n e s s o f t h e D e p a r t m e n t o f B i o c h e m i s t r y , had been i s o l a t e d f r o m r a b b i t m u s c l e ( 4 3 ) a s a c l a s s e x p e r i m e n t . U n f o r t u n a t e l y , t h e m a t e r i a l i s o l a t e d b y t h e w r i t e r was n o t a v a i l a b l e a n d one c o u l d q u e s t i o n t h e p u r i t y o f t h e sample o b t a i n e d . T h e sample h a d n o t been p r o p e r l y d r i e d hence a n a r b i t r a l amount o f t h i s m o i s t p r e p a r a t i o n was a d d e d t o g i v e a f i n a l c o n c e n t r a t i o n o f 1 p e r c e n t . T h e a d d i t i o n o f t h e ATP t o t h e medium c a u s e d a n i m m e d i a t e s l i g h t p r e c i p i t a t i o n w h i c h was u n d o u b t e d l y due t o t h e p r e s e n c e o f b a r i u m w h i c h had n o t been p r o p e r l y removed f r o m t h e enzyme . I f ATP i s b e i n g p r o d u c e d b y t h e b a c t e r i u m a t a r a t e w h i c h i s l e s s t h a n o p t i m a l f o r g l y c o l y s i s , one w o u l d e x p e c t t h a t t h e a d d i t i o n o f t h e enzyme w o u l d r e s u l t i n i n c r e a s e d g r o w t h . T h i s , h o w e v e r , was n o t t h e c a s e , and g r o w t h i n t h e t e s t medium was t h e same a s t h a t i n t h e c o n t r o l . L e c i t h i n a s e was p r o d u c e d t o a t i t e r o f 1 / 2 4 • W h e t h e r o r n o t t h e t o x i g e n i c f a c t o r p r e s e n t i n t h e ATP was t h e enzyme i t s e l f o r some c h e m i c a l i m p u r i t y i n t h e m a t e r i a l , i s n o t k n o w n . No c o m m e r c i a l ATP was i m m e d i a t e l y a v a i l a b l e t o t e s t f o r a c t i v i t y so t h a t one c a n n o t make a r e l i a b l e a s s e s s m e n t o f t h e a c t i o n o f a d e n o s i n e t r i p h o s p h a t e i n t h e p r o d u c t i o n o f l e c i t h i n a s e . A s t h i s was t h e l a s t e x p e r i m e n t o f t h e e n t i r e i n v e s t i g a t i o n , 33. only one test ( in triplicate ) was carried out. 3. The Effect of Yeast Extract on Growth, and Toxin Production. Numerous fractions of yeast extract had been prepared while attempting to devise a chemically reproducible medium for CI. perfringens. Following successful results with the medium of Boyd et al ( basal medium ) this work was discontinued. However, since the fractions were available it was decided to determine the effect of yeast extract in the basal medium. The concentrations of yeast extract ( Difco ) used were 1 and 2 per cent. Results are shown in Table Vll . Table Vll Addition of Yeast Extract to Basal Medium Addition to Basal Medium Growth mgm/ml Hot Hemolytic Titer Lecithinase Activity Nil 1.79 1/1280 0 l^ Yeast Extract 1.46 1/1280 0 2$Yeast Extract 1.96 1/1280 0 Yeast extract did not influence theta toxin or lecithinase production. It did, however, have a marked influence on growth in that in the lower concentration it depressed growth while in the higher concentration growth was stimulated. 3 4 -4 . Lecithinase production i n G.P.B.I. o The medium commonly employed i n t h i s laboratory to produce CI. perfringens l e c i t h i n a s e i s glucose-peptone-beef-infusion medium (G.P.B.I.) ( 7 ) . B e l l ( 4 4 ) found that by adsorbing.G.P.B.I. broth on F u l l e r ' s E a r th at pH 1 .2 the toxigenic f a c t o r was removed whereas growth progressed normally. T h i s f i n d i n g shows without a doubt that l e c i t h i n a s e production requires at l e a s t one f a c t o r more than i s required f o r optimal growth. The h i s t o r i c work of Mueller ( 4 5 ) i n devising a chemically reproducible medium f o r C_. diphtheriae u t i l i z e d a technique of i d e n t i f y i n g the e s s e n t i a l components i n a complex medium. Th i s i s , of course, the only s c i e n t i f i c mode of pe r f e c t i n g such a medium. To i s o l a t e a l l the f a c t o r s i n G.P.B.I. e s s e n t i a l f o r growth would have been a d u p l i c a t i o n of the work of the C i n c i n n a t i group, and the e a r l y part of t h i s work on Duff's medium had been discontinued f o r that very reason. I f one could f r a c t i o n a t e G.P.B.I. and add one or more of the r e l a t i v e l y pure f r a c t i o n s to the basal medium, thereby allowing the organism to form l e c i t h i n a s e , the problem as a whole would be g r e a t l y s i m p l i f i e d . G.P.B.I. i t s e l f contains three complex f a c t o r s : d r i e d meat, peptone, and meat i n f u s i o n broth. In order that the above scheme be p r a c t i c a l , one of these f a c t o r s must, when added to the basal medium, r e s u l t i n the production of CI. perfringens l e c i t h i n a s e . 3 5 . There was some doubt as to whether the 11 G.P.B.I. broth " o f . B e l l ( 4 4 ) was the meat i n f u s i o n broth alone or t h i s i n f u s i o n broth plus peptone. Where the term G.P.B.I. broth i s used i n t h i s report i t w i l l r e f e r to the meat broth plus peptone while the broth without peptone w i l l be r e f e r r e d to as beef i n f u s i o n broth(B.I.B.) N a t u r a l l y , the more simple the s t a r t i n g material, the e a s i e r the i d e n t i f i c a t i o n of the components. The beef i n f u s i o n broth was prepared as follows: To one pound of f a t - f r e e beef, i n a chemically clean beaker, was added one l i t e r of glass double d i s t i l l e d water. T h i s was l e f t at four degrees C- f o r 36 hours. A l l the excess f a t on the surface was removed and the mixture infused at 60 to 65 degrees C. f o r 2 hours. The meat was removed by f i l t r a t i o n through gauze and #1 Whatman f i l t e r paper. The meat p a r t i c l e s were heated to dryness i n a hot a i r oven and stored i n a s t e r i l e f l a s k at four degrees. The f i l t r a t e was N / adjusted t o H.8,7 with / 1 NaOH and autoclaved. T h i s material was IT again f i l t e r e d through #1 Whatman paper. The pH was 7.8 hence d i d not require f u r t h e r adjustment. The broth (BIB) was reautoclaved and stored at four degrees C. In order to f a c i l i t a t e future additions to the basal medium, i t was necessary to determine whether or not the buffer used i n the basal medium was suitable f o r the B.I.B. The concentration of glucose i n the basal medium and i n G.P.B.I. i s also d i f f e r e n t so that t h i s too was t i t r a t e d to determine the optimal concentration. T^yptone and proteose peptone are very s i m i l a r to peptone bujr are generally considered to be r i c h e r sources of n u t r i e n t s . 36 Hence, the addi t i o n of a l l three of these materials to the BIB was tested. In each case the concentration was 1 per cent. The r e s u l t s of these experiments are shown i n Table IX. All these experiments were c a r r i e d out so that one t r i p l i c a t e s e r i e s was incubated without s p e c i a l anaerobic precautions, while the other was incubated i n an atmosphere of hydrogen. As there were no s i g n i f i c a n t d i f f e r e n c e s i n r e s u l t s , only the former seri e s i s reported here. Table IX Toxin Production i n G.P.B.I. F r a c t i o n s . Media Growth mgms/ml Hemolytic T i t e r a f t e r Hot Incubation Hemolytic T i t e r a f t e r R e f r i g e r a t i o n Lecithinase A c t i v i t y G.P.B.I. - - — 1/384 G.P.B.I. Supernatant - - - 1/96 B.I.B.less Glucose . l e s s Buffer 0 1/160 1/160 0 B.I.B.less Buffer 1.05 1/160 I / 6 4 O 0 B.I.B.less Glucose 0 1/80 1/80 0 B.I .B.-0.5$ (Jlucose 0.70 1/160 I / 6 4 O 0 B.I.B.-0$ Glucose 1.10 1/160 I / 6 4 O 0 B.I.B.-2.0$ Glucose 1.22 1/160 1/320 0 B.I.B-l$Peptone 1.80 1/1280 1/1280^ 1/96 B.I.B.-l$Tryptone 2.06 1/1280 1/2560 1/384 B.I.B.-l$Proteos Peptone e 1.96 1/1280 l/2560 # 1/384 Basal Medium + 2../o B . l . B . 1.52 1/2560 - 0 - Basal Medium-4$ B.I.B. 1.90 1/2560 0 # hemolysis on primary incubation, not increasing on r e f r i g e r a t i b n . 37 The removal of the meat from G.P.B.I. g r e a t l y reduces the amount of l e c i t h i n a s e produced. This might mead that the G.P.B.I. broth contains an e s s e n t i a l toxigenic f a c t o r ( hereafter c a l l e d TF ) while the meat contains a stimulatory f a c t o r f o r toxin production. Or i t may be that some of the a c t i v e material i n the meat has been soluble i n the broth. B.I.B. i s not su i t a b l e f o r l e c i t h i n a s e production unless a complex f a c t o r such as peptone i s added. Both tryptone and proteose peptone are superior to peptone i n t h i s regard as well as i n stimulating growth., I t seems reasonable to assume that these f a c t o r s a l l contain the TF and the next step i s to t e s t the ac t i o n of these materials i n the basal medium, together with the usual energy source consisting of one per cent glucose. The f a c t that the hemolysis increased a f t e r cold incubation where there was no l e c i t h i n a s e a c t i v i t y makes one speculate as to whether or not alpha hemolysis and l e c i t h i n a s e are one and the same. Hov/ever, the evidence here i s not s u f f i c i e n t to postulate that they are separate substances. I t might well be that the l e c i t h i n a s e t e s t i s not s u f f i c i e n t l y s e n s i t i v e to detect very small amounts of the enzyme which are measurable by hemolytic methods. 5. E f f e c t of Tryptone and Proteose Peptone i n the Basal Medium These two f a c t o r s only were tested on the basis of the r e s u l t s i n Table IX. Both f a c t o r s were added i n a 1 per cent concentration. When they were added to B.I.B. t h e i r concentration was again 1 per cent and the f i n a l concentration of the broth i n the basal medium was 1 per cent so that the v a l i d i t y of comparing such t e s t s could be questioned. However, the problem was to f i n d any complex substance which when added to the basal medium would 38 allow the organism to synthesize l e c i t h i n a s e . These r e s u l t s are shown i n Table X. Table X Tryptone and Proteose Peptone i n the Basal Medium. Addition to Basal Medium Growth mgm/ml Hemolytic [l .Tit e r a f t e r Hot Incubation emoljrtic ] T i t e r a f t e r R e f r i g e r a t i o n [•ecithinase A c t i v i t y Control 1.61 1/640 1/3207" 0 1% B.I.B. 1.05 1/1280 I / 6 4 O ' 1/12 1% Tryptone 2.19 1/2560 1/1280'" 1/48 1% Proteose Peptone 2.00 1/5120 I/2560' 1/48 1% B.I.B.- Tryptone 0.89 1/2560 1/640" 1/24 1% B.I.B. -Proteose Peptone 0.70 1/2560 1/1280 1/48 Tryptone and proteose peptone both g r e a t l y increase growth and contain the l e c i t h i n a s e TF absent i n the basal medium. Why the addit i o n of B.I.B. to the basal medium a c t u a l l y r e s u l t e d i n a decrease of growth would be an i n t e r e s t i n g i n v e s t i g a t i o n , however, i t does not have a d i r e c t bearing on the problem at hand. I t should a l s o be noted that while growth i s e s s e n t i a l l y equivalent to that i n Table IX, the l e c i t h i n a s e a c t i v i t y i s gr e a t l y reduced. One can only postulate the presence of some f a c t o r i n the basal medium which depresses the synthesis of the enzyme. However, j u s t because a substance contains the TF does not mean that i t i s the most su i t a b l e to be f r a c t i o n a t e d . To be p r a c t i c a l , the stock s o l u t i o n f r a c t i o n a t e d should be about twenty times as concentrated as f i n a l concentration required i n the medium. 39. For example, i f one per cent tryptone i s required to stimulate lecithinase production, a 20 per cent solution of tryptone i s suitable for fractionation. Otherwise, more time w i l l be spent in concentrating fractions for testing than w i l l be spent i n obtaining these fractions. Both tryptone and proteose peptone have a solubility of about 2 grams per 100 ml at room temperature. Thus, unless they can exert TF activity in concentrations of 0.1 per cent or less, i t i s not practical to attempt to fractionate them. The two factors were added to the basal medium in varying concentrations between 0.01 and 0.5 per cent. There was no evidence of lecithinase production in any of the test media. I t was therefore decided to discard tryptone and proteose peptone as being impractical starting materials for the isolation of the lecithinase TF. 6. Effect of Particulate Material The single active factor of G.B.B.I, remaining to be tested i s the dried meat. While i t i s highly unlikely, i t i s always possible that at least one effect of the meat i s to cut down a i r currents in the medium thereby maintaining a negative Eh. In case the effect of the meat in G.P.B.I. was solely due to i t s particulate nature, controls were set up in which glass beads or quartz sand replaced the meat particals. Both the beads and the sand were cleaned in the same manner as glassware (31), except that the sand was washed in cleaning solution 48 hours with stirring and dried in' a" hot a i r oven. 40. The presence of p a r t i c u l a t e material made the micro technique impractical, hence to 5 ml. of basal medium, one inch l a y e r of p a r t i c u l a t e material was added. In order to measure t u r b i d i t y of growth, 1 ml. of culture was transferred to the micro tubes and measured i n the turbidimeter. The r e s u l t s of these t e s t s are shown i n Table XI. Table XI.  E f f e c t of P a r t i c u l a t e M a t e r i a l P a r t i c u l a t e M a t e r i a l Growth mgms/ml Lecithinase A c t i v i t y Dried Meat 2.99 1/384 Glass beads 2.12 0 Quartz sand 2.22 0 Not only does the dr i e d meat g r e a t l y increase the growth, but i t contains a high concentration of l e c i t h i n a s e TF. Thus the toxigenic f a c t o r which i s present i n small amounts i n peptone, tryptone and proteose peptone i s present i n water extracted d r i e d meat. While no actual t i t r a t i o n was done, 1 inch of meat i n the t e s t tubes used weighs approximately O.lg. T h i s d r i e d meat i s u s u a l l y about 20 per cent by weight of the o r i g i n a l f r e s h meat. 4 1 . 7. S o x h l e t E x t r a c t i o n I f t h e t o x i g e n i c f a c t o r i s i n a f r e e s t a t e i n meat i t i s n o t w a t e r s o l u b l e . T h i s i m m e d i a t e l y s u g g e s t e d e t h e r e x t r a c t i n g j t h e s m a l l e r t h e p a r t i c l e s o f m e a t , t h e more e f f i c i e n t w i l l be t h e e x t r a c t i o n . F o r t h i s r e a s o n , t h e d r i e d meat was g r o u n d i n a c h e m i c a l l y c l e a n b a l l m i l l f o r f o u r h o u r s . I t was t h e n r e g r o u n d i n a m o r t a r t o b r e a k u p c l u m p s o f s m a l l p a r t i c l e s . A v e r y f i n e p o w d e r , a b o u t t h e t e x t u r e o f q u a r t z s a n d was o b t a i n e d . T h i s i s r e f e r r e d t o a s d r i e d p u l v e r i z e d m e a t ( D P M ) . B e f o r e e x t r a c t i o n w i t h e t h e r , e x t r a c t i o n w i t h m e t h a n o l i s d e s i r a b l e so a s t o remove a n y t r a c e s o f w a t e r r e m a i n i n g . 25 grams o f DPM was p l a c e d i n t o t h e t h i m b l e o f a s o x h l e t e x t r a c t o r and e x t r a c t e d w i t h 100 m l . o f a c e t o n e - f r e e r e a g e n t g r a d e , a b s o l u t e m e t h y l a l c o h o l f o r 8 h o u r s . T h e a l c o h o l was t h e n r e p l a c e d w i t h 100 m l . o f r e a g e n t g r a d e e t h e r a n d e x t r a c t e d f o r f o u r h o u r s . T h i s p r o c e s s was r e p e a t e d u n t i l a l l t h e DPM had been e x t r a c t e d . The a l c o h o l was e v a p o r a t e d f r o m t h e a l c o h o l e x t r a c t l e a v i n g a y e l l o w o i l a n d a w h i t e w a x . A s i m i l a r p r o c e d u r e was f o l l o w e d w i t h t h e e t h e r e x t r a c t . A g a i n a y e l l o w o i l a n d a w h i t e wax were o b t a i n e d . T h e s e e x t r a c t s were p o o l e d b y r e d i s s o l v i n g them i n e t h e r a n d d i s t i l l i n g a t 35 d e g r e e s C - f o r 8 h o u r s and t h e n a t 65 t o 70 d e g r e e s C . u n t i l t h e r e was n o s m e l l o f a l c o h o l . The r e s i d u e was washed i n t o a 100 m l . b e a k e r w i t h a m i n i m a l amount o f e t h e r a n d t h e e t h e r removed o v e r a s t eam b a t h . T h e o r e t i c a l l y a l l t h e f r e e w a t e r s o l u b l e s u b s t a n c e s a s w e l l a s a l l f r e e l i p i d s ( i n c l u d i n g l e c i t h i n ) s h o u l d now have been removed f r o m t h e D P M . A l l fractions were added in a quantity equivalent to 0.1 gram of dried meat to 5 ml.of basal medium. Again 5 ml. amounts of medium had to be used and the growth was measured as before. The effect of these various fractions on growth and lecithinase production i s given in Table 111. Table X l l Effect of Alcohol and Ether Extraction. Addition to Basal Medium Growth mgms/ml Lecithinase Activity Control 1.79 0 Dried Meat 2.58 1/192 Extracted Dried Meat 2.75 1/192 D.P.M. 2.70 1/192 Extracted D.P.M. 2.58 1/96 Alcohol Ether Extract 2.11 0 Extracted D.P.M. & Extract 2.52 1/96 There appears to be a slight loss in ac t i v i t y i n the extraction of DEM which cannot be restored by the addition of the extract. This suggests that the TF may be a protein which has been part i a l l y inactiv^ated by the alcohol. However, there was s t i l l sufficient TF in the extracted meat particles to jus t i f y purification before considering the l i p o i d a l fraction. 8. Hydrolysis of Extracted Meat If the TF i s not soluble in water or in the fat solvents i t i s quite possible that i t i s present in the meat in a conjugated form. This, of course, suggests treatment by hydrolysis. The 43 three common methods of hydrolysis employed are a c i d , a l k a l i and emzymatic. I t was decided f i r s t to t r y d r a s t i c conditions of hydrolysis and i f no a c t i v e f r a c t i o n was obtained to repeat the procedure under l e s s severe conditions. The procedures are outlined below. a) A c i d Hydrolysis. 10 grams of extracted DPM were suspended i n 100 ml.of water and 6 ml. of 36 N H £ S0^ added. This was autoclaved at 121 degrees C. f o r 3 hours, cooled, and 0.2N Ba ( OH ) 0 added u n t i l there was no f u r t h e r p r e c i p i t a t i o n of barium s u l f a t e . A small sample of the n e u t r a l i z e d hydrolysate was removed and the remainder concentrated t o 20 ml.so that 1 ml. was equivalent to 0.5 grams of dri e d meat. b) A l k a l i h y d r o l y s i s . To 10 grams of extracted DPM, 93.5 grams of Ba ( OH ) 2 and 100 ml. of water were added. This gives an a l k a l i n e normality equivalent to that of the a c i d h y d r o l y s i s . The suspension was autoclaved three hours at 121 degrees C.} and cooled. S u l f u r i c a c i d was added u n t i l there was no f u r t h e r p r e c i p i t a t i o n of barium s u l f a t e and the p r e c i p i t a t e removed by c e n t r i f u g a t i o n . A sample was taken and the remainder concentrated so that 1 ml.was the equivalent of 0.5 grams of DPM. c) T r y p t i c h y d r o l y s i s . 30 grams of extracted DPM and 500 mgms.of t r y p s i n ( D i f c o 1: 250 ) were suspended i n 300 ml.of water, adjusted to pH 8:3 with Na OH ( with phenolphthalein ) and incubated i n a water bath a t 40 degrees C.under toluene. The pH was adjusted d a i l y . 44. However, a f t e r a large drop the f i r s t day, l i t t l e adjustment was required. On the eighth day, another 250 mgms-of t r y p s i n was added as d i g e s t i o n appeared to have stopped. A f t e r twelve days incubation, the digest was removed from the water bath and f i l t e r e d through three l a y e r s of #1 Whatman f i l t e r paper under suction. The volume of the f i l t r a t e obtained was 420 ml-so that 1 ml.was equivalent to 71 mgms.of DPM or 7 ml.was equivalent to 0.5 grams of DPM. The toluene was removed by allowing i t to separate from the .hydrolysate f o r 6 hours i n a separatory funnel. On cooling, some f a t t y material separated out of the hydrolysate, and t h i s was removed by extracting with equal volumes of ether four times i n a separatory funnel A small volume of the t r y p t i c hydrolysate was concentrated so that 1 ml-was equivalent to 1 gram of DPM. Further concentration r e s u l t e d i n the formation of a p r e c i p i t a t e . In' t e s t i n g the a c t i v i t y of a t r y p t i c hydrolysate, one must consider the p o s s i b i l i t y that t r y p s i n i t s e l f might contain a TF, or that the enzyme may act on the egg yolk i n such a way as to mask the l e c i t h i n a s e a c t i v i t y ; hence controls were set up to r u l e out these two p o s s i b i l i t i e s . The e f f e c t of the three hydrolysates on l e c i t h i n a s e production i s shown i n Table X l l l . In each case, the f a c t o r added to the medium was equivalent to 0.5 grams DPM, unless otherwise stated. 45 Table X l l l E f f e c t of Hydrolysis Addition to Basal Medium Growth mgms/ ml Lecithinase A c t i v i t y Control 1.80 0 Soxhlet Extracted DPM 2.58 1/96 Unconcentrated a c i d hydrolysate - 1/6 Concentrated a c i d hydrolysate - 0 Unconcentrated a l k a l i hydrolysate - 1/6 Concentrated a l k a l i hydrolysate - 1/12 T r y p t i c hydrolysate 2.52 1/96 Concentrated t r y p t i c hydrolysate 1.88 1/96 Ether extracted t r y p t i c hydrolysate 2.35 1/48 Aqueous t r y p s i n - 0 Aqueous t r y p s i n ( Uninoculated ) 0 0 T r y p t i c Hydrolysate ( Uninoculated ) 0 0 There was no l o s s of e i t h e r growth or toxin promoting properties on hydrolysis with t r y p s i n . Trypsin i t s e l f does not contain the TF. By using l e s s d r a s t i c conditions, the TF might po s s i b l y have been recovered i n an acid or an a l k a l i hydrolysate. 46 I t should be noted that concentrating the acid hydrolysate by b o i l i n g destroyed what l i t t l e TF was present, showing that a l i t t l e more than a three hour hydrolysis would destroy a l l the f a c t o r s . Thus, f a c t o r s to be i s o l a t e d from the t r y p t i c hydrolysate w i l l a l s o be more stable i n a l k a l i than i n a c i d . A t i t r a t i o n of the amount of t r y p t i c hydrolysate required to allow the production of l e c i t h i n a s e by CI. perfringens i n d i c a t e s that a 5 per cent concentration i n the basal medium was required. Since t h i s i s the approximate equivalent of 1.5 per cent of the o r i g i n a l pulverized meat p a r t i c l e s , i t appears that there was no destruction of the TF during enzymatic h y d r o l y s i s . 9) D i a l y s i s of the Trypic Hydrolysate. E s s e n t i a l l y the hy d r o l y t i c procedure i s an attempt to reduce molecular s i z e . A f u r t h e r separation can be obtained by d i a l S s i s which allows materials of small molecular si z e to di a l y z e out while l a r g e r molecules are retained. No information i s a v a i l a b l e as to the pore s i z e of the v i s k i n g sausage casing used i n t h i s procedure. A 50 ml.quantity of the t r y p t i c hydrolysate was dialyzed i n 925 ml. of double d i s t i l l e d water f o r 60 hours at 4 degrees C Both the d i a l y z a b l e and the non d i a l y z a b l e f r a c t i o n s were evaporated i n vacuo to dryness, and resuspended i n 25 ml. of water. The rate of evaporation was such that the approximately 250 ml. of water was removed per hour although the temperature was maintained below 50 degrees C. As can be seen from the r e s u l t s i n table XIV the toxigenic f a c t o r i s completely d i a l y z a b l e . L a t e r work i n which an increased volume of the hydrolysate was dialyzed i n the same volume of water re s u l t e d i n some a c t i v i t y remaining i n the non-dialyzable f r a c t i o n s . 4 7 Table XIV. D i a l y s i s of T r y p t i c Hydrolysate. Addition to Basal Medium Growth mgms/ml Lecithinase A c t i v i t y Control 1.68 0 t r y p t i c hydrolysate. 2.41 1/96 d i a l y z a b l e f r a c t i o n 2.18 1/96 non d i a l y z a b l e f r a c t i o n 1.68 0 d i a l y z a b l e and non d i a l y z a b l e f r a c t i o n 2.22 1/96 10) Adsorption of the Toxigenic F a c t i o n • The a c t i v e material i n the pancreatic f a c t o r of Adams, Hendee, and Pappenheimer (26 ) i s a l s o reported to be f r e e l y d i a l y z a b l e . The pancreatic f a c t o r could be adsorbed on charcoal from an a l c o h o l i c s o l u t i o n at pH 4.0 and eluted with water at pH 9«0. B e l l ( 4 4 ) found that h i s toxigenic f a c t o r was adsorbed by F u l l e r ' s earth at pH 2.6 and could not be eluted by eluants which he employed. The e f f e c t of adsorption with N o r i t e charcoal and F u l l e r ' s earth at various hydrogen ion concentrations was determined. Four 20 ml. samples of the d i a l y z a b l e f r a c t i o n were adjusted to pH 3.1 with methyl orange, pH 4.5 with methyl, pH 7.0 with brom thymol blue and pH 8.3 with phenolphthalsin. To an 8 ml.portion of each was added 1 gram of Norite and to a second 8 ml.amount was added 2 grams of F u l l e r ' s earth. The remainder of each sample served as a c o n t r o l . A f t e r adsorption at room temperature f o r 16 hours, the adsorbant was centrifuged o f f and the supernatants adjusted to pH 7.0. The toxigenic potency of these f r a c t i o n s i s given i n Table XV. 48 I t should be noted that only a single adsorption was c a r r i e d out i n each case. Time d i d not permit attempts to elute the a c t i v e substance from F u l l e r ' s earth. However, i t i s c l e a r l y shown that the TF i s l a r g l y removed by adsorption on F u l l e r * s earth at a c i d pH l e v e l s , and t h i s f i n d i n g agrees with that of B e l l (44 ) . Table XV Adsorption of Toxigenic Factor. Method of Adsorption Lecithinase A c t i v i t y Control pH 3.1 1/96 Control pH 4.5 Control pH 7.0 1/96 1/96 Control pH 8.3 1/96 Norite pH 3.1 1/96 No r i t e pH 4.5 I / 4 8 Norite pH 7.0 1/96 Norite pH 8.3 1/96 F u l l e r ' s earth pH 3.1 1/6 F u l l e r ' s earth pH 4.5 I / 2 4 F u l l e r ' s earth pH 7.0 1/96 F u l l e r ' s earth pH 8.3 1/96 I # Since the ether extracted d i a l y z a b l e f r a c t i o n s was pH 7.0 t h i s sample had no treatment and serves as a c o n t r o l . 49 11) Chemical Tests to Determine the Nature of the  Adsorbed substances. As stated above, time f o r experimental work had ended with the establishment of a successful adsorption procedure. I t was quite apparent that the toxigenic f a c t o r could not be i d e n t i f i e d i n the time remaining. In view of t h i s , i t was f e l t that an attempt to characterize the adsorbed substance would be of great benefit to an i n v e s t i g a t o r carrying on t h i s p r o j e c t . I f one knows the types of compounds adsorbed then much of the 11 t r i a l and er r o r method " w i l l be removed from the formulation of an elution'procedure. The procedures employed are those found i n the laboratory manual of the Biochemistry Department of t h i s U n i v e r s i t y . In a l l cases the ether extracted d i a l y z a b l e f r a c t i o n was tested before and a f t e r adsorption with F u l l e r ' s earth at pH 3.1. Both B i a l ' s and Benedict's t e s t s were negative i n d i c a t i n g that n e i t h e r pentoses nor reducing sugars were present i n e i t h e r case. The Hopkins- Cole g l y o x y l i c a c i d t e s t was p o s i t i v e i n the non- adsorbed f r a c t i o n and negative i n the adsorbed f r a c t i o n . T h i s i n d i c a t e s that tryptophan or a substance with a tryptophan grouping has been removed by the adsorption. I f the toxigenic f a c t o r i s a protein d e r i v a t i v e , one method of'separation and i d e n t i f i c a t i o n i s by paper chromatography ( 23 )• Chroma^graphy, using 1 i n 4 i s o l u t y ^ i c a c i d and water as the developer, showed that numerous ninhydrin p o s i t i v e substances were present i n both f r a c t i o n s . No dif f e r e n c e s were found i n the chromatograms. However, i n view 50 of the f a c t that only one solvent was t r i e d , these negative r e s u l t s are not s i g n i f i c a n t . Spectrographic a n a l y s i s f a i l e d to show adsorption of any inorganic substance. The spectrographic procedure involved the use of a carbon arc so that carbon and nitrogen could not be determined. An unexpected f i n d i n g was that both aluminum and calcium were eluted from the F u l l e r ' s earth during the adsorption procedure. In adsorption experiments such a phenomenon i s not u s u a l l y considered, however, i t i s quite reasonable t o expect basic ions to be eluted by an a c i d i c f l u i d . . While i t appears improbable, i t i s therefore nevertheless possible that the toxigenic f a c t o r may not have been a c t u a l l y adsorbed but that the apparent l o s s of t o x i g e n i c i t y was due to an i n h i b i t o r y concentration of calcium or aluminum, obtained from the adsorbing material, and present i n the f i l t r a t e s . 5 1 . DISCUSSION. I t should be pointed out that the experiments above are not reported i n the same sequence i n which they were c a r r i e d put unless s p e c i f i c a l l y so stated. In general, the sequence was as above except that the e f f e c t of inorganic ions, previously reported toxigenic f a c t o r s , and the carbon balance ( see appendix C ) were determined during the long slow f r a c t i o n a t i o n of the meat. Thus i t was not a case of turning to a n a l y t i c a l procedures when the t r i a l and e r r o r method had been unsuccessful. Unless otherwise stated, a l l t e s t s were c a r r i e d out i n t r i p l i c a t e and each s e r i e s of t e s t s repeated at l e a s t once. The r e s u l t s presented represent modal averages. In most cases, the modal and median averages are the same. B i o l o g i c a l v a r i a t i o n made the use of arithmetic averages i m p r a c t i c a l . The s e n s i t i v i t y of the method f o r determing growth cannot . be questioned. The amount of growth i n the basal medium under standardized conditions was consistant. However, t h i s i s to be expected i n a chemically reproducible medium where the components of each batch of medium are i d e n t i c a l . N a t u r a l l y there was b i o l o g i c a l v a r i a t i o n whereby one culture i n s i x or nine would give much l e s s or much more growth than the others, however, by taking modal averages t h i s v a r i a b l e has a minimal influence on r e s u l t s . The methods f o r determining the theta t o x i n and the l e c i t h i n a s e a c t i v i t y are r e l a t i v e l y tfti@R§itivej. The t i t e r of l e c i t h i n a s e i s never high and i t i s quite possible that a low t i t e r l e c i t h i n a s e would give negative r e s u l t s by the egg yolk t i t r a t i o n . However the primary object of t h i s i n v e s t i g a t i o n 52 was the production of l e c i t h i n a s e with an a c t i v i t y equal to that produced i n G.P.B.I. In view of t h i s , i t was not deemed neaessary t o use the more s e n s i t i v e and more time consuming methods of Gale and van Heyningen ( 44 ) f o r theta t o x i n and of Zamecnik, Brewster and Lipmann ( 14 ) f o r l e c i t h i n a s e a c t i v i t y . Some recent tentative work by Duff ( 46 ) has suggested that l e c i t h i n a s e may be produced h i n minute qua n t i t i e s i n the basal medium. Whether his findings were the r e s u l t of a more se n s i t i v e technique or whether they were the r e s u l t of carry over i n the inoculum has not been determined. Most reports i n the l i t e r a t u r e state that theta toxin i s l e t h a l i n large q u a n t i t i e s . However, a l l such reports dealt with theta toxin produced i n complex media, without reference to the p r o b a b i l i t y that l e s s e r amounts of alpha toxi n may a l s o have been produced, and that deaths r e s u l t i n g from large volume inocula may well have been due to t h e i r content of alpha t o x i n . As an example, i n one of our own s e r i e s where in o c u l a t i o n of 0.5 ml. of the basal medium toxin was l e t h a l to mice, the toxin was found to have a l e c i t h i n a s e a c t i v i t y of 1/12. Subsequent generations of the same serie s had n e i t h e r l e c i t h i n a s e a c t i v i t y nor l e t h a l p r o p e r t i e s . In spite of the f a c t that the t o x i n produced i n the basal medium i s not l e t h a l i n q u a n t i t i e s up to 1 mL, i t appears to have the other c h a r a c t e r i s t i c s of CI. perfringens theta t o x i n . The f a c t that l e c i t h i n a s e was not produced when previously reported toxigenic f a c t o r s .of known chemical composition were added to the basal medium shows that while these substances may be stimulatory or may act i n conjunction with other f a c t o r s , they are not themselves the u n i d e n t i f i e d toxigenic f a c t o r . I t 53 must be remembered that the substances, so claimed as toxigenic f a c t o r s , were i n f a c t added, not to a basal reproducible medium as recorded i n the present work, but were added to a medium of o r i g i n a l organic complexity. I t may be f a i r l y claimed that an advance has been achieved i n t h i s laboratory since the t o x i c f a c t o r concentrates c l e a r l y r e s u l t e d i n alpha toxin production i n a n u t r i t i o n a l l y adequate, chemically reproducible medium, which i n t h e i r absence was unable to elaborate t h i s product. The l a c k of enzyme formation when choline chloride and sodium glycerophosphate were added to the basal medium does not n e c e s s a r i l y imle out p o s s i b i l i t y that glycerophosphocholine i s a toxigenic f a c t o r . I f t h i s substance i s , i n r e a l i t y , a toxigenic f a c t o r then l e c i t h i n a s e could be classed as an adaptive enzyme by Stanier's ( 47 ) theory of simultaneous adaption. I f , however, Stanier's theory i s v a l i d , one would expect at l e a s t trace amounts of the enzyme being formed even though the choline and a glycerophosphate were not joined i n t o a sin g l e molecule. A. synthesis f o r producing glycerophosphocholine was devised i n which sodium glycerophosphate and ethylamine were methylated and brominated i n an eight step process* .. Assuming 50 per cent y i e l d i n a l l steps, a kilogram of s t a r t i n g material would give four grams of f i n a l product. A more serious d i f f i c u l t y i s the nature of the molecule i t s e l f . By reference to f i g u r e 1 i t can be seen that the p o s i t i v e l y charged nitrogen atom i s the s i t e of greatest r e a c t i v i t y . I t would be extremely d i f f i c u l t even i f the N were blocked to ensure a nieibhyl ester rather than an N-0 linkage. The r e a c t i v i t y of the nitrogen means that i f the 54 choline chloride and the sodium glycerophosphate had been united by some process during b a c t e r i a l growth the bond would have been an N-0 linkage. As pointed out previously, the chief d i f f i c u l t y i n i s o l a t i n g the toxigenic f a c t o r from any type of peptone i s i t s low s o l u b i l i t y . I t i s quite probable that the TF i n the t r y p t i c hydrolysate and the TF i n tryptone are one and the same. However, i t appears to be e i t h e r i n a more concentrated,more a c t i v e , o r more soluble form i n the hydrolysate. The a c t i v i t y found with the ATP could a l s o have been due to the same TF present as a contaminant. ATP i s now a v a i l a b l e commercially so that i t would be a simple matter to determine whether or not the l e c i t h i n a s e production was due to the ATP i t s e l f or not. I t should be noted that the basal medium contains a l l of the components of adenosine triphosphate, and since the organism grows well i n t h i s medium, i t must be able to synthesize ATP because of the part these enzymes pla y i n the t r a n s f e r of high energy phosphate bonds. The s l i g h t drop i n the toxigenic a c t i v i t y of DPM a f t e r a l c o h o l and ether extraction cannot be accounted f o r . Recombination of the extract and the extracted material does not restore the e'r±&r^tMn&i€ production completely. Two explanations are p o s s i b l e . F i r s t , i f the toxigenic f a c t o r i s of the nature of a protein d e r i v a t i v e , as the work so f a r suggests, the a l c o h o l i c extraction might r e s u l t i n some of the f a c t o r . However, i f the DPM i s not f i r s t extracted with alcohol,the r e s i d u a l moisture i n the meat could reduce the e f f i c i e n c y of the ether. Secondly, and l e s s probable, the l i p o i d f r a c t i o n may contain two f a c t o r s , one which stimulates 55 l e c i t h i n a s e formation, and a second which i n h i b i t s the a c t i v i t y of the stimulatory material. However, the f a c t that the t r y p t i c hydrolysate had been extracted with ether to remove l i p i d s which had presumably been freed by the hydrolysis, suggests that the soxhlet extraction before hydrolysis i s not an e s s e n t i a l step. On the other hand, i t may be that i f t h i s f i r s t extraction procedure i s omitted, the e f f i c i e n c y of the hydrolysis w i l l be reduced. Just what reactions take place during the t r y p t i c h ydrolysis i s not known. I t i s quite c e r t a i n that amino acids are not formed, as p u r i f i e d t r y p s i n s p l i t s proteins only to the proteose peptone stage by attacking peptide linkages i n v o l v i n g the carboxyl groups of ar g i n i n E and.lysine. I f , however, the t r y p s i n used ( D i f c o ) contains other enzymes i t i s quite possible that the proteins may have been p a r t i a l l y s p l i t to dipeptides or amino acids. C e r t a i n l y the hydrolysate contained several iiMhydrin p o s i t i v e substances vvhich must have a free carboxyl and free alpha amino jiot group to give t h i s r e a c t i o n . I t is Aunknown f o r a peptide linkage to involve a d e l t a carboxyjh group ( eg. glutathione ) and i t might be possible f o r a diamino acid to have a linkage leaving the alpha amino group f r e e , so that these ninhydrin p o s i t i v e substances need not be amino ac i d s . One can only speculate as to the s i z e of the molecules i n the di a l y z a b l e f r a c t i o n . No information i s a v a i l a b l e as to the pore si z e of the v i s k i n g sausage casing used as the d i a l y z i n g membrane. 56 Some cellophane membranes are reported by t h e i r manufactures, to have pore s i z e of 4O Angstroms. N a t u r a l l y , also the shape of the molecule w i l l be more importanjr than i t s actual s i z e i n determining whether or not i t w i l l pass through the membrane. As well as pore s i z e , surface charge must also be considered ( 48 ) . As iin'the case of f i l t e r paper, the retention of any material may be due to opposite charges between the paper and the material. Such could be the case i n d i a l y s i s . However, t h i s would be an immediate e f f e c t rather than a long term e f f e c t as the f i r s t a t t r a c t i o n would n e u t r a l i z e charges and the remaining material would pass through. The nature of the toxigenic f a c t o r f o r CI. perfringens l e c i t h i n a s e has not been determined. The f a c t that a f t e r a c i d adsorption on F u l l e r ' s earth the l o s s of a c t i v i t y was accompanied by the disappearance of trytophan or some tryptophan- containing substance as determined by the Hopkins- Cole t e s t i s s i g n i f i c a n t . The l o s s of a c t i v i t y a f t e r a c i d hydrolysis lends weight to t h i s hypothesis, as tryptophan i s destroyed by such a procedure. A l k a l i n e h y d r o l y s i s , which reduced but did not destroy the toxigenic f a c t o r , does not destroy tryptophan. While the degradation of tryptophan to indole i s well k n o w n ; l i t t l e i s known about i t s r o l e i n other metabolic reactions. I t i s known that tryptophan has a part i n maintaining normal l i p i d metabolism and that t h i s amino a c i d and n i c o t i n i c a c i d show a mutually sparing a c t i o n . I t i s also r e a d i l y deaminated and 57 reaminated. Since the formation of indole involves the opening of the pyrrole r i n g of indolepyruvic a c i d by oxidation, innumerable reactions are p o s s i b l e . Whether or not tryptophan has any part i n the composition of the toxigenic f a c t o r can only be speculated upon at t h i s time. In e a r l i e r work where yeast extract was adsorbed on F u l l e r ' s earth at pH 1.4, i t was found that amino acids were not adsorbed but were present i n the F u l l e r ' s earth f i l t r a t e . One ninhydrin p o s i t i v e substance was found i n the barium hydroxide eluate and although t h i s material was never p o s i t i v e l y i d e n t i f i e d i t was thought to be p r o l i n e or hydroxyproline. By a process of elimination, i t appears that the toxigenic f a c t o r should be a protein d e r i v a t i v e or a conjugate thereof. A n a l y t i c a l procedures have ruled out the p o s s i b i l i t i e s of inorganic s a l t s and of reducing sugars and pentoses ( hence n u c l e i c acids ) while the ether soluble l i p i d s have been removed. This leaves the p o s s i b i l i t y of a non reducing polysaccharide such as glycogen, a vitamin , or a protein d e r i v a t i v e . The s i n g l e procedure which has not been attempted ( because of the l a c k of time ) i s the development of a s a t i s f a c t o r y e l u t i o n method. For only a f t e r the TF has been obtained i n tan eluate w i l l i t be possible to proceed with i t s i d e n t i f i c a t i o n . Throughout t h i s paper, l e c i t h i n a s e has been r e f e r r e d to as both an enzyme and a t o x i n ; however, i t i s f e l t that the time i s f a s t approaching when i t w i l l be necessary to r e v i s e our d e f i n i t i o n of a t o x i n . No longer w i l l we be able to think of a l l t o x i c metabolic products of bacteria as a s i n g l e type of substance, 58 a t o x i n . Instead i t w i l l be necessary to consider toxins i n f i n e r subdivisions, f o r example, as enzymes and enzyme a c t i v a t o r s or as i n h i b i t o r s of enzyme systems or other e s s e n t i a l p h y s i o l o g i c a l processes. Such a system would g r e a t l y f a c i l i t a t e an understanding of what we now c a l l S f i n general, toxins. 59 CONCLUSIONS. 1 . If theulMgbatjon nte^ ,-de.grees^C,to 37 degrees, CI. perfringens produces theta toxin in the chemically reproducible medium of Boyd,Logan;and T y t e l l . 2. As well as the metabolites required for growth, an additional factor present in the meat particles of G.P.B.I. medium i s required for lecithinase production. 3. This toxigenic factor i s destroyed by acid hydrolysis, reduced in a c t i v i t y by alkaline hydrolysis, but i s f u l l y active after tryptic hydrolysis of aqueous extracted dried meat. A . The active fraction of the tryptic hydrolysate i s not soluble in alcohol or ether, i s freely dialyzable, and i s adsorbed on Fuller's earth in acid but not in alkaline or neutral solution. / It i s not adsorbed by Norite charcoal from either acid or alkaline solution. 5. The toxigenic factor does not f u l l y resemble any previously reported factors and i t i s suggested that i t i s a protein derivative. 60 APPENDIX. A SOURCES OF STRAINS OF CL. PERFRINGENS  TYPE A. BP6K Received by the American Type Culture C o l l e c t i o n from Dr. M.J. Boyd, U n i v e r s i t y of C i n c i n n a t i , as BP6K. Received by The Department of Bacteriology aid Preventive Medicine, U n i v e r s i t y of B.C., from The American Type Culture C o l l e c t i o n as 1054-3, May 1950. PB6H Received by Dept. Bact. Prev. Med., U.B.C, from The United States P u b l i c Health Service, 1940 ( ? ) SR 12 US Received by Dept. Bact. Prev. Med., U.B.C, from Dr. J.W. Hornibrook, United States P u b l i c Health Service, February 1946. SR 12 Eng Received by Dept. Bact. Prev Med., U.B.C, from Dr.J. Keppie, L i s t e r I n s t i t u t e , J u l y 1945. S 10 7F Received by Dept. Bact. Prev. Med., U.B.C. from Dr. J . Keppie, L i s t e r I n s t i t u t e , J u l y 1945. ACT3624 Received by The American Type Culture C o l l e c t i o n from Dr. I . C H a l l , U n i v e r s i t y of Colorado as S t r a i n 26, Wilsdon's Type A. Received by The Dept. Bact. Prev Med., U.B.C, from The American Type Culture C o l l e c t i o n as 3624, 1 9 a ( ? ) LH 119 Received by Laboratory of Hygiene, Ottawa from Dr. G.B. Reed, Queenss U n i v e r s i t y , May 1940, as S t r a i n 29, Becently Isolated Type A. Received by Dept. Bact. Prev. Med., U.B.C, from Dr. L. Greenberg, Laboratory of Hygiene, October 1948. 61 APPENDIX.B. CONSTITUENTS OF THE DOUBLE STRENGTH BASAL MEDIUM (19) Glucose 2.0 gm. Ri b o f l a v i n 100.0 X. Ascorbic a c i d 50.0 mgm. Ca d-pahtothenate 200.0 " DL- Alanine L- Arginine 100.0 " 50.0 " am Pyridoxine dihydro-chloride 100.0 " DL- Aspa r t i c a c i d 100.0 " B i o t i n 1.0 " L- Cystine 20.0 " MgSO^. 7H20 40.0 mgm Glycine 100.0" FeS0^.7H 20 2.0 " L- Glutamic a c i d 150.0 " MnS0^.4H20 2.0 " L- H i s t i d i n e 50.0 " NaCl 2.0 " Hydroxy-L-proline 25.0 " Adjust to pH 7.2 with DL- Isoleucine 50.0 " NaOH L - Leucine 75-0 " K 2HP0 2 1.66 gm L - Lysine 100.0 " KH 2P0 A 0.32 " DL - Methionine 50.0 » L - P r o l i n e 25.0 " Add d i s t i l l e d water to 100 ml. DL - Phenylalanine 50.0 » DL - Serine 150.0 " DL - Threonine 50.0 " L - Tryptophan 50.0" L - Tyrosine 50.0" DL - Valine 75.0" U r a c i l 2.5" Adenine s u l f a t e 3-4" 62 APPENDIX. C.  UTILIZATION OF CARBON. The portion of the i n v e s t i g a t i o n reported here was undertaken i n the hope that a d i f f e r e n c e i n the end products of glucose metabolism i n a medium producing theta t o x i n and one producing l e c i t h i n a s e might give some clue as to the nature of the toxigenic f a c t o r f o r l e c i t h i n a s e production. Had a chemically reproducible medium f o r l e c i t h i n a s e production been obtained, the carbohydrate metabolism was to have been investigated. The interconnections between proteins and carbohydrate metabolism make i t e s s e n t i a l to trace both pathways i f the mode of l e c i t h i n a s e synthesis i s to be demonstrated. CI. perfringens was grown i n both the basal medium and the basal medium with added meat, under anaerobic conditions f o r seven days, and the c e l l f r e e media analyzed f o r r e s i d u a l glucose, v o l a t i l e acids, flon v o l a t i l e acids, and v o l a t i l e neutral products. As well as t h i s , the carbon dioxide produced during growth was determined. a) A n a l y t i c a l Methods. Carbon dioxide was adsorbed i n a l k a l i and calculated from the amount of Na H C O 3 produced. Glucose was determined by the method of Shaffer and Somogyi ( 4 9 ). V o t a t i l e n e u t r a l products were determined by the method of Friedeman and Kless (50) and calculated as e t h y l a l c o h o l . The t o t a l v o l a t i l e acids were determined by the method of Friedemann ( 51 ) and calculated as a c e t i c and butyric a c i d s . A s a t i s f a c t o r y method f o r l a c t i c a c i d determinations was not found. 63 Neither the method of Barker and Summerson ( 52) nor the method of Friedemann and Graeser ( 53 ) gave s a t i s f a c t o r y recovery using a standard l a c t i c a c i d s o l u t i o n f o r a n a l y s i s . F i n a l l y the residue, a f t e r d i s t i l l a t i o n of the v o l a t i l e products, was extracted with ether and the ether extract t i t r a t e d f o r t o t a l a c i d i t y . This ether extract would contain not only l a c t i c a c i d but al s o any other higher f a t t y a c i d present, b) Experimental Results. The r e s u l t s which are presented i n Table XVI, do not show any s i g n i f i c a n t d i f f e r e n c e i n glucose metabolism i n the two media. Table XVI End Products of G l y c o l y s i s End Product Basal Medium Basal Medium plus Meat mgms/lOOml mM/lOOml mgms/lOOml mM/lOOml O r i g i n a l Glucose 1021.000 56.722 1021.100 56.728 Residual Glucose 27.000 1.500 30.400 1.689 Carbon dioxide 394.944 89.737 378.400 86.000 E t h y l Alcohol 6.177 1.343 5.746 1.248 Ac e t i c Acid 67.320 11.220 43 . 9 9 2 7.332 Butyric Acid 485.936 55.220 460.712 52.332 L a c t i c Acid 37.590 5.012 86.250 11.500 The a c e t i c a c i d and butyric a c i d were calculated on the basis of balancing the number of moles of C-4 compounds against number of moles of C-2 and C - l end products. That i s a mole of a C-6 compound w i l l give 1 mole of a C-4 together with 1 mole of a C-2 6 4 or 2 moles of a c-1 compound. In the basal medium, 66. 44O &M of v o l a t i l e acid was produced vriaile i n the meat medium 59. 664 mM was formed from 1 gram of glucose. The recovery of carbon i s 99-8 per cent i n the basal medium and 98.5 per cent i n the basal medium with added meat. Exception may be taken to the calculation of a l l the alcohol as ethyl alcohol and obf the v o l a t i l e acids as acetic and butyric acids, thereby accounting f o r the high recovery of carbon. However, the recovery i s based on weight whereas the empirical r a t i o of acetate to butyrate i s based on molar concentration. I t i s quite possible that the nearly complete recovery i s due rather to compensating errors, c) Discussion. This section of the investigation has no immediate bearing on the remainder of the problem. However, i t i s not easy to f i n d suitable a n a l y t i c a l procedures and the data has been included f o r fixture references. I t cannot be overemphasized that a satisfactory method f o r l a c t i c acid determination must be found before a complete carbon balance can be done. I t could well be that i n t h i s work some of the reagents were at f a u l t since Pappenheimer and Shaskan ( 22 ) report 96 per cent recovery of l a c t i c acid by the method of Friedemann and Graeser ( 53 ) Here again the negative results obtained are not s i g n i f i c a n t . Alpha toxin i s produced i n maximal amounts af t e r eight to sixteen hours growth and f a l l s off ra p i d l y with continued incubation ( 33 ). Therefore one would not expect the metabolic processes involved i n toxin production to continue during a seven 65 day incubation period. That i s , i t s t i l l seems reasonable to postulate that l e c i t h i n a s e production i s a r e s u l t of an a l t e r a t i o n of metabolic processes. I f such i s the case, one would expect t h i s s h i f t to occur during the production of the t o x i n . Quite p o s s i b l y the change i s i n the nature of a so-called " metabolic shunt ". Such a p o s s i b i l t y i s not hard tcjiionceive since the great majority of b a c t e r i a l syntheses are enzymatic and enzymes have, i n some cases, a rather narrow range of phy s i c a l conditions f o r optimal a c t i v i t y . Thus i t could well be that as g l y c o l y s i s advanced, the pH of the reaction mixture was lowered i n t o the optimal range, f o r the enzyme system catalyzing the metabolic shunt. The intermediates ' shunted 1 i n t o the alternate pathway take part i n the reactions r e s u l t i n g i n toxi n production. While t h i s i s going on, the intermediates following the normal metabolic pathway are continuing to form a c i d i c end products so that the pH i s now lowered s u f f i c i e n t l y to make the enzyme system involved i n the shunt inoperative. Thus toxin production i s halted. 66 BIBLIOGRAPHY. 1. • Woods, D.D. Ann. Rev. M i c r o b i o l . , 1,115, 1947. 2. Duff, D.C.B., Can. Jour. Pub. Health, 36, 84, 1945. 3. Oakley, C.L., Warrack, G.H., and van Heyningen, W.E., Jour. Path. Bact., 58, 229, 1946. 4. Evans, D.G., Jour. Path. Bact., 55, 429, 1943-5- MacFarlane, M.G., and Knight, B.C.J.G., Biochem. Jour., 35, 884, 1941. 6. Morton, J.?/., " The Toxic A c t i v i t y of Supernates from CI. w e l c h i i Grown i n a medium of varying Composition "., Unpubl. B.A. Thesis, U n i v e r s i t y of B.C. 1944• 7. Airey, F. " A Study of Alpha Toxin of Clostridium Welchii Type A Toxic Superna,tes.," Unpubl. B.A. Thesis, U n i v e r s i t y of B.C. 1945. 8. Grabar, P., Ann. Rev. Biochem., 19, 453, 1950. 9. Gale, E.F., " The Chemical A c t i v i t i e s of Bacteria.," New York, Academic Press, 1948, pp 161-168. 10. MacFarlane, M.G., Biochem. Jour. 47, 270, 1950. 11. Woolridge, W..,and Higginbottom, R., Biochem. Jour., 32, 1718, 1938. 12. Kiehley, W.W. and Meyerhoff, 0. Jour. B i o l . Chem., 183, 391, 1950 13. MacFarlane, M.G., Biochem. Jour., 47, xxix, 1950. 14. Zamecnik, P.C, Brewster, L.E., and Lipmann, F., Jour. E x p t l . Med., 85, 381, 1947. 15. van Heyningen, W.E., Biochem. Jour., 35, 1246, 1941. 16. Taumura, J.T., T y t e l l , A.A. Boyd, M.J., and Logan, M.A., Proc. Soc. E x p t l . B i o l . Med., 47, 284, 1941. 17. Taumura, J.T., T y t e l l , A.A., Boyd, M.J., and Logan, M.A., Jour. Bact., 42, 148, 1941. 18. B a l l e n t i n e , R., Tuck, G.M., and Oser, B.L., Jour. Am. Chem. S o c , 66, 1990, 1944. 67 19. Boyd, M.J., Logan, M.A., and T y t e l l , A.A., Jour. B i o l . Chem., 174, 1013, 1948. 20. Gale, E.F., and van Heyningen, W.W., Biochem. Jour. 36, 624, 1942. 21. Pappenheimer, A.M., and Shaskan, E., Jour. Bact. 47, 413, 1944. 22. Pappenheimer, A.M., and Shaskan, E., Jour. B i o l . Chem., 155, 265, 1944. 23. Walbum, L.E., and Reymann, C.G,. Jour. Path. Bact., 36, 469, 1933. 24. Seal, S.C., Indian Jour. Med. Res. 30, 229, 1942. 25. Rogers, H.J., and Knight, B.C.J.G., Biochem. Jour. 40, 400, 1946. 26. Adams, M.H., Hendee, E.D., and Pappenheimer, A.M., Jour. E x p t l . Med.,85, 701, 1947. 27. Orr, J.H., Josephson, J.E., Baker, M.C, and Reed, G.B., Can. Jour. Res., 9, 350, 1933. 28. McGaughey, CA., Jour. Path. Bact., 36, 263, 1933. 29. Livesay. H.R., Jour. I n f . Dis., 53, 125, 1933. 30. Stevens, F.A., Jour. Inf. Dis., 57, 275, 1935-31. C u r r i e , J.F., " An Investigation of some of the Amino Acid Requirements of Clostridium Perfringens"., Unpubl. B.A. Thesis, U n i v e r s i t y of B.C., 1949. 32. Reed, G.B., Orr, J.H., and Campbell, W.A., Jour. I n f . Dis., 41, 434, 1927. 33. Todd, M.D., " C h a r a c t e r i s t i c s of Certain Strains of CI. w e l c h i i with S p e c i a l Reference to t h e i r Toxic Components"., Unpubl. M.A. Thesis, U n i v e r s i t y of B.C., 1941. 34. Chapman, G.H., Jour. Bact., 16, 49, 1928. 35- van Heyningen, W.E., Biochem. Jour., 35, 1257, 1941. 36. MacFarlane, M.G., and Knight, B.C.J.G., Biochem. Jour., 35, 884, 1941. 37. Stoner, H.B., and Green, H.N., Biochem. Jour. 39, 474, 1945. 38. Lerner, E.M., and Mueller, J.H., Jour. B i o l . Chem., 181, 43, 1949. 68 39. Mueller, J.H., Jour. Immunol., 42 , 343, 1941 40. Bard, R.C., and Gunsulus, I . C , Jour. Bact. 59, 387, 1950. 41. L e i t h , A.R., " An Investigation of Some Phy s i c a l and Chemical Conditions Influencing Growth of, and Toxin Production by CI. w e l c h i i i n a Simple Medium "., Unpubl. B.A. Thesis, U n i v e r s i t y of B.C. 1945. 42. Porter, J.R., " B a c t e r i a l Chemistry and Physiology "., New York, John Wiley and Sons, 1946, p. 697. 43. Carter, H.E., ED., 11 Biochemical Preparations " New York, John Wiley and Sons, 1949, vol.1,pp 5-9. 44« B e l l , G., Unpublished Data. 45. Mueller, J.H., Bact. Rev., 4, 97, 1940. 46. Duff, D.C.B., Unpublished Data. 47. Stanier, R.Y., Jour. Bact., 54, 339, 1947. 48. Dean, R.B., " Modern C o l l o i d s . , " New York, van Nostrand, 1948, pp 26-29. 49. Shaffer, P.A., and Somogyi, M., Jour. B i o l . Chem., 100, 695, 1933. 50. Friedemann, T.E., and Klass, R., Jour. B i o l . Chem., 115, 47, 1936. 51. Friedemann, T.E., Jour. B i o l . Chem., 123, 161, 1938. 52. Barker, S.B., and W.H. Summerson, Jour. B i o l . Chem., 138, 535, 1941. 53. Friedemann, T.E., and Graeser, J.B., Jour. B i o l Chem., 100, 291. 1933. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0106583/manifest

Comment

Related Items