In vitro Quantification of Intracellular Mycobacterial Growth

Macrophage cultures were infected with each strain at a multiplicity of infection (MOI) of 1:1 (bacteria/macrophage). On the first and fourth day post-infection (p.i.), cells were lysed with 0.1% saponin solution and 20 min of incubation. The culture lysate was collected, treated in an ultrasonic bath and vortexed to break up any bacterial clumps, serially diluted in PBS, and submitted to the CFU (colony-forming unit) test. For this, 50 μL aliquots of each dilution were seeded on Middlebrook 7H10 agar. The plates were incubated for 3 weeks at 37°C and colonies were quantified. Bacterial counting was expressed in log₁₀; intracellular growth was quantified, subtracting the average CFU obtained from macrophages on day 4 and day 1. Growth values of each isolate were calculated as a proportion of the reference strain M. kansasii ATCC 12478 growth value set as 1.0 for comparison (index A).