Transmission electron microscopy (TEM)

Transmission electron microscopy (TEM) was performed as previously described with slight modifications [23]. Overnight cultures of S. aureus were collected by centrifugation at 4,000 × g for 5 min to avoid cell damage. The cells were immediately resuspended in 0.1 M phosphate buffer (pH 7.4) containing 2.5 % (v/v) glutaraldehyde overnight at 4 °C for fixation. Samples were collected by centrifugation at 4,000 × g for 5 min, and agar embedding was performed to easily process the following operation. After being washed with 0.1 M phosphate buffer (pH 7.4) for three time at 4 °C, they were fixed again with 1 % (v/v) OsO4 for 1 h at 4 °C. Afterwards, specimens were washed with 0.1 M phosphate buffer (pH 7.4) for three times and stained with 3 % (v/v) uranyl acetate for 3 h at 4 °C. Before being infiltrated and embedded in acetone and resin, the samples underwent dehydration in a series of graded ethanol solutions. After polymerization at 60 °C for 48 h, thin slices were cut with a diamond knife using an LKB ultramicrotome (LKB Instruments). The slices were positioned on uncoated 200-mesh copper grids prior to staining with lead citrate. Sections were subsequently observed under a Tecnai F20 G2 (FEI Company) Transmission Electron Microscope operated at 100 kV.