Salmonella Isolation and Identification Protocol

The techniques used to isolate and identify Salmonella were recommended by the International Organization for Standardization (ISO-6579, 2002) and the World Health Organization [27 ]. Global foodborne infections network (formerly WHO global Salmonella Surveillance) [27 ]. In a nonselective liquid medium (buffered peptone water (BPW) (Oxoid CM509, Basingstoke, England), a 10 ml milk sample was mixed with 90 ml of pre-enrichment, and the sample mixture was thoroughly shaken before being incubated at 37°C for 24 hours. Following incubation, the culture was mixed, and a portion (0.1 ml) was transferred to a tube containing 10 ml of selective enrichment liquid medium (Rappaport Vassiliadis (RV)) broth and incubated at 41.5°C for 24 hours. A 10 µl of loop full inoculum from selective enrichment media was streaked onto Xylose Lysine Deoxycholate (XLD) (Oxoid CM0469, Basingstoke, England) agar and Salmonella Shigella (SS) agar plates prepared on petri-dishes and incubated at 37 ± 1°C for 24 ± 3 hours. After proper incubation, the plates were examined for the presence of typical Salmonella colonies. The typical colonies of Salmonella grown on Xylose Lysine Deoxycholate agar medium produce black centers with distinct red colonies due to the color change of phenol red in medium and colorless transparent colonies on Salmonella Shigella agar. The presumptive Salmonella colonies on the XLD (Oxoid CM0469, Basingstoke, England) and SS agar medium were transferred onto the surface of predried nutrient agar plates in a manner that allow isolated colonies to develop and incubated at 37°C for 24 hours in further confirmation with biochemical tests. Thus, all suspected Salmonella colonies were picked from the nutrient agar and inoculated into the biochemical test including Triple Sugar Iron (TSI) agar (Oxoid CM0277, Basingstoke, England) for the TSI test, Simmons's Citrate agar (Oxoid CM53, Basingstoke, England) for the citrate utilization test, Tryptone Soya Broth (Becton Dickinson, USA) for the indole test and Methyl red-Voges Proskauer (MR-VP) (Micromaster Thane, India) for methyl red and Voges Proskauer test and incubated for 24 or 48 hours at 37°C. Colonies producing an alkaline (red) slant, with acid (yellow) but on TSI with blackening or hydrogen sulfide production, negative for Tryptophan utilization on indole test (yellow-brown ring), positive for Methyl red (produce red color on the surface of medium), negative for Voges–Proskauer (yellow color), and positive for Citrate utilization (deep blue slant) were consider to be Salmonella positive [28 (link), 29 ].