Study area, sampling and sample processing. Eight different types of fresh cow dung samples of Bangladeshi cow breed were randomly collected from different cow farm located at khillgaon, Savar, Gabtoli and Rampura of Dhaka city, Bangladesh following standard protocol. For the identification and enumeration of bacteria including pathogenic ones and fungi, 10g of each sample was added with 90 ml of normal saline and diluted up to 10-7 for all the samples following standard guidelines.
Isolation and identification of bacteria Estimation of total viable bacteria, Escherichia coli, Klebsiella spp., Staphylococcus spp. Bacillus spp. and Pseudomonas spp. For each of the cases, 0.1 ml of samples from the dilution 10-5 and 10-7 was introduced on to the nutrient agar and sabouraud dextrose agar for the isolation of total viable bacteria and fungi, respectively. Likewise, 0.1 ml of each sample from the dilution 10-3 and 10-5 was introduced onto MacConkey agar, mannitol salt agar (MSA), starch agar and Pseudomonas agar for the isolation of coliforms (Escherichia coli and Klebsiella spp.), Staphylococcus spp., Bacillus spp. and Pseudomonas spp., consecutively. All the plates were then incubated at 37oC for 24 hours.
Isolation of Salmonella spp., Shigella spp. and Vibrio spp. after enrichment. By considering the possible occurrence of viable but nonculturable (VBNC) cells 10 ml of sample was transferred into 90 ml of and alkaline peptone water (APW) for the enrichment of Salmonella, Shigella, and Vibrio spp., respectively and incubated at 37oC for 6 hours. After incubation, the samples were diluted up to 105 and then 0.1 ml of samples from each of the dilutions were spread onto Salmonella-Shigella (SS) agar and thiosulfate citrate bile salt sucrose (TCBS) agar for the isolation of Salmonella spp. & Shigella spp., and Vibrio spp., consecutively. Plates were incubated at 37oC for 48 hours for the detection of typical colonies.
Biochemical identification of the bacterial isolates. Finally, all the isolates were biochemically examined for their identification following standard procedures as described earlier.
Assay for the In vitro antimicrobial activity of the cow dung samples. For the determination of antimicrobial activity, modified agar well diffusion method was followed using Mueller-Hinton agar plate. Suspensions of different bacteria such as E. coli, Pseudomonas spp., Bacillus spp., Vibrio spp., Klebsiella spp. and Salmonella spp. introduced on to the MHA agar were prepared using normal saline, consisting of 105 cfu/ml with a turbidity equivalent to that of the 0.5 ml McFarland standard, and each suspension was then subjected to lawn on the Muller-Hinton agar (MHA). The wells were dug (8 mm3) on the inoculated Muller Hinton agar medium and 100μl or 11mg/ml o of each sample were introduced. Normal saline was used as negative controls whereas antibiotic disk of Gentamycin (GEN, 10 μg) was used as positive control. The plates were incubated at 37oC overnight and examined for the zone of inhibition. The diameter of the inhibition zone was measured in mm using slide calipers.