Figure 3. Accumulation of lipid bodies and loss of acid-fastness in Mtb cells under multiple-stress.

(A), Acid-fast staining cells (green) decreased and lipid body staining cells (red) increased with time under multiple-stress. Cells were stained with Auramine-O (acid-fast stain) and Nile Red (neutral lipid stain) and examined by confocal laser scanning microscopy (Leica TCS SP5) at the same laser intensity for all the samples with Z-stacking to get the depth of the scan field. Scanned samples were analyzed by LAS AF software for image projection. Overlaid images of the dual-stained Mtb are shown. Bar = 4 µM. (B), Magnified view of three different Mtb cells, representing three different subsets of Mtb cells in terms of acid-fast and neutral lipid staining property, observed in the Mtb population under multiple-stress: only acid-fast positive without any Nile Red stain (green), both acid-fast and lipid stain positive (orange yellow) and acid-fast negative cells with only Nile Red staining lipid bodies (right). The only acid-fast stain (green) positive cells gradually decreased and the other two types steadily increased during multiple-stress treatment. These cells selected from a day 9 sample were stained with both dyes and examined by confocal scanning as stated above in (A). Bar = 5 µM.