Bbl middlebrook 7h9 broth

The methods were carried out in accordance with the relevant guidelines and were approved under institutional biosafety protocols under IBC 12–160 and HSC-MS-15-0548. The M. tuberculosis (H37Rv) (ATCC-27294) strain was obtained from the ATCC and was grown in BBL Middlebrook 7H9 broth with OADC enrichment (BD Biosciences, CA, #211886) at 37 °C and 5% CO₂. Green-fluorescent-protein-expressing M. tuberculosis H37Rv (gfpMtb) strains and M. bovis BCG were constructed as previously described^{52 (link)}. Red-fluorescent-protein-expressing M. tuberculosis H37Rv (rfpMtb) was a kind gift from Dr. Malini Madiraju (University of Texas, Tyler campus, TX). All mycobacterial strains were grown for 7 days in 7H9 broth with (for gfp, rfp strains) or without 25 µg/mL kanamycin, and aliquots containing ~10⁸ viable bacterial cells were frozen for subsequent use. Before use, aliquots were thawed, washed three times in PBS (x12,000 rpm; 15 min), and sonicated at 4 watts with a sonicator (60 Sonic Dismembrator, Fisher Scientific) to prepare a uniform single-cell suspension of the bacteria.

Methods to prepare Mtb (H37Rv) and BCG (Pasteur) strains for infection have been described earlier (29). MΦs were infected (MOI = 1) using Mtb or BCG for 4 h prepared as follows. M. tuberculosis (H37Rv) (American Type Culture Collection-27294) (Mtb) was grown in BBL™ Middlebrook 7H9 broth with OADC enrichment (BD Biosciences, 211886) at 37 °C and 5% CO₂. Green-fluorescent-protein-expressing M. tuberculosis H37Rv (gfpMtb) and red-fluorescent-protein-expressing M. tuberculosis H37Rv (rfpMtb) were a kind gift from Dr. Malini Madiraju (The University of Texas at Tyler, Tyler, TX). All mycobacterial strains were grown for 7 days in 7H9 broth with (for gfp/rfp strains) or without 25 µg/mL kanamycin, and aliquots containing ~10*8 viable CFUs were frozen for subsequent use. Before use, aliquots were thawed, washed three times in phosphate-buffered saline (×12,000 rpm; 15 min), and sonicated at 4 W with a sonicator (60 Sonic Dismembrator, Fisher Scientific) to prepare a uniform single-cell suspension of Mtb without loss of viability.

Bacterial strains used in this study are listed in Table 2. E. coli strains were cultivated in Luria broth (Difco, Detroit, MI). For LB agar, 1.5% (wt/vol) agar was added to liquid. M. smegmatis was cultivated on BBL Middlebrook 7H9 broth or 7H10 agar (BD, Franklin Lakes, NJ, USA) supplemented with 0.2% (vol/vol) glycerol and 10% (vol/vol) OADC (BD). Additionally, 7H9 broth was supplemented with 0.05% Tween 80 (vol/vol).
If required, antibiotics were added to the medium at the following concentrations: ampicillin, 100 μg/ml (E. coli); kanamycin, 30 μg/ml (E. coli and M. smegmatis); and hygromycin, 200 μg/ml (E. coli) or 90 μg/ml (M. smegmatis). Unless stated otherwise, E. coli strains and M. smegmatis mc²155 were cultivated at 37°C with aeration at 200 rpm. When required for protein induction, tetracycline or isovaleronitrile was added at a final concentration of 30 ng/ml or 50 μm/ml, respectively.