Simmons citrate agar

The samples for Salmonella isolation and identification were tested using the ISO 6579 protocol. After incubation, the pre‐enriched media (TSB) were cultured in a ratio of 1:10 on tetrathionate broth (HiMedia, LOT no. M032) and in a ratio of 1:100 on Rappaport Vassiliadis medium (HiMedia, LOT no. M880), followed by incubation at 37 and 42°C, respectively. After the incubation period, the cultures were streaked on xylose–lysine–deoxycholate agar (Merck, LOT no. 105287) and Salmonella‐Shigella agar (BD, LOT no. 274500) media. Suspected colonies for Salmonella spp. (H₂S‐producing and non‐lactose fermenter colonies) were selected for confirmatory biochemical tests. Isolates were cultured on Triple Sugar–Iron agar (HiMedia, LOT no. 211825), SIM medium (BD, LOT no. 211578), urea agar (Merck, LOT no. 108492), lysine iron agar (BD, LOT no. 211363), Simmons citrate agar (HiMedia, LOT no. M099) and Methyl Red‐Voges Proskauer broth (HiMedia, LOT no. GM070) for further confirmation of Salmonella spp. ONPG disks (HiMedia, LOT no. DD008) were used to detect the beta‐galactosidase activity of late lactose fermenters (Bahramianfard et al., 2021 (link)).

A loop full of inoculums from each RVB and SFB cultures were plated onto Xylose lysine desoxycholate (XLD) (Oxoid) and Brilliant green (BGA) (Oxoid) agar plates and incubated at 37°C for 24 hours for identification. Characteristic red colonies with black centers on XLD and pink colonies on BGA plates were examined and considered as presumptive Salmonella colonies. Five typical colonies from both XLD and BGA were streaked onto the surface of pre-dried nutrient agar plates (Oxoid) and incubated at 37°C for 24 hours.
Then, typical colonies from nutrient agar were inoculated into the following biochemical tests for further identification as per ISO-6579 guidelines12 and incubated for 24 hours at 37°C. The biochemical tests conducted include triple sugar iron (TSI) agar (Hi Media), lysine iron agar (Hi Media), Simmon’s citrate agar (Hi Media), urea broth (Hi Media), and indole reaction (motility indole ornithine, MIO) medium (Pronadisa)

The biochemical identification of the organism was done by performing the biochemical tests. The biochemical tests were done as stated on Bergey's Manual of Determinative Bacteriology. Each identified colony with typical Salmonella morphology was confirmed biochemically by triple sugar iron (TSI) agar (Oxoid CM0277), Urease (Himedia M111A), Simmons’ citrate agar (Himedia M099, India), Indole (Oxoid CM0129), lysine iron agar (LIA; Oxoid CM0579), methyl red (MR) and Voges–Proskauer (VP) (Himedia M070) tests. Colonies producing red slant (alkaline), yellow butt (acidic) on TSI agar with H₂S production and bubbles formation/cracking at the butt (gas production), negative urea utilisation (yellow), positive citrate utilisation (deep blue slant), negative for indole production from tryptophan, positive LIA agar (alkaline slant/alkaline butt), positive for MR test and negative for VP test were considered Salmonella positive (ISO 6579, 2002). LIA (Oxoid CM0579) was used to demonstrate hydrogen sulfide production and the decarboxylation or deamination olysine. These Salmonella positive samples showed alkaline slant/alkaline butt. Isolates presumptive of Salmonella for all tests were cultured on nutrient agar (Oxoid CM0003) for antimicrobial susceptibility testing.