Zen software version 8

To estimate the fraction of dead cells in the entire colony and at the tips of the extending arms we used cells expressing GFP (green), and grew them in the presence of PI (red), which labels dead cells. At the time point of interest the total number of green and red pixels (above the background noise level) was calculated using Zen software version 8.0 (Zeiss). The fraction of dead cells was calculated as the number of red pixels divided by the number of green pixels. In some cases where the phase contrast image clearly showed that cells adjacent to PI labeled cells seemed to be dying, as indicated by the loss of their green signal, these cells were also counted as dead cells. Estimating the fraction of dead cells near the tips was conducted in a similar manner. In this analysis only cells, located up to 50 μm from the tip of the arm, were examined.

A custom designed construct was used to grow bacterial colonies under the microscope as previously described (Mamou et al., 2016 (link)). Bacterial cells were spotted at a concentration of ~1 cell/ μl on solid LB medium. When indicated, PI (Sigma) was added to a final concentration of 5 μg/ml. Construct was covered with a 35 mm cultFoil membrane (Pecon) to reduce dehydration, and incubated at 37°C in Lab-Tek S1 heating insert (Pecon) placed inside an incubator XL-LSM 710 S1 (Pecon). Developing colonies were visualized and photographed by CLSM LSM700 (Zeiss) with Plan-Aporchromat x10/NA0.45 (Zeiss). Cells expressing GFP or Dronpa were irradiated using 488 nm laser beam, while cells stained with PI were irradiated using 555 nm laser beam. For each experiment, both transmitted and reflected light were collected. System control and image processing were carried out using Zen software version 8.0 (Zeiss).