Microplate-based alamar blue assay (MABA)

The engineered Mtb strains WT-VC, Rv0062 and Rv1090 were grown in Middlebrook 7H9 media supplemented with 10% ADS (Albumin-Dextrose-Sodium chloride). Exponential cultures were freshly diluted to OD₆₀₀ 0.02 and 50 μl of dilutes cultures were added to 50 μl of media containing 2× MIC concentration of antibiotics (Rif and INH). The assay included appropriate negative (culture without antibiotics) and positive (Rif in case of INH plate and INH in case of Rif plate) controls. The plates were incubated at 37 °C for 7 days. Following incubation, 0.02 ml of 0.02% Resazurin (sodium salt, MP Biomedicals), prepared in sterile 7H9 media was added to each of the wells of the plates and color change was monitored after incubation of approximately 20 h at 37 °C. Excitation was at 530 nm and fluorescence emission was recorded at 590 nm. The efficiency of inhibition by the drugs for each of the three strains was calculated using earlier established methods⁶².