Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Summary

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Abstract

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity.

In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

Introduction

Transmission electron microscopy (TEM) is an effective tool for viewing the morphology and ultrastructure of biological specimens that are too small to be seen with a traditional light microscope ^1,2,3,4. TEMs shoot electrons through a very thin specimen producing a higher resolution image as electrons have a much shorter wavelength than light. Regions of the sample that bend or block electrons appear dark, while regions that are electron lucent appear white.

Lack of electron dense matter makes viruses difficult t....

Protocol

1. Experiment Preparation in a BSL-2 Environment Prior to Working with the Virus Samples

1. Prepare or purchase Formvar and carbon coated TEM copper grids, usually 200-400 mesh.
2. Insert the coated TEM grids into capsules.
   1. Use a magnified lens to make this process easy to perform. One or two grids may be inserted into each capsule. Pre-loaded capsules can be purchased to eliminate this step if desired.
3. Transfer the capsules with inserted TEM coated grids, together with other supplies and reagents, to biocontainment where the viruses will be negatively stained.

2. The....

Results

The capsule method produce good quality negative staining for TEM imaging:

First, we evaluated the quality of images generated by using both the manual droplet method and the capsule methods for negative staining Zaire ebolavirus. Ebolaviruses are members of the Filoviridae family, along with Marburg virus. Ebolavirus is typically 80 to 100 nm in diameter and can be over 1,000 nm in length. Ebolavirus must be handled in a .......

Discussion

Negative staining is a valuable TEM technique for evaluating and sizing viruses, protein complexes and nanoparticles. Droplet preparation of these specimens by manual moving of grids from reagent to negative stains has been the classic protocol for more than half a century. It is a simple process, but requires expertise gained through training for successful completion. Excellent negative staining is still considered a state-of-the-art skill set and highly desired in many TEM labs. The capsule method has several distinct.......

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