Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment
July 19th, 2017
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.
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 useful 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 Formvar 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 suspension 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 container. 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% UA 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 virion 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.
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Overview
This protocol provides a simple and effective negative staining procedure for viral samples within biocontainment. It includes an innovative processing capsule that enhances handling of electron microscopy grids in BSL-2, -3, or -4 laboratories.
Key Study Components
Area of Science
- Virology
- Electron Microscopy
- Biocontainment Techniques
Background
- Negative staining is crucial for studying virus morphology.
- Transmission electron microscopy (TEM) is a key tool in virology.
- Handling samples in biocontainment presents unique challenges.
- This method aims to simplify those challenges.
Purpose of Study
- To provide a reliable TEM negative staining method for viral samples.
- To facilitate the investigation of virus structures.
- To improve handling of samples in high-containment environments.
Methods Used
- Negative staining of viral samples.
- Use of a processing capsule for grid handling.
- Application in BSL-2, -3, or -4 laboratories.
- Training for new users to minimize grid damage.
Main Results
- Effective handling of TEM grids in biocontainment.
- Successful application to various viral samples.
- Potential for broader use in nanoparticle investigations.
- Improved user experience with the processing capsule.
Conclusions
- This method enhances the study of viruses in secure environments.
- It provides a practical solution for researchers in virology.
- Further training may be required for optimal use.
Frequently Asked Questions
Executive Industry Relevance
Handling electron microscopy grids in high-containment laboratories presents significant operational challenges due to environmental turbulence and restricted dexterity from personal protective equipment. This capsule-based method improves sample preparation reliability and throughput for virology discovery workflows in BSL-3 and BSL-4 settings. By minimizing manual grid manipulation, the technique supports consistent ultrastructural characterization of viral targets, enabling more confident go/no-go decisions in early-stage antiviral target validation.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables morphological interrogation of viral targets to confirm structural integrity and support hypothesis-driven target selection.
- Operational Value: Reduces grid handling errors in biocontainment, improving data quality for downstream target de-risking.
Screening & Assay Development
- Scientific Value: Produces standardized, high-contrast TEM specimens suitable for quantitative morphology assessment in screening cascades.
- Operational Value: Streamlines sample preparation for nanoparticle and macromolecule analysis, increasing assay readiness and reproducibility.
Translational & Preclinical Research
- Scientific Value: Supports translational continuity by providing reliable ultrastructural data from discovery through preclinical evaluation of viral candidates.
- Operational Value: Facilitates cross-functional collaboration between virology, microscopy, and toxicology teams through standardized, transferable sample preparation.
Pipeline & Workflow Integration
The method integrates into the discovery continuum by enabling reliable negative staining of viral samples after inactivation and prior to imaging, supporting hypothesis testing and lead confirmation stages.
- Discovery Biology: Supports viral target validation by providing clear ultrastructural readouts that help clarify morphology and inform mechanistic understanding.
- Screening: Enhances assay standardization by producing consistent, artifact-minimized TEM grids for comparative analysis across conditions.
- Analytics: Generates quantitative morphometric outputs (e.g., particle size, shape, surface features) that enable objective comparison of viral variants or treatment effects.
- Translational Research: Ensures sample preparation continuity from BSL-3/4 discovery to BSL-2 imaging, maintaining data integrity across containment levels.
- Enterprise Reuse: Functions as a platform capability applicable to diverse specimens including viruses, nanoparticles, and macromolecules, maximizing infrastructure utility.
Operational & Enterprise Impact
- Scientific Value: Improves predictive confidence in target validation by reducing variability in ultrastructural data acquisition.
- Operational Value: Increases reproducibility and scalability of TEM workflows in high-containment environments through encapsulated grid handling.
- Strategic Value: Supports better go/no-go decisions by minimizing false negatives from grid damage or staining inconsistency.
- Portfolio Impact: Enables risk-adjusted prioritization of viral targets based on reliable structural data, reducing late-stage attrition due to unexpected morphology.
Implementation Considerations
- Requires training in aseptic grid loading and unloading to prevent capsule or grid damage.
- Dependent on access to TEM-compatible capsules, pipettes, and biocontainment-approved reagents (glutaraldehyde, stains, osmium tetroxide).
- Necessitates standardized timing and orientation protocols across users to ensure uniform staining and washing.
- Requires adaptation of wash and staining times based on specimen density and contaminant load.
- Limited by the need for post-staining decontamination and transfer protocols to move samples from BSL-3/4 to BSL-2 imaging facilities.
Biopharma FAQs
Chapters in this video
0:05
Title
1:08
Experiment Preparation in a BSL-2 Environment Prior to Working with the Virus Samples (Biocontainment Procedures were Filmed in BSL-4 Training Lab)
7:14
Conclusion
5:50
Results: Analyzing the Capsule Method of Negative Staining Viral Samples
1:45
The Capsule Method for Negative Staining in Biocontainment Using Aqueous Glutaraldehyde and 1% Osmium Tetroxide Vapor Inactivation
4:24
The Capsule Method for Inactivation in Biocontainment 2% Glutaraldehyde Followed by Negative Staining in BSL-2 Laboratory
