Disc Diffusion Method

A bacteria culture (which has been adjusted to 0.5 McFarland standard), was used to lawn Muller Hinton agar plates evenly using a sterile swab. The plates were dried for 15 minutes and then used for the sensitivity test. In this method, 6 mm sterilized filter papers disks (Whatmann No. 1) are saturated with filter sterilized (Salie et al., 1996) plant extract of desired concentration. The discs which had been impregnated with a series of plant extracts. The drying time of impregnated paper disk varies from 2 h to overnight under a laminar flow cabinet (Basri and Fan, 2005). It is then placed on the Mueller Hinton agar surface which has been pre-inoculated with test organisms. Each test plate comprises of five discs. One positive control, which is a standard commercial antibiotic disc, one negative control, and three discs impregnated with plant extracts of different concentrations. The standard antibiotic discs were Vancomycin 30 µg for Staphylococcus aureus and Amikacin 30 µg was for P. aeruginosa and E. coli. The negative control was DMSO (100%). Plates are then incubated for 24 h at 37°C (Salie et al., 1996; Baris et al., 2006). After the incubation, the plates were examined for inhibition zone. The inhibition zones were then measured using calipers and recorded. The tests were repeated three times to ensure reliability.