Fei tecnai t20

Bacterial samples were similarly prepared as described in the above paragraph for SEM analysis, and treated with AuNPs@Esc(1-21) for 15 min. Approximately 4 x 10 8 bacterial cells in a total volume of 1 mL NaPB were incubated with AuNPs@Esc(1-21) for 15 min in the thermomixer with agitation. Controls were buffer-treated bacteria. After incubation, the samples were centrifuged at 6,000 x g for 10 min and washed three times with NaPB, fixed with 2.5% glutaraldehyde (1 mL) in NaPB for 2 h at room temperature. Finally, the samples were centrifuged at 12,000 x g for 10 min and washed three times as above. The pellets were subsequently embedded in 1.5 % agar (Panreac); post fixed in 2% osmium tetroxide for 1 h at room temperature and stained in 1% uranyl acetate in the dark for 2 h at 4 °C. Then, they were rinsed in distilled water, dehydrated in ethanol and infiltrated overnight in Durcupan resin (Fluka). Following polymerization, embedded cultures were detached from the wells and glued to araldite blocks. Finally, ultra-thin sections (0.08 µm) were cut with a diamond knife (Leica), stained with lead citrate (Reynolds solution) and examined under a 200 keV transmission electron microscope FEI Tecnai T20 (FEI Europe) operating at 60 keV.

Cyro-EM as performed according to standard procedure. 3 µL of suspension was placed on a glow-discharged holy carbon coated grid (Quantifiol 3.5/1, Quantifiol GmbH, Jena, Germany) blotted and vitrified in a Vitrobot (FEI Company, Eindhoven, The Netherlands). Samples were observed in a Gatan 626 cryo-stage (Gatan, Pleasanton, CA) in a Philips CM120 (Philips, Eindhoven, The Netherlands) operating at 120 keV or in a FEI Tecnai T20 (FEI Company, Eindhoven, The Netherlands) operating at 200 keV. Images were recorded under lowdose conditions on a slow-scan CCD camera.