Uranyl acetate

Hemocytes were fixed in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde (Thermo Fisher Scientific, Frederick, MD, USA) in a culture medium. Postfixation was carried out with 1% osmium tetroxide (Sigma-Aldrich, St. Louis, MO, USA) in 0.05 M cacodylate buffer (Vekton, Russian Federation), pH 7.4, using a low-temperature automatic water replacement system Leica EM AFS2 (Leica Microsystems, Germany).
The samples were infiltrated with epoxy resin (Agar Scientific Ltd, UK) / acetone (Vekton, Russian Federation) (1:3) for 30 min, followed by resin / acetone (1:2) for 30 min, followed by 1 h with resin / acetone (1:1) under room temperature. The samples were then transferred into the 100% resin; the polymerization was carried out over 24 h at +60°C. Resin blocks were carefully trimmed using a Leica EM UC7 trimmer. Ultrathin slices were collected on the mesh copper grids coated by carbon film (SPI Supplies, West Chester, PA, USA), post-stained with 1% uranyl acetate (Agar Scientific Ltd, UK) in water for 5 min, and with a lead solution for 7 min, and washed in distilled water. The material was then analyzed with a transmission electron microscope JEM– 1400 (Jeol, Japan).

For TEM analysis, samples were fixed in Karnovsky’s Fixative consisting of 8% PFA, 25% glutaraldehyde (Agar Scientific, Stansted, UK), and 0.2 M cacodylate buffer (Agar Scientific) for 1 hour before washing in 0.1 M cacodylate buffer. Samples were further fixed in 1% osmium tetroxide (Agar Scientific) for 1 hour, washed in 0.1 M cacodylate buffer before being dehydrated through an ethanol series before embedding in Epon resin (Agar Scientific) containing dodecenylsuccinic anhydride (DDSA), methyl nadic anhydride (MNA) and benzyldimethylamine (BDMA). Ultra-thin (70 nm) sections were obtained using a diamond knife (Agar Scientific) and loaded onto formvar-coated slot grids (Agar Scientific). Grids were then stained with uranyl acetate (Agar Scientific) and lead citrate (Sigma Aldrich) prior to imaging using a Hitachi H7600 electron microscope.

Human primary muscle cells were fixed for 24h in 4%PFA and 1% glutaraldehyde (Sigma) in 0.1 M phosphate buffer (pH 7,2). Cells were then washed (PBS) and post-fixed with 2% osmium teteroxide for 1h. Cells were then dehydrated in ethanol solutions of growing concentrations and propylene oxide. Samples were impregnated with a mixture propylene oxide/Epon resin (Sigma), (1:1), and left overnight in pure resin. After embedding, polymerization was allowed for 48 h at 60°C. Ultra-thin sections (70 nm) with a Leica EM UC7 ultramicrotome. Sections were stained with 5% uranyl acetate (Agar Scientific) and 5% lead citrate (Sigma), and observed with a JEOL 1011 transmission electron microscope.

Samples were incubated for 30 seconds on carbon coated 400 mesh copper grids (Electron Microscopy Sciences, Pennsylvania, USA) that had been glow discharged at 30 Volts for 30 seconds. Grids were then washed in ddH₂O for 10 seconds before negative staining with 2% (w/v) uranyl acetate (Agar Scientific, Essex, UK) for 1 minute. TEM data were recorded using a Tecnai BioTwin microscope at 100 Kv, and micrographs acquired using a Gatan Orius CCD camera. The nominal magnification was 23000 times, giving a sampling of 3.5 Å/pixel. Images were recorded between 0.5 and 5.0 nm defocus.

For negative staining, 5 μl of each sample were applied onto glow-discharged carbon-coated grids (Agar Scientific, Stansted, UK) and incubated for 1 min. The grids were washed with 5 μl of deionized water before incubating for 30 sec in 1% (w/v) of Uranyl Acetate (Agar Scientific, Stansted, UK). CCD images were collected using a Tecnai Spirit operated at 120 Kv and a 2Kx2K CCD camera.

Phage EC3a particles, before and after 6 h contact with honeys PF2 and U3 at 25% (w/v) concentration, were sedimented by centrifugation (25,000 × g, 60 min, 4°C) and washed twice in tap water by repeating the centrifugation step. Subsequently, the suspension was deposited on copper grids with carbon-coated Formvar films, stained with 2% (w/v) uranyl acetate (pH 4.0) (Agar Scientific), and examined using a Jeol JEM 1400 (Tokyo, Japan) transmission electron microscope (TEM).
Escherichia coli cells challenged with phage for 2 h, honey and the combination of both for 12 h, were also visualized by TEM along with the respective control samples. In brief, samples were fixed with 2.5% (v/v) glutaraldehyde (Electron Microscopy Sciences, Hatfield, United States) and 2% (v/v) paraformaldehyde (Merck, Darmstadt, Germany) in phosphate buffer 0.1 M with 0.5 mM MgCl₂ (pH 6.5), dehydrated and embedded in Epon resin (TAAB, Berks, England). Ultrathin sections (40–60 nm thickness) were prepared on a RMC Ultramicrotome (PowerTome, United States) using diamond knives (DDK, Wilmington, DE, United States). The sections were mounted on 200 mesh copper or nickel grids, stained with 2% (w/v) uranyl acetate and 3% (w/v) lead citrate for 5 min each, and examined under by TEM (Jeol JEM 1400, Tokyo, Japan). Images were digitally recorded using a CCD digital camera Orious 1100W, Tokyo, Japan.

Fetal bovine serum (FBS), hexamethyldisilazane (HMDS), and phosphate buffer saline were obtained from SIGMA-ALDRICH, USA. Roswell Park Memorial Institute medium (RPMI), Dulbecco's Modified Eagle Medium (DMEM), 1% penicillin and streptomycin, and 0.25% Trypsin-EDTA were purchased from Gibco, USA. Sodium cacodylate buffer, glutaraldehyde fixative, 1% osmium tetroxide, Resin Toluidine Blue, uranyl acetate, lead citrate, and Al stubs were obtained from Agar Scientific, UK, and acetone (25%, 50%, 75%, 95%, and 100%) was obtained from Fisher Scientific, UK.