5.3. Minimal Inhibitory Concentration (MIC)

The broth microdilution assay format following guidelines established by the Clinical and Laboratory Standards Institute (CLSI) [22] was utilized for MIC determination. Testing was conducted using 96-well, U-bottom microplates with an assay volume of 0.2 mL/well, as follows. First, the test media, Middlebrook 7H9 broth supplemented with Oleic Albumin Dextrose Catalase (OADC) Enrichment (BD BioSciences; Sparks, MD, USA), was added (0.1 mL/well) to each well. The tested compounds, solubilized in DMSO and subsequently diluted in test media, were added (0.1 mL/well) to appropriate wells at twice the intended starting concentration and serially diluted two-fold across the plate. The plates were then inoculated (0.1 mL/well) with a targeted concentration of 1.0 × 10⁶ CFU/mL Mtb and incubated at 37 °C for 7 days in approximately 90% humidity. After incubation, the plates were read visually and individual wells scored for turbidity, as partial clearing or complete clearing. Testing was conducted in duplicate and the following controls were included in each test plate: (i) medium only (sterility control); (ii) organism in medium (negative control); and (iii) rifampin or isoniazid (positive control). The MIC represents the lowest concentration (μM) of drug that visually inhibits growth of the microorganism.