2.8.3. Minimum Inhibitory Concentration (MIC)

A tetrazolium microplate assay was used to determine the minimum inhibitory concentrations (MICs) of the test organisms [21]. A 96-well clear microtiter plate was used for the experiment. Each well of the 96-well plate was inoculated with a suspension of freshly isolated bacteria (0.1 mL) at a concentration of 5 × 10⁵ CFU/mL. Different concentrations, 15 to 0.25 mg/mL, of the test extract were diluted in series with Muller–Hinton broth (Becton Dickinson, Sparks, MD, USA). A volume of 200 µL of each concentration was added in triplicate to the wells and the plates were then incubated for 18–24 h at 37 °C ± 0.5. After incubation, in each well, 50 µL of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), with a concentration of 0.2 mg/mL, was added and the plate was incubated at 37 °C for 30 min. The bacterial suspension without extract served as the positive control, while the corresponding solvent blank (DMSO) served as the negative control. The percentage reduction of the dye (representing the inhibition of bacterial growth) was determined by measuring the absorbance at 570 nm relative to a reference wavelength of 650 nm, which was accomplished by introducing DMSO to the spectrophotometer [22].