2.3. Antagonism study
Agar well diffusion test was conducted to study antagonistic
activity of B. coagulans, B. licheniformis and P. polymyxa following a
previously described method [17,5] with some modification. The
antagonistic activity was examined against indicator pathogenic
bacteria like A. hydrophila (MTCC 1739) and Vibrio harveyi (MTCC
7771). Briefly, a culture of the indicator bacteria at the cell density
of 104 CFU/ml was independently spread on nutrient agar plates,
and then wells of 5mm diameter were made on solidified agar. The
base of the wells was sealed with molten agar. Wells were then
filled with 15 ml of overnight live suspension of probiotic culture
with cell density of 105 CFU/ml [18]. The plates were incubated at
37 C and zones of inhibition around the wells were observed and
recorded after 24e48 h.