2.4 M.tb preparation for in vitro infection

Lyophilized M.tb H₃₇R_v (ATCC 25618) was obtained from American Tissue Culture Collection (Manassas, VA), reconstituted, and used as described previously [12, 13]. For each experiment aliquots of M.tb frozen stocks were plated on Difco™ Mycobacteria 7H11 agar plates (BD, Difco™) with 0.5% of glycerol and 5% Middlebrook OADC enrichment and bacteria were grown for 9–14 days at 37°C, 5% CO₂ and 95% relative humidity.

Single cell suspensions of M.tb H₃₇R_v were prepared as we previously described [12]. Briefly, bacteria were scraped from agar plates, suspended in cell culture media, briefly vortexed (five, 1 sec pulses) with two glass beads (3 mm), and allowed to settle for 30 min. The upper bacterial suspension (devoid of clumps) was then transferred to a second tube and let rest for an additional 5 min to obtain the final single cell suspension. Bacterial concentrations were determined by counting using a Petroff-Hausser chamber and subsequent plating for colony-forming units (CFUs). The concentration of bacteria was 1–2 × 10⁸ bacteria/ml and the degree of clumping was ≤10%. Bacteria prepared in this fashion are ≥90% viable by CFU assay.