Graduate Thesis Or Dissertation
 

Replica-plating and computer analysis for rapid identification of microorganisms in seafoods

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  • A method was devised and tested for rapid and quantitative identification of microbial flora in fresh seafoods. The rapid identification of large numbers of isolates was made possible by (1) a simplified identification scheme established by reference culture studies and from the known reactions of microorganisms reported in the literature, (2) the multiple transfer of large numbers of isolates by means of replica-plating, and (3) the use of an electronic computer to analyze data. For the identification of microbial isolates, colonies developing on initial isolation plates were picked by sterile toothpicks and inoculated on a master-plate in prearranged spacing and order. Growth on the master-plate was replicated on a series of solid agar plates containing differential or selective agents. Identifying characteristics consisted of growth responses of the isolates on media containing penicillin, tylosin, vancomycin, streptomycin, chloramphenicol, neomycin and colistin; growth responses on Bacto-SS, Bacto-S-110, Bacto-potato dextrose agar; and culture pigmentation, cell morphology and the Gram-reaction. Information was processed by an IBM 1410 digital computer which sorted and grouped each isolate into one of ten microbial genera or groups, according to a programmed identification key. The identification system was tested by analyzing the microbial flora of dover sole fillets (Microstomas pacificus) and ground beef. This rapid identification method was employed in an investigation designed to determine the nature of the microbial flora shifts in dover sole resulting from irradiation and storage at 6°C. The relationship between the microorganisms which initially survive irradiation, and those making up the final spoilage flora, was determined. A total of 2,723 isolates were examined in this study. The spoilage of unirradiated control samples during storage at 6°C was almost entirely due to the growth of Pseudomonas. This group, which occupied 25 percent of the fresh flora, grew up to nearly 100 percent in two days storage. In contrast, irradiation doses of 0.1, 0.2, 0.3, and 0.4 megarad favored the growth of Achromobacter and yeasts. Micrococcus species, which survived radiation, did not grow at 6°C. At 0.5 megarad, spoilage of fish samples at 6°C was due entirely to yeasts.
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