Vibriophage Induction by Mitomycin C

Induction of vibriophage by mitomycin C was performed according to the methodology mentioned by Castillo et al. [48 (link)], with slight modification. In brief, preserved Vibrio species in LB agar stab were aseptically transferred with a sterile inoculum loop to 10 mL of autoclaved LB media and incubated at 37 °C overnight at 200 rpm in an incubator shaker (ThermoFisher Scientific, Waltham, MA, USA). The overnight grown culture was sub-cultured in LB medium and incubated again at approximately 37 °C for 2 h at 200 rpm to reach the Vibrio culture optical density (OD₅₅₀) of ≥0.2 at 550 nm. Phage induction was initiated by the addition of mitomycin C (at a final concentration of 0.5 µg/mL) (ThermoFisher Scientific, MA, USA) in grown culture (MMC+), except for the control (MMC−) without mitomycin C. Then, the optical density (OD₆₀₀) of samples was measured at a time interval of 20 min for 8 h.