Antibiotic Susceptibility Testing of Mycobacteria

Mab, Msm, and Mtb were grown to mid-log phase and diluted to an OD₆₀₀=0.003 in each well of non-treated 96-well plates (Genesee Scientific) containing 100 μl of antibiotic serially diluted in 7H9+OADC + 5 μg/ml clavulanate (Sigma-Aldrich). For MICs on Mab knockdown cells, cultures were induced for knockdown 18 hr prior with 500 ng/μl ATc. Wells with knockdown bacteria also contained 500 ng/μl ATc. Msm media contained ADC rather than OADC. Cells were incubated with drug at 37°C with shaking for 1 day (Mab, Msm) or 7 days (Mtb). Afterward, 0.002% resazurin diluted in ddH₂O (Sigma-Aldrich) was added to each well. Plates were then incubated for 24 hr. MICs were determined by the concentration of antibiotic that turned wells blue signifying no metabolic activity (Kieser et al., 2015b (link)).