Type of Document	Master's Thesis
Author	Farmer, Bradley Donovan
URN	etd-11112004-113024
Title	Improved Methods for the Isolation and Characterization of Flavobacterium columnare
Degree	Master of Science (M.S.)
Department	Veterinary Microbiology & Parasitology (Veterinary Medical Sciences)
Advisory Committee	Advisor Name Title John Hawk Committee Chair Allen Rutherford Committee Member Richard Cooper Committee Member
Keywords	flavobacterium columnare columnaris disease rapd analysis isolation
Date of Defense	2004-10-26
Availability	unrestricted
Abstract Columnaris disease, caused by the bacterium Flavobacterium columnare, is an economically significant problem in many warmwater fish species. Difficulties encountered in the isolation and culture of F. columnare have been an impediment to research on the organism and the disease it causes. The goal of this study was to improve the methods for isolation, culture, identification and maintenance of F. columnare. Following the evaluation of different culture media selective cytophaga agar was determined to be the optimum isolation medium, Flavobacterum columnare growth medium proved to be the optimum culture medium, and tryptone yeast extract agar with increased moisture was best for maintenance of cultures. Biochemical characterization of 49 F. columnare isolates was accomplished utilizing the method of Griffin et al. (1992), and the API ZYM system from BioMérieux (Hazelwood Missouri). Using these methods 48 of the 49 strains were presumptively identified as F. columnare. Ten representative F. columnare isolates were further characterized by the API NE system, and all isolates yielded identical phenotypes. Molecular characterization of various strains of F. columnare was accomplished by random amplified polymorphic DNA analysis (reference). Strains evaluated were confirmed as F. columnare by polymerase chain reaction (PCR) using primers and methodology published by Bader et al (2003). The data from RAPD analysis was used to construct three groups based on similarity comparisons. Methods for antimicrobial susceptibility testing by disk diffusion were evaluated on different formulations of dilute Mueller Hinton agar to address the problem of non distinct zones of inhibition, and to reduce the variability of resulting zone data seen when using the published dilute Mueller Hinton formulation (Hawke and Thune 1992). The improved dilute Mueller Hinton medium formulation reduced variability by 40%, increase overall bacterial growth, and also improved the zones of inhibition by producing distinct margins.
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