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. 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 (18). For the identification and enumeration of pathogenic bacteria and fungi, at first 10 g of each sample was taken, then blended with 90 ml normal saline (pH 7.8) and diluted up to 10-4 and then dilutions were used for plating purposes according to the standard guideline.
Isolation and enumeration of spoiling microorganisms. The primary inoculation was performed by the conventional culture technique with the addition of 0.1 ml of each sample onto nutrient agar (NA) and Sabouraud’s Dextrose Agar (SDA) for the determination total viable bacterial count (TVBC) and total fungal load respectively, following the spread plate technique. The plates were incubated at 37oC for 24 hours and at 25oC for 48 hours for TVBC and fungal load respectively. From the dilutions 10-3 and 10-5, the sample (0.1 ml) was spread onto MacConkey agar for the enumeration of coliforms (especially, Escherichia coli and Klebsiella spp.) and fecal coliform, respectively. Afterward, the MacConkey agar plates were incubated at 37oC for 24 hours. An aliquot of 0.1 ml of diluted sample was spread onto Mannitol Salt Agar (MSA) and Pseudomonas agar for the isolation of Staphylococcus spp. And Pseudomonas spp. respectively and incubated at 37oC for 24 hours. For the enumeration of Listeria spp., 0.1 ml of suspension was spread onto the Listeria identification media and plates were incubated at 37oC for 24 hours. Listeria spp. was identified as blue-green colonies on Listeria identification agar media with a further confirmation by biochemical tests.
Enrichment for enumeration of Salmonella spp., Shigella spp. and Vibrio spp. Prior to quantify the relatively stressed cells or the viable but nonculturable (VBNC) microbial cells, 1 ml of sample was transferred into 9 ml of selenite cysteine broth (SCB) 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 10-5 and then 0.1 ml of samples 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 and Shigella spp, and Vibrio spp. respectively. Plates were incubated at 37oC for 48 hours for the detection of typical colonies. Finally, all the isolates were biochemically examined following standard procedures.
Antibiotic susceptibility test. All the bacterial isolates were examined for antibiotic susceptibility traits (either drug resistant or sensitive) against 16 antibacterial drugs (including first, second and third generation drugs) by disc diffusion assay on Mueller-Hinton agar (Difco, Detroit, MI) against commonly used antibiotics following the standard protocol. Antibiotic discs included trimethoprime/sulfamethoxazole (25 µg), erythromycin (15 µg), amoxicillin (30 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), streptomycin (10 µg), ampicillin (10 µg), tetracycline (30 µg), chloramphenicol (30 µg), cefixime (5 µg), polymixin b (300 units), kanamycine (30 µg), vancomycine (30 µg), gentamicin (10 µg), nalidixic acid (30 µg), azythromycine (15 µg) and penicillin G (10 µg).
Determination of antibacterial activity of apple samples. Antibacterial activity was determined by using the agar well diffusion methods. Normal saline suspensions of test organisms (Pseudomonas spp., Listeria spp., Aeromonas spp., Vibrio spp., Salmonella spp., Klebsiella spp., Staphylococcus aureus, E. coli) consisting of 106 cells/mL (compared with McFarland standard) were introduced on separate Muller- Hinton agar (MHA) and lawns were prepared. After drying, sterile cork borers were used to create 8 mm3 wells. A volume of 100 µL of each sample (with a concentration of 11µg/µL) was poured into separate wells, dried and then incubated at 37oC for 12-18 hours. Normal saline and Chloramphenicol (10 µg) were used as positive and negative control, respectively. Presence of clear zone around the sample solution (if any) indicated the presence of antibacterial activity.