Mmushi T MSc (Microbiology).pdf

susceptibility is determined by the absence or presence of a clearing around the test
organism.
1.4.2. Disc diffusion
The disc diffusion method commonly known as Kirby-Bauer is widely used for
antibacterial activity tests (Kelmanson et al., 2000). In this method, one species of
bacteria is uniformly swabbed onto a nutrient agar plate while plant extract is applied to
sterile filter paper discs which are allowed to dry before being placed onto the top layer
of the agar plates (Rasoanaivo and Ratsimamanga-Urveg, 1993). Each extract is tested
in quadruplicates. The relative effectiveness of a compound is determined by comparing
the diameter of the zone of inhibition of bacterial growth around the discs with values in
a standard table.
1.4.3. Well diffusion
The agar well diffusion assay uses a similar method as disc diffusion assay, except that
the extracts are placed into wells made on the solid agar after the inoculation with
standardized bacterial culture. The antibacterial activity is also measured as the
diameter (mm) of clear zone of growth inhibition around the well (Hufford et al., 1975).
Most researchers use agar diffusion assays to determine the antibacterial activity of
extracts (Eloff, 1998b) however there are limitations to this method, mainly because of
the sensitivity of the test to change in operator technique and also in the subsequent
interpretation of zone diameter.
The antimicrobial effect of plant extracts may be inhibited or increased by external
factors or contaminants. The agar type, salt concentration, incubation temperature and
molecular size of the antimicrobial component influence results obtained with agar
diffusion assays. This technique does not distinguish between bactericidal and
bacteriostatic effects and the minimum inhibitory concentration cannot be determined
and it only detects toxicants that can pass through agar (Eloff, 1998b). The technique
works well with defined inhibitors but when examining extracts containing unknown
17