Mycobacterium smegmatis Stress Response

M. smegmatis mc²155 was grown at 37°C in Middlebrook 7H9 broth supplemented with 11 mM glucose, 14.5 mM NaCl, and 0.05% (vol/vol) Tween 80. Dibucaine (Sigma-Aldrich) was added to a log-phase culture at the final concentration of 200 μg/mL, and an equivalent volume of water was used as a vehicle control. After a 3-h incubation at 37°C, the culture was washed with phosphate-buffered saline (PBS) containing 0.05% Tween 80 (PBST) three times and resuspended in the same volume of Middlebrook 7H9 broth for recovery. Benzyl alcohol treatment was identical to Dibucaine treatment except that the treatment was for 1 h at the final concentration of 100 mM, as described previously (14 (link)). CFU were determined by serially diluting cell culture using Middlebrook 7H9 broth and spotting 5 μL on Middlebrook 7H10 agar supplemented with 11 mM glucose and 14.5 mM NaCl. The agar plates were incubated at 37°C for 2 to 3 days before the number of microcolonies was determined.