Formvar film

Shape and surface morphology of nanoparticle formulation were examined with a transmission electron microscopy (TEM) (TEM 208 S, Philips NL, Eindhoven, The Netherlands). 15 µL of Nps suspension was placed on a 300 mesh copper grid covered with Formvar film (AGAR Scientific, Stansed, UK). The excess liquid was removed with filter paper, and then 10 μL of 1% uranyl acetate was added on to grids and left standing for 10 s, after that, liquid in excess was removed by filter paper and sample analyzed.

Arabidopsis roots were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde (both EM-grade, EMS, USA) in 0.1 M PHEM buffer (pH 7) for 2 hr at RT, then overnight at 4°C. The fixed roots were embedded in 12% gelatin and cut into 1 mm³ blocks which were immersed in 2.3 M sucrose overnight at 4°C. These blocks were mounted onto a Leica specimen carrier (Leica Microsystems, Austria) and frozen in liquid nitrogen. With a Leica UCT/FCS cryo-ultramicrotome (Leica Microsystems, Austria) the frozen blocks were cut into ultra-thin sections at a nominal thickness of 60 nm at −120°C. A mixture of 2% methylcellulose (25 centipoises) and 2.3 M sucrose in a ratio of 1:1 was used as a pick-up solution. Sections were picked up onto 200 mesh Ni grids (Gilder Grids, UK) with a carbon coated formvar film (Agar Scientific, UK). Fixation, embedding and cryo-sectioning was conducted as described by Tokuyasu, 1973 (link).

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.

Phage PAO1-D particles 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). Images were digitally recorded using a CCD digital camera Orious 1,100 W, Tokyo, Japan.