, from cultures grown for 20 h at 37 C,
cells were harvested by centrifugation at 9700g for 15 min,
washed twice with 0.1 M sodium phosphate buffer containing
10 mM dithiothreitol (DTT), pH 6.8 and re-suspended in the
same buffer to obtain a suspension with an optical absorbance
(A600 nm) of 3.0. Cell suspension was sonicated for 60 s. with
cooling on ice with two cycles of 16 mm using a sonicator
(Sonics and Materials Inc., Vibro cell), followed by centrifugation
at 9700g for 15 min. The reaction mixture consisted of
180 mL of 0.1 M sodium phosphate buffer, pH 6.0, 10 mL of
a 200 mM appropriate conjugated bile salt, 10 mM DTT and
10 mL of cell-free extract. The reaction mixture was incubated
at 37 C for 30 min., then a sample (100 lL) was taken and
200 lL of 15% (w/v) trichloroacetic acid was added to terminate
the reaction. The sample was centrifuged (9700g for
15 min) and 200 lL of the supernatant was added to 200 lchecked for the appearance of an inhibition zone. Clear zones
around the spots indicate the antimicrobial activity of isolated
bacteria.