2.2. V. alginolyticus
The fish pathogenic bacterium, V. alginolyticus (AUMOFP2) was isolated from the infected A. sebae and maintained on marine zobell agar (Himedia, Mumbai) in slopes at 5 °C in the laboratory under standard conditions (Dhayanithi et al., 2012). The virulence of the pathogen was routinely checked and before inoculation into A. sebae. The stock culture was stored at −70 °C in 0.85% NaCl with 20% glycerol (v/v) to provide stable inoculum throughout the experiment. The broth culture was centrifuged at 1000 g for 10 min at 4 °C and the bacterial pellet was collected carefully. The pellet was washed thrice and re-suspended with phosphate-buffered saline (PBS) at pH 7.4. The optical density (OD) of the solution was adjusted to 0.5 at 456 nm which corresponded to
1 × 107 cells ml−1. After the identification and confirmation, the bacterium,
was grown onto thiosulfate citrate bile salt sucrose agar plates
(TCBS, DifcoTM), specific to Vibrionaceae for 24 h at 30 °C; the colonies
were isolated on tryptone soy agar (TSA) to obtain a pure culture. The
pathogen was identified using API 20E (Biomeriux®) kit.