Aeromonas hydrophila

The Microbial Type Culture Collection (MTCC), Chandigarh, India, provided pathogenic bacterial strains such as Aeromonas hydrophila (MTCC 646), Vibrio alginolyticus (MTCC 4182), Vibrio harveyi (MTCC 3438), Vibrio parahaemolyticus (MTCC 451), Vibrio vulnificus (MTCC1145).

The Aeromonas hydrophila pure culture was obtained from the Microbial Type Culture Collection (MTCC-12301). The bacteria were stored in BHI (Brain Heart Infusion Broth, HiMedia, Kennett Square, PA, USA) medium with 30% glycerol (sterile) at −80 °C. For the experiment, an aliquot of 20 µL was added to 5 mL of autoclaved BHI medium and cultured in a bacteriological incubator at 90 rpm for 24 h at 30 °C. Following that, 100 mL of autoclaved BHI medium was added and incubated under the same conditions. This bacterial culture was centrifuged for 20 min at 12,000× g, and the supernatant was discarded. Following that, the residual pellet was washed twice with PBS buffer (0.01 M) and centrifuged for 20 min at 12,000× g. Finally, the pellet was resuspended in PBS (0.01 M) and adjusted according to the McFarland turbidity standard to generate a suspension of 1 × 10⁷ CFU mL⁻¹, which was inactivated for 30 min in a water bath at 100 °C (optimized). Inactivation was verified by streaking the culture on nutrient agar plates and incubating it at 37 °C for 48 h. The plates with no colonies were confirmed to be inactivated in comparison to the control plates. The preparation was stored at 4 °C [10 (link)].