Alkaline peptone water

Fish swabs and water samples were enriched in alkaline peptone water (Oxoid, Hampshire, UK) that were incubated at 25-30°C for 24 h before being plated onto Aeromonas agar medium (Oxoid) that had been incubated aerobically at 37°C for 18-24 h [27 (link)]. Based on colony characteristic morphology, suspected typical colonies (convex translucent, pale green, 0.5-3 mm in diameter) were obtained and purified for further testing. A complete set of biochemical tests was performed for colonies that were negative in Gram staining, motile, and positive for oxidase. Aerokey II was used for complete identification and biotyping of isolates down to the species level, where each species has its unique pattern in biochemical reactions [28 (link)].

The outer surfaces of the fish were rinsed with sterile distilled water prior to bacteriological analysis. The skin, gill and gut were aseptically excised with a scalpel and scissors and homogenised. A 1:10 broth dilution enrichment of homogenate into alkaline peptone water (Oxoid, Basingstoke, UK), pH 8.6, was carried out and this was incubated at 37 °C for 4–6 h, and further sub-cultured on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Oxoid, Basingstoke, UK). To enhance detection of Vibrio species from water samples, analysis was done according to American Public Health Association (APHA) [28 ], where 100 mL each of water samples were filtered through a 0.45-μm membrane, with the membrane inoculated on TCBS (Oxoid, Basingstoke, UK) as well as pre-enrichment of water samples in APW prior to culture on TCBS. Following 24–48 h incubation, 2–5 distinctive presumptive Vibrio colonies per TCBS plate were inoculated in brain heart infusion (BHI) agar (Oxoid, Basingstoke, UK) for purity and incubated at 37 °C for 24 h. Preliminary tests on presumptive isolates included gram staining, oxidase (using of oxidase test strips (MB0266A, MICROBACT™ Oxoid, Basingstoke, UK) and catalase tests.