Capsule-Based Negative Staining of Virus Samples on TEM Grids: A Heavy Metal Staining Technique to Visualize the Ultrastructure of Enveloped Viruses

Protocol

1. The Capsule Method for Inactivation in Biocontainment with 2% Glutaraldehyde, Followed by Negative Staining in a BSL-2 Laboratory

1. Virus inactivation procedure. 
   1. 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.
   2. Inactivate viruses with fixative for a minimum of 24 h before packaging, decontamination, and transfer it to the BSL-2 EM facility.
2. In the BSL-2 EM facility, aspirate the virus and fixative mixture into the capsule, containing two TEM grids, attached to a pipette.
3. 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.
4. 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.
5. 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.
6. 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.

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Materials

Name	Company	Catalog Number	Comments
Formvar/carbon coated TEM grids	SPI	3420C-MB	200 mesh Cu Pk/100
mPrep/g capsules	EMS	85010-01	Box
mPrep/f couplers	EMS	85010-11	Standard 16/Pk
Glutaraldehdyde	EMS	16320	50% solution, EM grade
Uranyl Acetate	EMS	22400	Powder
Potassium phosphotungstic acid	EMS	19500	Powder
Filter paper	Whatman	1450-090	Size 50
Tranmission Electron Microscope	JEOL	JEM-1011	TEM