Numerical aperture objective

Cell morphology was investigated either under the light microscope (Leica MC170 HD) or a fluorescence microscope (Nikon). The viability of cells was analyzed with the LIVE/DEAD BacLight fluorescence stain from Life Technologies (Thermo Fisher Scientific). For all measurements, live cells were mounted onto 1% agarose pads supplemented with TB medium prior to microscopy analysis. Phase-contrast and fluorescence images were captured using a Ti eclipse inverted research microscope (Nikon) with a ×100/1.45 numerical aperture (NA) objective (Nikon). Image processing and cell length measurement was conducted by Fiji ImageJ 1.8.0^{84 (link)}.

For living-cell analysis and time-lapse microscopy, cells were transferred onto a PYE 1% agarose pad with supplementation as required. Otherwise, cells were fixed with 1% formaldehyde and mounted onto 1% agarose pads. Phase-contrast and fluorescence images were taken using a Ti eclipse inverted research microscope (Nikon) with a 100×/1.45 numerical aperture (NA) objective (Nikon). Fiji (ImageJ) was used for image processing. Cells were classified as filamentous if they were more than twice the average length of exponentially growing C. crescentus cells.
Cells were measured using MicrobeJ 5.13l (15) beta (50 (link)). Cells were detected using the “Default” setting for thresholding and the “Filament” setting for detection of bacteria. Cell detection was manually corrected where necessary. To determine the cell width, the median width for each cell was used.

Cells were fixed by addition of formaldehyde (1% final) to culture samples and stored at 4°C. For visualization, fixed cells were transferred onto 1% agarose pads attached to glass slides, covered with a coverslip, and transferred to the microscope. A Ti eclipse inverted research microscope (Nikon) with 100×/1.45 numerical aperture (NA) objective (Nikon) was used to collect phase-contrast images. The images were processed with Fiji (ImageJ).