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 grids, together with other supplies and reagents, to biocontainment where the viruses will be negatively stained.</li>
</ol>
2. The Capsule Method for Negative Staining in Biocontainment using Aqueous Glutaraldehyde and 1% Osmium Tetroxide Vapor Inactivation
<ol>
	<li>Inside the biocontainment BSC, aspirate 40 &#181;L of virus suspension into the capsule attached to a pipette. 
	NOTE: The pipette remains attached to the capsule until the process is completed. Virus preparation is according to our previous publication14.</li>
	<li>Place the pipette on its side for 10 min with grids oriented horizontally. This is to promote an even distribution of virus particles onto the coated grids.</li>
	<li>Inactivate virus within the capsule inside the biocontainment BSC.
	<ol>
		<li>Pick up the pipette and depress the plunger to dispense the virus solution into a waste container.</li>
		<li>Aspirate 40 &#181;L of 2% glutaraldehyde fixative into the capsules. 
		Caution: Glutaraldehyde is a hazardous chemical and requires appropriate protection. Glutaraldehyde can be used for brief periods in a normal BSC, but extended open reagent requires working in a ducted BSC or chemical fume hood.</li>
		<li>Place the pipette on its side for 20 min. This is to ensure samples are well fixed.</li>
		<li>Expel the fixative and aspirate 40 &#181;L of dI water into the capsules to wash away the fixative. Repeat this wash step for a total of 3 rinse cycles.</li>
	</ol>
	</li>
	<li>Aspirate 40 &#181;L of either 1% uranyl acetate (UA) or 1% potassium phosphotungstic acid (PTA) into the capsules and allow to sit for 30 s. 
	NOTE: Staining time may vary from 10 s to 1 min based on virus sample. 
	Caution: UA is an alpha emitter, and a cumulative toxin. Handle it with appropriate protection.</li>
	<li>Remove the capsule from the pipette and blot dry the grids by touching a piece of filter paper to the edge of the grids while the grids remain within the capsule.</li>
	<li>Osmium Tetroxide Vapor inactivation procedure.
	<ol>
		<li>Place the capsule, with the lid open, into a 50 mL centrifuge tube containing filter paper soaked in a 1% osmium tetroxide solution . 
		Caution: Osmium tetroxide is extremely toxic with low vapor pressure. It must be used in a ducted BSC or chemical fume hood. Handle it with appropriate protection. Post warning information in the working area.</li>
		<li>Seal the 50-mL centrifuge tube for 1 h to allow complete permeation of the osmium tetroxide vapor. Then, subsequently decontaminate and transfer the tube out of the biocontainment to the BSL-2 EM facility.</li>
	</ol>
	</li>
	<li>Remove EM grids from the capsule.
	<ol>
		<li>In the BSL-2 EM facility, remove the capsule from the centrifuge tube and place it onto a pipette.</li>
		<li>Aspirate 40 &#181;L of dI water into the capsule and dispense the water into a waste container three times.</li>
		<li>Remove the capsule from the pipette and blot dry the grids using filter paper to touch the edge of the grids.</li>
		<li>After air drying, store the grids for subsequent TEM imaging.</li>
	</ol>
	</li>
</ol>
3. The Capsule Method for Inactivation in Biocontainment with 2% Glutaraldehyde, Followed by Negative Staining in a BSL-2 Laboratory
<ol>
	<li>Virus inactivation procedure.
	<ol>
		<li>Inside the biocontainment BSC, mix the virus suspension well with the same volume of 4% glutaraldehyde to achieve a final concentration of 2% glutaraldehyde. 
		Caution: Glutaraldehyde is a hazardous chemical and requires appropriate protection. Glutaraldehyde can be used for brief periods in a normal BSC, but extended open reagent requires working in a ducted BSC or chemical fume hood.</li>
		<li>Inactivate viruses with fixative for a minimum of 24 h before packaging, decontamination, and transfer it to the BSL-2 EM facility.</li>
	</ol>
	</li>
	<li>In the BSL-2 EM facility, aspirate the virus and fixative mixture into the capsule, containing two TEM grids, attached to a pipette.</li>
	<li>Place the pipette horizontally for 10 min with the grids oriented horizontally. 
	NOTE: This is to promote an even distribution of virus particles onto the TEM grids.</li>
	<li>Pick up the pipette and depress the plunger to expel the virus to a waste container. Aspirate 40 &#181;L of dI water into the capsules and expel it into the waste container for 3 rinse cycles.</li>
	<li>Aspirate 40 &#181;L of either 1% UA or 1% PTA into the capsules for 30 s. 
	NOTE: Staining time varied from 10 s to 1 min based on virus sample. 
	Caution: UA is an alpha emitter, and a cumulative toxin. Handle it with appropriate protection.</li>
	<li>Remove the capsule from the pipette and blot dry the grids by touching the edge of the grids to a piece of filter paper. Air dry the grids and store them for subsequent TEM imaging.</li>
</ol>

<ol>
	<li>Gentile, M., Gelderblom, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+in+rapid+viral+diagnosis:+an+update.">Electron microscopy in rapid viral diagnosis: an update.</a> New Microbiol. 37 (4), 403-422 (2014).</li><li>Kruger, D. H., Schneck, P., Gelderblom, H. 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. 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=A+thermostable,+chromatographically+purified+Ebola+nano-VLP+vaccine.">A thermostable, chromatographically purified Ebola nano-VLP vaccine.</a> J Transl Med. 13, 228 (2015).</li><li>Rein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+leukemia+viruses:+objects+and+organisms.">Murine leukemia viruses: objects and organisms.</a> Adv Virol. , 403419 (2011).</li><li>Barland, M., Rojkind, <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+with+osmium+tetroxide+vapour.">Negative staining with osmium tetroxide vapour.</a> Nature. 212 (5057), 84-85 (1966).</li></ol>

Opinions, interpretations, conclusions, and recommendations stated within the article are those of the authors and are not necessarily endorsed by the U.S. Army or the Department of Defense.

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><tbody><tr><td>Formvar/carbon coated TEM grids</td><td>SPI</td><td>3420C-MB</td><td>200 mesh Cu Pk/100</td></tr><tr><td>mPrep/g capsules</td><td>EMS</td><td>85010-01</td><td>box</td></tr><tr><td>mPrep/f couplers</td><td>EMS</td><td>85010-11</td><td>standard 16/Pk</td></tr><tr><td>glutaraldehdyde</td><td>EMS</td><td>16320</td><td>50% solution, EM grade</td></tr><tr><td>Osmium Tetroxide</td><td>EMS</td><td>19190</td><td>4% aqueous solution</td></tr><tr><td>Uranyl Acetate</td><td>EMS</td><td>22400</td><td>powder</td></tr><tr><td>Potassium phosphotungstic acid</td><td>EMS</td><td>19500</td><td>powder</td></tr><tr><td>filter paper</td><td>Whatman</td><td>1450-090</td><td>size 50</td></tr><tr><td>Tranmission Electron Microscope</td><td>JEOL</td><td>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 ...

utilization-capsules-for-negative-staining-viral-samples-within

Research

JoVE Journal

Immunology and Infection

10.2K Views.  United States Army Medical Research Institute of Infectious Diseases (USAMRIID).  Dit protocol bevat instructies voor negatieve kleuring virusmonsters die gemakkelijk in BSL-2, -3 of -4 laboratoria kunnen worden gebruikt. Het omvat het gebruik van een innovatieve verwerkingscapule die het transmissie-elektronenmicroscopie rooster beschermt en de gebruiker makkelijker in de meer turbulente omgevingen in biocontainer vergemakkelijkt. 

Video: Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay.

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

Genetics

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

Biochemistry

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
First, a threshold image is generated based on intensity values uniquely calculated for each image. Then, circles are detected based on contrast between the object and background using the well-established Circle Hough Transformation (CHT) algorithm. Finally, the detected cell capsules and bodies are matched according to center coordinates and radius size, and data is exported to the user in a manageable spreadsheet.
The advantages of this technique are simple but significant. First, because these calculations are performed by an algorithm rather than a human both accuracy and reliability are increased. There is no decline in accuracy or reliability regardless of how many samples are analyzed. Second, this approach establishes a potential standard operating procedure for the Cryptococcus field instead of the current situation where capsule measurement varies by lab. Third, given that manual capsule measurements are slow and monotonous, automation allows rapid measurements on large numbers of yeast cells that in turn facilitates high throughput data analysis and increasingly powerful statistics.
The major limitations of this technique come from how the algorithm functions. First, the algorithm will only generate circles. While Cryptococcus cells and their capsules take on a circular morphology, it would be difficult to apply this technique to non-circular object detection. Second, due to how circles are detected the CHT algorithm can detect enormous pseudo-circles based on the outer edges of several clustered circles. However, any misrepresented cell bodies caught within the pseudo-circle can be easily detected and removed from the resulting data sets.
This technique is meant for measuring the circular polysaccharide capsules of Cryptococcus species based on India Ink bright field microscopy; though it could be applied to other contrast based circular object measurements.

Automated Measurement of Cryptococcal Species Polysaccharide Capsule and Cell Body

This technique describes an automated batch image processor designed to measure polysaccharide capsule and body radii. While initially designed for Cryptococcus neoformans capsule measurements the automated image processor can also be applied to other contrast based detection of circular objects.

The purpose of this technique is to provide a consistent, accurate, and manageable process for large numbers of polysaccharide capsule measurements.
...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Biology

Microscopy and Staining: Gram, Capsule, and Endospore Staining

Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1 
1 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America
Bacteria are diverse microorganisms found nearly everywhere on Earth. Many properties help distinguish them from each other, including but not limited to Gram-staining type, shape and arrangement, production of capsule, and formation of spores. To observe these properties, one can use light microscopy; however, some bacterial characteristics (for example size, lack of coloration, and refractive properties) make it hard to distinguish bacteria solely with a light microscope (1, 2). Staining bacteria is necessary when distinguishing bacterial types with light microscopy. The two main types of light microscopes are simple and compound. The main difference between them is the number of lenses used to magnify the object. Simple microscopes (for example a magnifying glass) have only one lens to magnify an object, while compound microscopes have several lenses to enhance magnification (Figure 1). Compound microscopes have an objective lens close to the object which collects light to create an image of the object. This is then magnified by the eyepiece (ocular lens) which enlarges the image. Combining the objective lens and eyepiece allows for higher magnification than using a single lens alone. Typically, compound microscopes have multiple objective lenses of varying powers to allow for different magnification (1, 2). Here, we will discuss visualizing bacteria with Gram stains, Capsule stains, and Endospore stains.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10513/10513fig1.jpg" /> 
Figure 1: A typical compound microscope. The most important parts of the microscope are labeled.
The Gram stain, developed in 1884 by the Danish bacteriologist Hans Christian Gram (1), differentiates bacteria based on the composition of the cell wall (1, 2, 3, 4). Briefly, a bacterial smear is placed on a microscope slide and then heat-fixed to adhere the cells to the slide and make them more readily accepting of stains (1). The heat-fixed sample is stained with Crystal Violet, turning the cells purple. The slide is flushed with an Iodine solution, which fixes the Crystal Violet to the cell wall, followed by a decolorizer (an alcohol) to wash away any non-fixed Crystal Violet. In the final step, a counterstain, Safranin, is added to color cells red (Figure 2). Gram-positive bacteria stain purple due to the thick peptidoglycan layer which is not easily penetrated by the decolorizer; Gram-negative bacteria, with their thinner peptidoglycan layer and higher lipid content, destain with the decolorizer and are counterstained red when Safranin is added (Figure 3). Gram staining is used to differentiate cells into two types (Gram-positive and Gram-negative) and is also useful to distinguish cell shape (spheres or cocci, rods, curved rods, and spirals) and arrangement (single cells, pairs, chains, groups, and clusters) (1, 3).

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10513/10513fig2.jpg" /> 
Figure 2: Schematic of the Gram Staining Protocol. The left column shows how Gram-negative bacteria react at each step of the protocol. The right column shows how Gram-positive bacteria react. Also, shown are two typical bacterial cell shapes: the bacilli (or rods) and the cocci (or spheres).

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10513/10513fig3.jpg" /> 
Figure 3: Gram Staining Results. A Gram stain of a mixture of Staphylococcus aureus (Gram-positive purple cocci) and Escherichia coli (Gram-negative red rods).
Some bacteria produce an extracellular viscous outer layer called a capsule (3, 5). Capsules are protective structures with various functions, including but not limited to adherence to surfaces and other bacteria, protection from desiccation, and protection from phagocytosis. Capsules are typically composed of polysaccharides containing more than 95% water, but some may contain polyalcohols and polyamines (5). Due to their mostly non-ionic composition and tendency to repel stains, simple staining methods do not work with capsule; instead, capsule staining uses a negative staining technique which stains the cells and the background, leaving the capsule as a clear halo around the cells (1, 3) (Figure 4). Capsule staining involves smearing a bacterial sample into an acidic stain on a microscope slide. Unlike Gram staining, the bacterial smear is not heat-fixed during a Capsule Stain. Heat-fixing can disrupt or dehydrate the capsule, leading to false negatives (5). Furthermore, heat-fixing can shrink cells resulting in a clearing around the cell which can be mistaken as a capsule, leading to false positives (3). The acidic stain colors the slide background; while follow up with a basic stain, Crystal Violet, colors the bacterial cells themselves, leaving the capsule unstained and appearing as a clear halo between the cells and the slide background (Figure 5). Traditionally, India ink has been used as the acidic stain because these particles cannot penetrate the capsule. Therefore, neither the capsule nor the cell is stained by India ink; instead, the background is stained. Congo Red, Nigrosin, or Eosin can be used in place of India ink. Capsule staining can help doctors diagnose bacterial infections when looking at cultures from patient samples and guide appropriate patient treatment. Common diseases caused by encapsulated bacteria include pneumonia, meningitis, and salmonellosis.

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10513/10513fig4.jpg" /> 
Figure 4: Schematic of the Capsule Staining Protocol. The top panel shows the slide smear prior to any stain application. The middle panel shows how the slide and bacteria look after the primary stain, Congo Red. The final panel shows how the slide and bacteria look after the counterstain, Crystal Violet.

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/10513/10513fig5.jpg" /> 
Figure 5: Capsule Staining Results. Capsule staining of encapsulated Acinetobacter baumannii (denoted with black arrows) and non-encapsulated Escherichia coli (denoted with white arrows). Notice the background is dark and A. baumannii cells are stained purple. The capsule around A. baumannii cells is evident as a halo, while E. coli has no halo.
In adverse conditions (for example nutrient limitation, extreme temperatures, or dehydration), some bacteria produce endospores, metabolically inactive structures that are resistant to physical and chemical damage (1, 2, 8, 9). Endospores allow the bacterium to survive harsh conditions by protecting the genetic material of the cells; once conditions are favorable for growth, spores germinate, and bacterial growth continues. Endospores are difficult to stain with standard staining techniques because they are impermeable to dyes typically used for staining (1, 9). The technique routinely used to stain endospores is the Schaeffer-Fulton Method (Figure 6), which uses the primary stain Malachite Green, a water soluble stain that binds relatively weakly to cellular material, and heat, to allow the stain to break through the cortex of the spore (Figure 7). These steps color the growing cells (termed vegetative cells in the context of endospore biology), as well as endospores and any free spores (those no longer within the former cell envelope). Vegetative cells are washed with water to remove Malachite Green; endospores retain the stain due to heating the Malachite Green within the spore. Finally, the vegetative cells are counterstained with Safranin to visualize (Figure 8). Staining for endospores helps differentiate bacteria into spore formers and non-spore formers, as well as determines whether spores are present in a sample which, if present, could lead to bacterial contamination upon germination.

<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/10513/10513fig6.jpg" /> 
Figure 6: Schematic of the Endospore Staining Protocol. The left column shows how spore forming bacteria react at each step of the protocol. The right column shows how non-spore forming bacteria react.

<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/10513/10513fig7.jpg" /> 
Figure 7: Diagram of Endospore Structure. Bacterial cell containing an endospore with the various spore structures labeled.

<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/10513/10513fig8.jpg" /> 
Figure 8: Endospore Staining Results. A typical staining of endospores of Bacillus subtilis. The vegetative cells (denoted with the white arrows) are stained red, while the endospores (denoted with the black arrows) are stained green.

Microscopy and Staining Techniques in Bacteria

Source: Rhiannon M. LeVeque1, Natalia Martin1, Andrew J. Van Alst1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan ...

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Microbiology

Capsule-Based Negative Staining of Virus Samples on TEM Grids: A Heavy Metal Staining Technique to Visualize the Ultrastructure of Enveloped Viruses

Source: Blancett, C. D., et al. <a href="https://www.jove.com/v/56122">Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the negative staining of enveloped virus samples using a processing capsule within the biocontainment environment. The negatively stained viruses can be visualized using transmission electron microscopy to investigate viral structure and morphology with nanometer resolution.

Source: Blancett, C. D., et al. Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment. J. Vis. Exp. (2017)
In this video, ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

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