Oxidase reagent

Surface water samples were collected from 5 fresh water multi-purpose recreational lakes in Selangor, i.e., Tasik Aman (lake 1) (N 03.10261 °, E 101.62477 °), Tasik Taman Jaya (lake 2) (N 3.10418 °, E 101.64929 °), Tasik Varsiti Universiti Malaya (lake 3) (N 3.11941 °, E 101.65808 °) and two unnamed lakes in Rawang (lake 4 and lake 5) (N 03.36606 °, E 101.63717 ° and N 03.36752 °, E 101.63043 °). All samples were kept at 4°C and analysed within 30 hours of collection.
The samples were pre-filtered to remove residue and subsequently filtered through a 0.45 μm nitrocellulose membrane (Sartorius, Germany) using a vacuum system. The membranes were then suspended in broth and plated onto m-Aeromonas selective media (Biolife, Italia Srl) supplemented with ampicillin (10 mg/l) [25 (link)]. Yellow colonies on the agar plates due to dextrin fermentation, after 18–24 hours of incubation at 30°C were presumed to be Aeromonas species and tested with oxidase reagent (bioMérieux, France), checked for growth on MacConkey agar and 6.5% (w/v) NaCl-Luria Bertani (LB) broth. Oxidase-positive colonies growing on MacConkey agar but not in 6.5% NaCl-LB broth were further confirmed to genus level by the API 20E system (bioMérieux, France), then grown in LB broth, cryopreserved in 20% (v/v) glycerol at -80°C and maintained in LB agar and broth as working cultures.

Growth in R2A agar (Becton Dickinson, NJ, USA) was assessed at different temperatures (4, 10, 15, 20, 25, 30, 37, 40, and 45°C), at various pH values (pH 3–11 at intervals of 1.0 pH units), and with different NaCl concentrations (0, 0.5, 1–20% NaCl at intervals of 1.0%) after a 24hr incubation period. Catalase activity tests using 3% H₂O₂ solution and oxidase tests using oxidase reagent (bioMérieux, Inc.) were performed. To test the ability to ferment sugars and to produce hydrogen sulfide (H₂S), bacteria were inoculated into a Triple Sugar Iron (TSI) agar slant. Other biochemical characteristics and enzyme activities were assessed using the Vitek 2 GN ID Card (bioMérieux, Inc.).

Gram staining was performed with a Gram-staining kit (Himedia, India). Test for starch hydrolysis, IMViC (Indole, Methyl Red, Voges Proskauer, Citrate utilization), cellulase, pectinase, and lipase was performed following the standard protocol (Harley and Prescott, 2002 ). Catalase and oxidase activity was performed by using 3% (v/v) H₂O₂ and using an oxidase reagent (BioMerieux, France), respectively. The test of growth at different temperatures was done between 20°C to 45°C in sterile LB-medium. pH of LB-medium was adjusted with suitable buffers and tolerance to different pH (5.0 to 9.0) was examined. The ability to utilize different carbon sources was tested by the Himedia Carbohydrate utilization kit (KB009). The selected isolate was tested for its susceptibility against different antibiotics following CLSI (Clinical and Laboratory Standards Institute) instruction. The freshly grown culture of OS-1 was spread on an LB-agar plate and a paper disk containing different antibiotics namely ampicillin, kanamycin, erythromycin, tetracycline, ciprofloxacin, gentamicin, and streptomycin were placed, and incubated for 24–48 h. The result was interpreted by measuring the zone of inhibition (ZOI) created by the antibiotics disk. The isolate was tested for various motility behaviors such as swimming, swarming, and twitching following standard protocol (Connelly et al., 2004 (link)).