Laboratory and duration of the experiment
This investigation was performed in the bacteriological laboratory of Bangladesh Livestock Research Institute (BLRI) and the duration of the experiment was 6 months (January/2014 to June/2014).
Sample collection sites
The milk samples were collected from different districts (Dhaka, Joypurhat, Rangpur, Chittagong, Gazipur, Pabna and Sirajgonj) of Bangladesh.
Sample collection
The vendor and brand milk samples were collected from different districts such as, Joypurhat, Rangpur, Pabna, Sirajganj, Dhaka, Gazipur and Chittagong of Bangladesh. The milk samples were collected and transported aseptically in laboratory cool box and subsequently allowed for laboratory tests immediately upon arrival for the identification of bacterial species. Sometimes the samples were preserved at 40C in the refrigerator for 24 hours.
Laboratory tests for identification of bacteria
(i) Culture in bacteriological media
After ten fold dilution, the collected samples were allowed for culture in bacteriological media by pour plate method. The media used were MacConkey agar, mannitol salt agar, blood agar, nutrient broth, nutrient agar, Salmonella-Shigella (SS) agar thiosulfate citrate bile salt sucrose (TCBS) agar, eosine methylene blue (EMB) agar, etc. and the amount of sample taken in each plate was 0.5 ml. All plates were incubated for 24 hours at 37oC temperature.
(ii) Gram’s staining
Gram’s staining was done as per standard procedure to study the morphological properties of bacteria.
(iii) Biochemical tests
Different biochemical tests like oxidase test, catalase test, indole test, methyl red test, Voges Proskauer test, citrate test and carbohydrate fermentation test were performed with the isolated bacteria. The biochemical characteristics of bacterial isolates were observed on the basis of color, bubble, acid and/or gas formation.
Antibiotic sensitivity test
The antibiotic sensitivity test was done by using disc diffusion method. The commercially available antibiotic discs such as, oxytetracycline, amoxicillin, ciprofloxacin, cefotaxime, ceftriaxone, azithromycin and erythromycin were used to know the sensitivity of the identified bacteria to these drugs.