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1. The Capsule Method for Inactivation in Biocontainment with 2% Glutaraldehyde, Followed by Negative Staining in a BSL-2 Laboratory
- Virus inactivation procedure.
- Inside the biocontainment BSC, mix the virus suspension well with the same volume of 4% glutaraldehyde to achieve a final concentration of 2% glutaraldehyde.
Caution: Glutaraldehyde is a hazardous chemical and requires appropriate protection. Glutaraldehyde can be used for brief periods in a normal BSC, but extended open reagent requires working in a ducted BSC or chemical fume hood.
- Inactivate viruses with fixative for a minimum of 24 h before packaging, decontamination, and transfer it to the BSL-2 EM facility.
- In the BSL-2 EM facility, aspirate the virus and fixative mixture into the capsule, containing two TEM grids, attached to a pipette.
- Place the pipette horizontally for 10 min with the grids oriented horizontally.
NOTE: This is to promote an even distribution of virus particles onto the TEM grids.
- Pick up the pipette and depress the plunger to expel the virus to a waste container. Aspirate 40 μL of dI water into the capsules and expel it into the waste container for 3 rinse cycles.
- Aspirate 40 μL of either 1% uranyl acetate (UA) or 1% potassium phosphotungstic acid (PTA) into the capsules for 30 s.
NOTE: Staining time varied from 10 s to 1 min based on virus sample.
Caution: UA is an alpha emitter, and a cumulative toxin. Handle it with appropriate protection.
- Remove the capsule from the pipette and blot dry the grids by touching the edge of the grids to a piece of filter paper. Air dry the grids and store them for subsequent TEM imaging.