A Ninjaflex 2 mm thick gasket was 3D printed onto a 50 mm × 75 mm × 1 mm glass slide (VWR) using a Makerbot 2× printer. Following printing, the slides were cleaned of particles and fibers using lab tape. An additional slide was cleaned with ethanol and placed over the gasket so the space between the slides defined by the gasket could be filled with molten agarose media. The assembled agar mold was held together with binder clips.

Agarose was prepared as described previously (Herricks et al., 2017). Bulk agarose was prepared by dissolving 2 g of agarose in 150 g of 18 MΩ H2O. The solution was microwaved in 15 s intervals for about 3 min or until the agarose boiled and all agar particles were dissolved by visual inspection. The 3.1 g of molten agarose was then aliquoted into 15 ml Falcon tubes and stored at 4 °C. On the morning of an experiment, 0.4 ml of 10× 7H9 G media, 0.2 ml of sterile 18 MΩ H20 were added to five previously prepared 3 g aliquots of agarose tubes. The solution was boiled for 8 min in a covered boiling flask. After fully melting the agarose, the solution was placed into a 45 °C stirred bath to cool for 2–10 min. Once cooled, 0.4 ml of Middlebrook OADC and 4 μl of 1000× concentration of drug or drug vehicle were added to make a total of 4 ml of 7H9 glycerol oleate (7H9-GO) agarose media. The media was then injected into the single or each of the 5 glass-gasket-glass chambers using a 20-gauge syringe needle. The agarose was allowed to set for about 30 min and one glass slide was removed to expose the agar surface. The agarose slide was then placed in a sterile pipette tip box and stored at room temperature for about 2 hrs before spotting Mtb cultures.

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