Safe Viral Specimen Preparation for Transmission Electron Microscopy

Abstract

Source: Blancett, C. D., et al. Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment. J. Vis. Exp. (2017).

This video demonstrates the safe viral specimen preparation for transmission electron microscopy. The viral particles are fixed with glutaraldehyde, stained with an electron-dense agent, and subjected to vapor fixation with osmium tetroxide to stabilize structures and ensure complete inactivation, ensuring safe handling of viral samples while improving contrast for high-quality TEM imaging.

Protocol

1. The Capsule Method for Negative Staining in Biocontainment using Aqueous Glutaraldehyde and 1% Osmium Tetroxide Vapor Inactivation

1. Inside the biocontainment bio-safety cabinet (BSC), aspirate 40 µL of virus suspension into the capsule attached to a pipette.
   NOTE: The pipette remains attached to the capsule until the process is completed.
2. Place the pipette on its side for 10 min with grids oriented horizontally. This is to promote an even distribution of virus particles onto the coated grids.
3. Inactivate the virus within the capsule inside the biocontainment BSC.
   1. Pick up the pipette and depress the plunger to dispense the virus solution into a waste container.
   2. Aspirate 40 µL of 2% glutaraldehyde fixative into the capsules.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.
   3. Place the pipette on its side for 20 min. This is to ensure samples are well fixed.
   4. Expel the fixative and aspirate 40 µL of DI (deionized) water into the capsules to wash away the fixative. Repeat this wash step for a total of 3 rinse cycles.
4. Aspirate 40 µL of either 1% uranyl acetate (UA) or 1% potassium phosphotungstic acid (PTA) into the capsules and allow to sit for 30 s.
   NOTE: Staining time may vary from 10 s to 1 min based on the virus sample.
   CAUTION:: UA is an alpha emitter and a cumulative toxin. Handle it with appropriate protection.
5. Remove the capsule from the pipette and blot dry the grids by touching a piece of filter paper to the edge of the grids while the grids remain within the capsule.
6. Osmium Tetroxide Vapor inactivation procedure.
   1. Place the capsule, with the lid open, into a 50 mL centrifuge tube containing filter paper soaked in a 1% osmium tetroxide solution.Caution: Osmium tetroxide is extremely toxic with low vapor pressure. It must be used in a ducted BSC or chemical fume hood. Handle it with appropriate protection. Post warning information in the working area.
   2. Seal the 50-mL centrifuge tube for 1 h to allow complete permeation of the osmium tetroxide vapor. Then, subsequently, decontaminate and transfer the tube out of the biocontainment to the BSL-2 EM (electron microscope) facility.
7. Remove EM grids from the capsule.
   1. In the BSL-2 EM facility, remove the capsule from the centrifuge tube and place it onto a pipette.
   2. Aspirate 40 µL of DI water into the capsule and dispense the water into a waste container three times.
   3. Remove the capsule from the pipette and blot dry the grids using filter paper to touch the edge of the grids.
   4. After air drying, store the grids for subsequent TEM (transmission electron microscopy) imaging.

Materials

List of materials used in this article

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
Osmium tetroxide	EMS	19190	4% aqueous solution
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