The agar was allowed to solidify at ambient temperature. In each plate, using a sterile borer of 6 mm diameter, 4 wells were cut in the agar plate. A sterile cotton swab was immersed into the standardized bacterial suspension and pressed against the wall of the tube to remove excess fluid. MHA plates were inoculated by streaking with that swab. Streaking was done successively in 3 different directions to obtain even spread of inoculum.The concentrated leaf and stem extracts of the wild and tissue cultured callus were weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare extract solution of 1 mg in 1 mL of DMSO (1000 g/L concentration). To each well, concentration ranging from 50 to 300 L of this extract solution was dispensed using a sterile micropipette. The inoculated plates were incubated within 15 min of inoculation at 37°C for 24 h. Then the plates were examined for any zone of growth inhibition. Inhibition zones were recorded as the diameter of growth free zones including the diameter of the well in mm at the end of incubation period.
Melvin