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The bacterial culture sample taken before filtration and centrifugation and the isolated EcO83-OMVs were imaged with TEM. The bacterial culture was centrifuged at 5 000 × g and the pellet was washed twice with PBS before microscopy. EcO83-OMVs were dissolved in NaCl. Ultrastructural visualization was carried out by single-droplet negative stain. Briefly, 5 µl of each OMV preparation were left to adhere on formvar- and carbon-coated grids for 20 s. Simultaneous addition of 5—10 µl 1% uranylacetate and absorption of liquid was repeated twice to contrast the samples. Dried grids were imaged in an FEI Tecnai20 electron microscope (FEI, Eindhoven, NL) equipped with a 4 K Eagle-CCD camera.

Starved and Gal-8S-treated as well as untreated cell cultures were fixed with 2.5% glutaraldehyde and 2% PFA, post-fixed with Palade’s buffered osmium tetroxide and embedded in Epon according to standard protocols. 1 µm semi-thin sections were stained with toluidine blue and imaged under a ZEISS light microcope (AxioImager.A2) equipped with a ZEISS AxioCam MRc5 color camera. 70 nm ultra-thin sections were contrasted with lead citrate and uranyl acetate before being imaged with an FEI Tecnai20 electron microscope equipped with a 4 K Eagle-CCD camera. Images were processed with Adobe Photoshop.

All isolates were incubated overnight on MHA plates at 36 °C ( ± 1 °C) and re-suspended in 0.1 M phosphate buffer (pH 7.2). Cell pellets were obtained by centrifugation (10,000× g for 10 min at 4 °C) and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 20 min for scanning electron microscopy (SEM) or 24 h for transmission electron microscopy (TEM). For SEM, samples were dehydrated in ascending ethanol series and re-suspended in hexamethyldisilazane (HMDS). After complete evaporation of HMDS samples were sputter-coated with gold (Sputter Coater, SC 502, Polaron, Fisons Instruments^®, England) and examined using a scanning electron microscope (JSM 6310, Jeol Ltd.^®, Japan). Images were taken at an acceleration voltage of 15 kV^{51 (link)}.
TEM analysis was performed as described previously^{52 (link)}. Briefly, samples were incubated in 1% OsO₄ and embedded in Epon resin. 70 nm ultra-thin sections were imaged with an FEI Tecnai20 electron microscope equipped with a 4 K Eagle-CCD camera. Images were processed with Adobe Photoshop. For measurements of the thickness of the cell walls, ~100 non-dividing cells each were imaged. The ImageJ software package was used for measurements. Cell cycle stages of the three isolates (DR-I1, DR-I3 and DR-I4) were assessed by counting approximately 200 cells each.