Overnight culture of C. difficile or C. perfringens was diluted tenfold
up to 103 to achieve semi-confluent growth. Two ml of the
dilution were applied to BHI plates and the surface covered by
carefully rotating the plate. The excess liquid was removed and the
plates were dried shortly at 37 C. Five 9-mm wells were made in
each agar plate containing 20 ml of medium (Fig. 1) and 130 ml of
probiotic supernatant was added to the four outer wells whereas
sterile MRS was inoculated into the central well (control well).
When the inhibitory potential of probiotic supernatant without
neutralization was assessed, MRS broth adjusted to pH 3.9 (using
5 mM hydrochloric) was added to the control well. When using
neutralized supernatant, MRS broth pH 6.8 was used instead. The
presence of an inhibition zone >1 mm was assessed visually
following 24 h of anaerobic incubation at 37 C and classified as
present or absent. Each test was performed in duplicate.