Antibiotic Treatment and Cell Imaging

Exponential-phase cell cultures (OD₆₀₀, ∼0.1) were treated with antibiotics (CZ and/or ETP) and grown at 37°C in a roller. Samples were collected for imaging after 3 h. Eight microliters of cells was added to 2 μl of dye mix consisting of 2.5 μM Sytox Green (Life Technologies, Eugene, OR), 10 μg ml⁻¹ DAPI (4′,6-diamidino-2-phenylindole) (Life Technologies, Eugene, OR), and 20 μg ml⁻¹ WGA-647 (Life Technologies, Eugene, OR) in 1× Tris base and transferred to an agarose pad (10% MHB, 1% agarose). The exposure time of each wavelength was maintained constant for all images. Microscopy was performed as previously described (20 (link), 21 (link)). Quantification of the mean DAPI and Sytox Green intensities was performed using CellProfiler (22 (link)) as previously described (21 (link)).