Positive staining of Viruses

What is positive staining?

Positive staining of viruses is similar to negative staining. The difference is that the virus image is darker formed on a light background, unlike the negative staining where a light viral particle image is formed on a dark background. The technique is simple and it is used widely to study the diverse morphologies of viruses from aquatic environments.

Principle for positive staining of viruses

• This procedure is light sensitive and therefore should be performed in an environment that is protected from light.
• Similar to negative staining, the sample is incubated with a heavy metallic solution, which reacts with the cell organelles and structures.
• In positive staining, uranyl acetate and/or lead citrate are the most commonly used salts.
• Ultra-thin sections of the samples are incorporated into a grid and the uranyl acetate salt added.
• Washing which is a very critical process of the stain is done to eliminate excess salts from outside the stain.
• Airdried grids of the sample are viewed under the Transmission Electron Microscope.

Procedure of Positive Staining of Viruses

Materials and reagents

• 2-3 pairs of EM grid-grade tweezers
• Uranyl acetate
• Ultra-purified water
• Petri plate
• 4 pieces of 2ml microcentrifuge tubes with screw caps
• 4ml plastic culture tube with cap
• stir bar
• stir plate
• Filter paper (cut into wedges)
• 0.02 µm syringe filter and 3-5 ml syringe
• Lab coat, respiratory protection (mask), eye protection
• Timer
• waste container for uracyl acetate

Preparation of 2% Uranyl acetate

• 0.04 gm uranyl acetate + 2 ml Q-water = 2% uranyl acetate

Procedure using uranyl acetate

1. Prepare 2ml of 2% uranyl acetate with ultra-purified water in a 4ml culture tube and stir with a stir bar to mix the sample on a stir plate for approximately 30 minutes to 1 hour.
2. Filter the uranyl acetate solution using a 0.02 µm syringe filter into a 2 ml screw-cap tube to remove any undissolved particles.
3. Fill the 2 ml screw-cap tube with ultra-purified water
4. Immerse the grid into the 25 uranyl acetate solution for 30 seconds
5. Immerse the grid into the 1 tube of ultra-purified water for 10 seconds
6. Immerse the grid into the 2nd tube of ultra-purified water for 10 seconds
7. Immerse the grid into the 3rd tube of ultra-purified water for 10 seconds
8. Wick off liquid from the grid using a wedge of filter paper placed at the edge of the grid.
9. Place the grid in a grid box and allow it to dry overnight.
10. Observe under a Transmission electron Microscope.

Procedure using Uranyl acetate and lead citrate

This procedure should be performed in an environment protected from light and free of CO2.

1. The ultra-thin section of the sample is added to the grid.
2. The grid with the sample is incubated in uranyl acetate for 15 minutes.
3. Wash the grids in ultra-purified water
4. Incubate the grids in lead citrate for 4-5 minutes in a CO2 free environment, by placing NaOH tablets during the experiment to absorb and CO2 that may react with the salts.
5. The grids are then airdried and observed under the Transmission microscope.

Results and interpretation

Image Source: https://cpb-us-w2.wpmucdn.com/u.osu.edu/dist/e/20087/files/2015/08/Positive_and_Negative_Stainging_of_Viruses_on_TEM_Grids-239c21p.pdf

• Positive staining under a Transmission Electron Microscope (TEM) shows a dark image of the virus and a light background, showing the physical morphology of the virus, revealing any structural elements such as pikes and envelope of the virus.
• For example, the use of uranyl acetate to study Hepatitis B antigen showed the spread of viral particles all over the grid.

Advantages of Positive staining

• It is simple to perform.
• It is rapid to perform.

Limitations of Positive staining of viruses

• External reaction with CO2 may affect the result and orientation of the morphology of the viruses.
• Exposure to small light rays may affect the outcome of the results.

Applications of Positive Staining of Viruses

• It has been used to visualize various morphologies of viruses such as viral spikes, and envelopes, for viruses such as orthomyxoviruses, adenoviruses, hepatitis, rhinoviruses, influenza viruses.
• It is used to study the morphologies and physiological features of viruses.
• It has been used to identify and differentiate various viruses such as animal viruses from human viruses.

Progress in Positive staining of viruses

• New techniques are being developed to positively identify viruses using the Transmission electron Microscope to observe novel morphologies and physiological features of viruses.
• For example,  the Tokuyasu staining procedure (TSP), is a positive staining method using Uranyl acetate, glutaraldehyde, and polyvinyl alcohol to identify and visualize non-enveloped and enveloped viruses.
• This includes rotaviruses, rubella viruses, HIV-1, Human T cell Lymphotropic virus.
• This stain has been used in identifying novel features of rotavirus and rubella viruses as well.

References and Sources

• 3% – https://microbenotes.com/negative-staining-of-viruses/
• 2% – https://www.sciencedirect.com/topics/materials-science/transmission-electron-microscopy
• 2% – https://www.researchgate.net/publication/291354740_Negative_and_Positive_Staining_in_Transmission_Electron_Microscopy_for_Virus_Diagnosis
• 1% – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789507/
• 1% – https://www.microscopemaster.com/transmission-electron-microscope.html
• 1% – https://en.m.wikipedia.org/wiki/Uranyl_acetate
• <1% – https://www.researchgate.net/post/Does_anybody_have_a_protocol_for_uranyl_acetate_staining_of_nanocellulose
• <1% – https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978762/
• <1% – https://greenbioresearch.com/product/2ml-screw-cap-microcentrifuge-tubes-conical-bottom-freezer-micro-tubes/