Broth microdilution MIC method.

The 7H9 broth microdilution MIC method was conducted as described in the companion paper (9). In brief, M. tuberculosis H37Rv was grown in 7H11 or Lowenstein-Jensen medium and resuspended in saline Tween with glass beads. The turbidity of the resulting suspension was adjusted with sterile deionized water to match that of a McFarland standard 1 suspension, corresponding to ∼5 × 10⁷ CFU/ml. There is good concordance between the McFarland scale and CFU per milliliter for M. tuberculosis (12). A 2× inoculum was to be prepared by inoculating 12.5-ml sterile deionized water tubes with 255 μl of the 1 McFarland suspension to give a 50-fold dilution from 1 McFarland (∼1 × 10⁶ CFU/ml). The 2× M. tuberculosis H37Rv suspension was then poured into a disposable inoculum reservoir, and then 100 μl was transferred to the microtiter plate wells using an 8- or 12-channel micropipette and sterile tips with filters. The final inoculum size in each well was ∼5 × 10⁵ CFU/ml. The growth control well, but not the well containing the negative control, was also inoculated with M. tuberculosis H37Rv. After inoculation, the plates were sealed in plastic bags (10 plates per bag) and incubated at 35 to 37°C. Each drug was tested over a 5- to 7-fold range, taking into consideration the CLSI guidance that whenever possible, the low end of the QC range for dilution testing should only include concentrations that can be accurately prepared and that dilutions should extend to no more than five dilutions below a drug's susceptibility breakpoint (10). The final drug concentration ranges in the wells following inoculation are given in Table 1; e.g., for isoniazid the concentrations were 1, 0.5, 0.25, 0.12, 0.06, and 0.03 μg/ml.