Measurement of cell dimensions

Overnight cultures were diluted in LB to an OD₆₀₀ = 0.01 and grown at 37°C with vigorous shaking to an OD₆₀₀ = 0.5. Cells were imaged before and 30 min after adding rifampicin (150 µg/mL). Cells (1–2 µL) were mounted on glass microscope slides with 1.2% (w/v) agarose pads. Acquisitions were made with a Nikon Eclipse TI-E/B wide field epifluorescence microscope using a phase contrast objective (CFI Plan Fluor DLL 100× oil NA1.3). Images were captured immediately at room temperature and edited using Nis-Elements AR software (Nikon). Images were analyzed using ImageJ v.1.38 software (National Institutes of Health) (Collins 2007; Schneider et al. 2012). Line scans were drawn over the long and short axis of cells to produce phase-contrast intensity profiles. Sharp intensity changes occurred where the lines crossed the boundary between the interior and exterior of the cell. Cell length and width were calculated from the distance between the points where the intensity change was half maximum (FWMH method, full width at half maximum). Statistical significance of the differences between untreated and rifampicin treated cells was calculated using the nonparametric Mann−Whitney test (GraphPad Prism version 8.0).