Preparation of phage particles for electron microscopy has been described elsewhere [19 (link)]. In brief, bacteriophage particles were applied to parafilm to produce a spherical drop. Carbon-coated nitrocellulose films were fabricated on copper grids, which were placed face down on the sample drop for 1 min to absorb the particles. After being briefly washed twice in 10 μl of 10 mM Tris buffer (pH 8) (Amresco, USA), the samples were then rinsed twice with freshly prepared 2% uranyl acetate (UA; Sigma-Aldrich, USA) in Tris–HCl (pH 8.0) and stained for 60 seconds. Following each step of absorption, washing, and UA staining, the grids were blotted with filter paper until almost dry. The finished grids were dried in vacuo overnight. Images of phage particles were taken at a magnification of 40,000× and a defocus of 3 μm, using a 200-kV electron microscope (JEOL JEM-2010, equipped with a Gatan-832 CCD camera).
Jem 2010
Manufactured by Ametek
The JEM-2010 is a transmission electron microscope (TEM) designed and manufactured by Ametek. It is a high-performance instrument used for the examination and analysis of materials at the nanoscale level. The JEM-2010 provides high-resolution imaging, diffraction, and analytical capabilities to support a wide range of scientific and industrial applications.
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4 protocols using jem 2010
1
Electron Microscopy Imaging of Bacteriophages
2
Staphylococcus aureus FtsZ Polymerization Inhibitor
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Staphylococcus aureus FtsZ (12 μM) was incubated in the absence and in the presence of 3 μg/mL of 1 in 50 mM MOPS buffer (pH 6.5) at 25°C. After 10 min, 5 mM MgCl2, 50 mM KCl, and 1 mM GTP were added to the reaction mixtures and incubated at 37°C for 15 min. Then, 10 μL of the sample mixtures were placed on a glow-discharged Formvar carbon-coated copper grid (400 mesh) for 10 min. The grids were subsequently subjected to negative staining using 10 μL of 0.5% phosphotungstic acid (PTA) for 30 s, air-dried and digital images of the specimen were observed with a transmission electron microscope (JEOL model JEM 2010) operated at 200 kV and equipped with a Gatan MSC 794 CCD camera.
3
FtsZ Polymerization Visualization Protocol
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S. aureus FtsZ (12 µM) was incubated in the absence and in the presence of different concentrations of the test compounds in 50 mM MOPS buffer (pH 6.5) at 25°C. After 10 min, 5 mM MgCl2, 50 mM KCl, and 1 mM GTP were added to the reaction mixtures and incubated at 37°C for 15min. Then, 10 µL of the sample mixtures were placed on a glow-discharged Formvar carbon-coated copper grid (400 mesh) for 10 min. The grids were subsequently subjected to negative staining using 10 µL of 0.5% phosphotungstic acid (PTA) for 30s, air-dried and digital images of the specimen were observed with a transmission electron microscope (JEOL model JEM 2010) operated at 200 kV and equipped with a Gatan MSC 794 CCD camera.
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S. aureus FtsZ (12 µM) was incubated in the absence and in the presence of different concentrations of the test compounds in 50 mM MOPS buffer (pH 6.5) at 25°C. After 10 min, 5 mM MgCl2, 50 mM KCl, and 1 mM GTP were added to the reaction mixtures and incubated at 37°C for 15min. Then, 10 µL of the sample mixtures were placed on a glow-discharged Formvar carbon-coated copper grid (400 mesh) for 10 min. The grids were subsequently subjected to negative staining using 10 µL of 0.5% phosphotungstic acid (PTA) for 30s, air-dried and digital images of the specimen were observed with a transmission electron microscope (JEOL model JEM 2010) operated at 200 kV and equipped with a Gatan MSC 794 CCD camera.
4
Characterization of Nanoemulsion Formulation
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The nano-emulsion’s size and surface morphology images were examined via the high-resolution electron microscopy (HR-TEM) model, JOEL JEM-2010, connected with a Gatan digital camera model Erlangshen ES500 and adjusted at an acceleration voltage of 200 kV, a drop of the nanoemulsion was directly deposited on the copper coated film grid and a drop of sodium phosphotungstate (2% w/v) was then placed over the sample after drying the images was taken via the HR-TEM. The zeta potential that reflects the surface charges and the size distribution of the prepared sample was determined by dynamic light scattering using a Malvern Zetasizer model Nano ZS-90, working at 25oC. To estimate the structural composition of the nano-emulsion, Fourier-transform infrared spectra were detected using a JASCO spectrometer in a scanning range of 4000–400 cm-1 using KBr as a reference. The UV-Vis absorption spectra of the free drug and nanoemulsion-containing drug were recorded in the range of 800–200 nm using a UV-visible spectrophotometer (V-630 UV–vis –Jasco, Japan).
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