The overall goal of this method is to provide a simple and effective TEM negative staining procedure for viral samples within biocontainment. This method can help answer key questions in virology about the morphology and structures of viruses being investigated. The main advantage of this technique is that it provides easy and effective handling of electron microscopy grids within biocontainment environments.

This technique is used for as part of TEM diagnosis of an unknown virus because it can be easily applied to viral samples in all environments. Though we have primarily used this method to aid in virus TEM studies, it has much wider application and can be used in EM investigation of most nanoparticles. Generally, individuals new to this method will struggle because it takes some practice to ensure TEM grids are not damaged when loaded and unloaded from the capsules.

We first had the idea for this method when we were having problems handling small EM grids in biocontainment. To begin this procedure, prepare or purchase form viral and carbon coated TEM copper grids. Insert the coated TEM grids into capsules.

Next, transfer the loaded capsules along with other supplies and reagents to biocontainment. To begin, attach the capsule to a pipette. Aspirate 40 microliters of virus expansion into the capsule.

Place the pipette on its side, with the grids oriented horizontally for 10 minutes. Next, pick up the pipette and dispense the virus solution into a waste containment. Aspirate 40 microliters of 2%glutaraldehyde fixative into the capsules.

Place the pipette on its side for 20 minutes. After this, expel the fixative and aspirate 40 microliters of de-ionized water into the capsule, to wash away the fixative. Repeat this wash three times.

Next, aspirate 40 microliters of either 1%uranyl acetate or 1%potassium phosphotungstic acid into the capsule. Let the capsule sit for 30 seconds. Then, remove the capsule from the pipette.

Touch a piece of filter paper to the edge of the grids to blot dry. Transfer filter paper into a 15 milliliters centrifuge tube. Next, soak the filter paper in 1%osmium tetroxide solution.

Place the capsule with the lid open into the tube. Seal the tube for one hour to allow complete permeation of the osmium tetroxide vapor. After this, decontaminate the tube and transfer it out of the biocontainment to the BSL-2 EM facility.

Remove the capsule from the centrifuge tube, then place it onto a pipette. Aspirate 40 microliters of de-ionized water into the capsule and then dispense the water into a waste container. Repeat this water wash three times.

Next, remove the capsule from the pipette. Using filter paper, blot dry the grids. Allow the grids to air dry and then store them in a grid box, until ready to perform TEM imaging.

To begin, mix the virus suspension with an equal volume of 4%glutaraldehyde, to achieve a final concentration of 2%glutaraldehyde. Using fixative, inactivate the virus for at least 24 hours. Decontaminate the tube and transfer it to the BLS-2 EM facility.

After this, attach a grid loaded capture to a pipette. Aspirate the mixture into the capsule. Place the pipette on its side with the grids oriented horizontally for 10 minutes.

Next, pick up the pipette and expel the virus mixture into a waste container. Aspirate 40 microliters of de-ionized water into the capsule and then dispose of the water in a waste container. Repeat this wash three times.

After this, aspirate 40 microliters of either 1%QA or 1%PTA into the capsule for 30 seconds. Remove the capsule from the pipette. Using filter paper, blot dry the grids.

Then, allow the grids to air dry. In this study, Zaire Ebola virus samples undergo negative staining, using both the manual droplet method and the capsule method. The TEM images generated from these methods, both show clearly defined details, with nucleocapsid structures in the center of the varion and visible glycoproteins on the surface.

Thus, the capsule method is able to produce TEM images of similar quality to those produced by the manual droplet method. Next, different methods of sample inactivation are compared using the Chikungunya virus. Sample inactivation is achieved either rapidly, by using 2%glutaraldehyde for 20 minutes with subsequent exposure to 1%osmium tetroxide for one hour, or overnight, by using 2%glutaraldehyde for 24 hours.

As can be seen, the longer inactivation process without osmium tetroxide allows for more ultrastructural detail to be seen. Then, utilizing the capsule method, two stains were compared using aldehyde fixed virus-like-particles. Both uranyl acetate and phosphotungstic acid display high quality results, with clearly defined borders and visible glycoproteins.

Once mastered, this technique can be done in about an hour and a half, depending on the number of samples. While attempting this procedure, it's important to remember to select proper fixative and staining reagents for your experimental goals. Following this procedure, other methods like particle counting can be performed in order to answer additional questions by calculating virus concentrations.

This technique improves the way virologists can explore virus morphology and structure information with greater ease, especially in BLS-3 and 4 level viruses. After watching this video, you should have a good understanding of how to easily handle TEM grids for negative staining within biocontainment. Don't forget that working with aldehyde, osmium tetroxide and uranyl acetate can be extremely hazardous and precautions such as, proper personal protective equipment and a chemical fume hood should always be used while performing this procedure.