Determination of the minimum inhibitory concentration (MIC)

The MIC values were defined as the lowest concentration of extracts that inhibited growth. Extract MIC values was evaluated using a resazurin-based microtiter dilution assay according to Elshikh et al. (15), with a slight modification: assays were carried out under aseptic conditions in 96 well microtiter plates (Nunc, Roskilde, Denmark). The first column of each microtiter plate was filled with 100 μL of test materials (from 1 mg/mL extract stock solution), and the 2nd to 10th wells were filled with 50 μL of sterile water. A two-fold serial dilution (throughout 2nd to 10th wells) was achieved by transferring 50 μL of test material wells in the first column to subsequent wells of each row, so that each well had 50 μL of test material in serially descending concentrations. From wells in the 10th columns, 50 μL solution was removed. The working solution of extracts was diluted across the 96-wells using a two-fold serial dilution to give final testing concentrations of 1,000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.9, and 1.95 μg/mL. Each microtiter plate had a set of 2 controls: (a) test organisms without test extract as a (positive control; 11th wells), and (b) solutions without test organisms (12th wells), as a control for contamination during plate preparation. Aliquots (20 μL) of bacterial, yeast, and fungal suspensions (test organisms) were added to each well. The plates were incubated in a temperature-controlled incubator at 37°C for 24 h for bacteria, and at 48 h for yeast and fungi. After the incubation period, 80 μL resazurin dyes were added and reincubated for 2 h for color development, and the color changes in each well were observed. Following addition of resazurin, blue coloration indicated pathogen inhibition, and a change from blue to red/pink indicated the presence of live micro-organisms. All experiments were performed in triplicate. The average values were calculated for the MIC of the test material.