Tcbs agar plates

Standard determination and identification of V. cholerae was performed via a triplicate MPN method in accordance with the British national standard method (Public Health England 2005). Triplicate 1‐ml samples were serially diluted (1 : 1, 1 : 10 and 1 : 100) in APW and the triplicate 1‐ml samples of each dilution were inoculated into 9 ml APW enrichment broth. Each tube was incubated for 18–24 h at 30 ± 1°C and observed for the presence or absence of growth. Surface aliquots from the microaerophilic pellicle layer (Huq et al. 2012) of each tube were streaked out on TCBS agar plates (Oxoid, Wesel, Germany, CM0333). After 24 h at 37 ± 1°C presumptive V. cholerae colonies from all positive MPN tubes were transferred to nutrient agar without NaCl (Columbia agar+5% sheep blood; BioMérieux, Marcy l'Etoile, France). When growth was observed, the isolates were identified with specific tests: oxidase, API 20 E, O1/O139 serum agglutination test and MALDI‐TOF (Bruker Daltonik). Final MPN results were obtained from using a three‐tube MPN table according to the methods recommended by the US Food and Drug Administration (FDA 2010).

Fish body surfaces were disinfected using 70% alcohol (Al-Goumhoria Co., Egypt) and aseptically dissected using a 3-cut incision. Loopful from kidney, liver, and brain were collected under complete aseptic conditions and inoculated into tryptic soy broth TSB (Oxoid, UK) supplemented with 1.5% NaCl, incubated at 28°C for 24–48 h. A loopful of the incubated broth was streaked onto thiosulfate citrate bile salt (TCBS) agar plates (Oxoid, UK). All plates were incubated at 28°C for 24–48 h, and colonies were purified by multiple re-streaking on TSA plates and stored in 30% glycerol (v/v) at ‒80°C.