Phagolysosomal trafficking is an important innate defense pathway that clears 
microbes by delivering them to lysosomes, the degradative compartment of the 
cell. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, 
subverts this host defense mechanism by arresting maturation of the phagosome. 
The ability of Mtb to arrest its delivery to the lysosome can be demonstrated by 
the prolonged co-localization of bacteria containing phagosomes/vacuole with 
early phagosomal markers [such as, Ras-related proteins in the brain 5 (Rab5) 
and Transferrin receptor (TfR)], and a failure to acquire late phagosomal and 
lysosomal markers (such as Rab7 and LAMP1) (Deretic and Fratti, 1999, Mehra et 
al., 2013). Here, a protocol is outlined for infection of macrophages with 
mycobacterial species like pathogenic Mtb, vaccine strain Mycobacterium bovis- 
bacillus Calmatte- Guérin (BCG) and rapidly dividing non-pathogenic 
Mycobacterium smegmatis (Msmeg), followed by indirect-immunofluorescence 
microscopy to visualize host vacuolar markers. Thereafter, automated 
quantification of degree of co-localization between mycobacteria and host 
vacuolar markers like TfR and LAMP1 is done by processing the binary images of 
bacteria using mathematical tools. This results in quantification of the mean 
fluorescence intensity (MFI) of these host markers directly around the 
bacteria/bacterial clusters with increased sensitivity relative to when done 
manually. By manipulating host or pathogen, this assay can be used to evaluate 
host or bacterial determinants of intracellular trafficking. The basic method 
can be applied to studying trafficking of other bacteria or particles like 
beads, although the kinetics of infection and phagosome maturation will depend 
upon the phagocytic cargo. The mathematical analysis tools are available in many 
standard imaging analysis programs. However, any adaption for similar analysis 
should be confirmed by the individual user with their imaging and analysis 
platform.