SEM Analysis of T. forsythia Morphology

T. forsythia WT and ΔTf_murK::erm mutant were cultivated as described above, using MurNAc concentrations of 0.1, 0.5, 1.0, and 20.0 μg/ml, respectively. Briefly, at an OD₆₀₀ of ∼0.5 (exponential growth phase) or ∼1.5 (stationary growth phase), 1 ml of bacterial culture was harvested, each, and centrifuged at 5000 g for 7 min. Cell pellets were washed twice with phosphate-buffered saline (PBS), suspended in 500 μl of ethanol (25% in PBS), incubated for 7 min at RT and centrifuged. This step was repeated using solutions of 35, 50, 60, 70, 80, 90, and 95% ethanol in PBS and finally 100% ethanol. Finally, the samples were sputter-coated with gold (EM SDC005 apparatus; Leica, Wetzlar, Germany) and imaged with an Inspect S50 scanning electron microscope (FEI, Eindhoven, Netherlands). A detailed description of sample preparation for sputter-coating and SEM is published elsewhere (Tomek et al., 2014 (link)).