disinfection, 70 GL ethanol was sprayed on the fish surface, and
the proximal intestine (length of 2.0 cm) was aseptically removed,
macerated, and serially diluted up to 104 in sterile 0.7% saline
solution. One hundred-microliter samples of each dilution were
cultured in duplicate on tryptic soy agar (TSA plate e [22]), and
incubated for 48 h at 30 C. The B. subtilis colonies were counted by
observing their morphological characteristics of commercial probiotic
(compared to pure probiotic colony). All colonies of B. subtilis
observed from intestine analyses were considered from diet
supplementation.