Rappaport vassiliadis enrichment broth rvb

Isolation and identification of Salmonella was conducted as described previously [7 (link)]. Briefly, fecal swabs in buffered peptone water (BPW) pre-enrichment broth were homogenized using vortex mixer for 30 s and incubated at 37 °C for 24 h. A 100 μl pre-enriched suspension was added into 9.9 ml of Rappaport-Vassiliadis enrichment Broth (RVB) (Oxoid, USA) and incubated at 42 °C for 24 h. At the same time, 1 ml of suspension was also transferred to 9 ml of Tetrathionate broth (TTB) (Oxoid, USA) and incubated for 24 h at 37 °C. It was then streaked from both RVB and TTB to Xylose Lysine Tergitol 4 (XLT-4) (Oxoid, USA) and Brilliant Green Agar (BGA) (Difco Becton Dickinson, USA) selective media and the plates were then incubated at 37 °C for 24 h. XLT-4 plates were incubated for additional 24 h and second reading was conducted at 48 h. Presumptive Salmonella colonies were further investigated biochemically using Triple Sugar Iron agar, Urea, Citrate and Lysine Iron Agar slants. Those colonies with typical Salmonella biochemical properties were then further confirmed by genus specific polymerase chain reaction (PCR) [10 (link)]. Reference strain of S. Typhimurium (ATCC 14028) was used as a positive control during biochemical analysis and PCR. Confirmed Salmonella isolates were stored at −80 °C in 20% glycerol until further testing.

Salmonella isolation was conducted according to Global Foodborne Infections Network, laboratory protocol [17 (link)]. Briefly, 1 g of stool sample was suspended in 9 ml of buffered peptone water (Himedia, India) and incubated for 24 hours at 37°C. Then, 100 μl of the suspension was transferred to 10 ml of Rappaport Vassiliadis enrichment broth (RVB), (Oxoid, UK) and incubated for 24 h at 42°C. Suspension of 1 ml of each sample was also transferred to 9 ml of Muller-Kauffmann-Tetrathionate broth (Himedia, India) and Selanite-F broth (Becton-Dickinson, USA) and incubated for 24 h at 37°C. Samples from these three enrichment broths were streaked on to Xylose-Lysine Deoxycholate Agar (Himedia, India), and the plates were incubated at 37°C for 24-48 hours.
Biochemical tests were conducted for presumptive Salmonella colonies using Urea, Triple Sugar Iron Agar, Lysine Iron Agar, and Citrate as described elsewhere. Typical Salmonella colonies were confirmed using genus specific PCR as described previously [18 (link)]. Serotyping of Salmonella isolates was conducted at the National Microbiology Laboratory, Office International des Epizooties (OIE) Salmonella Reference Laboratory, Public Health Agency of Canada. Somatic (O) antigens were determined using slide agglutination tests whereas flagellar antigens using microplate agglutination technique [19 , 20 (link)].