title: Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining.
abstract: 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.
authors.affiliation: Pathology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 21702, United States.
authors.forename: Mitchell K
authors.initials: MK
authors.lastname: Monninger
authors.affiliation: Pathology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 21702, United States.
authors.forename: Chrystal A
authors.initials: CA
authors.lastname: Nguessan
authors.affiliation: Pathology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 21702, United States.
authors.forename: Candace D
authors.initials: CD
authors.lastname: Blancett
authors.affiliation: Pathology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 21702, United States.
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Kuehl
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 21702, United States.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Rossi
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 21702, United States.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Olschner
authors.affiliation: Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 21702, United States.
authors.forename: Priscilla L
authors.initials: PL
authors.lastname: Williams
authors.affiliation: Microscopy Innovations LLC, Marshfield, WI, United States.
authors.forename: Steven L
authors.initials: SL
authors.lastname: Goodman
authors.affiliation: Pathology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, MD, 21702, United States. Electronic address: mei.g.sun.ctr@mail.mil.
authors.forename: Mei G
authors.initials: MG
authors.lastname: Sun
mesh_terms: D002646:Chikungunya virus / Q000648:ultrastructure; D003264:Containment of Biohazards / Q000379:methods; D029043:Ebolavirus / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D016624:Negative Staining / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D014780:Viruses / Q000302:isolation & purification / Q000648:ultrastructure*
keywords: Biocontainment; Chikungunya virus; Ebolavirus; Negative staining; TEM; mPrep