Visualization of M. tuberculosis in Animal Models Figure 1. Numbers of viable M. tuberculosis organisms in lungs of infected IFN-c knock-out and C57BL/6 mice after drug treatment. Mice were infected with M. tuberculosis strain Erdman and treated with drugs starting 18 to 21 days after aerosol infection; with isoniazid (INH), rifampin (RIF), ethambutol (EMB), gatifloxacin (Gati) or moxifloxacin (Moxi). Sacrifice times were at 2, 5 and 7 days of drug treatment. Data points represent mean log10 viable bacilli +/2 standard error present in whole lung homogenates. doi:10.1371/journal.pone.0017550.g001 without effective drug intervention, multiple lesions coalesce and exhibit extensive lesion necrosis (Fig. 2E, F). Necrotic foci contained high numbers of both intracellular bacteria [within mW and multinucleated giant cells (55%)] (Fig. 2G) as well as extracellular AR+ bacilli [residing in alveolar spaces filled with necrotic cellular debris (40%)] (Fig. 2H). Few AR+ bacilli (5%) were visible in and around blood vessels and non-granulomatous lung parenchyma (Fig. 2I). In the GKO mouse model, drug treatments start 14 to 18 days post aerosol exposure of M. tuberculosis and lasts for 9 to 14 consecutive days. After 4 days of drug treatment with INH or RIF in the GKO mice, there was a minimal effect observed on lesion histology as well as on the distribution of almost exclusively intracellular bacilli in the lesions. After 7 days of treatment, an initial reduction in lesion size and of AR+ bacilli number became evident. After 10 days of drug treatment, there was minimal inflammation remaining in the lung pathology. At this time, a reduction in AR+ bacilli was clearly evident and this reduction was seen uniformly across the entire tissue section. The AR+ bacilli were mainly located either within the few remaining inflammatory lesions or were situated peripherally within the adjacent alveolar spaces (Fig. 2J). A minority of AR+ bacilli was dispersed in non- inflamed lung parenchyma (5–10%). The remaining bacilli were primarily intracellular, which is as expected as treatment was initiated prior to the onset of necrosis (Fig. 2K, L). Progression of necrosis and development of hypoxia in late stage GKO The necrotic granulomas found in the later stages of M. tuberculosis infected GKO mice showed similar morphology to necrotic, hypoxic granulomas observed in M. tuberculosis infected guinea pigs [22,23,26]. Staining with hematoxylin and eosin showed a distinct increase in cellular necrosis over time. Early pathology (around 22 days post-infection) shows the aggregation of predominantly granulo- cytes within alveolar spaces (Fig. 3A, B). These cellular accumulations then begin to degenerate as observed by the progression from intact cells to necrotic, cellular debris (Fig. 2E, F). The accumulation of nuclear material within the alveolar spaces lead to an increase in basophilic staining during the progression of disease. Confocal microscopy was utilized to confirm whether the bacteria were intra- or extracellular in the eukaryotic cells. Immunohistochemistry using anti-tubulin antibodies visualized the cytoskeleton, in combination with DAPI staining which stained the nuclei. The integrity or degeneration of the viable eukaryotic cells was thereby shown by the intactness of the nuclei via the DAPI staining, as well as by the organization of the tubulin structure via immunohistochemistry. Since every section is 5 microns only, the bacilli observed in a microscopy section will be in the same plain of focus as the viable cells, and the proximity of the bacillary location to the intact nuclei indicates their intracellular nature. At 18 days post infection, intact nuclei and highly organized tubulin network can be observed (Fig. 3C), whereas at day 28 the nuclei are ill- defined and the cytoskeleton structure is lost (Fig. 3D) indicating loss of cell viability. Confocal microscopy confirmed this observation by showing the transition from healthy appearing nuclei at day 18 (Fig. 3E) to accumulated nuclear debris within alveoli at day 28 in GKO mice (Fig. 3F). To determine whether lesions in the lungs of GKO mice infected with M. tuberculosis are hypoxic, mice were injected i.p. PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17550