Formvar nickel grid

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.