Tryptose soy broth

DNA extraction was carried out using the boiling method. Briefly, the culture of each isolate was inoculated in Tryptose soy broth (Merck, Germany) and incubated at 37°C for 24 hours. Bacteria were harvested from an overnight broth culture and suspended in sterile water (DDW), after which the bacterial suspension was boiled for 10 minutes and chilled on ice and finally the debris were separated by centrifugation. The supernatant was taken as the DNA template for PCR.

From each of the enriched samples, 0.1 mL was aseptically transferred into 10 mL of Rappaport Vassiliadis (RV) broth and incubated for 24 h at 42 °C (Sigma-Aldrich, Mumbai, India). RV is a selective medium that is enriched with malachite green which inhibits the growth of microorganisms other than Salmonella. A previously identified and confirmed S. enterica was used as a positive control for this experiment [63 (link)]. Microbiological isolation was performed on Xylose-Lysine-Deoxycholate (XLD) agar (Sigma-Aldrich, Buchs, Switzerland) by aseptically streaking a loopful of the culture from RV broth onto the XLD plates. S. enterica is differentiated from Escherichia coli and Shigella spp. by producing red colonies with black centers on XLD agar. After 24 h of incubation at 35 °C, the plates were observed for the growth of the expected colonies. Single colonies were picked from each plate and transferred into tubes containing 10 mL of tryptose soy broth (Merck, Johannesburg, South Africa) and incubated at 37 °C for 18–24 h. A 2 mL of the culture was used for DNA extraction. Equal amounts of 0.5 mL each of 60% glycerol and Salmonella pure culture were mixed in 1.5 mL cryotubes and stored at −80 °C for future use.