Sampling and sample processing. Imported samples of five types of fresh fruits including Guava (Psidium guajava), malta (Citrus sinensis), apple (Malus domestica), orange (Citrus reticulate) and dragon fruits (Hylocereus polyrhizus) were randomly collected from super shops within the city of Dhaka, Bangladesh. Samples were collected early in the morning and transported quickly to the laboratory according to the standard method. Microbiological analyses were carried out according to standard procedures as described earlier. Briefly 10 g of each fruit sample was homogenized with 90 ml normal saline and serial dilutions were prepared up to 10-6.
Quantification of microorganisms. For the enumeration of total viable bacteria (TVB) and fungi, 0.1 ml of each sample from the dilutions 10-3 and 10-5 was spread onto the nutrient agar (NA) and Sabouraud’s dextrose agar (SDA) plates, respectively. The NA plates were incubated at 37oC for 24 hours and the SDA plates were incubated at 25oC for 48 hours. For the estimation of specific bacterial pathogens, 0.1 ml from each of the 10-3 and 10-5 dilutions of all samples were spread onto the membrane fecal coliform (MFC) agar and MacConkey agar for the enumeration of total fecal coliform (TFC), and coliforms (i.e., Escherichia coli and Klebsiella spp.), followed by incubation at 44.5oC and 37 oC for fecal coliform and coliforms, respectively for 24 hours. Likewise, Staphylococcus spp. was isolated by spreading 0.1 ml of the diluted samples onto the mannitol salt agar (MSA). The plates were incubated at 37oC for 24 hours.
For the isolation and quantification of Salmonella, Shigella, and vibrio spp., the enrichment procedure was applied as described earlier (9). For this purpose, after completing homogenization, 1 ml of samples were transferred into 9 ml of selenite cysteine broth (SCB) and alkaline peptone water (APW) for the enrichment of Salmonella, Shigella, and vibrio spp., consecutively and incubated at 37oC for 4-6 hours. An aliquot of 0.1 ml of each of the sample from 10-3 and 10-5 dilutions were spread onto Salmonella-Shigella (SS) agar and thiosulfate citrate bile salt sucrose (TCBS) agar for the isolation of Salmonella spp. and Shigella spp., and Vibrio spp., consecutively. Followed by incubation at 37oC, the appearance of typical colonies (if any) was noticed within for 24-48 hours. Finally, a series of biochemical tests were conducted to confirm the identity of all the isolates as described previously.
Determination of anti-bacterial activity. Determination of anti-bacterial activity was performed by using the agar well diffusion method as previously described. Culture suspensions of 9 laboratory bacterial strains (Bacillus spp., Pseudomonas spp., Vibrio spp., Escherichia coli, Klebsiella spp., Staphylococcus spp., Listeria spp., Salmonella spp., and Aeromonas spp.) were prepared in normal saline equivalent with the turbidity of the McFarland standard. Each of the test bacterial lawn was made by separately spreading evenly over the separate Muller-Hinton agar (MHA). Wells with volumes of 8 mm3 were made through the MHA. Each of the crashed fruit blends (100 µl) was added to the wells along with the disc of gentamicin 10 five areas of Dhaka city such as Rampura, Khilgao, Shantinagar, Moghbazar and Mailbagh areas.µg as the positive control and an aliquot of 100 µl normal saline was used as the negative control. After drying the plates were then incubated at 37°C for 12-18 aa hours. Presence of clear zone (if any) around the samples was analytical for the existence of the anti-bacterial traits of the samples studied.