A Medium for Presumptive Identification of Vibrio parahaemolyticus.

A medium has been devised for rapid, presumptive identification of Vibrio parahaemolyticus. Fermentation of mannitol, sucrose and lactose, arginine dihydrolase, production of indole, gas from carbohydrate and H 2S can be recorded in a single tube of the medium. Because of the ability to test seven characteristics suitable for identifying this species in a single step, the biochemical screening of large numbers of isolates from seafood, water and sediment samples is greatly facilitated. Vibrio parahaemolyticus is an important human pathogen, and has frequently been isolated from seafood. A major cause of food poisoning in Japan, it has also been associated with outbreaks in the United States and other countries (3). In addition to having been recognized as an important foodborne pathogen, V. parahaemolyticus has also been shown to be an autochthonous estuarine bacterial species. Thus it is an organism of interest for microbial ecologists as well as medical and food microbiologists. Because of extensive studies carried out in recent years, the isolation and identification of V. parahaemolyticus is relatively uncomplicated, but reliable identification requires that a number of biochemical tests be done to avoid confusion of V. parahaemolyticus with related marine vibrios. Several enrichment and isolation media have been reported for V. parahaemolyticus (3), with the isolation medium most commonly employed being thiosulfate citrate bile salts sucrose agar (TCBS). In TCBS, V. parahaemolyticus colonies are bluish or blue-green in color. Unfortunately, a number of other Vibrio species produce a similar reaction, necessitating the employment of additional biochemical tests for reliable identification. Because screening, followed by extensive biochemical characterization of large numbers of isolates is laborious and time-consuming, as well as expensive, a multi-test, presumptive identification medium was devised to reduce time, labor and cost Present address: Department of Microbiology, University of Washington, Seattle, Washington98195. involved. MATERIALS AND METHODS The basis of the medium, hereafter referred to as VP medium, rests upon several key biochemical characteristics of V. parahaemolyticus, i.e. mannitol fermentation and production of indole, negative sucrose and lactose fermentation reactions, positive arginine dihydrolase reaction, lack of gas production from carbohydrates and production of hydrogen sulfide from sodium thiosulfate. The ingredients for VP medium are listed in Table 1. To prepare VP medium. the ingredients must be thoroughly mixed in I liter of distilled water and the pH adjusted to 6.7. The medium is heated to a boil, dispensed in 5-ml quantities in 13 x 100 mm tubes and autoclaved at 121 C for 12 min. The tubes are cooled in a slanted position to allow a generous butt. Colonies of suspect V. parahaemolyticus can be picked directly from isolation plates or membrane filters and inoculated into the medium by stabbing to the base of the tube with a straight needle, and then withdrawing the needle to inoculate the slant. The inoculated tubes are incubated at 35 C for 18-24 h, after which the reactions are recorded. To detect indole production, 3-4 drops of Kovacs reagent (p-dimethylaminobenzaldehyde,lO g, amyl or isoamyl alcohol, ISO ml; HCI (concentrated), SO ml) are added to each tube. TABLE 1. Formula of the medium employed for presump· tive identification ofVibrio parahaemolyticus.