2.1. Study Area and Sample Collection Thirty water bodies (10 ponds, 10 tube-wells and 10 supply water samples) were randomly selected and collected from Nakla Paurosova, Sherpur district of Bangladesh. The study was carried out from July to November 2018. Approximately, 300 ml of water samples were collected in 500 ml of plastic bottles and before sample collection, all the plastic bottles were properly sterile by autoclave and cleaned by distilled water. Bottles were immersed below the water surface, filled, brought out of the water and properly closed. Then they were properly labeled with sample no, source, date, time etc. and samples were carried to the laboratory within six hours of collection.
2.2. Primary Data Collection To investigate the alternation of physicochemical parameters and source of bacterial contamination of different water sources, literature based related primary data (such as-color, odor and surroundings of pond water bodies, deepness of tube-well, types of latrine and its distance from tube-well etc.) were collected with a semi-structured based questionnaire and further analysis.
2.3. Physiochemical Parameters Analysis pH was determined by the digital pH meter (Hanna instruments). The Dissolved Oxygen (DO) was determined by digital DO meter (Model: D.46974; Taiwan). Digital Electrical Conductivity (EC) meter (Model: HM digital and made in Germany) was used to determine EC. Temperature was measured by the digital thermometer.
2.4. Detection of Heavy Metal Arsenic, Lead and Iron were detected with Detection Kit (Hach Company, USA) and was carried out as the manufacturer’s instruction.
2.5. Microbial Analysis 2.5.1. Total Viable Count (TVC) For direct counting, spread plate technique was performed as described previously (APHA, 2003). Firstly, ten-fold serial dilution was carried out of every raw pond water samples. 0.1 ml of each sample is transferred by a micro pipette and spread on agar plates with a sterile bent glass rod. All the plates were inoculated at 370C for 18 hours. Total count is expressed as colony forming unit per 100 ml (cfu/ml). Nutrient Agar media was used as culture media for enumeration of total viable bacteria in sample.
2.5.2. Heterotrophic Plate Count (HPC) of Drinking Water (Jar and Tube-Wells) For the determination of heterotrophic plate count, 100 μl of serial tenfold dilution of jar and tube-well water samples were transferred and spread on Plate Count Agar (PCA) media using micro pipette for each dilution. The diluted samples were spread as quickly as possible on the surface of plate count agar with a sterile glass spreader. One sterile glass spreader was used for each plate. The plates were then taken in an incubator at 370C for 18 hours. After incubation at 370C for 18 hours plates exhibiting 30 - 300 colonies were counted. The average number of colonies in a particular dilution was multiplied by the dilution factor to obtain the heterotrophic plate count (HPC). The heterotrophic plate count was calculated according to International Organization for Standardization (ISO) (1995). The result of total bacterial count was expressed as the number of organism or colony forming units per 100 milliliter (cfu/100ml) of water samples.
2.5.3. Total Coliform Count (TCC) Most probable number (MPN) test was done to identify the presence of coliforms in jar and tube-well water samples. In presumptive MPN procedure, 15 lactose broth tubes were inoculated with the water samples. Five tubes received 10 ml of water, 5 tubes received 1 ml of water and 5 tubes received 0.1 ml of water. The number of tubes showing gas production and color change was compared to a standard table developed by American Public Health Association. The number of coliform was the MPN of coliforms per 100 ml of the water sample. For pond water, 0.1 ml of ten-fold serial dilution of every sample was spread with a sterile glass spreader and incubated them at 370C for 18 hours. After incubation the colony was counted by standard plate count method described as before.
2.5.4. Detection of Fecal Coliforms The positive presumptive cultures were transferred to lactose broth, which is specific for fecal coliform bacteria. Any presumptive tube which showed gas production after 24 (±2) hours incubation at 44.50C (±0.20C), confirmed the presence of fecal coliform bacteria in that tube and was recorded as positive.
2.5.5. Isolation of Pathogenic Bacteria For the identification of pathogenic bacteria, 100 µl of each sample were transferred into Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS) media, Eosin methylene blue Agar (EMB) and Shigella Salmonella Agar (SS) media with ten-fold dilution. The diluted samples were spread as quickly on the surface of the plate with a sterile glass spreader and incubated at 370C for overnight. The presence of pathogenic bacteria were observed and counted.
2.5.7. Antibiotic Sensitivity Test Antibiotic susceptibility test was performed by disk diffusion method (Kirby-Baur method) using the commercially available antibiotic disk on Mullar-Hinton agar to assess the susceptibility and resistance pattern of the isolates. 10 different types of common antibiotic disks (Himedia, India and Oxoid Ltd. England) were used in this study.
2.6. Evaluation of Public Health Impact A field investigation and semi-structured questionnaire-based survey was conducted among randomly selected 300 respondents (10 from each sampling sight, 50% male and 50% female) of the study area to determine the health status of people who were the consumers of these water.
2.7. Statistical Analysis MS Excel 2013 was used for data analysis and presentation of graphs.