Selection of study area & collection of fruit samples for investigation. Samples were collected from some popular super shops from five areas of Dhaka city such as Rampura, Khilgao, Shantinagar, Moghbazar and Mailbagh areas. Samples were collected in different time intervals and transported to the laboratory as soon as possible according to the method suggested by American Public Health Association. For the identification & enumeration of pathogenic bacteria & fungi, at first 10g of each sample was taken, then blended with 90ml normal saline (pH 7.8) & diluted up to 10-4 & then dilutions were used for plating purposes according to the standard guideline.
Enumeration of total viable bacteria & fungi. The enumeration was performed by using 0.1ml of each sample onto nutrient agar (NA) & Sabouraud Dextrose Agar (SDA) for the determination of total viable bacterial count (TVBC) and total fungal load respectively by the spread plate technique. The plates were incubated at 37oC for 24 hours and at 25 oC for 48 hours for TVBC & total fungal load respectively.
Estimation of total coliform. From the dilution 10-2 & 10-3, the 0.1ml sample was spread onto MacConkey agar for the detection of total coliform. The plates were incubated at 37oC for 24 hours.
Isolation of Salmonella spp., Shigella & Vibrio spp. The 1ml sample was transferred into 9ml of seleniote cysteine broth (SCB) and alkaline peptone water (APW) for the enrichment of Salmonella, Shigella and Vibrio spp. respectively & incubated at 37oC for 6 hours. After enrichment the samples were diluted up to 10-4 & then 0.1ml of samples from 10-2 & 10-3 were spread onto SalmonellaShigella (SS) agar & Thiosulfate Citrate Bile Salt Sucrose (TCBS) agar followed by incubation at 37oC for 24 hours for the detection of the colony characteristics. For the identification, the biochemical trait of the isolates was tested following standard biochemical methods.
Isolation of Staphylococcus spp., Pseudomonas spp. Staphylococcus & Pseudomonus spp were isolated from the Mannitol Salt Agar (MSA) & Pseudomonas agar respectively by spreading 0.1ml of the diluted samples on these media & then incubated at 37oC for 24 hours. Biochemical tests for the confirmative identification. Finally, the standard biochemical tests were performed to confirm the identification of all the pathogenic isolates found in all 5 types of pear samples by the previously described methods.
Antibiotic susceptibility test. The pathogenic isolates were examined for antibiotic susceptibility traits (either drug resisistant or sensitive) by disc diffusion assay on Muller-Hinton agar (MHA) (Difco, Detroit, MI) against commonly used antibiotics following the standard protocol. Antibiotics used in the study are, Ampicillin (10µg), Amoxicillin (10µg), Ciprofloxacin (5µg), Ceftriaxone (30µg), Nalidixic acid (30µg), Imipenem (30µg), Erythromycin (15µg), Chloramphenicol (10µg), Trimethoprime-sulfomethoxazole (25µg), Gentamicin (10µg) & Piperaciline (10µg).
Determination of antibacterial activity of the fruit sample. The antibacterial activity of the fruit samples was performed by using agar well diffusion method. Briefly, fruit blends were used directly on the Mueller-Hinton agar (MHA) media. At first, the bacterial pathogens (Pseudomonas spp, Bacillus spp, Salmonella spp, E. coli) were introduced evenly over the MHA separately using cotton swab, followed by making hole in the MHA by cork borer. Each of the blends was then introduced separately in the specified hole with a positive control (antibiotic discGentamicin (GEN) 10µg) & a negative control (normal saline). Presence of clear zone around the sample solution (if any) indicated the presence of antibacterial activity.