A Novel Agar Formulation for Isolation and Direct Enumeration of Vibrio vulnificus from Oyster Tissue

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Coastal Sciences, Gulf Coast Research Laboratory


A new selective and differential medium, Vibrio vulnificus X-Gal (VVX), was developed for direct enumeration of V. vulnificus (Vv) from oyster samples. This agar utilizes cellobiose and lactose as carbon sources, and the antibiotics colistin and polymyxin B as selective agents. Hydrolysis of 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (x-gal), used in the agar as a lactose analog, produces an insoluble blue dye that makes lactose positive colonies easily distinguishable from any non-lactose fermenting bacteria. Various bacterial species were spot plated onto thiosulfate-citrate-bile salts-sucrose agar (TCBS), and CHROMagar Vibrio, two vibrio-specific selective agars, nonselective agar, and VVX to compare selectivity of VVX to other widely used media. A V. vulnificus pure culture was serially diluted on VVX and non-selective agar to determine the VVX percent recovery. Water and oyster samples were spread plated on VVX agar and allowed to incubate for 16-18 h at 33 degrees C. Blue and white colonies from VVX agar were picked and screened by end point PCR for the Vv hemolysin vvhA. VVX agar showed a significant improvement over TCBS and CHROMagar at preventing non-target growth. There was an 87.5% recovery compared to non-selective plating and a 98% positivity rate of blue colonies picked from oyster tissue plating. The findings suggest that this new agar is a fast, distinctive, and accurate method for enumeration of V. vulnificus from the environment. (C) 2013 Elsevier B.V. All rights reserved.

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Journal of Microbiological Methods





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