2.1. Sample Collection A total of 88 fresh fruits and vegetables samples (FFV) comprising 12 types of fruit such as, guava (Psidium guajava), hog plum (Spondius dulcis), date palm (Phoenix sylvestris), pineapple (Ananas comosus), mango (Mangifera indica), lemon (Citrus lemon), Indian gooseberry (Phyllanthus emblica), pomelo (Citrus grandis), apple (Malus pumila), grape (Vitis vinifera), Burmese grapes (Baccaurea ramiflora), starfruit (Averrhoea carambola) and 10 types of vegetables such as, yard-long bean (Vigna sinensis), teasle gourd (Momordica dioica), ribbed gourd (Luffa actangula), bitter gourd (Momordica charantia), ladies finger (Hibiscus esculentus), pointed gourd (Trichosanthes dioeca), carrot (Daucus carota), tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), and brinjal (Solanum melongena) were collected and kept in sterile ice bags from four different locations (Mohammadpur, Jatrabari, Uttara, and Gulshan) in Dhaka City. The microbiological tests were carried out in the food microbiological laboratory, Institute of Food Science and Technology (IFST), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka, Bangladesh.
2.2. Sample Processing Twenty grams of each sample was aseptically mixed (1:10) with 180 mL Ringer solution into a sterile conical flask. The sample was homogenized with a blender at 6000 rpm for 5–10 min. The samples were diluted six times (10−1–10−6 ) to reduce the concentration of microorganisms in the ringer solution. The number of colonies in an appropriate dilution was multiplied by the dilution factor to obtain the total viable bacteria count (TVBC), which was expressed as a mean colony-forming unit (CFU) per gram.
2.3. Microbiological Analysis of Fresh Fruit and Vegetables A total of 0.1 mL of each decimal dilution was distributed over 20–25 mL of plate count agar (PCA) for the calculation of total viable bacteria (TVBC), and the plates were incubated at 37 0C for 24–48 h. Triplicate agar plates were counted between 30 and 300 colonies. A colony counter was used to count the colonies. The samples were analyzed with the most probable number (MPN) technique to detect the total coliform and fecal coliform counts (TCC and TFC). Lauryl tryptose broth (LTB) and brilliant green lactose bile broth (BGLBB) were used for this test. As a presumptive measure, the LTB was used, and for the confirmatory and completed test, the BGLBB was used. The tubes with lauryl sulphate tryptose broth (LTB) were inoculated with (10−1, 10−3, 10−6 ) diluted samples for 48 h at 35 0C for the TCC and TFC count. In each broth, Durham tubes were inserted to observe the gas formation [39]. After 24 h of incubation, each set of positive tubes that displayed positive acid and gas were counted and calculated using the standard MPN table. The positive sample for the completed test was suspended in BGLBB medium at 1 mL and kept at 37 0C at 24–48 h, and the positive results for the tube were determined with the MPN table.
2.4. Preparation of Low-Cost Disinfectant Solution and Examination of the Washing Effect Four types of disinfectant were used in this study, namely, distilled water, vinegar, salt water, and blanched. In a laminar flow biosafety cabinet, the whole fruit and salad vegetables were placed on a sterile surface. Both fruit and salads were cut to pieces aseptically (around 5 by 5 cm). In a 500 mL volumetric flask or beaker, the required quantity of decontaminated agents was tested. In a 500 mL volumetric flask or beaker, the selected 20 g sample was rinsed thoroughly with 180 mL of the various decontaminated solutions and was immersed in a disinfectant solution. Without washing, a portion of each tested sample was counted. The dip sample was shaken several times in diluents with hand- gloves to ensure complete solution coverage and to be fully settled. The species were assumed to have been washed off and distributed in diluents. The total viable bacterial count (TVBC/g) in tested unwashed samples was identified only as being dipped into sterile ringer solution, but TVBC was first reported in treated samples with four low-cost disinfectants and then dipped into sterile ringer solution and identified. The fruit and vegetable samples were dipped in sterile ringer solution (1:10) and plated for bacterial load enumeration with washed and unwashed samples. A 0.1 mL solution was taken from each of them and inoculated to count the microbes in the corresponding selective media. The FFV samples were washed for three minutes with distilled water, blanching (80–100 0C) for 1 min, vinegar (4.5 percent acetic acid) for a few seconds, and salt solutions (0.09 percent NaCl or 900 ppm) for 3 min to assess the effect of washing on the microbial load. Then, CFU/g enumerated the microbial load of the samples.
2.5. Statistical Analysis All experiments were replicated three times. Total viable bacterial count (TVBC) was enumerated, and the microbial counts were expressed as log CFU/g. The log CFU/g reduction in the bacterial population was calculated. The results of TVBC level from the surface of FFVs were analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by LSD’s post hoc multiple comparison test using statistical software (SPSS) package version 21 (SPSS 21.0, US). p ≤ 0.05 was considered to be statistically significant.