Positive staining of Viruses

Positive staining of viruses is a technique that uses heavy metal salts, such as uranyl acetate and lead citrate, to create a contrast between the virus particles and the background. The virus particles appear dark on a light background, revealing their shape, size and structure. This technique is useful for studying the diverse morphologies of viruses from different environments.

Positive staining of viruses is similar to negative staining, which is another technique that uses heavy metal salts to create a contrast. However, in negative staining, the virus particles appear light on a dark background, while the background is stained by the metal salts. This technique is useful for studying the surface features of viruses, such as spikes and envelopes.

Both positive and negative staining are simple and rapid methods that do not require any fixation or embedding of the samples. They can be performed directly on the grids that are used for observing the samples under a transmission electron microscope (TEM). However, they have some limitations, such as sensitivity to light and carbon dioxide, which can affect the quality of the images. Also, they do not preserve the internal structures of the viruses, such as nucleic acids and proteins.

Positive and negative staining are complementary techniques that can provide different information about the viruses. By using both techniques, one can obtain a more comprehensive view of the viral morphology and physiology. For example, positive staining can show the size and shape of the virus particles, while negative staining can show the surface features and arrangement of the virus particles. Together, they can help identify and characterize different types of viruses.

Positive staining of viruses is based on the principle that heavy metal salts, such as uranyl acetate and lead citrate, can bind to the viral components and form a dark precipitate. This creates a contrast between the virus and the background, allowing the visualization of the viral morphology and structure under a transmission electron microscope (TEM).

The heavy metal salts act as electron-dense agents that scatter the electrons in the TEM beam. The more electrons are scattered, the darker the image appears. The viral components that have a high affinity for the metal salts, such as proteins and nucleic acids, will appear darker than the surrounding medium. This allows the identification of different viral features, such as capsids, envelopes, spikes, and nucleocapsids.

Positive staining of viruses requires ultra-thin sections of the sample to be prepared and mounted on a grid. The grid is then incubated with the metal salt solution for a specific time and washed with water to remove excess salt. The grid is then air-dried and observed under a TEM.

Positive staining of viruses can be performed with either uranyl acetate or lead citrate alone, or with a combination of both. Uranyl acetate is more commonly used for positive staining of viruses because it has a higher affinity for proteins and nucleic acids than lead citrate. Lead citrate can enhance the contrast of uranyl acetate by binding to phosphates and carboxylates in the sample. However, lead citrate is sensitive to carbon dioxide and light, which can affect the quality of the stain.

Positive staining of viruses can reveal various aspects of viral morphology and structure, such as size, shape, symmetry, surface features, and internal components. However, positive staining of viruses also has some limitations, such as distortion of the viral shape due to dehydration and shrinkage during staining, loss of some viral components due to solubilization by the metal salts, and difficulty in distinguishing between different types of viruses with similar morphology. Therefore, positive staining of viruses should be complemented by other techniques, such as negative staining, immunoelectron microscopy, or molecular methods, to obtain a comprehensive characterization of viruses.

Positive staining of viruses can be done using either uranyl acetate or a combination of uranyl acetate and lead citrate. Both methods require ultra-thin sections of the samples to be mounted on a grid and incubated with the metallic salts. The grids are then washed, dried and observed under a transmission electron microscope (TEM). The following are the detailed steps for each method:

Procedure using uranyl acetate

• Prepare 2 ml of 2% uranyl acetate solution by dissolving 0.04 g of uranyl acetate in 2 ml of ultra-purified water in a 4 ml plastic culture tube. Stir the solution with a stir bar on a stir plate for about 30 minutes to 1 hour until the salt is completely dissolved.
• Filter the uranyl acetate solution using a 0.02 µm syringe filter into a 2 ml screw-cap tube to remove any undissolved particles. Label this tube as UA.
• Fill three other 2 ml screw-cap tubes with ultra-purified water and label them as W1, W2 and W3.
• Immerse the grid with the sample into the UA tube for 30 seconds using a pair of EM grid-grade tweezers. Make sure the grid is fully submerged in the solution and avoid touching the sample with the tweezers.
• Transfer the grid to the W1 tube and immerse it for 10 seconds. Repeat this step with the W2 and W3 tubes to wash off any excess uranyl acetate from the grid.
• Wick off the liquid from the grid using a wedge of filter paper placed at the edge of the grid. Do not touch the sample with the filter paper.
• Place the grid in a grid box and allow it to dry overnight in a dark place.
• Observe the grid under a TEM.

Procedure using uranyl acetate and lead citrate

• This procedure should be performed in an environment protected from light and free of CO2. Place some NaOH tablets in a Petri plate near the work area to absorb any CO2 that may react with the salts.
• Immerse the grid with the sample into a tube containing uranyl acetate solution for 15 minutes using a pair of EM grid-grade tweezers. Make sure the grid is fully submerged in the solution and avoid touching the sample with the tweezers.
• Wash the grid in three tubes of ultra-purified water as described above.
• Immerse the grid into a tube containing lead citrate solution for 4-5 minutes using another pair of EM grid-grade tweezers. Make sure the grid is fully submerged in the solution and avoid touching the sample with the tweezers.
• Wash the grid in three tubes of ultra-purified water as described above.
• Wick off the liquid from the grid using a wedge of filter paper placed at the edge of the grid. Do not touch the sample with the filter paper.
• Place the grid in a grid box and allow it to dry overnight in a dark place.
• Observe the grid under a TEM.

Depending on the type of positive staining method used, the materials and reagents required may vary slightly. However, some of the common items needed are:

• 2-3 pairs of EM grid-grade tweezers
• Uranyl acetate
• Lead citrate
• 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 uranyl acetate

Some of these items are used to prepare the staining solutions, while others are used to handle and wash the grids with the samples. It is important to use clean and sterile equipment to avoid contamination and interference with the staining results.

The uranyl acetate and lead citrate are the main staining agents that react with the viral structures and produce a dark image on a light background. These salts are light-sensitive and should be stored in a dark place. They are also toxic and should be handled with care and disposed of properly.

The ultra-purified water is used to dilute the staining solutions and to wash the grids after staining. The water should be free of any impurities or contaminants that may affect the staining quality.

The EM grid-grade tweezers are used to hold and transfer the grids with the samples. They should be clean and free of any dust or grease that may interfere with the staining.

The filter paper wedges are used to wick off excess liquid from the grids after washing. They should be soft and absorbent and not leave any fibers on the grids.

The syringe filter and syringe are used to filter the uranyl acetate solution before use to remove any undissolved particles that may affect the staining quality.

The petri plate, culture tube, stir bar, and stir plate are used to prepare and mix the uranyl acetate solution.

The timer is used to monitor the incubation time of the grids in the staining solutions.

The lab coat, mask, and eye protection are used to protect the user from exposure to the toxic staining agents.

The waste container is used to collect and dispose of the used staining solutions safely.

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 spikes and envelope of the virus. The contrast between the virus and the background is due to the electron density of the stain that binds to the viral components.

For example, the use of uranyl acetate to study Hepatitis B antigen showed the spread of viral particles all over the grid. The image below shows the Hepatitis B surface antigen (HBsAg) particles stained with uranyl acetate. The particles are spherical, about 20 nm in diameter, and have a smooth surface.

Another example is the use of uranyl acetate and lead citrate to study influenza A virus. The image below shows the influenza A virus particles stained with uranyl acetate and lead citrate. The particles are pleomorphic, ranging from 80 to 120 nm in diameter, and have an envelope with surface projections. The envelope contains a matrix protein (M1) that forms a shell under the lipid bilayer. The nucleocapsid (NC) consists of eight segments of RNA wrapped by a nucleoprotein (NP). The surface projections are hemagglutinin (HA) and neuraminidase (NA), which are glycoproteins involved in viral attachment and release.

Positive staining can also be used to compare different types of viruses or different strains of the same virus. For example, the image below shows the morphological differences between human rhinovirus (HRV) and bovine rhinovirus (BRV) stained with uranyl acetate. Both viruses belong to the Picornaviridae family and have icosahedral capsids, but HRV has a larger diameter (30 nm) than BRV (25 nm). HRV also has a more prominent depression at each of the 12 vertices of the capsid, called a canyon, which is involved in receptor binding.

Positive staining can provide valuable information about the morphology and structure of viruses, but it also has some limitations. For instance, positive staining may cause artifacts or distortions in the viral image due to factors such as sample preparation, staining conditions, or microscope settings. Some examples of artifacts are aggregation, precipitation, deformation, or shrinkage of the viral particles. Therefore, positive staining should be performed carefully and interpreted cautiously.

Positive staining of viruses has some advantages and limitations that should be considered when using this technique. Some of the advantages are:

• It is simple to perform. The procedure does not require complex steps or equipment, and can be done in a short time.
• It is rapid to perform. The staining and observation can be completed within an hour, making it suitable for quick diagnosis or identification of viruses.
• It reveals the physical morphology of the virus. The dark image of the virus on a light background shows the shape, size, and structure of the virus, such as spikes, envelope, or capsid.

Some of the limitations are:

• It may alter the natural state of the virus. The heavy metal salts used for staining may react with the viral components and change their orientation or conformation, affecting the accuracy of the results.
• It may damage the virus. The high concentration of salts and the drying process may cause dehydration or denaturation of the virus, reducing its viability or infectivity.
• It may not distinguish between different types of viruses. The staining may not show enough details or contrast to differentiate between viruses that have similar morphology or size. For example, positive staining may not be able to tell apart coronaviruses from paramyxoviruses.

Therefore, positive staining of viruses is a useful technique for visualizing the general morphology and structure of viruses, but it has some drawbacks that may affect the quality and reliability of the results. Other techniques, such as negative staining, immunostaining, or molecular methods, may be needed to complement or confirm the findings from positive staining.

Positive staining of viruses has been widely used to study the diverse morphologies and physiological features of viruses from various sources. Some of the applications and progress in this technique are:

• It has been used to visualize the structural elements of viruses, such as spikes, envelopes, capsids, and nucleic acids, for viruses such as orthomyxoviruses, adenoviruses, hepatitis B virus, rhinoviruses, influenza viruses, and coronaviruses .
• It has been used to identify and differentiate various types of viruses, such as animal viruses from human viruses, enveloped viruses from non-enveloped viruses, and RNA viruses from DNA viruses .
• It has been used to study the interactions of viruses with host cells, such as entry, replication, assembly, and release .
• It has been used to monitor the effects of antiviral agents and vaccines on viral morphology and infectivity .

New techniques are being developed to improve the positive staining of viruses using the transmission electron microscope. Some of the recent advances are:

• The Tokuyasu staining procedure (TSP), which is a positive staining method using uranyl acetate, glutaraldehyde, and polyvinyl alcohol to identify and visualize non-enveloped and enveloped viruses. This technique has been used to study rotaviruses, rubella virus, HIV-1, and human T cell lymphotropic virus .
• The cryo-electron microscopy (cryo-EM), which is a technique that preserves the native structure of viruses by freezing them in a thin layer of vitreous ice. This technique allows high-resolution imaging of viral particles without the need for staining or fixation. This technique has been used to study Zika virus, Ebola virus, SARS-CoV-2, and many other viruses .
• The immunogold labeling (IGL), which is a technique that uses antibodies conjugated with gold particles to specifically target viral antigens. This technique enhances the contrast and specificity of viral images by binding gold particles to viral structures. This technique has been used to study herpes simplex virus, human papillomavirus, hepatitis C virus, and dengue virus .

Positive staining of viruses is a simple and rapid technique that provides valuable information on the morphology and physiology of viruses. However, it also has some limitations, such as sensitivity to light and CO2 exposure, distortion of viral structures by heavy metals, and low resolution compared to other techniques. Therefore, new methods are being developed to overcome these challenges and improve the positive staining of viruses. These methods include TSP, cryo-EM, and IGL, which offer higher resolution, better preservation, and more specificity for viral imaging.

Positive staining of viruses has been applied to various types of viruses, both enveloped and non-enveloped, to reveal their structural and morphological features. Some examples are:

• Orthomyxoviruses: These are enveloped viruses with segmented RNA genomes that cause influenza in humans and animals. Positive staining with uranyl acetate and lead citrate has shown the presence of surface spikes composed of hemagglutinin and neuraminidase proteins, which are important for viral attachment and release.
• Adenoviruses: These are non-enveloped viruses with double-stranded DNA genomes that cause respiratory, gastrointestinal, and ocular infections in humans and animals. Positive staining with uranyl acetate has shown the icosahedral capsid structure with 12 penton fibers at the vertices, which mediate viral entry into host cells.
• Hepatitis B virus: This is an enveloped virus with a partially double-stranded DNA genome that causes chronic liver disease in humans. Positive staining with uranyl acetate has shown the spherical or filamentous forms of the virus, as well as the presence of a nucleocapsid core containing the viral DNA and polymerase.
• Rhinoviruses: These are non-enveloped viruses with single-stranded RNA genomes that cause the common cold in humans. Positive staining with uranyl acetate has shown the icosahedral capsid structure with four structural proteins (VP1-VP4), as well as the presence of a deep canyon on the surface that binds to cellular receptors.
• Influenza A virus: This is an enveloped virus with segmented RNA genome that causes seasonal and pandemic influenza in humans and animals. Positive staining with uranyl acetate and lead citrate has shown the surface spikes composed of hemagglutinin and neuraminidase proteins, as well as the presence of a matrix protein layer under the envelope and ribonucleoprotein complexes inside the nucleocapsid.

These are just some examples of how positive staining of viruses can provide valuable information about their structure, morphology, and function. By using this technique, researchers can gain insights into the viral life cycle, pathogenesis, and evolution. Positive staining of viruses is therefore a useful tool for virology research and diagnosis.

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