Authors would like to acknowledge Dr. Frank Scholle for his helpful comments in the manuscript, Chloe Mariant for her help with the microscopy images and Teresa M. Tiedge for her helpful English revision.

1. Titration protocol
NOTE: Use a cytopathic virus infecting adherent cells. For this demonstration, Influenza A Virus (IAV) of swine origin (A/California/07/2009/(H1N1)) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 2, strain NC 1-7-4 were used.
<ol>
	<li>Titrate these viruses in 96 well plates for 7 days in a biosafety cabinet located in a Biosafety Level 2 (BSL-2) laboratory.
	<ol>
		<li>To perform these titrations, seed 96 well plates with the required cell line. For PRRSV, use MA-104 cell line and for IAV use MDCK cell line. For the cell culture, use DMEM medium supplemented with 10% FBS, L-Glutamine and Penicillin-Streptomycin and grow the cells to confluency.</li>
		<li>Before infection, wash the cells using 200 &#181;L of PBS.</li>
		<li>Dilute the virus stocks using 10 fold dilutions series by mixing 900 &#181;L of media and 100 &#181;L of virus. Make sure to properly vortex the tube to ensure proper mixing of the medium and virus and to avoid dilution errors.</li>
		<li>Mark the layout of the plate on the lid. Wash the wells with 1x phosphate saline buffer (PBS). Add 50 &#181;L of the inoculum in the corresponding wells following previously described titration methods2,3.</li>
		<li>Incubate at 37 &#176;C in a 5% CO2 incubator for 7 days.</li>
	</ol>
	</li>
</ol>
2. Cytopathic effect (CPE) assessment&#160;via microscopy
<ol>
	<li>After the 7-day incubation, wash all wells with 200 &#181;L of 1x PBS twice.</li>
	<li>Assess all wells of the plate under a light microscope to visually detect CPE. In the case of both PRRSV and IAV, their CPE consists of cell death and subsequent detachment from the plate, leading to monolayer disruption. However, other viruses may present different types of CPE.</li>
</ol>
3. Staining protocol
NOTE: Cytopathic effect (CPE) was assessed via crystal violet staining.
<ol>
	<li>After the 7-day incubation, wash all the wells using 200 &#181;L of 1x PBS twice.</li>
	<li>Fix the cells by adding 50 &#181;L/well of 4% paraformaldehyde (PFA) in 1x PBS and incubate for 15 min at room temperature (RT).</li>
	<li>After incubation, wash the cells twice with 200 &#181;L of 1x PBS. Then, add 50 &#181;L/well of crystal violet diluted to 4% in water and incubate for 5 min at RT. 
	NOTE: Crystal violet chemical stains the cells that remain attached to the plate at the time of fixation, leaving the sections of the well where the cells detached as unstained.</li>
	<li>Finally, aspirate crystal violet from the wells and optionally leave the plates to air dry for 2-5 min at RT or wash the plate with 200 &#181;L of water to remove excess stain prior to visualization.</li>
	<li>Use previously described methods to mathematically calculate the titer. Here, Karber formula and Muench formula were applied for PRRSV and IAV, respectively2,3. Details of these equations are presented in the representative result section.</li>
</ol>
4. Immunocytochemical (ICC) labeling
NOTE: Immunocytochemistry labeling for both viruses was performed following previously described methods9,10,11.
<ol>
	<li>After the 7-day incubation of the titration, fix the cells using PFA as described in step 3.2.</li>
	<li>Wash the plates with a solution composed by 100 mL of 1 M Tris hydrochloride, 1 g of Saponin, and 8.5 g of NaCl in 900 mL of H2O. Then, complete two additional washes with a mixture of 1x PBS and 5% Fetal Bovine Serum (FBS) and incubate with that Tris-Saponin-NaCl solution at Room temperature (RT) for 20 min.</li>
	<li>Incubate all the wells with the primary antibody for 2 h at RT. 
	NOTE: The volume of primary antibodies for each virus was 100 &#181;L and was prepared using a 1:300 dilution of 2% FBS in 1x PBS.</li>
	<li>Wash the cells twice with the Tris, Saponin, and NaCl solution prepared in step 4.2.</li>
	<li>Incubate all the wells with the secondary antibody for 1 h at RT. 
	NOTE: The volume of secondary antibody was 100 &#181;L and was prepared using a 1:250 dilution of 2% FBS in 1x PBS.</li>
	<li>Wash the cells with the Tris, Saponin, and NaCl dilution as described in step 4.2.</li>
	<li>Aspirate the previous dilution and incubate the plates with 200 &#181;L/well of aminoethyl carbazole (AEC) solution at a final dilution of 1:50 in water following the manufacturer&#39;s directions for 30 min at RT.</li>
	<li>After incubation, discard the AEC solution and add 100 &#181;L/well of 1x PBS for imaging via microscopy.</li>
	<li>Mathematically derive the titer as described in step 3.5.</li>
</ol>

<ol>
	<li>K&#228;rber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+to+the+collective+treatment+of+pharmacological+series+experiments.">Contribution to the collective treatment of pharmacological series experiments.</a> Naunyn-Schmiedeberg's Archive for Experimental Pathology and Pharmacology. 162 (4), 480-483 (1931).</li><li>Ramakrishnan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+50%+endpoint+titer+using+a+simple+formula.">Determination of 50% endpoint titer using a simple formula.</a> World Journal of Virology. 5 (2), 85-86 (2016).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+per+cent+endpoints.">A simple method of estimating fifty per cent endpoints.</a> American Journal of Epidemiology. 27 (3), 493-497 (1938).</li><li>Spearman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+method+of+right+and+wrong+cases+(constant+stimuli)+without+Gauss's+formulae.">The method of right and wrong cases (constant stimuli) without Gauss's formulae.</a> British Journal of Psychology. 2 (3), 227 (1908).</li><li>Darling, A. J., Boose, J. A., Spaltro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+assay+methods:+accuracy+and+validation.">Virus assay methods: accuracy and validation.</a> Biologicals. 26 (2), 105-110 (1998).</li><li>Kim, J., Chae, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+virus+isolation,+polymerase+chain+reaction,+immunohistochemistry,+and+in+situ+hybridization+for+the+detection+of+porcine+circovirus+2+and+porcine+parvovirus+in+experimentally+and+naturally+coinfected+pigs.">A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs.</a> Journal of Veterinary Diagnostic Investigation. 16 (1), 45-50 (2004).</li><li>Suchman, E., Blair, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytopathic+effects+of+viruses+protocols.">Cytopathic effects of viruses protocols.</a> American Society of Microbiology. , (2007).</li><li>Karakus, U., Crameri, M., Lanz, C., Y&#225;ng&#252;ez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Influenza Virus. , 59-88 (2018).</li><li>Tingstedt, J. -. E., Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+immune+responses+in+the+lungs+of+pigs+infected+in+utero+with+PRRSV:+an+immunohistochemical+study.">Cellular immune responses in the lungs of pigs infected in utero with PRRSV: an immunohistochemical study.</a> Viral Immunology. 17 (4), 558-564 (2004).</li><li>Guarner, J., 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=Immunohistochemical+and+in+situ+hybridization+studies+of+influenza+A+virus+infection+in+human+lungs.">Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs.</a> American Journal of Clinical Pathology. 114 (2), 227-233 (2000).</li><li>Nicholls, J. M., 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=Detection+of+highly+pathogenic+influenza+and+pandemic+influenza+virus+in+formalin+fixed+tissues+by+immunohistochemical+methods.">Detection of highly pathogenic influenza and pandemic influenza virus in formalin fixed tissues by immunohistochemical methods.</a> Journal of Virological Methods. 179 (2), 409-413 (2012).</li></ol>

The authors have nothing to disclose.

Viral titrations are routinely used in virology research, with PFU detection and TCID50 assays most commonly used1,2,3,4. Both methods rely on the detection of CPE in infected cells, and even though they can be visually assessed via microscopy, a staining is usually applied to obtain a more objective result or to even reduce incubation times. In the case of TCID50, one of the most commonly used alternatives to visual CPE detection that offers an accurate and objective measure is ICC staining6, which is time consuming and often expensive due to the utilization of specific antibodies9,10. However, even though this type of assay also benefits from the application of crystal violet staining for visualization, there are no comparative studies assessing the differences in sensitivity between ICC and crystal violet staining. Thus, the protocol proposed here is a suitable option for TCID50-based viral stock titrations, as the application of crystal violet showed a comparable level of accuracy to the sensitive immunocytochemical intracellular staining targeting specific virus protein. This method is less time consuming, generally easier to execute and more accurate than CPE detection via microscopy, demonstrating that this approach is beneficial to use for virus titrations that are performed on a regular basis.
The critical steps for this crystal violet staining are a proper fixation of the cells after the 7-day incubation period for the titration and the application of enough crystal violet to cover the entire surface of the wells. To achieve successful staining, ensure that non-infected cells were properly covered with crystal violet, and that all wells are stained. This indicates that the cells were healthy, and the protocol worked as expected.
In conclusion, the utilization of crystal violet staining is proposed as a faster and less costly alternative for the objective detection of CPE on TCID50 assays, with similar accuracy to the specific ICC intracellular labeling commonly applied during this type of titration. However, there may be cases where the pathogen-specific antibodies are able to detect positive wells that would go undetected by our crystal violet method, since ICC positives will show successfully infected cells, even before the infection leads to CPE. Therefore, even though we did not find significant differences between the two, it is strongly recommended to test both methods to assess the level of sensitivity in each virus titration to determine potential differences and subsequent adjustments before using this staining routinely.

Viral titration via TCID50 assay is a commonly used technique in infectious disease research1. Although variations on the math behind this method have been proposed over time1,2,3,4, the currently applied methods of infection detection rely on visual confirmation through the presence of cytopathic effect (CPE) using microscopy5. To confirm CPE visualization more objectively on TCID50 assays, immunocytochemical (ICC) intracellular staining targeting the proteins of the virus is one of the most-commonly used methods6 as different viruses can produce varying forms of CPE. In our case, the cell morphological changes are similar when infected with both Influenza A virus (IAV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), where infected cells round up and detach from the plate. In the case of PRRSV, it causes a CPE known as &#34;total destruction&#34;, where all cells end up detaching from the well. IAV on the other hand, can present both total destruction and an additional CPE known as &#34;subtotal destruction&#34; where a small number of cells do not detach after infection7. However, this technique is time consuming to perform and requires the use of relatively costly reagents. It is important to note that ICC does not label CPE, but rather the number of cells successfully infected by the virus. This implies that the cells that were successfully infected by the end of the incubation will be seen as positive even if the infection has not caused CPE yet, and thus, a higher percentage of ICC positive cells compared to CPE is expected. For that reason, in this study we describe a complementary method of visual detection of CPE in a TCID50 assay based on crystal violet, a chemical with a positive charge that attaches to cell membranes and is used to stain adherent cells. Crystal violet is often utilized in virology research to measure plaque-forming units, among others8.
In this study, we compare the sensitivity of non-stained microscopy CPE detection with crystal violet staining and immunocytochemical staining based on viral protein recognition, which is known to be more objective due to its high sensitivity. This study shows that both crystal violet and immunocytochemical staining are more accurate than visual microscopy-based CPE detection and can be used to objectively identify infected wells in a TCID50 titration. Given their ability to reach nearly identical level of accuracy on the cytopathic viruses tested in cell lines, crystal violet is presented as a faster and more affordable way to determine viral titrations on a TCID50 assay. The proposed method using crystal violet staining takes a total time of 40 min to perform, with 15 min for paraformaldehyde (PFA) incubation, 5 min for crystal violet incubation and a maximum of 15 min for material preparation, buffer washes, and drying. The immunocytochemistry protocol applied for comparison takes an average time of 4 h 30 min and was performed as previously described9,10. The method proposed aims to help visualize a completed viral titration. Infection and incubation times can be performed with different layout depending on the virus. Here we tested two RNA viruses with cytopathic effect on cell lines.

<table><tbody><tr><td>96-well cell culture plates</td><td>Genesee</td><td>25-221</td><td>Clear, flat bottom</td></tr><tr><td>AEC solution</td><td>Thermo Fisher</td><td>1122</td><td></td></tr><tr><td>Crystal violet</td><td>Thermo Fisher</td><td>C581-25; C581-100</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>Fetal bovine serum</td><td>BioWest</td><td>S1480</td><td></td></tr><tr><td>Paraformaldehide</td><td>Thermo Fisher</td><td>J19943</td><td></td></tr><tr><td>Primary Influenza Antibody</td><td>Bioss</td><td>BS-0344R</td><td></td></tr><tr><td>Primary PRRSV Antibody</td><td>Bioss</td><td>BS-10043R</td><td></td></tr><tr><td>Saponin</td><td>Thermo Scientific</td><td>AAA1882014</td><td></td></tr><tr><td>Secondaty antibody</td><td>Invitrogen</td><td>31460</td><td></td></tr><tr><td>Tris Hydrochloride</td><td>Thermo Scientific</td><td>AM9856</td><td></td></tr></tbody></table>

use of crystal violet to improve visual cytopathic effect-based reading for viral titration using tcid50 assays

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are the two main methods to calculate the titer of a virus stock and are often based on microscopy detection or cell staining for visualization. In the case of TCID50 assay, objective visualization is commonly based on immunocytochemical (ICC) staining of intracellular virus to calculate titers combined with visual CPE detection via microscopy. However, ICC staining is costly and time consuming. In this study, we compared visual CPE observation via microscopy, ICC staining and crystal violet staining to determine the titers of two CPE-forming viruses, Influenza A virus (IAV) of swine origin and Porcine Reproductive and Respiratory Syndrome virus (PRRSV). We show that both crystal violet and ICC staining are more accurate than visual CPE detection, presenting nearly identical levels of precision on both IAV and PRRSV. For this reason, here we present crystal violet staining as a faster and more affordable way to determine viral titrations on a TCID50 assay for CPE-forming viruses titrated in cell lines.

The equations used for mathematically calculating the titer were previously described2,3.
Briefly, for PRRSV, we apply the Karber method:
Titer (TCID50) = 10 T + 1.3 where:
<img alt="Static equilibrium equation T formula, symbols, mathematical concept, education research." src="/files/ftp_upload/63063/63063eq01.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1">
In this formula d = negative log of the last dilution with complete positive virus response: five positive replicates; r = log of dilution range; N = number of replicates by dilution; n = number of wells with positive virus response on the next dilutions.
In the case of IAV, we use the Muench formula:
<img alt="Titer calculation formula with TCID50, CPE, proportionate distance parameters for virology analysis." src="/files/ftp_upload/63063/63063eq02.jpg" data-altupdatedat="1747911167000" data-alt="Equation 2">
For details on each mathematical method, refer to Ramakrishnan et al., (2016) and Reed et al., (1938)2,3.
As a visual example, the titer obtained for PRRSV shown in Figure 1 would be calculated as follows:
<img alt="Mathematical formula for calculating T with nested fractions, used in algebraic equation solving." src="/files/ftp_upload/63063/63063eq03.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1"> 
<img alt="Titer calculation formula, Titer = 10^(5.7+1.3), displayed as text equation." src="/files/ftp_upload/63063/63063eq04.jpg" data-altupdatedat="1747911167000" data-alt="Equation 4"> 
<img alt="Virus titer formula, Titer = 10⁷ TCID50/ml, used in virology experiments." src="/files/ftp_upload/63063/63063eq05v2.jpg" data-altupdatedat="1747911167000" data-alt="Equation 5">
The number of positive wells obtained when ICC (left half of the plate) and Crystal Violet staining (right part of the plate) were compared was highly similar for both PRRSV (Figure 1) and IAV (Figure 2) and was in both cases able to detect between one and two positive wells more than the CPE observed prior to staining through microscopy, even though these differences were not statistically significant (Table 1). While the output obtained from crystal violet staining is usually a positive-negative well, using ICC there is a gradual decrease on the number of positive cells that get labeled as the virus gets more diluted. Furthermore, ICC requires the use of a microscope for visualization, while crystal violet can be easily performed by eye.
<img alt="Crystal violet assay plate; staining process; microtiter layout; cell proliferation; comparative analysis." class="xfigimg" src="/files/ftp_upload/63063/63063fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Comparison of crystal violet staining and immunocytochemical labeling (ICC) for PRRSV titration. (A) Visualization of the titration plate by eye, with the left-half corresponding to crystal violet staining and right-half corresponding to ICC; (B) Schematic representation of the plate with positive wells indicated with a '+' sign using crystal violet and ICC; (C) Microscopy image (10x objective) of crystal violet staining; (D) Microscopy image (10x objective) of positive wells detected by ICC. Ten-fold dilutions of the virus from -1 to -8 log. were performed. Images of positive wells from -1 log to -5 log. Uninfected cells were used as negative control (referred as negative). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63063/63063fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microplate assay results with crystal violet and ICQ; microscopy images show viral cytotoxicity." class="xfigimg" src="/files/ftp_upload/63063/63063fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Comparison of crystal violet staining and immunocytochemical labeling (ICC) for IAV titration. (A) Visualization of the titration plate by eye, with the left-half corresponding to crystal violet staining and right-half corresponding to ICC; (B) Schematic representation of the plate with positive wells indicated with a '+' sign using crystal violet and ICC; (C) Microscopy image (10x objective) of crystal violet staining; (D) Microscopy image (10x objective) of positive wells detected by ICC. Ten-fold dilutions of the virus from 10-1 to 10-8. were performed. Images of positive wells from -1 log to -5 log. Uninfected cells were used as negative control (referred as negative). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63063/63063fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Comparison of the titers obtained for both PRRSV and IAV using the three visualization approaches. Titers expressed as the average ± SEM of the log10 TCID50/mL in n = 5 replicates. No statistically significant differences were found between the groups (p &gt; 0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63063/Frias%20&amp;%20Crisci%20(2021)_Table%201.docx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays at JoVE.com

Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

This protocol shows an accurate and objective approach to visualize viral titrations using crystal violet, by comparing it with optical microscopy and immunocytochemical staining.

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are ...

use-crystal-violet-to-improve-visual-cytopathic-effect-based-reading

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.6K Views.  North Carolina State University. Het doel van dit protocol is om een virusvoorraadtitratie te produceren en deze te visualiseren met behulp van kristalvioletkleuring. Om dat te doen, zal een algemene virale titratie worden aangetoond, gevolgd door de kleuring. Deze titratie wordt uitgevoerd in een 96-well plaat gedurende een periode van zeven dagen in een bioveiligheidskast binnen een bioveiligheidsniveau 2 laboratorium.Om dit te doen, zal een 96-well plaat van de gewenste cellijn worden gezaaid met behulp van de over...

Video: Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Biology

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for the identification of candidate viral entry inhibitors. Here, we present the experimental approaches for the identification of small molecules capable of blocking infection by the non-enveloped coxsackievirus A16 (CVA16) through targeting the virus particles or specific steps in early viral entry. Assays include the time-of-drug-addition analysis, flow cytometry-based viral binding assay, and viral inactivation assay. We also present a molecular docking protocol utilizing virus capsid proteins to predict potential residues targeted by the antiviral compounds. These assays should help in the identification of candidate antiviral agents that act on viral entry. Future directions can explore these possible inhibitors for further drug development.

The goal of the protocol is to illustrate the different assays relating to viral entry that can be used to identify candidate viral entry inhibitors.

Antiviral assays that mechanistically examine viral entry are pertinent to discern at which step the evaluated agents are most effective, and allow for ...

Virus Propagation and Cell-Based Colorimetric Quantification

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and potentially Guillain-Barr&#233; syndrome in adults. Research on ZIKV to understand the disease mechanisms is important to facilitate vaccine and treatment development. The method of quantifying viruses is crucial and fundamental in the field of virology. The focus forming assay (FFA) is a virus quantification assay that detects the viral antigen with antibodies and identifies the infection foci of cells using the peroxidase immunostaining technique. The current study describes the virus propagation and quantification protocol using both 24-well and 96-well (high throughput) formats. Compared with other similar studies, this protocol has further described foci size optimization, which can serve as a guide to expand the use of this assay for other viruses. Firstly, ZIKV propagation is performed in Vero cells for 3 days. The culture supernatant containing ZIKV is harvested and quantitated using the FFA. Briefly, the virus culture is inoculated onto Vero cells and incubated for 2-3 days. Foci formation is then determined after optimized staining processes, including cell fixation, permeabilization, blocking, antibody binding, and incubation with peroxidase substrate. The stained virus foci are visualized using a stereo microscope (manual counting in 24-well format) or software analyzer (automated counting in 96-well format). The FFA provides reproducible, relatively fast results (3-4 days) and is suitable to be used for different viruses, including non-plaque-forming viruses. Subsequently, this protocol is useful for the study of ZIKV infection and could be used to detect other clinically important viruses.

The present protocol describes the propagation of Zika virus (ZIKV) in Vero African green monkey kidney cells and the quantification of ZIKV using cell-based colorimetric immunodetection methods in 24-well and 96-well (high throughput) formats.

Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus. ZIKV infection has been associated with congenital brain abnormalities and ...

Assay to Determine the Virus Infectious Dose of Drosophila C Virus in Cultured Insect Cells

Source: Yang, S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58845">Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the cytopathic effect assay to determine the tissue culture infective viral dose of Drosophila C virus on Drosophila S2* cells. A range of viral concentrations are used to infect the cultured cells. The proportion of cells exhibiting structural changes is determined, and this data is used to calculate the tissue culture infective dose.

1. Drosophila C virus load measure by CPE assay

	Pick 5 random virus-containing tubes to determine the average virus tissue culture infective dose ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

A Solid Phase Adherence Assay to Assess Biofilm Formation

Source: Nickerson, K. P. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57322">Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification.</a>&#160;J. Vis. Exp.&#160;(2018)
The video demonstrates a solid-phase adherence assay for assessing biofilm formation. In a multi-well plate, bacteria in bile salt-supplemented media adhere to solid surfaces, forming biofilms, which is confirmed by colorimetry through crystal violet staining and ethanol treatment.

1. Preparation of Reagents

	Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile ...

Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

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