Determination of minimum inhibitory concentrations (MIC) (agar dilution)

The MIC of the extracts was determined for each of the test organisms using agar dilution method recommended by Clinical Laboratory Science Institute (CLSI 2017). Stock solutions of 2 mg/mL (2,000 µg/mL) of extract were prepared. Then, 2-fold serial-dilution was carried out to get a range of concentrations (1000, 500, 250 and 125 µg/mL). Thereafter, 10-fold dilution was obtained for each of the concentrations. Exactly 1 mL from each of these concentrations was transferred into sterile Petri dishes and 9 mL of molten agar cooled to 45 °C was added resulting to final concentrations of 100, 50, 25 and 12.5 µg/mL, respectively. The mixture was rocked clockwise and anticlockwise to ensure proper mixing. Following that, a loopful each of the test organisms previously standardized to McFarland turbidity was streaked on the solidified agar. A Petri dish containing only 1 mL of DMSO (Sigma Aldrich, Hamburg, Germany) mixed with the sterile molten agar and the organisms were prepared as above to serve as the negative control. The culture plates were then incubated at 37 °C for 24 h. After incubation, the plates were examined for visible microbial growth. The plates were observed for another 7 d.