Universal pre enrichment broth

When Aeromonas spp. were detected in faecal samples by the Seeplex Diarrhoea ACE system, 1 g portions were each suspended into 9 ml of Universal Pre-enrichment Broth (Acumedia, Michigan) and Alkaline Peptone Water (FDA-BAM media formulation) [11 ]. The broths were incubated at 37 °C for 24 h. A 10 μl loopful of each enriched broth was streaked onto SA agar (Himedia, India) and incubated at 30 °C for 24 h. Starch hydrolysis was observed by flooding each incubated plate with 5 ml of iodine solution (Oxoid, Hampshire). Presumptive yellow colonies with zones of clearing were confirmed using API 20E strips (bioMérieux, France).

When VTECs were detected by the Seeplex Diarrhoea ACE system, 1 g portions of the faecal samples were added into 9 ml of the Universal Pre-enrichment Broth (Acumedia, Michigan) and incubated at 37 °C for 24 h. A 10 μl loopful of the enriched broth was sub-cultured onto Eosin Methylene Blue, Levine agar (Acumedia, Michigan) and incubated at 37 °C for 24 h. Blue black colonies were confirmed as E. coli based on positive indole test reactions (Remel, Kansas). E. coli colonies were then screened for the presence of vtx 1 and/or vtx 2 genes using DEC PCR kit (Statens Serum Institut, Hillerød) according to the manufacturer’s instructions.

One gram of each faecal sample that was tested positive for Salmonella spp. using Seeplex Diarrhoea ACE system was suspended into 9 ml of the Universal Pre-enrichment Broth (Acumedia, Michigan) and incubated at 37 °C for 24 h. One millilitre of the enriched broth was then sub-cultured into 9 ml of the 2X Rappaport-Vassiliadis Enrichment Broth (Neogen, Michigan) and incubated at 42 °C for 24 h. After incubation, a 10 μl loopful of the enriched broth was sub-cultured onto Hektoen Enteric Agar (Oxoid, Hampshire). Presumptive green colonies with black centres on Hektoen Enteric Agar were confirmed using API 20E strips (bioMérieux, France). Serological groups of the isolates were determined using Wellcolex Colour Salmonella latex agglutination tests according to the manufacturer’s instructions (Remel Europe, UK).