Fish Microbiology and Quality Assurance

Part B: Confirmed test for the determination of the most probable number of fecal coliform bacteria

The purpose of the confirmed test is to confirm the presence of bacteria of faecal origin in a sample for which the presumptive test was positive. This is necessary since the positive presumptive may be result of organisms of noncoliform/ non-faecal origin. This is done by subjecting the tubes that are showing the positive result of acid and gas production in presumptive test to confirmatory test. 0.1 ml of the inoculum from each positive tube is transferred to 5 ml of the EC broth tubes containing durham tubes. The E.C. broth tubes are incubated at 44.5 ± 0.5⁰C for 24 h in the water bath. The tubes are observed for gas formation and acid production. This test is called as confirmatory test for faecal coliforms as only faecal coliforms such as E. coli are able to grow at elevated temperature. The MPN is estimated by determining the number of E.C. broth tubes positive in each group and referring to MPN table.

Further, the confirmed test requires streaking inoculums from the doubtful but positive EC broth onto Eosine Methylene Blue (EMB) agar plates, on which E. coli produces characteristic colonies. The plates are incubated at 37⁰C for 48 h and the colonies of E. coli on EMB agar plates appear as dark purple with metallic sheen. Suspected typical colonies of E. coli are counted, isolated and purified on Trypticase Soya Agar (TSA) plates and further subjected to biochemical tests for confirmation.