Application of confocal microscopy for quantification of intracellular mycobacteria in macrophages

This chapter describes the techniques used to prepare a uniform and consistent mycobacterial culture and for the infection of macrophages in vitro. Here, protocols are described for the achievement of a certain number of single cell bacilli per macrophage. Confocal microscopy in combination with the software ImageJ are highlighted, and these techniques will be correlated with quantification by FACS and confirmed by colony forming units (CFU) the classical method to validate the intracellular survival of Mycobacterium tuberculosis. Conventional CFU for quantification of intracellular slow growing mycobacteria is labour-intensive, with incubation requirements that can take up to several weeks. New alternatives and fast methods are required for a rapid assessment of the immune response as well to test new antibacterial drugs in high- throughput screens.