2.6.4. Antimycobacterial Activity

The antimycobacterial activity of the extract was assessed using the microtiter Alamar blue assay [21]. Briefly, *Mycobacterium smegmatis* (MC<sup>2</sup> 155) was cultured and maintained on 7H11 agar plates. A single colony was transferred into fresh 7H9 media supplemented with 2% glycerol and 0.5% Tween 80 and allowed to grow for 24 h at 37 ◦C. The bacterial inoculum was prepared by adjusting the 24 h logarithmic culture to a concentration of 1.5 <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL. The extract was tested at concentrations ranging from 15.6 to 1000 μg/mL. Ciprofloxacin (0.08–5 μg/mL) was used as the positive control and a solvent (2.5% DMSO), untreated bacterial and negative control were included in the assay. The final assay volume was 200 μL. The plates were covered and incubated at 37 ◦C for 18–24 h, followed by the addition of 20 μL of Alamar blue solution. The plates were left to incubate for an additional 30 min and the Minimum Inhibitory Concentration (MIC) was determined as the lowest concentration where no color change could be observed from blue to pink.
