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Fish Bacterial Flora Identification Via Rapid Cellular Fatty Acid Analysis

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Fish Bacterial Flora Identification Via Rapid Cellular Fatty Acid Analysis Fish bacterial flora identification via rapid cellular fatty acid analysis Item Type Thesis Authors Morey, Amit Download date 09/10/2021 08:41:29 Link to Item http://hdl.handle.net/11122/4939 FISH BACTERIAL FLORA IDENTIFICATION VIA RAPID CELLULAR FATTY ACID ANALYSIS By Amit Morey /V RECOMMENDED: $ Advisory Committe/ Chair < r Head, Interdisciplinary iProgram in Seafood Science and Nutrition /-■ x ? APPROVED: Dean, SchooLof Fisheries and Ocfcan Sciences de3n of the Graduate School Date FISH BACTERIAL FLORA IDENTIFICATION VIA RAPID CELLULAR FATTY ACID ANALYSIS A THESIS Presented to the Faculty of the University of Alaska Fairbanks in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE By Amit Morey, M.F.Sc. Fairbanks, Alaska h r A Q t ■ ^% 0 /v AlA s ((0 August 2007 ^>c0^b Abstract Seafood quality can be assessed by determining the bacterial load and flora composition, although classical taxonomic methods are time-consuming and subjective to interpretation bias. A two-prong approach was used to assess a commercially available microbial identification system: confirmation of known cultures and fish spoilage experiments to isolate unknowns for identification. Bacterial isolates from the Fishery Industrial Technology Center Culture Collection (FITCCC) and the American Type Culture Collection (ATCC) were used to test the identification ability of the Sherlock Microbial Identification System (MIS). Twelve ATCC and 21 FITCCC strains were identified to species with the exception of Pseudomonas fluorescens and P. putida which could not be distinguished by cellular fatty acid analysis. The bacterial flora changes that occurred in iced Alaska pink salmon ( Oncorhynchus gorbuscha) were determined by the rapid method. Fresh fish contained up to 7 genera in which the aerobic plate counts (APC) was 3.04 log colony-forming units (CFU)/cm2. As the fish spoiled, the APC increased to 6.60 log CFU/cm and the flora was composed of P. fluorescens I putida, Psychrobacter immobilis and Shewanella putrefaciens. The Sherlock MIS rapidly and accurately identified seafood bacteria in fresh fish and can be used to monitor quality changes during iced storage of fish. iv T able of Contents Page Signature Page .................................................................................................................................i Title Page ..................................................................................................................................... ii Abstract ....................................................................................................................................iii Table of Contents .......................................................................................................................... iv List of Figures ................................................................................................................................ix List of Tables .................................................................................................................................xi Acknowledgments .......................................................................................................................xiii C hapter 1: Review of the Bacteriology of Seafood Spoilage..............................................1 1.1. Introduction ............................................................................................................................1 1.1.1. Spoilage in seafood ........................................................................................................ 2 1.1.1.1. Physical spoilage ..................................................................................................... 2 1.1.1.2. Biochemical spoilage ..............................................................................................3 1.1.1.2.1. Adenosine triphosphate degradation .............................................................3 1.1.1.2.2. Proteins ............................................................................................................. 4 1.1.1.2.3. Lipids .................................................................................................................9 1.1.1.2.4. Non-protein nitrogen fraction ......................................................................12 1.1.1.3. Microorganisms in fish spoilage ......................................................................... 13 1.1.1.3.1. Pseudomonas spp ...........................................................................................14 1.1.1.3.2. Shewanellaputrefaciens...............................................................................15 1.1.1.3.3. Psychrobacter immobilis..............................................................................16 1.1.1.3.4. Interaction between microorganisms ..........................................................17 1.1.2. Rapid techniques for total bacterial count and specific bacteria ...........................18 1.1.2.1. Impedance technique for rapid APC detection .................................................18 1.1.2.2. ATP bioluminescence ...........................................................................................19 1.1.2.3. Substrate utilization ........................................................................................... 20 1.1.2.4. Nucleic acid based bacterial identification .......................................................22 1.1.2.5. Rapid technique using bacterial fatty acids ......................................................22 1.1.3. Fatty acids location in cell m em brane ...................................................................... 25 1.1.3.1. Role of fatty acids in bacteria ........................................................................... 25 V Page 1.1.3.2. Fatty acid as biomarkers ......................................................................................27 1.1.3.3. Stress related changes in cellular fatty acids .....................................................28 1.2. References ............................................................................................................................31 Chapter 2: Whole-Cell Fatty Acid Analysis for Bacterial Identification Using the Sherlock M IS .........................................................................................................63 2.1. Abstract .................................................................................................................................. 63 2.2. Introduction ............................................................................................................................64 2.2.1. Fatty acid synthesis ...................................................................................................... 64 2.2.2. Extraction methods ...................................................................................................... 67 2.2.3. Accuracy and reproducibility of Sherlock M IS ....................................................... 69 2.2.4. Objectives ......................................................................................................................70 2.3. Material and methods ...........................................................................................................71 2.3.1. Bacterial isolates ...........................................................................................................71 2.3.2. Characterization of microorganisms ..........................................................................72 2.3.3. Identification of bacteria using Sherlock Microbial Identification System (MIS)............................................................................................................................72 2.3.4. Definitions .....................................................................................................................73 2.3.5. Superimposing comparison charts ............................................................................. 75 2.4. Results and Discussion ........................................................................................................76 2.4.1. Identification capability of Sherlock M IS ................................................................76 2.4.2. Gram-positive bacteria .................................................................................................77 2.4.2.1. Arthrobacter sp. 5 B 6 ............................................................................................77 2.4.2.1.1. Fatty acid profile ............................................................................................77 2.4.2.1.2. Identification based on fatty acid profile ...................................................77 2.4.2.2. Bacillus subtilis 5D 1.............................................................................................78 2.4.2.2.1. Fatty acid profile ............................................................................................78 2.4.2.2.2. Identification based on fatty acid profile ...................................................79 2.4.2.3. Micrococcus varians 7F1.....................................................................................79 2.4.2.3.1. Fatty acid profile ............................................................................................80 2.4.2.3.2. Identification based on fatty acid profile ...................................................81 vi ige 2.4.2.4. Staphylococcus
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