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Xerox University Microfilms 300 NorthZ M b Rood Ann Aibor, Michigan 481 OS 77-2477 PARK, Yoong, 1944- QUANTIFICATION AND SELECTED PROPERTIES OF ALPHA TOXIN OF CLOSTRIDIUM PERFRINGENS. The Ohio State University, Ph.D., 1976 Food Technology Xerox University MicrofilmsAnn , Arbor, Michigan 48106 QUANTIFICATION AND SELECTED PROPERTIES OF ALPHA TOXIN OF CLOSTRIDIUM PERFRINGENS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yoong Park, B.Sc., M.Sc. ***** The Ohio State University 1976 Reading Committee 1 Approved By J. R. Chipley M. Mangino T. Kristoffersen E. M. Mikolajcik Advisor Department of Food Science and Nutrition ACKNOWLEDGMENTS I wish to express my sincere appreciation and deepest gratitude to my advisor, Professor E. M. Mikolajcik, for his patience and encouragement throughout my graduate program, and for his guidance and assistance during the research and in the preparation of the manuscript; to Professor T. Kris- toffersen, Chairman, Department of Food Science and Nutri­ tion, for providing the opportunity for graduate study; and to Frofessor W. J. Harper and other faculty members of the department for their cooperation and help in my training program. Special thanks are extended to Professor A. C. Ottolen- ghi, Department of Medical Microbiology, and Frofessor G. P. Royer, Department of Biochemistry for their helpful discus­ sion. I would also like to express my gratitude to my wife, Ok-rin, whose encouragement, sacrifice, and best companion­ ship have enabled me to complete my graduate study. Acknowledgment is made to the Ohio Agricultural Research and Development Center for financial support of the research. ii VITA June 6, 1 9 ^ .... Born - Seoul, Korea 1966 ............. B.Sci. in Food Technology Dongguk University, Seoul, Korea 1968-1970......... Research Associate, Food Resources Laboratory, Korea Institute of Science and Technology, Seoul, Korea 1970-1972......... Graduate Research Associate, Department of Nutritional Sciences, University of California, Berkeley, California 1972 ............. M.Sci. in Food Science University of California, Berkeley, California 1973-1976......... Graduate Research Associate Department of Food Science and Nutrition, The Ohio State University, Columbus, Ohio PUBLICATIONS Y, Park, E. M. Mikola,jcik, and T. Kristoffersen, 1975 Coenzyme A Activity of Lactic Streptococci J. Dairy Sci. , 581786 M. Plitman, Y. Park, R. Gomez, and A. J. Sinskey, 1973 Viability of Staphylococcus aureus in intermediate Moisture Meats J. Food Sci., 3 8 >100^ Y. Park, M. M. Morris, and G, Mackinney, 1973 On Chlorophyll Breakdown in Senescent Leaves J. Agr. Food Chem., 21*279 M. M. Morris, Y. Park, and G. Mackinney, 1973 On the Photodecomposition of Chlorophyll in vitro J. Agr. Food Chem., 21*277 iii T . VJ. Kwon, T . I . Mheen, Y . Park, and Y . R . Pyun, 1970 Production of Single-cell Protein on Petroleum Hydrocarbon, I, Isolation and Selection of Hydrocarbon Utilizing Microorganisms, Kor, J. Food Sci. Tech., 2*10? Y. Park, T. I. Mheen, Y. R. Pyun, and T. W. Kwon, 1970 Production of Single-cell Protein on Petroleum Hydrocarbon, II. On the Growth of Candida tropicalis KIST 359, Kor. J. Food Sci. Tech., 2ill2 FIELDS OF STUDY Major Field* Food Science Studies in Food Science and Nutrition. Professors W. J. Harper, T. Kristoffersen, E. M. Mikolajcik, P. M. T. Hansen, R. V. Josephson, and J. P. Kenyon Studies in Biochemistry. Professors R. A. Scott, G. P. Royer, and G. A. Barber Studies in Microbiology. Professors B. J. Kolodziej, G. D. Kagle, and D. A. Wolff Studies in Immunology. Professor M. C. Dodd Studies in Electron Microscopy. Professor R. M. Pfister Studies in Physical Chemistry. Professors Q. V. Winkle and M. H. Klapper iv TABLE OP CONTENTS Page ACKNOWLEDGMENTS.................. ii VITA ............................................ iii LIST OF T A B L E S...................................viii LIST OP FIGURES................................ X INTRODUCTION .................................. 1 OBJECTIVES .................................... 4 REVIEW OF LITERATURE ........................... 5 The Organism................................ 5 Habitat ................................ 6 Morphological characteristics ............ 7 Biochemical characteristics .............. 7 Factors affecting growth................ • 8 Resistance to hostile environment ......... 11 Enumeration of C. oerfringens in foods. 12 The Toxins.................................. 15 Alpha toxin . ......................... 16 C • oerfringens enterotoxin................ 22 MATERIALS AND METHODS.......................... 27 Toxins and Anti se r a ......................... 27 Electro immunodiffusion Technique.............. 27 Radial Immunodiffusion Technique.............. 28 Lecithovitellin Agar T e s t .................... 28 v Page Hemolysin Indicator Plate Test................ 29 Growth, Alpha Toxin Production and pH Development............................. 30 Polyacrylamide Gel Electrophoresis............ 32 Heat Inactivation of Alpha Toxin.............. 34 Treatment of D a t a ........................... 35 RESULTS ...................................... 3 6 Assay Procedures............................. 36 Electroimmunodiffusion.................... 36 Radial immunodiffusion.................... 37 Lecithovitellin agar...................... 37 Hemolysin indicator plate ................ 40 Factors Affecting Hemolvsin Indicator Test. 48 Diluting agents ......................... 48 Type of agar............................. 55 Minerals and sodium thioglycollate......... 60 Chelating agents................... 69 Red blood cell(rbc) concentration ......... 69 Incubation temperature and t i m e ........... 74 Commercial preparations .................. 74 Bacterial origins ....................... 85 Growth, Aloha Toxin Production■ and pH Development of C. oerfringens.............. 85 Thioglycollate medium .................... 83 Beef broth with ground beef and/or Promine-D............................. 93 vi Pa&£ Population levels of C. -oerfringens and amount of alpha toxin produced........... 99 Selected Chemical and Physical Properties .... 104 Polyacrylamide gel electrophoresis......... 10? Heat inactivation......... ............ 118 DISCUSSION..................................... 156 SUMMARY AND CONCLUSION.......................... 169 REFERENCES..................................... 1 ?4 LIST OF TABLES Distribution of four major lethal toxins among the five types of C. oerfringens. Comparison of concentration of alpha toxin required to produce a minimum detectable reaction zone diameter....... ........... Composition of the Brewer Thioglycollate medium. ............................... Mineral composition of the different types of agar ............................ Generation time, Gt; maximum yield of alpha toxin, Y; and time required for 90Jf reduction of alpha toxin, Dt, in Thioglycollate medium at different incubation temperatures .................. Comparison of generation time, Gt, and maximum alpha toxin yield, Y, in beef broth and 20% ground beef, and Thiogly­ collate medium at incubation temperature of 35 C ...................... Selected characteristics of fractions isolated by polyacrylamide gel electrophoresis of two different commercial preparations of alpha toxin. "D" values for alpha toxin in physiological saline and Thioglycollate medium................................. The percent residual activity of alpha toxin when heated in physiological saline and Thioglycollate medium for 18 min....... The percent residual activity at 18 min of