Transmission electron microscopy (Joel model JEM 10–10) was used to visualize cellwall thickening in a vancomycin-decreased susceptibility strain. Bacteria were exposure to sub-inhibitor concentrations of vancomycin and then analyzed during the exponential growth phase. As a control, ATCC 25923 and VISA Mu ATCC 700699 strains were also grown to the exponential phase. The bacteria were harvested, washed, and fixed with a glutaraldehyde/formalin (2.5%/10%) solution in 0.1 M phosphate-buffered saline (PBS), pH 6.0. After that, the bacteria were post-fixed with osmium tetroxide, contrasted with uranyl acetate, and dehydrated in graded concentrations of ethyl alcohol (20, 30, 40, 50, 60, 70, 80, 90, and 100%). Transverse thin sections from samples embedded in resin were mounted on grids, which was followed by treatment with lead citrate. The samples were stained with 1% phosphotungstic acid at pH 7.2 and visualized by transmission electron microscopy. Observation and acquisition was performed using a transmission electron microscope operated at 100 kV and equipped with a Hamamatsu C4742-95 digital camera. The images were processed at 100,000× and analyzed to determine the cellwall thickness from an average of 10 cells per bacterial strain [30 (link)].
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