Glucose Salt Teepol HiVeg™ Broth (Twin pack)

Principle And Interpretation

Glucose Salt Teepol Broth is a special media used to enrich Vibrio parahaemolyticus from sea foods (1) and also used to enumerate the bacteria by MPN technique (2). V.parahaemolyticus is a gram-negative marine bacterium, which causes seafood-borne gastroenteritis in humans (3). Fujino and co-workers were the first to isolate Vibrio parahaemolyticus as a causative agent of food-borne gastroenteritis, following a large outbreak in Japan (4). Glucose Salt Teepol HiVeg™ Broth (Twin pack) is prepared by completely replacing animal based peptone with vegetable peptones to avoid BSE/TSE risks associate with animal peptones. HiVeg™ peptone and HiVeg™ extract provide essential nitrogenous nutrients and the high percentage of sodium chloride (3%) helps for the better enrichment of halophilic V. parahaemolyticus. Glucose is utilized while teepol inhibits the growth of gram-positive organisms. The test sample should be held under moderate refrigeration (about 7 to 10°C) and should be analyzed as soon as possible, after collection as possible. This maximizes the survival and recovery of Vibrio's and reduces the tendency for overgrowth by indigenous marine microflora. Weigh 50 gram of seafood sample into a blender. Add 450 ml of PBS (Phosphate Buffer Saline) dilution water and blend for 1 min at 8000 rpm. This constitutes the 1:10 dilution. Prepare 1:100, 1:1000, 1:10000 dilutions or higher if necessary in PBS. Inoculate 3 x 10 ml portion of the 1:10 dilution into 3 tubes containing 10 ml of enrichment broth i.e. Glucose Salt Teepol Broth in 2x concentration. This represents the 1-gram portion. Similarly inoculate 10 ml of single strength enrichment broth as above. If high numbers of V. parahaemolyticus are expected, the examination may start at the 1:10 dilution of the product (2). After overnight incubation of Glucose Salt Teepol Broth at 35 ± 2°C, a loopful of culture from top 1 cm of the broth showing growth is streaked onto TCBS HiVeg™ Agar (MV189). After overnight incubation at 35 ± 2°C, V. parahaemolyticus colonies on TCBS HiVeg™ Agar appear as round, green or bluish measuring 2.3 mm in diameter, while V. alginolyticus colonies are larger and yellow coloured. These colonies are further identified by biochemical characterization. For biochemical tests in identification of V. parahaemolyticus, V. cholera, and V.vulnificus, appropriate positive control organisms have to be inoculated. When the blue green colonies are finally identified as V. parahaemolyticus, refer to the original positive dilution in the enrichment broth and apply the 3 tube MPN tables for final enumeration of the organism.