Samples and Sampling Sites. Total 24 vegetable samples of four categories (Lady’s finger, Coriander, Green chili and Tomato) were collected from three local markets (Karwan Bazar, Malibag, Shantinagar) and three super shops (near Bailey Road) early in the morning and transported to the laboratory as soon as possible according to the standard methods suggested by American Public Health Association.
Microbiological analysis of sample. Ten grams of each of the vegetable sample was homogenized in 90 ml saline and diluted up to 10-6 following the standard methods then the volume of 0.1 ml from each sample suspension was spread onto nutrient agar (NA) and incubated at 37oC for 24 hours for enumerating total viable bacteria (TVB). Sabouraud dextrose agar (SDA) (Oxoid Ltd., Hampshire, England) was inoculated followed by incubation at 25oC for 48 hours for isolation of fungi. On the other hand, for the isolation of coliform bacteria (Escherichia coli, Klebsiella spp.), 0.1 ml of each sample suspension was spread over MacConkey (Oxoid Ltd., Basingstoke, Hampshire, England) agar and incubated at 37oC for 24 hours (7, 10, 22). For enumerating total fecal coliform, 0.1 ml of each sample suspension was spread onto membrane fecal coliform (MFC) (Oxoid Ltd., Hampshire, England) agar and incubated at 45oC for 24 hours. 0.1 ml of each sample suspension was spread on mannitol salt agar (MSA) (Oxoid Ltd., Hampshire, England) for the estimation of Staphylococcus aureus, and the plates were incubated at 37oC for 24 hours. For the estimation of starch hydrolyzing bacteria (Bacillus spp.), 0.1 ml of each sample suspension was spread onto starch agar (SA) (Oxoid Ltd., Hampshire, England) and incubated at 37oC for 24 hours.
For the enumeration of Pseudomonas spp., 0.1 ml of each sample suspension was spread onto Pseudomonas agar (Oxoid Ltd., Hampshire, England) and plates were incubated at 37oC for 24 hours. For the estimation of Listeria spp., 0.1 ml of each sample suspension was spread onto Listeria identification agar (LA) (Oxoid Ltd., Hampshire, England) containing Listeria supplements and the plates were incubated at 37oC for 24 hours.
Enrichment of Salmonella spp., Shigella spp. and Vibrio spp. The in vitro cultivation of the species of Salmonella, Shigella and Vibrio often appears difficult or with faulty results (false-negative) due to their viable but nonculturable (VBNC) attributes. Therefore, enrichment was used prior to isolating these bacteria. Enrichment was performed for Salmonella spp. and Shigella spp., in the selenite cysteine broth (SCB). 1 ml of homogenized sample suspension was transferred to SCB followed by incubation at 37oC for 4 hours and serially diluted up to 10-5, and from 10-3 dilution, 0.1 ml was spread onto Salmonella Shigella (SS) agar (Hi media, India) followed by the incubation at 37oC for 24 hours. For the enrichment of Vibrio spp., 0.1 ml of the homogenized sample suspension was transferred to 10ml alkaline peptone water (APW) and incubated at 37oC for 4 hours and serial dilutions were made up to 10-5 and from 10-3 dilution, 0.1 ml was spread onto TCBS (Oxoid Ltd., Hampshire, England) agar followed by the incubation at 37oC for 24 hours. Finally, representative isolates were confirmed by the number of biochemical tests, like the triple sugar iron (TSI), motility indole urease (MIU), methyl-red (MR), Voges-Proskauer (VP), citrate utilization, catalase, and oxidase tests.