Epon resin mixture

Synthesized nanoparticle-treated and untreated biofilms of MSSA 6538 were examined by TEM. Initially, overnight grown cells in 96-well plates were incubated with nanodispersions for 24 h without shaking at 37°C. For prefixation, cells were then treated with aldehyde mixture (glutaraldehyde 2.5% and formaldehyde 2%) and kept overnight at 4°C. After washing, cells were harvested by sonication to disrupt biofilms, immediately postfixed using 2% osmium tetroxide overnight at 4°C, and washed with 0.2 M phosphate buffer. Cell blocks were made using 2% agarose in sterile tubes, which were then sliced to the desired size. Specimens were dehydrated in an ethanol series and embedded in an Epon resin mixture (Electron Microscopy Sciences, Hatfield, PA, USA), which was treated at different temperatures to ensure complete polymerization. Thin sections were obtained using a MT-X ultramicrotome (Boeckeler Instruments, Tucson, AZ, USA) and loaded onto TEM copper grids. Finally, sections were stained with 1% uranyl acetate followed by lead citrate to remove any traces of moisture. Microscopy was performed using an H-7600 electron microscope (Hitachi Ltd., Tokyo, Japan) at 120-keV.