Name: utilization of capsules for negative staining of viral samples within biocontainment
Uploaded: 2017-07-19T14:00:00
Description: Watch this Scientific Journal Video about Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment at JoVE.com

We would like to acknowledge and thank Dr. John Carra and Rowena Schokman for providing purified Ebola nano-VLPs, Dr. Rajini Mudhasani for providing Chikungunya virus, and Dr. Charles (Jason) Shoemaker for providing Murine Leukemia VLPs expressing Ebolavirus glycoproteins. We would also like to thank MAJ Carl Soffler for facilitating the Summer Internship Program (SIP) and the Science and Engineering Apprenticeship Program (SEAP) and Dr. Catherine Wilhelmsen for the lab safety training.

1. Experiment Preparation in a BSL-2 Environment Prior to Working with the Virus Samples
<ol>
	<li>Prepare or purchase Formvar and carbon coated TEM copper grids, usually 200-400 mesh.</li>
	<li>Insert the coated TEM grids into capsules.
	<ol>
		<li>Use a magnified lens to make this process easy to perform. One or two grids may be inserted into each capsule. Pre-loaded capsules can be purchased to eliminate this step if desired.</li>
	</ol>
	</li>
	<li>Transfer the capsules with inserted TEM coated gri...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helmut+Ruska+and+the+visualisation+of+viruses.">Helmut Ruska and the visualisation of viruses.</a> Lancet. 355 (9216), 1713-1717 (2000).</li><li>Curry, A., Appleton, H., Dowsett, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+transmission+electron+microscopy+to+the+clinical+study+of+viral+and+bacterial+infections:+present+and+future.">Application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future.</a> Micron. 37 (2), 91-106 (2006).</li><li>Goldsmith, C. S., Miller, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+uses+of+electron+microscopy+for+detection+of+viruses.">Modern uses of electron microscopy for detection of viruses.</a> Clin Microbiol Rev. 22 (4), 552-563 (2009).</li><li>Kiselev, N. A., Sherman, M. B., Tsuprun, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+staining+of+proteins.">Negative staining of proteins.</a> Electron Microsc Rev. 3 (1), 43-72 (1990).</li><li>Brenner, S., Horne, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+negative+staining+method+for+high+resolution+electron+microscopy+of+viruses.">A negative staining method for high resolution electron microscopy of viruses.</a> Biochim Biophys Acta. 34, 103-110 (1959).</li><li>Harris, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+staining+of+thinly+spread+biological+samples.">Negative staining of thinly spread biological samples.</a> Methods Mol Biol. 369, 107-142 (2007).</li><li>Bradley, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+bacteriophage+and+bacteriocins.">Ultrastructure of bacteriophage and bacteriocins.</a> Bacteriol Rev. 31 (4), 230-314 (1967).</li><li>Suzuki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+two+negative+staining+methods+on+the+Chinese+atypical+rotavirus.">Effects of two negative staining methods on the Chinese atypical rotavirus.</a> Arch Virol. 94 (3-4), 305-308 (1987).</li><li>Benmeradi, N., Payre, B., Goodman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easier+and+Safter+Biological+Staining:+High+Contrast+Uranyless+Staining+of+TEM+Grids+using+mPrep/g+Capsules.">Easier and Safter Biological Staining: High Contrast Uranyless Staining of TEM Grids using mPrep/g Capsules.</a> Microsc Microanal. 21, 721 (2015).</li><li>Goodman, S. L., Wendt, K. D., Kostrna, M. S., Radi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsule-Based+Processing+and+Handling+of+Electron+Microscopy+Specimens+and+Grids.">Capsule-Based Processing and Handling of Electron Microscopy Specimens and Grids.</a> Microscopy Today. 23 (5), 30-37 (2015).</li><li>Goodman, S. L., Kostrna, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+Reagent+Consumption+and+Improving+Efficiency+of+Specimen+Fixation+and+Embedding,+Grid+Staining+and+Archiving+using+mPrep+Capsule+Processing.">Reducing Reagent Consumption and Improving Efficiency of Specimen Fixation and Embedding, Grid Staining and Archiving using mPrep Capsule Processing.</a> Microsc Microanal. 17, 174-175 (2011).</li><li>Monninger, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+viral+samples+within+biocontainment+for+ultrastructural+analysis:+Utilization+of+an+innovative+processing+capsule+for+negative+staining.">Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining.</a> J Virol Methods. 238, 70-76 (2016).</li><li>Rossi, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+ViroCyt(R)+Virus+Counter+for+rapid+filovirus+quantitation.">Evaluation of ViroCyt(R) Virus Counter for rapid filovirus quantitation.</a> Viruses. 7 (3), 857-872 (2015).</li><li>Carra, J. 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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...

Transmission electron microscopy (TEM) is an effective tool for viewing the morphology and ultrastructure of biological specimens that are too small to be seen with a traditional light microscope 1,2,3,4. TEMs shoot electrons through a very thin specimen producing a higher resolution image as electrons have a much shorter wavelength than light. Regions of the sample that bend or block electrons appear dark, while regions that are electron lucent appear white.
Lack of electron dense matter makes viruses difficult to view under a TEM because they cannot scatter electrons. Negative staining is the most common method used to create contrast and view viruses with a TEM. The first negative staining procedure was proposed by Brenner and Horne in 1959, based on an experiment where Hall (1955) and Huxley (1957) observed the appearance of biological structures in reverse contrast when immersed in an electron-dense substance 5. The process of negative staining has been virtually unchanged over the past half century. Negative staining involves briefly applying a heavy metal salt solution to a sample on a TEM grid in an attempt to surround the virus with dense material without infiltrating the virus 6. This creates a dark border and reveals the particle&#39;s shape 5. This study uses two reagents for negative staining, uranyl acetate (UA) and potassium phosphotungstic acid (PTA). Both of these stains are commonly used to negatively stain small biological samples, such as viruses, protein complexes, and nanoparticles 7,8,9.
The conventional negative staining technique is the manual droplet negative staining technique7. This method requires precise handling of small, fragile TEM grids with forceps to apply small amounts of virus sample, stain, and rinses. The typical preparation protocol involves applying a droplet of sample suspension onto the surface of a film-coated TEM grid (Figure 1A). After attachment of the sample to the film surface, the grid is rinsed to remove non-adherent viruses and stained with either UA or PTA for a few seconds to a minute, depending on the type of sample. Excess liquid is wicked away from the grid by touching a piece of filter paper to the edge of the grid.
The manual droplet method requires that each grid be individually made. If not handled carefully, coated TEM grids are easily punctured, bent, or contaminated. Processing multiple samples can lead to difficulties in tracking the grids and ensuring consistent staining for each sample. This manual staining procedure is much more difficult when conducted in biosafety level (BSL)-3 and -4 biocontainment laboratories, due to the required personal protective equipment (PPE) required for these environments. PPE is cumbersome and the biocontainment environment is much more turbulent compared to a regular laboratory. Personnel working in BSL-3 biocontainment laboratories are required to wear 2 pairs of gloves and work in a biosafety cabinet (BSC). This double layer of gloves reduces tactile sensitivity and restricts fine motor movement. The airflow of the BSC that protects the user and helps prevent sample contamination can cause the samples and stains to dry too quickly thus affecting the stain quality. The strong turbulent airflow in the BSC can also quickly blow away a grid that is not well secured. In BSL-4 biocontainment laboratories, there are additional safety requirements. Personnel are required to wear a positive pressure suit, which further restricts physical movement and the ability to clearly see and manipulate grids. The technician working in BSL-4 also wears at least 2 pairs of gloves, with the outer pair being a thick glove which greatly reduces dexterity and tactile sensation. Finally, the forceps used to handle TEM grids are sharp, thereby posing a risk to the technician due to their ability to puncture gloves. With capsules containing grids, forceps are not necessary, thus providing a safe, forceps-free alternative for manipulating grids in biocontainment. Finally, the capsules also provide an effective way to store grids during processing, osmium vapor decontamination, and during storage; thereby keeping the grids organized and safe from damage.
In this report, we introduce a new method for negative staining TEM grids in biocontainment laboratories that utilizes mPrep/g capsules, a capsule-based device for grid handling and staining 10,11,12. The capsule accommodates two TEM grids, minimizes direct handling, and reduces the potential for grid damage. The capsule attaches directly to a single or multichannel pipette in the same manner as a pipette tip, allowing the application of various liquids to grids contained within. This enables simultaneous preparation of multiple samples with duplicate grids (Figure 1B). To negative stain with capsules the virus sample is aspirated into the capsule and held for 10 min to let the viruses adsorb onto the grid surfaces. The grids with adsorbed virus are subsequently washed with deionized (dI) water and stained with either UA or PTA for a few seconds to 1 min. This process uses the same protocol steps and reagents as the manual droplet method; the difference being that all work occurs inside the capsule with no physical handling of the grids. (Figures 1C, 1D).
The purpose of this study was to evaluate capsules as a new method for negative staining of virus samples in biocontainment environments. This study also examined the quality of TEM images produced from two different virus inactivation procedures: 1) rapid inactivation, with 1% osmium tetroxide vapor, and 2) a 24 h inactivation with 2% glutaraldehyde. Both of these were conducted using the capsules. Finally, we evaluated two commonly used negative stains, UA and PTA, for use in the capsule. 13

<table border="0" cellpadding="0" cellspacing="0" width="590">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Formvar/carbon coated TEM grids</td>
			<td style="width:71px;">SPI</td>
			<td style="width:119px;">3420C-MB</td>
			<td style="width:167px;">200 mesh Cu Pk/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">mPrep/g capsules</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">85010-01</td>
			<td>box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">mPrep/g couplers</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">85010-11</td>
			<td>standard 16/Pk</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">glutaraldehdyde</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">16320</td>
			<td>50% solution, EM grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Osmium Tetroxide</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">19190</td>
			<td>4% aqueous solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Uranyl Acetate</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">22400</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Potassium phosphotungstic acid</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">19500</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">1450-090</td>
			<td>size 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Tranmission Electron Microscope</td>
			<td style="width:71px;">JEOL</td>
			<td style="width:119px;">JEM-1011</td>
			<td>TEM</td>
		</tr>
	</tbody>
</table>

utilization of capsules for negative staining of viral samples within biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

The capsule method produce good quality negative staining for TEM imaging:
First, we evaluated the quality of images generated by using both the manual droplet method and the capsule methods for negative staining Zaire ebolavirus. Ebolaviruses are members of the Filoviridae family, along with Marburg virus. Ebolavirus is typically 80 to 100 nm in diameter and can be over 1,000 nm in length. Ebolavirus must be handled in a ...

Watch this Scientific Journal Video about Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment at JoVE.com

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

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.

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

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.

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