Em 300 electron microscope

For immunogold labeling, infected or uninfected macrophages were fixed in 0.1% glutaraldehyde + 4% paraformaldehyde in a cocodylate buffer at pH 7.2, and post-fixed in 1.3% osmium tetroxide in collidine buffer. After dehydration, samples were embedded using the ERL-4221 kit (Polysciences Inc) and placed in BEEM capsules (Pelco Int). After resin polymerization, samples were cut using an ultramicrotome system (Ultratome). The thin sections were placed on nickel grids, treated with sodium metaperiodate and blocked with 1% BSA in PBS. Grids were then incubated with primary antibodies, washed, and incubated in suitable 10 nm (anti-mouse) or 20 nm (anti-rabbit) gold particle-conjugated secondary antibodies (Abcam). After washing, samples were contrasted with uranyl acetate and lead citrate and subsequently visualized using a Philips EM 300 electron microscope. Matched and mismatched antibody combinations were done to assay for the presence of non-specificity and background (S11 Fig). Electron microscopy was also used to visualize vesicular structures in sucrose gradient-fractionated lysates. A 10 μl aliquot of each fraction was spotted onto a copper grid, spun, and negatively stained using 3% phosphotungstic acid at pH 6.0. Structures were then visualized.

The Xccφ1 stock (10⁸ PFU/mL) was purified by CsCl density gradient ultracentrifugation (Centrifuge for 2.5 h 24 K in the SW 28.1) and dialyzed against SM buffer overnight at 4°C. Phage particles were negatively stained with 2% phosphotungstic acid (pH 7.2) for 5 min. Phages were observed in a Philips EM 300 electron microscope.

TEM images were primarily obtained with a Philips EM300 electron microscope operated at an accelerating voltage of 300 kV. Elemental analysis was acquired with an energy-dispersive X-ray (EDX) equipped with the TEM. Scanning TEM (STEM) image was captured with an FEI Titan TEM with Schottky emitter operated at 200 kV. X-ray diffraction (XRD) patterns were collected by using a Bruker GADDS D8 Discover diffractometer with Cu Kα radiation (λ = 1.5418 Å). Fluorescence spectra were acquired with a SpectraPro 2150i fluorescence spectrometer equipped with a commercial 980 nm NIR laser. Room-temperature UV-visible absorption spectra were recorded with a Shimadzu UV-3150 UV/Vis spectrophotometer. Nano-zeta sizer measurements were performed on a Malvern zetasizer nano series.

To prepare phiAxp-1 for transmission electron microscopy studies, cell debris from 500 mL of A. xylosoxidans strain A22732 infected with phiAxp-1 was pelleted by centrifugation. Phage particles were precipitated with 1 M NaCl and 10% polyethylene glycol (PEG) 8000 at 4 °C with stirring for 60 min. The precipitated phage particles were harvested. Phage particles were resuspended in Saline - magnesium (SM) diluent plus gelatin (SMG) (50 mM Tris-HCl [pH 7.5] containing 100 mM NaCl, 8.1 mM MgSO₄ and 0.01% (w/v) gelatin) and extracted with an equal volume of chloroform. After low-speed centrifugation, the aqueous phase was sedimented at about 25,000 × g for 60 min. Phage particles were negatively stained with 2% (wt/vol) phosphotungstic acid (pH 7), air dried, and examined under a Philips EM 300 electron microscope operated at 80 kV and 120 KEv.