M.tb low-dose aerosol infection of mice

M.tb strain H37Rv (ATCC 27294; American Type Culture Collection, Rockville, MD) was a kind gift from the Colorado State University repository (Fort Collins, CO). Mouse infections were performed in accordance to the low-dose aerosol infection model using a Madison Aerosol Chamber (University of Wisconsin, Madison, WI) with the nebulizer pre-calibrated at ~200 CFU/mice. M.tb strain H37Rv was grown to mid-log phase (OD₆₀₀ ∼0.6) in Middlebrook 7H9 media (Difco, USA) with 0.1% Tween 80 (Sigma, USA), 0.2% glycerol and 10% Middlebrook albumin, dextrose and catalase (ADC) enrichment medium (Difco, USA). Bacteria were stored at −80°C in 20% glycerol stocks for further experiments. For aerosol infection, cultures were washed twice with PBS and made into a single cell suspension by passing through a 26-gauge syringe ten times followed by two passes through a 30-gauge syringe. Fifteen ml of the M.tb strain H37Rv single cell suspension (20X10⁶ cells per ml) was placed in the Nebulizer reservoir of the Madison Aerosol Chamber calibrated to deliver the desired CFUs of bacteria into the lungs of mice kept in the chamber in 15 minute cycles. At 24 hours after aerosol challenge 3 mice were euthanized for quantification of pathogen delivery to lungs by measuring CFUs in lung homogenates. Mice were found to be infected with ∼220 CFU of M.tb strain H37Rv in their lungs. The mice were maintained under BSL-3 containment thereafter.