The tubes were mixed
vigorously and dispensed immediately into petri dishes.
When the mixture had solidified, wells (7 mm diameter) were
made in the medium by using a drill bit. The agar plugs were
removed with a sterile needle. After drying (37°C for 24 h),
200 pul of the supernatant fluid was placed in wells as follows.
Wells were filled with 100 ,ul of supernatant fluid, and plates
were stored for 12 h at room temperature to allow supernatant
absorption. Wells were filled again with another 100 p.l
of supernatant fluid, and plates were stored for another 12 h
at room temperature. The nonabsorbed supernatant was
removed, and RBCTL was added. Further incubation of
plates to screen the hemolytic activities of the supernatants
was done as described above. The hemolytic activity was
measured as the diameter of the zones of hemolysis.