H 7100 transmission electron microscope

EVs isolated by differential centrifugation were stained with 2% phosphotungstic acid (pH 6.8) or aqueous uranyl acetate, together with aqueous bacitracin. The sEV or mEV samples were then transferred to a 400-mesh copper grid (Agar Scientific) with a Pioloform support film (Agar Scientific) where they were treated with aqueous Alcian blue 8GX (1%) for 10 min. The sections were examined using a Hitachi H-7100 Transmission Electron Microscope (Olympus, Tokyo, Japan) and digital images were then acquired with an AMT digital camera. CVB1-stimulated sEV uptake in HeLa cells was visualised by cross-sectional TEM. Here, cells were fixed by incubating in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 1 h at RT and then incubated for 1 h at 0 °C in osmium tetroxide (1:1 mixture of 2% osmium tetroxide and 0.2 M sodium cacodylate). After block staining in 1% uranyl acetate, samples in 1% hot agarose were dehydrated in an ascending ethanol series (70% to absolute ethanol) and then washed in propylene oxide. After infiltration with a 1:1 mixture of propylene oxide:agar resin, left rocking for 16 h at RT, samples were embedded in capsules and polymerised (24 h at 60 °C). After cutting sections on a Leica ultramicrotome and staining in Reynolds lead citrate, they were examined by TEM using a JEOL JEM-1200 EX II electron microscope (JEOL, Peabody, MA).

Cell suspensions of pure cultures of S. aureus and E. coli from photosensitising antibacterial experiments were processed for electron microscopic observations according to the procedures reported previously (Laue and Bannert, 2010) . In brief, 5 µL of cell suspension in PBS was placed on grids coated with plastic-carbon support film and incubated for 30 min under ultraviolet (UV) illumination. After washing four times with double-distilled water, the cells were stained with 0.5% uranyl acetate and then dried. Finally, the cells were observed with a Hitachi H-7100 transmission electron microscope (accelerating voltage, 100kV) equipped with an Olympus Megaview G2 TEM CCD camera.