Fish Microbiology and Quality Assurance

Part A : Presumptive test for the determination of the most probable number of total coliform bacteria

The presumptive test which is specific for the detection of total coliforms is performed by MPN method by adding measured aliquots of sample to lactose fermentation broth containing inverted durhams tube. The positive test is indicated by the growth of bacteria in broth and collection of gas in durahms tube. Only coliform bacteria ferment lactose while other enteric bacteria are not.

Procedure

Sample preparation: 25g of seafood sample to be tested is weighed aseptically and homogenized with 225 ml of physiological saline and suitable serial dilutions prepared using saline solution.

To fifteen test tubes containing inverted durhams tubes, first 5 tubes are added with 10 ml double strength (2xLSTB) and the remaining with single strength Lauryl Sulphate Tryptose (1x LST) broth each. The tubes with the media are sterilized at sterilized at 110⁰C for 10 minutes, and cooled and used for the inoculation of samples. Ten milliliter of appropriate dilution of the sample is inoculated in to each of the 5 tubes of double strength medium, 1 ml each to 5 tubes of single strength medium and 0.1 ml into the remaining 5 tubes of single strength medium. All the tubes are incubated at 37⁰C for 24 h. The tubes are observed for gas formation and acid production. This test is called as presumptive test, which answers for total coliforms. In the case of water samples, appropriate volumes may be added as described above without dilution. The development of gas in any of the tubes is considered presumptive evidence of presence of coliform bacteria in the sample. The MPN is estimated by determining the number of tubes positive in each group and referring to MPN table.