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The Toxigenic and Hemolytic Activity of Clostridium Perfringens Isolated from Foods Feces and Soil

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The Toxigenic and Hemolytic Activity of Clostridium Perfringens Isolated from Foods Feces and Soil University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 1968 The toxigenic and hemolytic activity of Clostridium perfringens isolated from foods feces and soil Judith Ann Schulze The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Schulze, Judith Ann, "The toxigenic and hemolytic activity of Clostridium perfringens isolated from foods feces and soil" (1968). Graduate Student Theses, Dissertations, & Professional Papers. 6864. https://scholarworks.umt.edu/etd/6864 This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. THE TOXIGENIC AND HEMOLYTIC ACTIVITY OF CLOSTRIDIUM PERFRINGMS ISOLATED FROM FOODS, FECES, AND SOIL By Judith Ao Schulze Bo Ao, Valparaiso University, 1965 Presented in partial fulfillment of the requirements for the degree of Master of Science UNIVERSITY OF M)NTANA 1968 Approved by; Chairman, Board of Examiners Dean,/waduate School ex'' May 27, 1968 Date Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: EP37665 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMI* Dissertation Publishing UMI EP37665 Published by ProQuest LLC (2013). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 481 06-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I wish to express my sincere appreciation to Dr» Kakamnra, Professor and Chairman, Department of Microbiology, University of Montana, to vftiom I am indebted to for his advice, guidance, and encouragement through the course of this investigation» I would also like to thank Mrs» Katherine Kelly for her encouragement and helpful suggestions» A major portion of this investigation was supported by a research grant (UI-00291-02) awarded to Dr» Nakamura from the National Center for Urban and Industrial Health, Public Health Service» 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS p a g e AclC nO W l© d.^G niG ïl'tf S o o o o ^ ooooooooqoooooooooovov ^ ooooooooooo Ü Ij X sij» o f Tctblo s OOOOOOOdOOOOOOOOOOOOOA4090«eO«OOdOOOÛO<l«0 1V" XiXSb of FX^Us oooooooi>QO«9ooooooooooooooooo<i9ooooooooe VXX CHAPTER X O XMTRODIÎC TXQN OOftOdOOOOOOOOO0OOO0«dl>âOd00O0OO«<!^0> X XX« STATEMEINT OF THE PROBLEM o*o»oooa*o@#»»o#oe*oeo X5 XXX* METHODS AND MATERXALS <,coo^o^eaoo«*^o*ooce*o«oc 16 X Vo RESIXÜj TS ee4eea««oo9oooooooftoo«oo«««o04ooo9oooeo 31 Vo DISCUSSION #****o#**oooooo***«e#**@#0eooce**e@@ 6Y VXo SUMMARX *«ee*e#*oo**»o*o*oeo*#*#A##»*#*o0o**ooo S3 BXELXOŒtAPHX #o#o*oeeo#e**oe#»oooo#oooe*eee#*#**##@e*ooA0 S5 AUT0BX0C21AP Hï ##o0Aeo*ee**@«*0OCOooe»oe^«#**»##@o***ooo*0 91 XXX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES T A B L E p a g e l o The toxins of Clostridium perfringens oooooooooooooooooooooo10 2o Composition of media used for toxin assay oooooooooooooooooo 20 3o Scheme for primary isolation of _Co perfringens from foods oo 23 ho Criteria for confirmation of Clostridium perfringens ooooooo 24 5* Protocol for assay of alpha to^cun oooooooooooaooooooooooooo 26 6o Ç» perfringens isolated from foods under various culture COndxtXOnS ooooooooooooaoaooooooooaooooooooooooooooo32 7* Biochemical reactions of perfringens isolated from fecesj soil^ and food ooooooooooooaooooooooooooooooooooooooo34 8o Biochemical reactions of Co perfringens isolated from feces^ SOXl^ and food ooaooooooooooooooooooooooooaoooooooooo35 9o Lecithinase activity of perfringens isolated from VarXOUS sources ooooooooooooooooooooaoooaoooooaoooaaoooaoooa36 10* A summary of the alpha toxin activity of Co perfringens xsolated from feces^ soxl and food oooaooooooooooooooooooooo ^7 l l o Lecithinase activity of mutants of _Co perfringens oooaoooooo 41 12o Biochemical reactions of mutants of ^o perfringens ooooooooo 42 13o Colonial dimensions and hemolytic activity of Co perfrxngens on horse blood ooooooaooooeoooooooooooooooooo 43 l4o Colonial dimensions and hemolytic activity of _Co perfringenS on horse blood oeooooeooaooooooooaoooooooeooo44 XV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T A 3 . E p a g e 15o Colonial dimensions and hemolytic activity of on sheep hlood oooooooo^oooooooooooooooooocioo A-5 160 Colonial dimensions and hemolytic activity of ~penfnxnffens on sheep hlood ooooooooo^oooooooooooooooooooo A*^ 17o Colonial dimensions and hemolytic activity of ^ezina^^en^ on nahhxt hlood ooooooooooooooooooooooooooooo 180 Colonial dimensions and hemolytic activity of on nahhit hlood eoooooooooooooooooooooooooooo A-8 19o Colonial dimensions and hemolytic activity of Co ^er^fn^j^^ens on human hlood oooooooooooooooooooooooooooooo A*9 2O0 Colonial dimensions and hemolytic activity of j^eTin^n^^ens on hnman hlood 000000000000000000000000000000 21o Effect of red hlood cell species on hemolysis due to alpha toxin 00000000000000000000000000000000000000000 53 22o Effect of red hlood cell species on hemolysis die to alpha toxin ooooooooooooooooooooooooooooooooooooooooo 5A 23o Effect of red blood cell species on hemolysis die to theta toxin 00000000000000000000000000000000000000000 55 2Ao Effect of red hlood cell species on hemolysis due to theta toxan ooooooooooooooooooooooooooooooooooooooooo 5^ 25o Hemolytic activity of Go perfringens ÜMJS-6 on erythrocytes of different species at various temperatures 000000000000000 58 260 Effect of time and temperature on the hemolytic activity of commercial phospholipase C on sheep blood 000000000000000 6l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T A B L E PAGE 27o Effect of time and temperature on the hemolytic activity of commercial phospholipase C on rabbit blood oooooooooooooo 62 28o Effect of time and temperature on the hemolytic activity of commercial phospholipase C on human blood ooooooooooooooo 63 VI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES F IG U R E p a g e lo Effect of time and temperature of incubation on alpha toxin activity of C«> perfringens ooo«oo»ooooooooo9o 39 IIo Hemolytic activity of C» perfringens UMJS-6 due to theta toxin on erythrocytes of different animal species «ooooo» 59 IIIo Effect of time and temperature on the hemolytic activity of commercial phospholipase C (80 pg/ml) «ooeeoooooeeoeoe 64 IVo Hemolytic activity of commercial phospholipase C (80 p,g/ml) at 46 C on erythrocytes of different animal species 00000099090090000000e00000«00900»«00e09d00 65 Vo Comparison of hemolysis due to aljha toxin of Co perfringens UMJS-6 and hemolysis due to commercial phospholipase C eooaoooaooa80o«oeooaoo«oosoooo66 via Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I INTRODUCTION Clostridiim perfringens is an anaerobic, gram positive, rod­ shaped bacterium, measuring approximately 5 p long and 1 p video It is encapsulated, nonmotile, and spore-forming, with central to subterminal sporeSo Six types. A, B, C, D, E, and F, are differentiated primarily on the basis of toxin productiono C,o perfringens was first isolated and described under the name Bacillus aerogenes capsulatus by Welch and Nuttal (65) in 1892 as a result of gas bubbles in blood vessels after deatho This organism is the most common of several members of the genus Clostridium associated with gas gangreneo ^0 perfringens also causes enterotoxemia of sheep and cattle, lamb dysentery, "struck" in sheep, and has been found to be the causative agent of enteritis necroticans, and enterotoxemic disease of man with a ease fatality rate of about 40/So Certain strains of this organism are also capable of causing gastroenteritis in man following ingestion of food grossly contaminated with £0 perfringens* Co perfringens is widely distributed over the earth* It is present in air, dust, soil, water, feces, and food products* _C* perfringens is found in the intestine of man and animals, and is part of the normal flora of soil, vÆiere it exists in both vegetative and spore state* The fact that Ç.O perfrirngens occurs universally in nature is well documented* Taylor and Gordon (6O) found C* perfringens in all of 196 soil samples examined0 Of these strains, 189 were found to be type A and 7 were Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. type Do Smith and Gardner (56) found soil samples in which the number of C,o perfringens varied from 110 to 56,700 organisms per
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