2.8. Anti-Mycobacterial Assay

Alamar blue assay (MABA) was used to determine the anti-TB activity of EO. Mycobacterium tuberculosis (RCMB 010126) strain was cultivated in complete 7H9-S medium in the dilution of 1:20. The inoculum (100 μL) was incubated for 7 days at 37 °C with and without several concentrations of EO and isoniazid (positive control) (100 μL) in 96-well plate. Following this, cells were incubated for further 24 h with alamar blue solution (30 μL). The alamar blue interacted with bacteria and changed from oxidized form (blue) to reduced form (pink). The color measured spectrophotometry and the inhibition % was calculated from the equation = 1 − (mean of test well/mean of blank wells) × 100. The lowest concentration of EO that prevents the color change is considered as MIC [35].