The study was performed to isolate and identify bacterial pathogen from stringing catfish, Shing (Heteropneustes fossilis) and to test the bacterial sensitivity towards commonly used antibiotics. The whole work was performed during the period of January 2018 to July 2018.
2.1 Study sample: The study sample was indigenous stinging catfish (Heteropneustes fossilis), locally known as Shing or Shingi. A diseased and a fresh Shing were collected from aquaculture farm in Bangladesh.
2.2 Collection and Transportation of Samples The collected fish (Heteropneustes fossilis) samples were transported to the laboratory aquarium of the Department of Fish Health Management, Sylhet Agricultural University (SAU), Sylhet.
2.3 Preservation of Samples The collected fish samples were preserved in refrigerator at - 20 °C to prevent further bacterial contamination.
2.4 Media and reagents used for bacterial culture 2.4.1 Preparation of Solid Media Nutrient agar medium was prepared by dissolving 28 g of nutrient agar powder in 1 liter of distilled water following standard methods. After sterilization, the medium was poured into sterile petridishes (5 ml in each petridish) and allowed to solidify and then incubated at 37 °C for overnight to check the sterility and used for cultural characterization or stored at 4 °C in refrigerator for future use.
2.5 Preparation of pathogen sample for culture The live fish was sacrificed for the collection of sub sample. For this purpose, six cotton bars and inoculating loop were taken to collect mucus and slime from whole skin, gill and body cavity of the infected and fresh fish. After that the cotton bars with sample were stricken three times on sterile solid nutrient agar media and inoculated with the loop into nutrient broth media and incubated at 37°C for 24 hours in incubator for observation of different bacterial colonies.
2.6 Isolation and identification of Bacteria For the isolation and identification of bacteria from the mentioned samples the Morphological (size, shape, arrangement, motility) study was performed by Gram’s staining reaction, colony characteristics, Biochemical reaction, catalase test, motility test. The suspected colony from these media was sub cultured in Nutrient agar and Nutrient Broth to promote the growth of a particular type of bacterium. Finally, the pure culture was obtained from the selective media. Staining with “Gram’s staining” method along with other tests was performed. Strict aseptic measures were maintained during the period of study. Striking on different solid agar was done under laminar air flow. After performing the above-mentioned tests, the results were analyzed and the isolated bacteria present in samples were identified.
2.7 Identification of bacteria Bacterial identification was performed on the basis of colony morphology; Gram’s staining reaction, motility and biochemical tests (including indole test, MR-VP test and sugar fermentation test).
2.8.1 Sugar Fermentation test The sugar fermentation test was performed by inoculating a loop full of NB culture of the organisms into each tube containing three basic sugars (e.g. sucrose, lactose, and mannitol) separately. Acid production was indicated by the color change of reddish to yellow in the medium and the gas production by the appearance of gas bubbles in inverted Durham’s tube.
2.9 Antibiotic sensitivity test Antimicrobial Susceptibility Testing (AST) was used to determine which specific organism or group of organisms were susceptible to which antibiotics. The standard procedure for assessing antimicrobial activity was the disc diffusion test. After incubation period, the diameters of the inhibition zones formed around each disc were measured. The zone radius was actually scaled from the centre of the antibiotic disc to the end of the clear zone where bacteria could be seen growing. The antibiotics, their codes and concentrations were as follows: Ciprofloxacin (5μg), Azithromycin (15μg), Ampicillin/Sulbactam (20μg), Tetracycline (30μg) and Erythromycin (15μg). Inhibition zone diameters were then interpreted into susceptibility categories based on the zone size (Susceptible, Intermediate, and Resistant). The sensitivity was identified as (a) Sensitive- S: zone inhibition wider than or equal to 18 mm, (b) Intermediate- I: zone inhibition between 13-17 mm and (c) Resistant- R: no zone of inhibition or less than 13 mm.
2.10 Statistical analysis The tables were prepared using MS Excel. Other statistical analysis and interpretations thereafter were done by using the computer software like Microsoft Excel.