Detection and enumeration of Vibrio vulnificus by direct colony immunoblot

A direct colony immunoblot method (DCI) for the enumeration of Vibrio vulnificus was developed. Bacterial colonies were transferred from agar plates to membranes, which were then dried and blocked with bovine serum albumin. Subsequently, the membranes were treated with anti-V. vulnificus H antibodies, washed and incubated with peroxidase-conjugated goat anti-rabbit IgG. After a final wash, the membranes were exposed to a substrate mixture containing H(2)O(2) which resulted in the development of a purple color by V. vulnificus colonies. The DCI detected all clinical and environmental V. vulnificus strains tested and did not cross-react with other Vibrio species including V. cholerae, V. parahaemolyticus, or V. fluvialis. The DCI was then compared to the DNA hybridization procedure (DNAH) using V. vulnificus agar plates inoculated with mixed cultures of V. vulnificus and V. parahaemolyticus and V. vulnificus-seeded oyster homogenates. Both DCI and DNAH detected 1 to 2 log colony forming units (CFU)/mL V. vulnificus mixed with 4 log CFU/mL V. parahaemolyticus. Both methods were comparable and demonstrated no significant statistical differences when enumerating V. vulnificus in mixed cultures or in oyster homogenates seeded with levels of V. vulnificus from 2 to 6 log CFU/mL. The DCI demonstrated clearer color development and was less time consuming than the DNAH.