Emb agar

Soft tissue samples; about 25 g of each shellfish species batch comprised of 7–15 individual shrimp, crab or oyster, were pooled to represent one sample, placed in a sterile stomacher bag with 225 ml E. coli broth (Biolife, Italiana) and homogenized in stomacher (Lab. Blender 400, Seward Lab, London) for 3 min. 1 mL of the homogenized tissue was incubated with 9 mL of E. coli broth at 37 ℃ for 24 h (Doyle et al. 2020 ). Moreover, sterile swabs were rolled over the outer surface of the pooled shellfish samples, immersed in E. coli broth (Biolife, Italiana) and incubated at 37 ℃ for 24 h. Loopfuls from each surface swab and tissue’s E. coli broth culture were sub-cultured on MacConkey agar (Himedia, India) and EMB agar (Himedia, India) that were incubated at 37 ℃ for 24 h. Typical E. coli colonies on EMB (greenish metallic sheen colonies in reflected light) were re-cultured until pure colonies were obtained (Doyle et al. 2020 ). Isolates were biochemically identified using IMVIC tests and isolation on TSI as previously described (Thampuran et al. 2005 (link)). For selective STEC detection, pure identified E. coli colonies were grown on CHROMagar STEC agar medium (CHROMagar Microbiology, Paris, France) and incubated at 37 ℃ for 24 h (Meng et al. 2012 ).

Primary culture was prepared by aseptically inoculating 1 g of the Suya sample in 10 ml of nutrient broth and incubated at 37°C for 24 hrs. To obtain pure cultures, samples from the primary culture were sub-cultured on Levine Eosin Methylene Blue (EMB) Agar (HiMedia, India) plates by streaking and incubated at 37°C. The plates were observed after 24 hrs incubation; greenish metallic sheen indicates the presence of Escherichia spp [12 ]. API 20E (BiomerieuxTM) kit was used for biochemical identification of E. coli following manufacturer´s instructions.

Bacteriological loops were dipped in a bottle containing the original samples, and then a loop full of the sample was streaked primarily on MacConkey agar (Sigma Aldrich, United States) and incubated aerobically at 37 °C for 24 h. On MacConkey agar colonies with round shapes, smooth surfaces and pink color were suspected to be coliforms and were subcultured on Eosin-methylene blue (EMB) (HiMedia, India) agar. A single colony with a large, blue-black color and with or without a green metallic sheen on EMB agar [27 ] was isolated and further subcultured on nutrient agar (Himedia, India). Fresh colonies from nutrient agar were inoculated into test tubes containing sterile tryptone broth and incubated for 24 h at 37 °C followed by Indole, Methyl red, Voges-Proskauer and Citrate (IMVIC) and Triple sugar iron agar (TSI) tests [28 (link), 29 ]. Finally, the bacterial isolates were cultured with 800 µl of tryptone soya broth (TSB) and incubated for 24 h at 37 °C and 35% glycerol was added. Then, the cultured samples were stored at -20 °C for further molecular characterization and antibiogram testing [30 (link)].