Five samples of each of six different categories of commercially available oral herbal drugs in liquid formulations were randomly collected from different medicinal stores in Dhaka City during September through November 2014. The medicaments used included Sample 1: Skin care (MXN Homooeo Laboratories), Sample 2: Decnar (MarcoUnani Pharma), Sample 3: Sharbat Ejaz (MarcoUnani Pharma), Sample 4: Cinkara (Hamdard, Waqf), Sample 5: Marbelus (Hamdard Unani Products) and Sample 6: Devas (Drug International Ltd.). The manufacturing and expiry dates at the time of sampling were found to be satisfactory. The samples were processed aseptically according to the guidelines of American Public Health Association1 followed by homogenization of 25 ml of each samples with 75 ml normal saline and then diluted up to 10-5 for the enumeration of potential pathogenic bacteria and fungi1-2.
An aliquot of 0.1 ml of each sample from the dilutions 10-2 and 10-4 was introduced employing the spread plate technique onto the nutrient agar (NA) and Sabouraud’s dextrose agar (SDA) plates for the estimation of total viable bacteria (TVB) and fungi respectively. Plates were incubated at 37°C for 24 hours and at 25°C for 48 hours for bacterial and fungal growth observations, respectively. Membrane faecal coliform (MFC) agar and MacConkey agar were used for the quantification of total faecal coliform (TFC), and coliforms ( Escherichia coli and Klebsiella spp.), respectively. Likewise, Staphylococcus spp. and actinomycetes were isolated on mannitol salt agar (MSA) and actinomycetes agar, respectively. All plates were incubated at 37°C for 24 hours, while for the observation of the growth of faecal coliforms, plates were incubated at 44.5°C for 24 hours1-2. An aliquot of 10 ml sample was transferred into 90 ml of selenite cysteine broth (SCB) and alkaline peptone water (APW) for the enrichment of Salmonella, Shigella and Vibrio , respectively and incubated at 37°C for 6 hours 1-2 . After incubation, 0.1 ml of samples (with optical density of 0.3, measured by spectrophotometer at 600 nm) from each of the 10 -2 and 10 -4 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. Plates were incubated at 37°C for 48 hours for the detection of the desired isolates. Finally, all the isolates were biochemically examined following standard procedures as described earlier 13 .
Antibiotic susceptibility traits of the pathogenic isolates were examined by using the commercially available laboratory grade antibiotic discs of ampicillin (10 μg), ciproofxacin (5 μg), streptomycin (10 μg), ceftriazone (30 μg), imipenem (30 μg), penicillin (10 μg), gentamicin (10 μg), azithromycin (15 μg), tetracycline (30 μg), cefuroxime (5 μg), erythromycin (15 μg), chloramphenicol (10 μg) and salfamethxazole (25 μg). Discs were aseptically placed over the surface of Mueller-Hinton agar plates (Difco) at spatial distance of 5 mm, which had been previously inoculated with the pathogenic test bacterial suspensions ( i.e ., bacterial lawn as previously described) 6. The in vitro antibacterial activity of the samples was detected as described in our recent study1. At first, the bacterial suspensions (with standard turbidity compared to that of the McFarland standard of 0.5) of Pseudomonas spp., Listeria spp., Bacillus spp., Vibrio spp., Salmonella spp., Klebsiella spp., Staphylococcus spp. and E. coli was spread evenly over the Mueller-Hinton agar using cotton swabs. Wells were made by means of the sterile cork-borer, and an aliquot of 100 μl of each samples was introduced into the specified wells with a residual concentrations of 3.4, 2.4, 0.6, 1.4, 0.5 and 0.8 mg/ml for samples 1, 2, 3, 4, 5, and 6, consecutively in the specified well along with a positive control (gentamicin, 10 μg) and a negative control (normal saline)1. Presence of clear zone was indicative of the active compound of the tested samples against the pathogens1. Estimation of the MIC of the samples was demonstrated as described in our study earlier1. This method could notice the minimum concentration of each herbal medicine capable to reduce the cell division rate of the tested bacteria1. Each of the test bacteria (100 μl of overnight culture adjusted with 0.5 McFarland standard) was inoculated into the appropriately labelled sterile tubes containing Mueller-Hinton broth with a final volume of 3 ml (Mueller-Hinton broth + samples + bacterial cells)1. The different volumes of samples were carefully used with 16, 32, 64, 128, 256, 512 and 1,024 μl1 . All tubes were incubated at 37°C for 24 hours.