Spores were purified by lysozyme treatment and salt and detergent washes as described previously (Nicholson and Setlow 1990 ). Briefly, sporulation-induced cells were harvested 24 h after T0, and incubated at 37 °C for 1 h in 50 mM Tris–Cl, pH 7.2 with 50 µg/ml lysozyme. Cells were then subjected to five washes in 1 M NaCl, deionized water, 0.05% SDS, 50 mM Tris–Cl, pH 7.2. Spores were fixed overnight with 3% glutaraldehyde. The fixed spores were dehydrated with a series of washes with increasing ethanol concentration (40%, 50%, 60%, 70%, 80%, 90%, and 100%) for 10 min each. Thin films were prepared by using dehydrated spores on cover glass and dried for 2 h at room temperature. The dried samples were sputter coated with gold (5 nm) before imaging. SEM imaging was performed with the Supra 60 VP microscope (Carl Zeiss AG).
Supra 60 vp microscope
Manufactured by Zeiss
Sourced in Germany
The Supra 60 VP microscope is a high-performance scanning electron microscope (SEM) designed for advanced materials analysis. It features a field-emission electron source and a range of detectors, providing high-resolution imaging and analytical capabilities. The Supra 60 VP microscope is suitable for a variety of research and industrial applications.
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5 protocols using supra 60 vp microscope
1
Purification and Preparation of Bacterial Spores for SEM Imaging
2
Fabrication of LDPE/Graphene Nanocomposites
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The low-density polyethylene (LDPE) graphene nanoplatelet nanocomposites were prepared as described previously [7 (link)]. LDPE was utilized as the composite matrix. M25 graphene nanoplatelets from 2D Fab (Sweden) with average particle diameter of 25 microns and thickness of 6–8 nm were used as a filler. LDPE pellets were cryogenically ground into powder form and mixed with well-dispersed and homogenized suspension of graphene nanoplatelets with acetone. This process was followed by drying at 60 °C. Then, the extrusion process of nanocomposites was carried out using a circular die by Brabender 19/25 D single-screw extruder (Duisburg, Germany) by means of a compression screw (diameter D = 19 mm and screw length of 25 × 19, compression ratio 2:1). Samples for antibacterial analysis and SEM imaging were collected. SEM imaging was performed randomly from the entire surface using Supra 60 VP microscope (Carl Zeiss AG, Oberkochen, Germany).
3
Characterization of TiO2-Coated α-Fe2O3 Samples
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The chemical phase of the prepared samples was determined by using a confocal Raman microscope (alpha300 R; WITec) with a 488 nm laser pulse as an excitation source. The surface morphology of the bare and TiO2 coated α-Fe2O3 samples was examined by field emission scanning electron microscope (FE-SEM) using a Zeiss Supra 60 VP microscope operated at an acceleration voltage of 10 kV. The optical absorption of all the samples was measured with the help of a Cary 5000 spectrophotometer (Varian). X-ray photoelectron spectroscopy (XPS) spectra were acquired in a PerkinElmer Phi 5500 setup (base pressure < 10−10 mbar) using AlKα radiation of 1.4866 keV. The XPS spectra were shifted using the Fe(2p3/2) peak corresponding to 710.9 eV as a reference.
4
SEM Imaging of Cell Treatments
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For SEM imaging, cells were seeded onto 12-well plates at a density of 2 × 105 cells per well and incubated for 24 h before treatment with GO, GO–Rg3, GO–Rg3–DOX, and DOX for 24 h. The cells grown in medium without any treatment were used as the control. After treatment, the cells were washed with PBS three times and fixed in 3% glutaraldehyde for 2 h. Finally, the fixed samples were dehydrated with a series of washes with increasing ethanol concentrations (40%, 50%, 60%, 70%, 80%, 90%, and 100%) for 10 min each and then dried for 2 h at room temperature. Before imaging, the dried samples were sputter coated with gold (5 nm). SEM imaging was performed with the Supra 60 VP microscope (Carl Zeiss AG, Jena, Germany).
5
Biofilm Inhibition by Silver Nanoparticles
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Three different methods analyzed the biofilm inhibition activity of AgNPs: (1) by checking the bacterial viability, (2) by observing the cells under the scanning electron microscope (SEM), and (3) by live/dead staining assay25 (link),28 (link). The inoculum of 2–5 × 106 CFU/mL was obtained by diluting overnight cultures of E. coli and P. aeruginosa and loading the samples on top of a 15 mm cover glass. The cover glass was further incubated at 37 °C for 24 h, which allowed for biofilm formation. After 24 h, old culture medium was replaced with fresh medium, containing sterile water (negative control) or 1 × MBC, 2 × MBC, 4 × MBC, or 8 × MBC of AgNPs. Sample were incubated for further 24 h. Then the biofilms were homogenized for 20 s in 0.89% NaCl. The obtained bacterial samples were serially diluted and plated on LB agar plates. Next, the plates were incubated overnight at 37 °C, followed by CFU counting. For live/dead cells staining, samples were stained for 20 min with a mixture of 6.0 μM SYTO 9 and 30 μM KI from Live/Dead BacLight Viability kit L13152, (Invitrogen, Molecular Probes, Inc. Eugene, Oregon, USA). Fluorescence microscopy were collected using a Zeiss fluorescence microscope (Axio Imager.Z2m Carl Zeiss, Zena, Germany). Further, control and AgNPs-treated biofilms were examined by SEM (Supra 60 VP microscope, Carl Zeiss AG)28 (link).
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