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Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining
- Monninger, Mitchell K., Nguessan, Chrystal A., Blancett, Candace D., Kuehl, Kathleen A., Rossi, Cynthia A., Olschner, Scott P., Williams, Priscilla L., Goodman, Steven L., Sun, Mei G.
- Journal of virological methods 2016 v.238 pp. 70-76
- air, biocontainment, biological safety cabinet, heavy metals, pathogens, plastic film, salts, staining, transmission electron microscopes, transmission electron microscopy, ultrastructure, viruses
- Transmission electron microscopy can be used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. In a transmission electron microscope (TEM), the image is created by passing an electron beam through a specimen with contrast generated by electron scattering from dense elements in the specimen. Viruses do not normally contain dense elements, so a negative stain that places dense heavy metal salts around the sample is added to create a dark border. To prepare a virus sample for a negative stain transmission electron microscopy, a virus suspension is applied to a TEM grid specimen support, which is a 3mm diameter fragile specimen screen coated with a few nanometers of plastic film. Then, deionized (dI) water rinses and a negative stain solution are applied to the grid. All infectious viruses must be handled in a biosafety cabinet (BSC) and many require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL) 3 and 4 is especially challenging because the support grids are small, fragile, and easily moved by air currents. In this study we evaluated a new device for negative staining viruses called mPrep/g capsule. It is a capsule that holds up to two TEM grids during all processing steps and for storage after staining is complete. This study reports that the mPrep/g capsule method is valid and effective to negative stain virus specimens, especially in high containment laboratory environments.