Determination of minimum inhibitory concentration (MIC)

The MIC values of plant extracts and antibiotics against the bacterial strains were determined using a rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay (Eloff, 1998). Stock solutions of plant extracts (100 mg/ml) and antibiotic (10 mg/ml) were prepared with absolute ethanol and sterilized distilled water, respectively. Prior to the assay, stock solutions (plant extracts) were subsequently diluted with sterilized distilled water to a concentration of 12.5 mg/ml. These (plant extracts and antibiotics) were then added to Mueller Hinton Broth (MHB), and serially diluted 2-fold in a 96-well-microplate to a final concentration range of 3120–24 μg/ml for plant extracts and 2500–0.61 μg/ml for antibiotics. Bacterial strains were cultured overnight at 37°C on MHB and adjusted to a final density of 10⁶ cfu/ml with MHB. These were subsequently used as inocula. One hundred microliters (100 μl) of inoculum was added to each well. The plates were covered with a sterile plate sealer and then incubated at 37°C for 20 h. Wells containing 20% aqueous ethanol, MHB and 100 μl of inoculum served as the negative controls. The total volume in each well was 200 μl. The MICs of samples were observed after 20 h incubation at 37°C, and subsequent 30 min incubation after the addition 40 μl of 0.2 mg/ml INT. Clear wells with INT after incubation indicate inhibition of bacterial growth. Minimum inhibitory concentration (MIC) values were recorded as the lowest concentration of the sample that completely inhibited bacterial growth.