2.3. Bacterial isolates preservation and recovery

From 2012 to 2018, bacterial cultures done on the chicken samples yielded bacteria, which upon identification as E. coli and Salmonella spp. were emulsified into uniquely labelled cryovials having preservation media; 10% skimmed milk or brain heart infusion broth supplemented with 20% glycerol. In the review period, a total of 155 isolates (63 E. coli and 92 Salmonella spp.) were archived at −30°C and −80°C. During the study, archived isolates were recovered as per the procedures by Scythes et al. [18]. Briefly, the procedure involved first inoculating 200 µl of the bacteria stocks in 2 ml of brain heart infusion broth (Oxoid, United States of America) and incubation at 37°C for 24 hrs. The broth was then streaked on MacConkey agar (Conda laboratories, Spain) and xylose lysine dextrose agar (Mastgroup, United Kingdom). An additional 24 hrs broth incubation was done for bacteria that did not grow after the first 24hr broth incubation. The two bacterial species colonies were then identified biochemically using methods described by Khan et al. [19] and Sarba et al. [20]. The following biochemical tests were used for identification of E. coli and Salmonella spp. (indole, methyl red, Voges-Proskauer, hydrogen sulphide production, urease, citrate and lactose fermentation).