Tyloxapol surfactant

Mycobacterium tuberculosis H37Rv (Mtb), Mper1 (ppe15; Rv1039c) deletion mutant of Mtb (d-mper1) and mper1-complemented strain (C-d-mper1) were grown and subjected to multiple-stress conditions as described earlier (Deb et al., 2009 (link)). Mtb, d-mper1 and C-d-mper1 were grown in complete Dubos medium containing 1.5% glycerol and 10% Dubos medium ADC supplement to an OD₆₀₀ nm of 0.2 in a roller bottle incubator at 37 °C. Cells were harvested and resuspended to obtain OD₆₀₀ nm of 0.2 in an acidic (pH 5.0), low nutrient Dubos medium (10% Dubos medium with Dubos albumin supplement and without glycerol) supplemented with 0.018% Tyloxapol surfactant (Sigma-Aldrich, MO, USA). This cell suspension (150 ml) was placed in an 800 ml vented tissue culture flask and incubated in a hypoxia incubator maintaining 10% CO₂ and 5% O₂ for time-periods up to 18 days. The d-mper1 strains were grown in media containing hygromycin (Hyg, 75 μg/ml) and C-d-mper1 strains were grown in the presence of 75 μg/ml hygromycin and 50 μg/ml kanamycin.