For static images of cells analyzed in S2 and S3 Figs, cells were grown in C+Y medium to an OD492 of 0.2, and 2 μL of these cultures was spotted onto a microscope slide. A polylysin-coated coverslip was placed on top to fix the cells in position. Images were captured and processed using the Nis-Elements AR software (Nikon). Analysis of cell dimensions was carried out using the MATLAB-based open source software MicrobeTracker [46 (link)]. Cell contours were obtained using the alg4 spneumoniae3 algorithm implemented in MicrobeTracker, a derivative of alg4 ecoli2 with parameters spliltTreshold, joindist and joinangle refined to fit the shape of S. pneumoniae.
Time-lapse microscopy was carried out as previously described [47 ], with modifications. Briefly, pneumococcal precultures were grown in liquid CAT medium at 37°C to an OD492 of 0.2, and 2 μL of these cultures was spotted onto a microscope slide containing a slab of 1.2% CAT agarose before imaging. Images were captured at 5 min intervals for 6 hours and processed using the Nis-Elements AR software (Nikon).
Time-lapse microscopy was carried out as previously described [47 ], with modifications. Briefly, pneumococcal precultures were grown in liquid CAT medium at 37°C to an OD492 of 0.2, and 2 μL of these cultures was spotted onto a microscope slide containing a slab of 1.2% CAT agarose before imaging. Images were captured at 5 min intervals for 6 hours and processed using the Nis-Elements AR software (Nikon).
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