Ultra cut c

To assess myelin sheath thickness, cocultures were fixed with 2% glutaraldehyde overnight at 4°C, followed by postfixation with 1% osmium tetroxide for 30 min at room temperature. After the samples had been washed three times with ultrafiltered water, they were dehydrated in an ethanol series (50, 70, 80, 85, 90, 95, and 100%). Using an ultramicrotome (Ultra Cut C; Leica, Wetzlar, Germany), samples were sectioned and mounted on copper grids. Grids were contrast stained with uranyl acetate and lead citrate. Images were captured by cryo-TEM (Tecnai F20 Cryo; FEI, Hillsboro, OR, USA). To quantitatively determine the relative thickness of individual remyelinated nerves, the g-ratio was analyzed with a g-ratio calculator plug-in for ImageJ software (available at https://imagej.nih.gov/ij/), which allowed semiautomated analysis of randomly selected nerve fibers (53 (link), 54 (link)). At least seven remyelinated axons per group were randomly selected for measurement, and the mean g-ratio was calculated for each group by averaging across all individual g-ratios.

For determining spores in the isolates, C. difficile strains positive for tcdA/B were directly examined by electron microscopy. The bacterial cells were diluted in 1.5 mL of 0.85% saline and washed thrice at 12,000 rpm. The bacterial sample was fixed in 2.5% glutaraldehyde/0.1 M phosphate-buffered saline (PBS; pH 7.4) for 2 hours and then transferred to 1% osmium tetroxide for 1 hour. The sample was washed with 0.1 M PBS, embedded into epoxy resin using propylene oxide, and dehydrated using alcohol. After embedding, the sample was polymerized in a dry oven at 60°C for 12 hours. In the subsequent steps, the bacteria-embedded resin was cut into 70 to 80 mm thick sections using an ultra-microtome (Ultra cut C; Leica, Wetzlar, Germany) and observed under an electron microscope (Cryo-TEM CryoTecnai F20; FEI, Hillsboro, OR, USA) after staining with uranyl acetate and lead citrate. Two standard strains, ATCC-43598 (tcdA−/tcdB+) and KCCH-12115 (tcdA+/tcdB+), were used for the comparison of spores.