Study area and sampling. Total five aquarium water samples (7 days, 20 days, 30 days, 60 days, and 90 days) were collected from a local supplier of ornamental fish of the Dhaka division from November 10 to November 15 in 2020. Samples were collected aseptically in sterile screw-capped bottles kept in a thermal stabilizing box maintained at 25°C, transported to the laboratory, and immediately subjected to microbiological analysis.
Physicochemical status of the water samples. Physicochemical properties such as, dissolved oxygen, temperature (DO), pH, electrical conductivity (EC), salinity, total dissolved solids (TDS) and turbidity were measured for all water samples following the standard methods of American Society for Testing and Materials (ASTM) using different calibrated standard instruments.
Enumeration of Total viable bacteria (TVC) and Total Fecal Coliform (TFC). For enumeration of TVC and TFC, 0.1 ml of each sample was spread onto Nutrient agar and membrane fecal coliform (mFC) agar, respectively. For total bacterial count plates were incubated at 37°C for 24 hours and for obtaining fecal coliform count plates were incubated at 44.5°C for 24 hours.
Identification of pathogenic microorganisms. For the isolation of E. coli, Klebsiella spp., S. aureus, and Pseudomonas spp., 0.1 ml of each sample was spread onto MacConkey agar, Mannitol salt agar (MSA), and Cetrimide agar (HiMedia), respectively. The plates were incubated at 37°C for 24 hours. Eosin methylene blue (EMB) agar media were further used for the observation of the production of green metallic sheen (if any) as the specific characteristic of E. coli strains. For the assessment of Salmonella spp., Shigella spp. and Vibrio spp., 0.1 ml of each sample was spread onto xylose lysine deoxycholate (XLD) agar and thiosulfate citrate bile salt (TCBS) sucrose agar respectively. For the final identification of all isolates, several biochemical tests were performed including the triple sugar iron test, motility, urease test, methyl red test, Voges Proskauer test, indole utilization test and the oxidase test.
Determination of antimicrobial susceptibility. The pathogenic isolates were examined for antibiotic susceptibility traits (either drug-resistant or sensitive) by disc diffusion assay on Mueller-Hinton agar (Difco, Detroit, MI) against commonly used antibiotics following the standard protocol. Antibiotics used in the study included erythromycin (15 µg), amikacin (30 µg), tetracycline (30 µg), ciprofloxacin (5 µg), gentamycin (10 µg), neomycin (10 µg). Plates were then inverted and incubated at 37°C. After 8 hours, plates were examined and the diameters of the zones of inhibition were measured in diameter.