To conduct this research microbial content of arsenic affected areas were selected based on prior study and the arsenic surveillance report published by Department of Public Health Engineering (DPHE). The areas from where samples were collected are:
Area 1: Bangladesh Agricultural University dairy farm of Mymensingh district
Area 2: Sujanagar thana of Pabna district
Area 3: Faridganj thana of Chandpur district
Area 4: Kulaura thana of Moulavibazar district
Area 5: Sadar thana of Bogra district
Area 1 comprises non arsenic affected area and taken as control and remaining 4 were arsenic affected area.
Collection of samples: The apparently healthy lactating cows were selected as the source of sample. All of these cows received drinking water from tube-well and without providing any arsenical feed supplements. From the selected areas milk samples were collected directly from the udder. Prior to collection the udder was washed with fresh water and the udder surface was dried by using a towel. A quantity of 25 ml milk was drawn from each cow and collected aseptically. For milk collection sterile screw capped tubes were used. From each area 5 milk samples were collected and brought from the experimental site as early as possible. Milk samples were transferred in a large wide mouthed thermo flask containing good quality ice thus maintaining the temperature to about 40C .
Preparation and examination of milk samples for the determination of sanitary quality: To determine the sanitary quality from each group only five representative milk samples were subjected to microbial quality examination. All steps of preparation of milk samples and determination of their sanitary quality were carried out using sterile sampling equipment and procedure as per recommendation of International Commission for the Microbiological Specification of Foods (1986 ).
Isolation of microorganisms from milk samples: Serial dilutions of samples were made up to 107 in sterile normal saline.
Enumeration of total viable count (TVC): The total viable bacterial count was carried out by the spread plate technique. This enables the number of living organisms or clumps of organisms (i.e. colony forming units) in a form sample to be counted, subject to the appropriate medium and incubation conditions being used. For the determination of total viable counts, 0.1 ml of each ten fold s dilution was transferred and spread on PCA agar using a sterile pipette for each dilution. The diluents samples were spread as quickly as possible on the surfaces of the plate with a sterile glass sp reader. The plates were kept in an incubator at 37 º C for 24 to 48 hours and then count colonies . The average number of colonies in a particular dilution was multiplied by the dilution factor to obtain the total viable count. The total viable count was calculated according to ISO (1995). The results of the total bacterial count were expressed as the number of organism or colony forming units per gram (CFU/gm) of milk sample.
Enumeration of total coliform count (TCC): For the determination of total coliform count the procedures of sampling, dilution and streaking were similar to those followed in total viable bacterial count. For the determination of total coliform count, o.1 ml of each ten-fold dilution was transferred and spread on MacConkey agar. The diluted samples were spread as quickly as possible on the surface of the plate with a sterile glass spreader. The plates were kept in an incubator at 370C for 48 hours and then count colonies. The results were expressed as the number of organism or colony forming units per gram (CFU/gm) of milk sample.
Enumeration of Total Staphylococcal Count (TSC): For the determination of TSC the procedures of sampling, dilution and streaking were similar to those followed in total viable bacterial count. Only in case of staphylococcal count, Mannitol salt agar was used. The calculation for TSC was similar to that of total viable count.
Morphological characterization by Gram stain: Respective bacteria colonies were characterized microscopically using Gram’s stain as per recommendation of (Merchant and Packer, 1967).
Use of statistical calculation for processing of data: Data on the total viable count, coliform count and staphylococcal count/ml of milk sample were analyzed statistically using one way analysis of variance and correlation with the help of the SPSS software to find out the level of significance and determine any significant correlation between the variable factors.