Mol Microbiol. septating BCG cells divided, the mode of division of the cells in the remaining low proportion (20%) of the septating mycobacterial cells in the population remained unknown. Therefore, the present study was initiated to find out how the (pathogen) cells in the low proportions of mycobacterial populace divided. Transmission and scanning electron microscopy and fluorescence microscopy of septum-stained live and fixed cells were used to find out whether cells were present with the septum deviated significantly more than the 5-10% found in the majority of the cells in the population. After ascer-taining the presence of cells with highly deviated asymmetric septum, the corresponding highly deviated asymmetric constriction and division were verified using live cell time-lapse imaging of the division process. Subsequently,the differences in the mode of division of the cells in the minority populace, as compared to the features of the symmetric division with minor deviation of the cells in the majority of the populace, were documented. The possible physiological significance of the highly deviated asymmetric division in the minority populace was then discussed. MATERIALS AND METHODS Bacterial Strains and Culture Conditions M. smegmatismc2155 [5] and and cells FR 167653 free base was performed, as described [7], but with minor modifications [8]. For scanning electron microscopy (SEM), mid-log FR 167653 free base phase cells were harvested, washed once with 1x PBS, fixed with 2% glutaraldehyde, treated with 0.5% osmium tetroxide for 2 hrs, dehydrated in ethanol series, 30%, 50%, 70%, and 100%. The samples were sputter-coated with gold and observed under SIRION scanning electron microscope at 4 kV, and the images were captured. Staining and Detection of Septum and Nucleoid in Fixed and Live Cells Vancomycin-BODIPY (VBP) was used to stain the septum of live cells, as described [9-11]. One g/ml of VBP FR 167653 free base (in PBS) was added to the cells and incubated with shaking at 170 rpm for 3 hrs at 37C. The cells were then adhered to poly-L-lysine coated slides for observation under Zeiss AXIO Imager M1 microscope. For staining with WGA-Alexa488 (2 g/ml in 1x PBS) [12], the cells were fixed in 4% para formaldehyde, adhered to poly-L-lysine coated slides, washed with 1x PBS for 1 min, treated with lysozyme (2 mg/ml) for 15 min, washed thrice with 1x PBS for 1 min each, stained for 15 min, mounted on 90% glycerol, and observed. DAPI staining for nucleoid was performed using 0.5 g/ml of DAPI in 1x PBS with 0.1% Triton-X100 for 5 min, and washed thrice with 1x PBS for 1 min each time. The cells were mounted in 90% glycerol and observed. A large number of septum-stained cells were analysed using fluorescence microscopy (FM). Documentation of Time-Lapse Live Cell Division (LCM) Live cell time-lapse microscopy of the asymmetric division of cells (n = 50) was performed in low melting point agarose (1.5% in Middlebrook 7H9 medium) pads, as described [13, 14], but with minor modifications [15], with Z-stacking at 37C. The cells were observed for about 8-9 hrs (for more than two generations), by taking DIC images at every 10 Gnb4 min time interval. The data were analysed and the cell length and cell constriction were decided around the images, using Axio vision 4 software.The tracking of the live cell time-lapse imaging movies was performed using the ImajeJ version 1.43m [16]. RESULTS Ultrastructural Analyses Reveal Cells with Highly Deviated Septum/Constriction Using transmission and scanning electron microscopy (TEM and SEM), a large number of cells in the mid-log phase populace were first screened for the presence of cells with division site position deviated more than that shown by the majority of the cells in the population. The position of the division site (septum/constriction), when deviated by more than 10% from the mid-cell site, irrespective of the sizes of the mother cells, was considered highly deviated asymmetric division in our studies. This higher cut-off was set up in order to differentiate the major deviation in the asymmetric septum/constriction position of the cells in.
