Albumin dextrose catalase enrichment

Mycobacterium tuberculosis (Mtb) H37Rv (American Type Culture Collection; catalog no. 27294) and BCG Danish 1331 (BCG SSI) (American Type Culture Collection; catalog no. 35733) were grown in Middlebrook 7H9 broth (BD) supplemented with albumin-dextrose-catalase enrichment (BD), 0.2% glycerol, and 0.05% Tween 80 or on Middlebrook 7H11 agar (BD) containing 10% (vol/vol) oleic acid-albumin-dextrose-catalase enrichment (BD) and 0.2% glycerol. BCG was grown to the mid-log phase, washed with phosphate-buffered saline (PBS), and stored at −80°C in PBS/10% glycerol. Prior to vaccination, BCG was thawed, washed in PBS, and prepared at a dose of 10⁶ colony forming units (CFU) in 100 µL PBS.

Mycobacterium tuberculosis H37Rv was cultured in a incubator for 3–4 weeks at 37°C in Middlebrook 7H9 medium (BD, Franklin Lakes, NJ) supplemented with albumin‐dextrose‐catalase enrichment (BD). Small aliquots of M. tuberculosis suspended in Middlebrook 7H9 medium containing 10% glycerol were stored at −80°C until use. The viable bacterial numbers were determined by plating serially diluted samples on Middlebrook 7H10 (BD) plates supplemented with oleic acid‐albumin‐dextrose‐catalase enrichment (BD). The colony forming unit (CFU) of bacteria was quantified by colony counting on the plates. The bacteria were re‐suspended in PBS before use.
The mice were inoculated intratracheally with 1 × 10³ or 1 × 10⁵ CFU of M. tuberculosis H37Rv in 50 μl of PBS.

M. tuberculosis H37Rv (American Type Culture Collection, #27294) or its derivative expressing pGFPHYG2 replicative plasmid (Addgene #30173; deposited by Lalita Ramakrishnan; Cosma et al., 2006 (link)) was grown in Middlebrook 7H9 broth (Becton Dickinson) supplemented with albumin-dextrose-catalase enrichment (Becton Dickinson), 0.2% glycerol, 0.05% Tween 80, or on Middlebrook 7H11 agar (Becton Dickinson) containing 10% v/v oleic acid-albumin-dextrose-catalase enrichment (Becton Dickinson) and 0.2% glycerol. Ten milligrams of FX11 (Merck Millipore) were dissolved in 1 ml of DMSO.

M. tuberculosis H37Rv (American Type Culture Collection, #27294) was grown in Middlebrook 7H9 broth (Becton Dickinson) supplemented with albumin-dextrose-catalase enrichment (Becton Dickinson), 0.2% glycerol, 0.05% Tween 80 or on Middlebrook 7H11 agar (Becton Dickinson) containing 10% v/v oleic acid-albumin-dextrose-catalase enrichment (Becton Dickinson) and 0.2% glycerol. Infection stocks were prepared from mid-log phase cultures. For c.f.u. determinations, serial dilutions were performed in PBS/0.05% Tween 80 and plated onto Middlebrook 7H11 agar. Plates were incubated at 37 °C for 3–4 weeks prior to c.f.u. counting.

M. tuberculosis H37Rv (American Type Culture Collection; catalog no. 27294), M. tuberculosis Beijing/W (RIVM catalog no. 17919; isolated in Mongolia), BCG Danish 1331 (BCG SSI) (American Type Culture Collection; catalog no. 35733), BCG ΔureC::hly (5 (link)), and derivatives were grown in Middlebrook 7H9 broth (Becton, Dickinson) supplemented with albumin-dextrose-catalase enrichment (Becton, Dickinson), 0.2% glycerol, and 0.05% Tween 80 or on Middlebrook 7H11 agar (Becton, Dickinson) containing 10% (vol/vol) oleic acid-albumin-dextrose-catalase enrichment (Becton, Dickinson) and 0.2% glycerol. Cultures were grown to mid-log phase in 1-liter roller bottles at 37°C and 2 rpm. For vaccine stock preparations, log-phase bacilli were harvested, washed with phosphate-buffered saline (PBS), and stored at −80°C in PBS-10% glycerol. Prior to vaccination, vials were thawed, and cells were pelleted and administered as PBS suspensions. For CFU estimation, serial dilutions were performed in PBS-0.05% Tween 80 (PBST) and dilutions were plated on Middlebrook 7H11 agar. Plates were incubated at 37°C for 3 to 4 weeks prior to CFU counting.

Mycobacterium bovis BCG Pasteur (ATCC #35734) was obtained from the American Type Culture Collection. M. bovis BCG Pasteur (ATCC #35734) piniBAC-RFP harbors the coding sequence of the red fluorescent protein mCherry under control of the iniBAC promoter and was constructed as previously described (Yang T. et al., 2017 (link)). Liquid cultures were grown in complete Middlebrook 7H9 medium (BD Difco) supplemented with 0.05% Tween 80, 0.4% glycerol, and 10% albumin-dextrose-catalase enrichment (Becton Dickinson) at 37°C and 80 rpm. To grow cultures on solid medium for CFU determination Middlebrook 7H11 agar (BD Difco) supplemented with 0.2% glycerol and 10% oleic-acid-albumin-dextrose-catalase enrichment was used. Drugs and chemicals were from Sigma-Aldrich, with the exception of BDQ which was from MedChem Express. All drugs were dissolved in 90% dimethyl sulfoxide and filter sterilized to prepare stocks that were kept at -20°C.

Mtb H37Rv (ATCC #27294) was cultured in Middlebrook 7H9 broth supplemented with 0.05% Tween-80, 0.2% glycerol and 10% albumin-dextrose-catalase enrichment (Becton Dickinson) at 37°C and 80 rpm or on Middlebrook 7H11 agar containing 0.5% glycerol and 10% oleic-acid-albumin-dextrose-catalase enrichment (Becton Dickinson) at 37°C. Colonies were counted after 3–4 weeks of incubation.