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

Overview

In this video, we demonstrate the negative staining of enveloped virus samples using a processing capsule within the biocontainment environment. The negatively stained viruses can be visualized using transmission electron microscopy to investigate viral structure and morphology with nanometer resolution.

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.

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