4.3. Determination of MICs of PZA Using Broth Microdilution Method

The broth microdilution method was used to determine the MICs as previously described [80]. In brief, the PZA concentration ranges in the well were twofold serial dilutions arranged from 800 mg/L to 12.5 mg/L or 1000 mg/L to 1 mg/L. A set of media with and without PZA control was included in each experiment. More than six colonies of M. tuberculosis H37Ra or PZA-resistant strains were scraped from the 7H11 agar plates using a sterile inoculating loop into a medium with 10 glass 0.5 mm beads. The sample was vortexed for 30∼60 s and settled for 15 min to remove clumps. The supernatant suspensions were diluted in the media to reach a 0.5 McFarland unit, standardized using the DEN-1B McFarland Densitometer (Grant Instruments (Cambridge) Ltd, Shepreth, Cambridgeshire, UK) according to the manufacturer’s instructions, corresponding to ∼1 × 10⁷ CFU/mL, and then diluted 1:50 and 1:100 with the PZA-S1 minimal medium used for PZA susceptibility testing via the broth microdilution method [80]. The standard inoculum size in each well was ∼1 × 10⁵ CFU/mL to ∼5 × 10⁵ CFU/mL. There is a good concordance between the McFarland scale and the CFU/mL for M. tuberculosis [81]. There was 0.2 mL of the bacterial suspension per well. The microtiter plates were sealed with parafilm to avoid drying and were incubated at 37 °C. The MICs—the lowest concentrations of the drug inhibiting visible growth—in liquid media were read at one, two and four weeks. The MIC results recorded by the first reader were considered to be the test results. All tests were carried out three times.