Cm100 biotwin transmission electron microscope

Prior to TEM, S. aureus cultures were grown as described for SR-SIM. Cells were harvested at 8,000 × g, and pellets were suspended in fixation solution [2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)] and incubated overnight at 4°C. The fixed cells were treated with 2% osmium tetroxide, followed by 0.25% uranyl acetate for contrast enhancement. The pellets were dehydrated in increasing concentrations of ethanol, followed by pure propylene oxide. Cells were finally embedded in Epon resin, and thin sections were stained with lead citrate. Images were acquired with a Philips CM100 BioTWIN transmission electron microscope fitted with an Olympus Veleta camera with a resolution of 2,048 by 2,048 pixels. Sample preparation and microscopy were performed at the CFIM.

Overnight cultures grown at 37°C were diluted 1:200 into 40 ml of fresh TSB and grown at 30°C or 37°C to an OD₆₀₀ of 0.5. Bacteria from a 10-ml culture aliquot were collected by centrifugation at 8,000 × g, and the cell pellets were suspended in fixation solution (2.5% glutaraldehyde–0.1 M cacodylate buffer [pH 7.4]; Electron Microscopy Sciences, PA) and incubated overnight at 4°C. The fixed cells were further treated with 2% osmium tetroxide, followed by 0.25% uranyl acetate for contrast enhancement. The pellets were dehydrated in increasing concentrations of ethanol, followed by pure propylene oxide, and were then embedded in Epon resin. Thin sections for electron microscopy were stained with lead citrate and observed using a Philips CM100 BioTWIN transmission electron microscope fitted with an Olympus Veleta camera with a resolution of 2,048 by 2,048 pixels. Sample processing and microscopy were performed at the Center for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen.

S. aureus overnight cultures grown at 37°C were diluted 1:200 into 40 ml of TSB and grown at 30°C or 37°C to an OD₆₀₀ of 0.5. Bacteria cells were collected from a 10-ml aliquot by centrifugation at 8,000 x g, and the cell pellets were suspended in fixation solution (2.5% glutaraldehyde in 0.1 M cacodylate buffer [pH 7.4]) and incubated overnight at 4°C. The fixed cells were further treated with 2% osmium tetroxide, followed by 0.25% uranyl acetate for contrast enhancement. The pellets were dehydrated in increasing concentrations of ethanol, followed by pure propylene oxide, and then embedded in Epon resin. Thin sections for electron microscopy were stained with lead citrate and observed in a Philips CM100 BioTWIN transmission electron microscope fitted with an Olympus Veleta camera with a resolution of 2,048 by 2,048 pixels. Sample processing and microscopy were performed at the Core Facility for Integrated Microscopy (CFIM), Faculty of Health and Medical Sciences, University of Copenhagen.