Ls fluoview fv3000 confocal laser scanning microscope

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 A₆₀₀ 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% 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 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.

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.