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The plates were observed for the presence of inhibition of bacterial growth that was
indicated by the clear zone around the wells. The size of the zone of inhibition was
measured and the antibacterial activity was expressed in terms of the average
diameter of zone inhibition in millimeters. The absence of zone inhibition was
interpreted as the absence of activity (Mathabe et al., 2006)
2.5.3.2 Disc diffusion assay
Three methods were carried out for antibacterial testing for comparison of efficacy of
each method and learning. The disc-diffusion technique was used for testing for
antibacterial activity. Mueller-Hinton broth culture was prepared and the McFarland
standard was maintained as described above. Fifty microliter (50 µl) of the respective
bacterial strains were spread over the surface of the plate containing 10 ml Mueller
Hinton agar in sterile 9 cm petri dishes. Different plant extracts (30 µl) were applied
to a sterile filter paper disc (Whatman No.1). The plates were allowed to dry before
the discs were placed on the agar. DMSO was used as the control and was prepared
in the same manner as the plate samples. Each plate contained 4 paper discs with
different plant extracts. A disc with 30 µl neomycin was placed on the agar and used
as a positive control. The plates were incubated at 37 ºC overnight, and the zones of
inhibition were measured in millimeters (Vlietinck et al., 1995).
2.5.3.3 Serial dilution assay for determination of the minimal inhibitory
concentration (MIC).
A micro-dilution technique using a sterile 96 well micro-plate, as described by Eloff
(1998) was used to obtain MIC values of the crude extracts against the bacteria. The
microbial cultures were diluted in fresh Mueller Hinton broth to a 0.5 McFarland
standard (approximate inoculum size of 1x10 6 CFU/mL) and 25 µl of the Mueller
Hinton broth was added to all wells. Each plant extract (50 µl), which was prepared
in the Dimethyl Sulphoxide (DMSO) at a concentration of 100 mg/ml, was serially
diluted from the first rows of the microtitre plate. The 96-well sterile plates were
prepared by dispensing 25 µl of inoculated broth, 50 µl of the plant extract and 25 µl
of the nutrient broth. Similar serial dilutions were performed for neomycin (0.1
mg/ml), a positive control, and DMSO, a negative control, instead of the plant
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