Visualization of M. tuberculosis in Animal Models decreased oxygen conditions associated with tuberculosis disease in humans and other animal models are present in the lungs of GKO mice. Intracellular M. tuberculosis in C57BL/6 lungs The location of bacilli was studied in lung sections from C57BL/6 mice after M. tuberculosis infection via a modified staining method combining AR for staining acid fast bacteria, hematoxylin QS for staining tissue and DAPI for staining nuclei. Four weeks after M. tuberculosis aerosol exposure, which is the start of drug treatment in our standard laboratory protocol for this mouse model [44], the inflammatory lung lesions consisted of large lymphocyte (LC) aggregates surrounding multiple, smaller accu- mulations of epithelioid mWs (Fig. 4A) as previously reported [18,49]. AR+ bacilli (,95%) were found intracellular in the multiple epithelioid mW aggregates located within the lymphocyte field (Fig. 4B, 4C). A small proportion of AR+ bacilli (,5%) were observed in foamy mWs located adjacent to areas of inflammation. At nine weeks post aerosol infection, most of the inflammatory lesions displayed a well-defined, highly organized lesion structure that consisted primarily of a central region of epithelioid mFs and neutrophils surrounded by a distinct, sometimes incomplete, lymphocyte rim (Fig. 4D) as previously reported [18,49]. These lesions sometimes exhibited a mild degree of tissue necrosis Figure 4. Intracellular M. tuberculosis bacilli in lungs from M. tuberculosis infected C57BL/6 mice, throughout infection and after 6 weeks of MXF drug treatment. (AR) auramine-rhodamine, hematoxylin QS and DAPI; (H&E) hematoxylin and eosin. (A) An inflammatory lesion from the lungs of an M. tuberculosis infected C57BL/6 mouse 4 weeks after aerosol infection. The cellular architecture of lesions shows a field of lymphocytes (L) surrounding multiple macrophage aggregates (W) (H&E staining, 2006magnification). (B) An inflammatory lesion from lungs of an M. tuberculosis infected C57BL/6 mouse 4 weeks after aerosol infection. Intracellular AR+ bacilli were predominantly found within macrophage rich regions surrounded by lymphocytes (AR, 4006 magnification). (C) High magnification of the inflammatory lesion shown in figure 4B (circle) showing intracellular AR+ stained bacilli within macrophages (AR, 10006 magnification). (D) An inflammatory lesion from the lungs of an M. tuberculosis infected C57BL/6 mouse 9 weeks after aerosol infection. The lesion shows a distinct rim of lymphocytes (L) surrounding a core of epithelioid macrophages (W). A layer of foamy macrophages (f) can be seen surrounding the lymphocyte rim (H&E staining, 2006 magnification). (E & F) Fluorescent images of low (E) and high (F) magnifications of the inflammatory lesion depicted in figure 4D taken from a serial tissue section. The majority of AR+ bacilli at this time are found in epitheloid mWs (W) located within the lymphocyte cuff (L), whereas a lower number of AR+ bacilli are located in foamy mWs (f) located at the peripheral edges of inflammation (AR, 1006and 2006magnifications). (G) Inflammatory lesion from the lungs of a C57Bl/6 mouse infected with M. tuberculosis and treated with moxifloxacin for 6 weeks. Cellular architecture of lesions after 6 weeks of drug treatment is similar to untreated controls and consist of a mW core (W) surrounded by lymphocytes (L) and foamy mWs (f) (AR, 2006 magnification). (H) High magnification of the peripheral edges of the inflammatory lesion shown in figure 4G. A few remaining AR+ bacilli are found within foamy macrophages located outside of the lymphocyte cuff (AR, 10006 magnification). doi:10.1371/journal.pone.0017550.g004 PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17550