title: A novel agar formulation for isolation and direct enumeration of Vibrio vulnificus from oyster tissue.
abstract: 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, non-selective 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 °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.
authors: The University of Southern Mississippi, Gulf Coast Research Laboratory, 703 East Beach Drive, Ocean Springs, MS 39564, United States.
authors: Kimberly J
authors: KJ
authors: Griffitt
authors: The University of Southern Mississippi, Gulf Coast Research Laboratory, 703 East Beach Drive, Ocean Springs, MS 39564, United States. Electronic address: jay.grimes@usm.edu.
authors: D Jay
authors: DJ
authors: Grimes
mesh_terms: D000818:Animals; D015169:Colony Count, Microbial / Q000295:instrumentation / Q000379:methods*; D003470:Culture Media / Q000378:metabolism; D005506:Food Contamination / Q000032:analysis; D010125:Ostreidae / Q000382:microbiology*; D012758:Shellfish / Q000382:microbiology*; D041261:Vibrio vulnificus / Q000254:growth & development* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D003470:Culture Media
keywords: Direct enumeration; Oysters; Selective agar; Vibrio vulnificus