Microscopy analysis of cell size

For microscopy experiments, JE2, pgl and pgl_comp were grown overnight at 37°C in CDMG w/wo 0.05 μg/ml OX. The next day, the cultures were washed and normalized to an OD₆₀₀ of 1 in PBS and 75 μl of these cultures were double stained for 30 mins at 37°C with vancomycin-BODIPY FL at a final concentration of 2 μg/ml and WGA Alexa Fluor 594 at a final concentration of 25 μg/ml. Bacteria were then collected by centrifugation for 2 mins at 14,000 xg. The cells were resuspended with 100 μl of PBS, pH 7.4, and 5 μl of this sample was spotted onto a thin 1.5% agarose gel patch prepared in PBS. Stained bacteria were then imaged at X1000 magnification using Olympus LS FLUOVIEW Fv3000 Confocal Laser Scanning Microscope. Cell size was measured as previously described [54 (link)] using ImageJ software (Fiji v.1.0). Images of cells from three/four biological replicates were acquired, 50 cells measured per biological replicate (150–200 cells in total per condition), and the average and standard deviations for the three/four biological replicates were plotted using GraphPad Prism version 9.2 and significant differences were determined using a Kruskal-Wallis test followed by a Dunn’s multiple comparison test. Only 60 cells could be measured for OX treated cells due to cell lysis.