Study area The milk and environmental samples were collected from BAU Dairy Farm, Mymensingh during the period from July 2012 to November 2012. The collected samples were transported immediately to the Bacteriology laboratory of the Department of Microbiology and Hygiene, BAU, Mymensingh, for bacterial isolation and characterization.
Collection of samples After complete milking the milk was mixed thoroughly and 5 ml of milk was collected with the help of clean, autoclaved pipette and taken into test tube. Water sample was collected from the water trough after complete mixing. Sample also collected from soiled feces at the standing area of animal. A total of 56 samples were collected aseptically from BAU dairy farm and immediately carried to the laboratory for the isolation, identification and characterization of V. cholerae.
Cultivation and isolation of V. cholerae After collection, each of the samples was inoculated into each of the freshly prepared NB. The tubes were identified properly and then incubated at 37°C for 24 hrs aerobically in bacteriological incubator. The incubated tubes were then examined for growth of bacteria. 500 µl samples were spread into the TCBS agar with sterile glass rod spreader and incubated at 37°C for 24 hrs aerobically in bacteriological incubator. Then the plates were examined and studied carefully for the presence of characteristic colonies of V. cholerae.
Morphological and physiological characterization All bacterial isolates were identified and Gram-stained initially, characterized biochemically and identified up to species level by performing standard tests according to Buchanan and Gibbons (1974). Motility was tested under light microscope of 100 X magnifications by using slide with a drop of fresh bacterial culture.
Identification of the isolated V. cholerae using biochemical tests For this study carbohydrate fermentation tests, TSI agar slant reaction, MR-VP test, oxidase test, catalase test and indole reaction test were carried out for identification of suspected V. cholerae. All the isolates from different sources were tested for the detection of V. cholerae described by Larsen et al. (1999). Examinations of characters were performed according to the principles of Cowan and Steels Manual.
Antimicrobial susceptibility test Susceptibility and resistance of different antibiotics was measured in vitro by employing the Kirby-Bauer method (Bauer et al., 1996). This method allowed for the rapid determination of the efficacy of a drug by measuring the diameter of the zone of inhibition that resulted from diffusion of the agent into the medium surrounding the disc. A suspension of test organism was prepared in nutrient broth by overnight culture for 24 hours at 37°C. The broths were streaked by using sterile glass spreader homogenously on the medium. Antibiotic disc were applied aseptically to the surface of the inoculated plates at an appropriate special arrangement with the help of a sterile forceps on MuellerHinton agar plates. The plates were then inverted and incubated at 37°C for 24 hours. The diffusion discs with antimicrobial drugs were placed on the plates and incubated for 24 hours at 37°C. The antimicrobial discs (Oxoid, Basingtoke, Hampshire, England) used were: ampicillin (10 µg), chloramphenicol (30 µg), tetracycline (30 µg), erythromycin (15 µg), azithromycin (15 µg), streptomycin (10 µg), gentamicin (10 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg) and norfloxacin (10 µg). Sterile glass spreader was used to spread the culture homogenously on the medium. Antimicrobial disc were applied aseptically with the help of a sterile forceps. The plates were then examined and diameters of the zone of complete inhibition were observed. Isolates were classified as susceptible, intermediate and resistant categories based on the standard interpretation table (Table 2) updated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2011).
Maintenance of stock culture The V. cholerae strains were grown in nutrient broth and maintained at −70°C in 20% (vol/vol) glycerol.