Dyeing assay of colony boundaries

The fate of boundary cells was determined using LIVE/DEAD® BacLightTM Bacterial Viability Kits, L7012 (Invitrogen, USA) following the manufacturer's fluorescence microscopy protocol with some modifications. SYTO 9 and propidium iodide dyes were mixed in equal volumes. Three microliters of the mixture were diluted in 1 ml TPM buffer and dropped onto a cover slip. The agar between two strain colonies where the boundary was formed was excised and placed upside-down on a drop of dye mixture in the dark for 15 min, and then observed under a fluorescence inverse microscope equipped with a B-2A standard filter (Nikon, Japan).