This article details a method for negative staining of enveloped viruses using glutaraldehyde as a fixative. The process preserves viral morphology while allowing for safe handling and visualization through transmission electron microscopy (TEM).
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.
To begin, mix the virus suspension with an equal volume of 4% Glutaraldehyde to achieve a final concentration of 2% Glutaraldehyde. Using fixative, inactivate the virus for at least 24 hours. Decontaminate the tube, and transfer it to the BSL2-EM facility.
After this, attach a grid-loaded capture to a pipette. Aspirate the mixture into the capsule. Place the pipette on its side with the grids oriented horizontally for 10 minutes.
Next, pick up the pipette, and expel the virus mixture into a waste container. Aspirate 40 microliters of deionized water into the capsule, and then, dispose of the water in a waste container. Repeat this wash three times.
After this, aspirate 40 microliters of either 1% UA or 1% PTA into the capsule for 30 seconds. Remove the capsule from the pipette. Using filter paper, blot-dry the grids, then, allow the grids to air-dry.
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