Tracking Cell Division and Lineages

To track the in vivo growth of dividing single cells and lineages, bacteria were tracked by time-lapse microscopy. Cells were grown on agar pads (64 (link), 67 (link)) containing 15 mg ml⁻¹ of electrophoresis-grade agarose (Fisher Scientific) in M9 medium with the desired concentration of streptomycin. All cells used to inoculate agar pads were grown exponentially in streptomycin-free M9 medium. Time-lapse images were capture on a Nikon Eclipse Ti microscope with the Nikon NIS Element AR software by 100× phase-contrast and fluorescence (Prior, Lumen 200) imaging. To visualize the distribution of cell wall during the formation of minicells, cells were pulse-labeled with Alexa Fluor 488 wheat germ agglutinin (WGA) (Life Technologies Corporation) (68 (link)). Phase-contrast images were taken at intervals of 2 min. To minimize phototoxicity and bleaching, fluorescence images were recorded at longer intervals of 10 and 30 min. Microscope and agar pads were kept at 37°C with a Nevtek ASI 400 heater.