1.1.1 Preparation of live Vibrio harveyi suspension
Virulent bacteria was grown in TCBS agar plates (Appendix 1).The bacterium was purified and sub-cultured in TCBS agar slant, Sub culture of Vibrio harveyi was inoculated in 5 ml TSB (Tryptic Soy Broth) with NaCl 1.5 % intest tube. The test tube was incubated at 30 oC for 24 hr, for challenge dilution, 24 hrcultured was further in order to produce sufficient amount of bacteria. After growthperiod, cells was harvested by centrifugation for 15 minutes at 2,700–2,800 rpm.