Epon resin

Samples were fixed in 2.5% glutaraldehyde (Ted Pella) + 1% paraformaldehyde (Merck) in PIPES pH 7.4 and stored at 4°C until further processed. Samples were rinsed with 0.1 M PBS for 10 min prior to 1 h incubation in 1% osmium tetroxide (TAAB; Berks, UK) in 0.1 M PBS. After rinsing in 0.1 M PBS, samples were dehydrated using increasing concentrations of ethanol (50%, 70%, 95% and 99.9%) for 10 min each step, followed by 5 min incubation in propylene oxide (TAAB). The samples were then placed in a mixture of Epon Resin (Ted Pella) and propylene oxide (1:1) for 1 h, followed by 100% resin and left overnight. Subsequently, samples were embedded in capsules in newly prepared Epon Resin and left for 1–2 h and then polymerised at 60°C for 48 h. Ultrathin sections (60–70 nm) were cut in an EM UC7 Ultramicrotome (Leica) and placed on a grid. The sections were subsequently contrasted with 5% uranyl acetate and Reynold's lead citrate and visualised with Tecnai™ G2 Spirit BioTwin transmission electron microscope (Thermo Fisher/FEI) at 80 kV with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

For TEM, the bacterial pellets obtained during OMV isolation were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in PBS and mixed thoroughly. Fixed samples were washed in 0.1 M sodium cacodylate (pH 7.24), postfixed with buffered 2% OsO₄, water washed, and dehydrated in a graded ethanol series (25%–100%) with 25% increments. The samples were then infiltrated in a graded series of Epon resin (Ted Pella, Redding, CA, USA) for 2 d, embedded in fresh Epon resin, and polymerized at 60°C for 48 h. Purified OMV pellets were resuspended in 2% paraformaldehyde and floated onto a 200-mesh carbon-coated Formvar nickel grid (EMS, Hsin An Instruments, Taiwan) for 5 min. Excess solution was removed with filter paper, and the sample grid was floated on a 10-µL droplet of 1% aqueous uranyl acetate for 30 s. The stain was removed with filter paper, and the sample was air dried and subsequently imaged using a Hitachi HT7700 transmission electron microscope (Hitachi, Tokyo, Japan).

CCNF, CCNF-DA, and CCNF-DA/AgNPs hydrogels with the concentration of 1 (w/v) % were analyzed by a cryogenic transmission electron microscopy (cryo-TEM; JEOL JEM 1011, Tokyo, Japan) and a polarized optical microscopy (POM; Nikon LV100 Pol) before the film casting. The samples were covered with the flat side of another B-type planchette and were rapidly frozen in a Bal-Tec HPM 010 high-pressure freezer (Boeckeler Instruments, Tucson, AZ, USA). After freeze substitution for 5 days at −80 °C in anhydrous acetone containing 2% OsO₄, the samples were warmed up to room temperature over 2 days (24 h from −80 °C to −20 °C, 20 h from −20 °C to 4 °C, 4 h from 4 °C to 20 °C). After washing 3 times with anhydrous acetone, the samples were embedded in a graduated Epon resin (Ted Pella Inc., Redding, CA, USA) and diluted in acetone (5%, 15%, 25%, 50%, 75% and 100% (v/v)) over 3 days. After polymerization in a 60 °C oven for 24 h, the samples were sectioned and post-stained with aqueous 2% (v/v) uranyl acetate (UA) and Reynolds lead citrate (LC) solution. The resin was cut into 200 nm-sections using Leica EM UC7 (Wetzlar, Germany) and applied onto copper grids. The TEM images of samples were recorded. The optical textures were characterized by depolarized transmitted light microscopy (DTLM).

The OMVs isolated from cultures grown with and without ampicillin, amikacin, chloramphenicol, colistin, meropenem, minocycline, imipenem, polymyxin B, and tigecycline were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in PBS and thoroughly mixed for TEM analysis. Fixed samples were washed in 0.1 M sodium cacodylate (pH 7.24), postfixed with buffered 2% OsO₄, washed with water, and dehydrated in a graded ethanol series (25 to 100%) with 25% increments. The samples were filtered in a graded series of Epon resin (Ted Pella Inc., Redding, CA, USA) for 2 days, embedded in fresh Epon resin, and polymerized at 60°C for 48 h. Purified OMV pellets were resuspended in 2% paraformaldehyde and allowed to free-float onto a 200-mesh carbon-coated Formvar nickel grid (Electron Microscopy Sciences, Hsin An Instruments Co. Ltd., Taiwan) for 5 min. The excess solution was dried off with filter paper, and the sample grid was then floated on a 10-μL droplet of 1% aqueous uranyl acetate for 30 s. The stain was removed with filter paper, and the sample was air dried. Finally, the samples were imaged using a Hitachi (Tokyo, Japan) HT7700 transmission electron microscope.