Salmonella Isolation Protocol

The samples were inoculated in 40 ml of nonselective tryptic soy broth (TSB) (Oxoid Ltd., Basingstoke Hampshire, UK) and incubated for two hours at 37°C with shaking at 100 rpm. Thereafter, 0.1 ml was inoculated into 9.9 ml of Rappaport-Vassiliadis broth (Oxoid Ltd., Basingstoke, UK), a selective enrichment media for Salmonella and incubated at 42°C for 24 hours [17 (link)]. A loopful of the sample was streaked onto Brilliance Salmonella Agar (Oxoid Ltd., Basingstoke Hampshire, England) and incubated at 37°C for 24 hours [18 (link)]. Single presumptive purple/pink colonies typical of Salmonella were picked and subsequently streaked onto Hektoen Enteric Agar (HEA) (Oxoid Ltd., Basingstoke Hampshire, England) and Salmonella Shigella agar (SSA) (Oxoid Ltd., Basingstoke Hampshire, England) plates and incubated at 37°C for 24 hours. Culture plates were examined for the presence of typical colonies based on morphological characteristics, i.e., clear colonies with a black centre on HEA and SSA. Single colonies were subcultured onto nutrient agar and subsequently stored in TSB supplemented with 10% glycerol (Merck, USA) at −60°C until further analysis. Salmonella enterica subsp. enterica serovar Choleraesuis ATCC 10708 (ATCC, Manassas, Virginia, USA) was included as a control strain.