Oadc enrichment medium

Mycobacterium tuberculosis strain H37Rv was cultured from frozen stocks stored at −80°C in Middlebrook 7H9 broth (BD Biosciences, Oxford, UK) supplemented with 10% OADC enrichment medium (BD Biosciences), 0.2% glycerol, and 0.02% Tween 20 with agitation at 37°C. Growth was monitored by measuring optical density (OD) using a Biowave cell density meter (WPA, Cambridge, UK). Infection experiments were performed using cultures at mid-log growth (at OD 0.55–0.65) corresponding to 1–2 × 10⁸ CFU/ml. Correlation with optical density was checked by performing colony counts in triplicate on Middlebrook 7H11 agar. Cells were infected at a multiplicity of infection (MOI) of 1 unless otherwise stated.

The BCG Danish strain 1311 used in this study was provided by Prof. Kanglin Wan from the Chinese Center for Disease Control and Prevention. The BCG vaccine was stationary cultured in 30 mL of Middlebrook 7H9 Broth (BD, Sparks, MD, USA) with an OADC enrichment medium (BD, Sparks, MD, USA) to a mid-log phase (OD₆₀₀ = 0.6) at 37 °C for 12–15 days. Subsequently, it was harvested via centrifugation (3000 rpm for 15 min), and the supernatant was discarded and replaced with HBSS at 1 mg and suspended with 3 mL of HBSS. The bacterial concentration (CFU/mL) was determined retrospectively by plating tenfold dilutions in Middlebrook 7H9 Broth with OADC and counted on 7H11 agar with OADC. The one-milligram moist weight of BCG was equivalent to 2.6–4.3 × 10⁶ CFU.