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Identification of Pathogenic Microorganisms in Seafood by Gas Chromatography Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1974 Identification of Pathogenic Microorganisms in Seafood by Gas Chromatography. William Earnest Mccullough Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Mccullough, William Earnest, "Identification of Pathogenic Microorganisms in Seafood by Gas Chromatography." (1974). LSU Historical Dissertations and Theses. 2621. https://digitalcommons.lsu.edu/gradschool_disstheses/2621 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. 74-24,790 NcCULLOUGH, William Earnest, 1937- IDENTIFICATION OF PATHOGENIC MICROORGANISMS IN SEAFOOD BY GAS CHROMATOGRAPHY. The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1974 Food Technology University Microfilms, A XEROX Company, Ann Arbor, Michigan © 1974 WILLIAM EARNEST McCULLOUGH ALL RIGHTS RESERVED IDENTIFICATION OF PATHOGENIC MICROORGANISMS IN SEAFOOD BY GAS CHROMATOGRAPHY A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Food Science by William Earnest McCullough B.S., Louisiana College, 1963 M.S., Louisiana Polytechnic University, 1965 May 1974 ACKNOWLEDGMENT The author wishes to express sincere appreciation to his Major Professor, Dr. M.R. Ramachandra Rao, for his direction, counsel, and assistance during this investigation and during the preparation of this dissertation. Appreciation is also expressed to Dr. Arthur F. Novak, Head of the Department of Food Science, for the opportunity to perform this investigation in the department. Gratitude is expressed to Dr. Arthur F. Novak, Dr. Ronald J. Siebeling, Dr. William H. James, Dr. Joseph A. Liuzzo, Dr. Robert M. Grodner and Dr. Fred H. Hoskins for serving on his examining committee. He wishes to thank Dr. Robert M. Grodner, Dr. James Rutledge, The National Shrimp Dreaders and Processors, Inc., and his fellow graduate students, John Kendall, Mike Moody, Dill Hopkins, Dill Hurst and Sidney Crow, for their helpful suggestions during the course of this research. The author wishes to express thanks to the Office of Naval Research and to the U.S. Public Health Service for financial support during this study. Deep appreciation is expressed to his wife, Jeannette, for her financial support and counsel throughout his education. ii TABLE OF CONTENTS ACKNOWLEDGMENT................................................. ii LIST OF TABLES................................................. vi LIST OF FIGURES................................................ vii ABSTRACT....................................................... ix INTRODUCTION................................................... 1 REVIEW OF LITERATURE........................................... 3 I. Breading as a Source of Contamination.................. 4 II. Staphlococcus in Seafood............................... 7 III. E. coli as an Indicator................................ II IV. Various Tests for Contamination........................ 13 V. Detection of Salmonella................................ 23 VI. Gas Chromatography..................................... 27 MATERIALS AND METHODS.......................................... 33 I. Materials.................... 33 A. Source of Sample................................ 33 B. Glassware....................................... 34 C. Reagents.................................. 36 D. Bacto-Brain Heart Infusion Agar................. 36 E. Bacto-Brain Heart Infusion Broth................ 36 F. Tryptone Glucose Extract Agar................... 36 G. Butterfield's Phosphate Buffer.................. 36 H. Nutrient Agar..... 3 ? I. Eugon Agar...................................... 37 iii J. Tryptic Mannitol Meat Broth...................... 37 K. Trypticase Soy Broth............................ 37 L. Vogel and Johnson Agar........................... 37 M. Coagulase Plasma................................ 38 N. Bacto-Lauryl Tryptone Broth...................... 38 0. Simmons Citrate Agar............................ 38 P. Bacto-Brilliant Green Bile 2 % . ................... 39 Q. Levine Eosin Methylene Blue Agar................ 39 R. M.R.-V.P. Medium................................ 40 S. Tryptone........................................ 40 T. SIM Medium...................................... 40 U. Bacto-Tetrathionate Broth Base .............. 41 V. Bacto-Triple Sugar Iron Agar..................... 41 W. Bacto-Salmonella Shigella Agar................ 41 X. Bacto-Urea Broth................................ 42 Y. Bacto-Bismuth Sulfite Agar....................... 42 Z. Growth Media.................................... 4^ AA. Gas Chromatograph............................... 43 II. Methods................................................ 44 A. Sterilization................... 44 B. Shrimp Homogenate............................... 44 C. Total Plate Count............................... 45 D. Isolation of E. coli from Shrimp................. 46 E. Completed Test on E. coli......................... 4^ a. Indole Test.................................. 48 b. Methyl Red Test.......................... 48 c. Vogus-Proskauer Teat.......................... 49 (I. Citrate Test.................. 49 iv F. Standardization of Inoculum-Staphylocoecus aureus.......................................... 49 G. Most Probable Number Technique for Coagulase- Positive S. aureus............................... 51 H. Standardization of Inoculum-Salmonella........... 53 I. Isolation of Salmonella.......... ............... 53 J. Gas Chromatography.............................. 56 RESULTS........................................................ 60 DISCUSSION..................................................... 95 SUMMARY........................................................ 101 LITERATURE CITED............................................... 103 VITA........................................................... 113 v LIST OF TABLES TABLE Page 1. Characteristics of Test Organisms.............. 35 2. Food Sample Retention Times................................. 61 3. Peak Retention Times. ............ 94 vi LIST OF FIGURES FIGURE Page 1. Coliform-Fecal coliform AOAC method.......................... 47 2. MPN technique for coagulase-positive S. aureus...............52 3. Gas chromatograms of Media I and Media II on Machine 1....... 63 4. Gas chromatograms of Media I containing n-butanol, Media II containing n-butanol and Media II containing redistilled Anisole..................................................... 65 5. Gas chromatograms of E. coli in Media I and Media II on Machine I and Machine II.................................... 66 6. Gas chromatograms of E. coli in Media II on Machine II after I hr, 2 hr, and 10 hr incubation at 35°G.................... 68 7. Gas chromatograms of Enterobacter aerogenes in Media II on Machine II.................................................. 69 8. Gas chromatograms of Enterobacter aerogenes in Media II on Machine II.................................................. 70 9. Gas chromatograms of Salmonella typhimurium in Media II on Machine II.................................................. 71 10. Gas chromatograms of Salmonella thompson in Media II on Machine II after 18 hr and 48 hr incubation at 35°C......... 72 11. Gas chromatograms of Salmonella senftenberg in Media II developed on Machine II.................................. 73 12. Gas chromatograms of Salmonella anatum in Media II on Machine II.................................................. 75 13. Gas chromatograms of a mixture of pure cultures of E. coli, Salmonella typhimurium, and Enterobacter aerogenes in Media II on Machine II. ................................. 76 14. Gas chromatograms of Staphylococcus aureus in Media II with 1 % dextrose and Media II w i t h l k mannitol on Machine I I..........................................................77 IS. Gas chromatograms of green headless shrimp in Media II on Machine II....................................... 78 16. Gas chromatograms of green headless shrimp sample, 401-1, inoculated with Enterobacter aerogenes in Media II on Machine II................................................. 79 17. Gas chromatograms of green headless shrimp sample, 401-1M, inoculated with Enterobacter aerogenes in Media II con­ taining 1 % mannitol on Machine II......... ................. 81 18. Gas chromatograms of green headless shrimp sample, 402-1, inoculated with Salmonella anatum in Media II on Machine II..82 19. Gas chromatograms of breaded shrimp sample, 500, in Media II on Machine II........................................... 83 20. Gas chromatograms of peeled and deveined shrimp sample, 502, in Media II on Machine II.............................. 84 21. Gas chromatograms of breaded shrimp sample, 503, in Media II on Machine II........................................... 85 22. Gas chromatograms of breaded shrimp sample, 600, in Media II on Machine II........................................... 87 23. Gas chromatograms of breaded shrimp sample, 603, in Media II on Machine II........................................... 88 24.
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