Middlebrook oadc enrichment medium

For BCG infection experiments, M. bovis BCG Pasteur strain was grown at 37°C in Middlebrook 7H9 broth (BD Biosciences) supplemented with 10% Middlebrook OADC enrichment medium (BD Biosciences), 0.002% glycerol (Roth), and 0.05% Tween 80 (Roth). Midlog phase cultures were harvested, aliquoted, and frozen at −80°C. Bacteria were prepared from frozen stocks by thawing at 37°C, resuspension in PBS supplemented with 0.025% Tween 80 (PBS-T), and passaged through a 27G needle. Mice were infected with 2×10⁶ CFU M. bovis BCG Pasteur i.v. diluted in PBS-T.

Mycobacterium bovis BCG overexpressing Ag85B (M. bovis BCG Ag85B) was kindly provided by Dr. Joel Ernst (NYU School of Medicine, USA), M. bovis BCG GFP by Dr. Camille Locht (University of Lille), and M. bovis BCG RFP by Dr. Nathalie Winter (French National Institute for Agricultural Research). All strains were grown at 37°C in Middlebrook 7H9 broth (BD Biosciences) supplemented with 10% Middlebrook OADC enrichment medium (BD Biosciences), 0.002% glycerol (Roth), and 0.05% Tween 80 (Roth). Midlog phase cultures were harvested, aliquoted, and frozen at −80°C. For in vitro infections BCG strains were prepared as previously described (44 (link)) and cells infected with different multiplicities of infection (MOI). Bacteria for in vivo infections were prepared from frozen stocks by thawing at 37°C, washing with PBS 0.025% Tween 80 (PBS-T), and passaging through a 27 gauge needle. Mice were infected intravenously (i.v.) with 2 × 10⁶ colony forming units (CFU). For Mtb infections, the Mtb strain H37Rv was grown and prepared as described previously (45 (link)). Mice were infected with a low (100 CFU) or a high dose (1,000 CFU) by aerosol exposure. Mtb-infected mice were scored according to Morton and Griffiths (46 (link)). According to the local animal welfare guidelines, mice that reached a score of >3.0 had to be euthanized.