3.6. In Vitro Antimycobacterial Assay

The minimum inhibitory concentration (MIC) was determined using the standard broth micro dilution method, where a 10 mL culture of Mycobacterium tuberculosis pMSp12:GFP [58], was grown to an optical density (OD600) of 0.6–0.7. The media used were: (i) Gaste-Fe (glycerol-alanine-salts) medium pH 6.6, supplemented with 0.05% Tween-80 and 1% Glycerol, and (ii) 7H9 supplemented with 10% Albumin Dextrose Catalase supplement (ADC), 0.05% Tween-80 [59,60]. Cultures grown in Gaste-Fe were diluted 1:100, and cultures grown in 7H9 ADC were diluted 1:500, prior to inoculation of the MIC assay. The compounds to be tested are reconstituted to a concentration of 10 mM in DMSO. Two-fold serial dilutions of the test compound are prepared across a 96-well micro titre plate, after which, 50 μL of the diluted M. tuberculosis cultures were added to each well in the serial dilution. The plate layout was a modification of the method previously described [61]. Assay controls used were a minimum growth control (Rifampicin at 2 × MIC), and a maximum growth control (5% DMSO). The micro titre plates were sealed in a secondary container and incubated at 37 °C with 5% CO₂ and humidification. Relative fluorescence (excitation 485 nM; emission 520 nM) was measured using a plate reader (FLUOstar OPTIMA, BMG LABTECH, Ortenberg, Germany), at day 7 and day 14. The raw fluorescence data were archived and analyzed using the CDD Vault from Collaborative Drug Discovery, in which, data were normalized to the minimum and maximum inhibition controls to generate a dose response curve (% inhibition), using the Levenberg-Marquardt (Burlingame, CA, USA www.collaborativedrug.com) damped least squares method, from which the MIC₉₀ was calculated. The lowest concentration of drug that inhibits growth of more than 90% of the bacterial population was considered the MIC₉₀.