Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining

• Published in: Journal of virological methods 2016-12, Vol.238, p.70-76
• Main Authors: 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.
• Format: Article
• Language: English
• Subjects: Biocontainment; Chikungunya virus; Chikungunya virus - ultrastructure; Containment of Biohazards - methods; Ebolavirus; Ebolavirus - ultrastructure; Microscopy, Electron, Transmission - instrumentation; Microscopy, Electron, Transmission - methods; Microscopy, Electron, Transmission - standards; mPrep; Negative staining; Negative Staining - methods; Specimen Handling - methods; TEM; Viruses - isolation & purification; Viruses - ultrastructure
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2016.10.005

•Recommend a revolutionary method for preparing virus sample in biocontainment.•Evaluate TEM image qualities according to different virus inactivation procedures.•Present good TEM image qualities using Uranyl Acetate (UA) and potassium phosphotungstic acid (PTA). 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.