Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

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 advantages over the manual droplet method, especially in biocontainment laboratories. First, the manual droplet method requires substantial training and experience before it can be performed successfully, while the easy to use the capsule method can be accomplished by entry level technicians by simple pipetting. The biggest challenge with the manual droplet method is the successful handling the TEM grids with forceps in BSL-3 and BSL-4 PPE due to the greatly reduced tactile sensation and dexterity. Fragile coated TEM grids are easily damaged or punctured by forceps. Damage to the coated TEM grid is often not revealed until it is viewed with a TEM. A second advantage of capsules is that TEM grids are only directly handled when they are inserted into capsules and when they are removed for insertion into the electron microscope. Whether working in the biocontainment laboratory or in the BSL-2 EM facility, there is no direct grid handling. A third advantage of the capsule method is that both sides of the grid are covered with viruses and stains. This can be useful when the sample concentration is low; however this could cause too much sample if the concentration is high, or result in dilution of the sample prior to use.

Negative staining multiple grids using the manual droplet technique is time consuming because each sample needs to be individually prepared and stained. This leads to difficulties obtaining consistent, reproducible staining. Furthermore, keeping track of many individual grids is always a challenge. Each capsule holds two grids and multiple capsules can be attached to a multichannel pipette, streamlining the process. Therefore, capsules ensure that grids are stained using a very similar process, under consistent sample conditions, and at the same time. The capsules can be labeled to aid in organization, thus reducing potential for mixing up or misidentification of grids. Another common problem faced when using the manual droplet method is grid contamination. This can occur when the grids are left in the open air for too long or are dropped. The capsule protects the TEM grids from the open air and holds them securely so they are never dropped. Capsules provide greater experimental control and repeatability when preparing and negative staining because the capsule is a more controlled environment.

The capsules can be purchased pre-loaded if desired. Manually loading grids into the capsules takes some practice to ensure grids are not damaged. The capsule method also requires slightly more sample and stain than the manual droplet method. The capsule method requires at least 40 µL of sample, compared to the manual droplet method, which can be done with as little as 8 µL.

Virus inactivation is an important part of negative staining in BSL-3 and BSL-4, as it allows the inactivated virus to be taken out of the biocontainment laboratories for imaging in the EM facility. Inactivation using fixatives has the added benefit of fixing the virus, preventing degradation. There are two different ways to inactivate the virus sample, both of which were examined in this study. The first adheres the virus to coated TEM grids, fixes the viruses for 20 min in a 2% glutaraldehyde solution, followed by 1 h exposure of the grid to osmium tetroxide vapor (Figure 1C). This inactivation method is advantageous because it saves time by enabling the sample to be taken out of biocontainment laboratory after 1 h and 20 min. It also prevents dilution of virus before application to the coated TEM grid, so it may be necessary for samples suspected to be near the TEM detection limit. However, 1% osmium tetroxide vapor reduces the quality of TEM images as the osmium can further stain the sample (Figure 3). Previous reports show a 5 min exposure to osmium tetroxide vapor provides great results when negative staining; however, longer exposure required for absolute viral deactivation generated a mixture of positive and negative staining¹⁷. The second inactivation method involves a minimum of 24 h in a 2% glutaraldehyde solution, and does not require osmium tetroxide vapor treatment (Figure 1D). This second method takes longer to complete as the sample is not removed from the biocontainment laboratory for 24 h, but can be advantageous because it allows the negative staining to be done outside of biocontainment in a BSL-2 environment, and eliminates the need for hazardous osmium tetroxide. Our results demonstrate that 24 h of glutaraldehyde inactivation produces higher quality TEM images than when samples are treated with osmium tetroxide vapor (Figure 3).

Two negative stains were used in this experiment: UA and PTA. Both stains are used as a 1% solution. UA, the acetate salt of uranium, works well as a negative stain because dense uranium atoms scatter electrons⁵. PTA, a heteropoly acid, similarly works well as a negative stain due to dense tungsten atoms⁵. PTA is sometimes preferred over UA because it is much less toxic, and only a mild irritant if inhaled or contacted. When negative staining unfixed samples, the lower pH of UA must be considered as it can cause damage to the sample. The viruses used in this experiment require fixation for inactivation, so both UA and PTA achieved similar results and no distinct advantage could be detected for either stain. Both stains are easy to work with, and they both produce similar quality TEM images (Figure 4).

Overall, the capsule method is easier to use in a biocontainment environment and can be used as an alternative to the manual droplet method. Using capsules eases sample manipulation defects and yields good quality TEM images. The images produced using the capsule method are comparable to images produced using the manual droplet method. Short virus inactivation with osmium tetroxide improves speed, but should only be used if reduced image quality is acceptable, or it is suspected diluting the virus will put it below the TEM detection limit for the procedure. Both UA and PTA provide similar results with the fixed virus samples used in this study; however, results may be different when staining unfixed viruses.