*4.6. Minimal Inhibitory Concentration (MIC) Assay*

The mycobacterial strains used were *M. tuberculosis* H37Rv ATCC 27294, *M. fortuitum* ATCC 6841, *M. abscessus* ATCC 19977 and *M. chelonae* ATCC 35752. The mycobacterial strains were cultured in Middlebrook 7H9 enriched (Difco, Becton, NJ, USA) 10% Oleic acid, Dextrose, 0.2% glycerol, BSA and 0.05% Tween-80 supplemented medium.

The antibacterial susceptibility testing was performed using a broth microdilution technique. Stock solutions of plant extracts and control substances at 10 mg/mL in DMSO were prepared and kept at −20 ◦C. Bacterial cultures were put into appropriate media and their absorbance was measured at OD600, before diluting the culture to reach a concentration of 10<sup>5</sup> CFU/mL. The plant extracts were evaluated in a two-fold serial dilution method from 64 to 0.5 mg/L, with 2.5 µL of every individual concentration added in each well of Elisa plate. Each well contained bacterial culture around 97.5 µL with the test drug and associated controls. Resazurin-based dye (Thermo Fisher, Waltham, MA, USA) was applied to visually identify active phytoconstituents. The lowest concentration of active substance that prevented observable development after an incubation period was established as the MIC of the active plant extracts. The MIC experiment was done in triplicate and independently on duplicate samples for each drug. The MIC 96 well (Elisa) microtiter plate was incubated for non-tuberculous mycobacteria for 24–48 h and slow growers for 7 days [41,42].
