Encyclopedia of Experiments: Biological Techniques
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Transcript
For negative staining of enveloped viruses, begin with the desired viral suspension. Incubate the viruses with glutaraldehyde, a chemical fixative. Upon penetration, glutaraldehyde crosslinks the viral proteins and nucleic acids, inactivating viruses while preserving morphology, thereby facilitating safe virus handling. Transfer this mixture into a multi-well plate.
Attach transmission electron microscopy, TEM, grids containing capsule to a pipette via a coupler. Using the pipette, aspirate the virus-fixative mixture into the capsule. Incubate the pipette assembly for viruses to adsorb onto the grid surfaces.
Remove the mixture. Wash the capsule, removing non-adherent viruses and fixatives. Pipette a solution of uranyl acetate, a heavy-metal-based stain, into the capsule. Incubate to enable the stain to contact the viruses.
Uranyl ions bind to specific membrane glycoproteins and phospholipids on viral surfaces. Ions can penetrate viruses and bind to the nucleocapsid proteins enclosing the genome. This binding induces heavy-metal ion aggregation and deposition around the virus and specific viral structures.
Separate the capsule. Remove excess stain from the grids and air dry.
During TEM imaging, high-energy electron beams traverse the virus, causing the virus’ electron-dense stained regions to scatter more electrons. However, the overall low-electron density of the virus allows electrons to traverse. The imaging system processes these differently scattered electrons, producing a high-contrast image.
Negatively-stained viruses appear lighter with dark nucleocapsid against a dark border, facilitating ultrastructure visualization.
