Simmons citrate agar

From each specimen, samples were inoculated onto MacConkey agar, Xylose lysine deoxycholate (XLD) agar, Cystine lactose electrolyte deficient agar (CLED), sheep blood agar, and chocolate agar (Oxoid, UK). The plates were incubated at 37°C aerobically for 24 hours [15 ].
For blood culture, blood samples were aseptically inoculated to Tryptic Soy Broth (Oxoid UK) in 1:5–1:10 proportions and incubated at 37°C. Overnight incubated culture bottles were sub- cultured onto sheep blood, chocolate and MacConkey agar. Culture tubes with negative results were examined for growth daily until fifth day and subsequently subculture when growth was observed. Species of Enterobacteriaceae were identified from pure culture colonies, based on cellular morphologies and biochemical tests as per the standards of microbiology procedure [15 ].
Isolation and identification of Enterobacteriaceae species from water samples was done using multiple tube fermentation method [15 , 17 ]. Phenotypic bacterial identification of Enterobacteriaceae species was made manually using the following culture media: Urea 40% broth, Simmons citrate agar, sulphide in dole motility medium, Triple Sugar Iron Agar and Klingler Iron Agar and Oxidase test reagents (Oxoid, UK) [17 ].

All Shigella strains were previously cultured in trypticase soy broth (Oxoid, England) for 6 to 8 hours; Subsequently, each strain was plotted onto Salmonella–Shigella agar (Oxoid, England) and incubated at 37°C from 18 to 24 h. Presumptive identification was carried out using biochemical tests consisting of triple sugar iron agar, lysine iron agar, mobility indole ornithine agar and Simmons citrate agar (Oxoid, England). The species and serotype were determined using commercially available polyvalent and monovalent antisera (Denka Seiken Co., Ltd, Japan).

Fecal and egg samples were washed with 0.5 mL phosphate buffer saline (PBS), and 0.1 mL of them was inoculated in selective enrichment broth Selenite F broth (HIMEDIA, IND) and incubated at 37 °C for 24 h. For meat and organ samples, 1 g of each sample was mixed with 5 mL PBS and homogenized using pestle and mortar, and 1 mL of homogenized sample was mixed with 9 mL Selenite F broth and incubated at 37 °C for 24 h. After selective enrichment was completed, a serial dilution of each sample was made up to 10⁻⁸. 100 μL of enriched samples were spread on Salmonella Shigella (SS) agar (Oxoid, UK) plates and incubated at 37 °C for 24 h. Two or three suspected Salmonella black colonies on agar plates were picked to obtain purified isolates by further streaking method. Biochemical tests including Triple Sugar Iron (TSI), Citrate utilization, Urease, Sulphate, Indole, and motility tests were performed for preliminary screening of Salmonella enterica identification [15 (link)]. Overnight grown bacterial cultures were streaked on Triple Sugar Iron agar (Oxoid, UK.) Simmons Citrate agar (Oxoid, UK), Urease agar (Oxoid, UK), and Sulphate, Indole Motility (SIM) agar (HIMEDIA, IND) and were incubated at 37 °C for 24 h for subsequent biochemical characterization.