Culture medium (1.5 ml) containing 0.6% NuSieve GTG agarose (no. 50081, Lonza) was plated into six-well plates and allowed to solidify at 4°C. A final concentration of 0.3% agarose containing 5000 22Rv1 cells was carefully plated on top of the solidified GTG agarose. Plates were incubated at 37°C with 5% circulating CO2, and the medium was changed every 3 days. After 2 to 3 weeks, colonies were stained with 0.005% crystal violet in 4% paraformaldehyde solution, and the number of colonies was counted.

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