A4224

Mycobacterium tuberculosis H37Rv was grown in Middlebrook 7H9 broth as described previously (29 (link)). Prior to infection, stock solutions of Mtb were thawed, washed once with phosphate-buffered saline and inoculum was prepared in sterile saline. Aerosol infection was performed using an inhalation exposure system (model A4224, Glas-Col). To infect mice with a low dose of 100 CFU/lung, animals were exposed for 40 min to an aerosol generated by nebulizing approximately 6 ml of a suspension containing 2.4x10⁷ live bacteria. Similarly, for intranasal infection, 25µl per nostril was administered in anaesthetized mice to achieve the indicated dose. After infection, the inoculum was also plated to determine the change in the inoculum. Infection dose was checked at one day post-infection by determining the bacterial load in the lungs of four infected mice.

A virulent Mtb H37Rv strain was cultured and stocks were prepared and stored at −80 °C, as described elsewhere [27 (link)]. Mice were exposed to aerosol infection for 40 min by nebulizing 6 mL of a suspension that contained 2.4 × 10⁷ live bacteria in an inhalation exposure system (model A4224, Glas-Col). One day following infection, four mice were euthanized to confirm the infection dose, which was 500 colony-forming units (CFU)/mouse.

Virulent Mtb H37Rv strain was cultured and stocks were prepared and stored at −80°C, as described previously (38 (link), 47 (link)). Mice were infected via aerosol by nebulizing with 6 ml of a suspension that contained 2.4 × 10⁷ live bacteria in an inhalation exposure system (model A4224, Glas-Col) for 40 min. The mice were each infected with approximately 50–70 Mtb colony-forming units (CFU).