One of the most important procedures in virology is measuring the virus titer – the concentration of viruses in a sample. A widely used approach for determining the quantity of infectious virus is the plaque assay. This technique was first developed to calculate the titers of bacteriophage stocks. Renato Dulbecco modified this procedure in 1952 for use in animal virology, and it has since been used for reliable determination of the titers of many different viruses.
To perform a plaque assay, 10-fold dilutions of a virus stock are prepared, and 0.1 ml aliquots are inoculated onto susceptible cell monolayers. After an incubation period, to allow virus to attach to cells, the monolayers are covered with a nutrient medium containing a substance, usually agar, that causes the formation of a gel. When the plates are incubated, the original infected cells release viral progeny. The spread of the new viruses is restricted to neighboring cells by the gel. Consequently, each infectious particle produces a circular zone of infected cells called a plaque. Eventually the plaque becomes large enough to be visible to the naked eye. Dyes that stain living cells are often used to enhance the contrast between the living cells and the plaques. Only viruses that cause visible damage of cells can be assayed in this way. An example of plaques formed by poliovirus on a monolayer of HeLa cells is shown at left. In this image, the cells have been stained with crystal violet, and the plaques are readily visible where the cells have been destroyed by viral infection.
The titer of a virus stock can be calculated in plaque-forming units (PFU) per milliliter. To determine the virus titer, the plaques are counted. To minimize error, only plates containing between 10 and 100 plaques are counted, depending on the size of the cell culture plate that is used. Statistical principles dictate that when 100 plaques are counted, the sample titer will vary by plus or minus 10%. Each dilution is plated in duplicate to enhance accuracy. In the example shown below, there are 17 plaques on the plate made from the 10-6 dilution. The titer of the virus stock is therefore 1.7 x 108 PFU/ml.
Next we’ll consider how the plaque assay can be used to prepare clonal virus stocks, a step that is essential for studying viral genetics.
Dulbecco, R., & Vogt, M. (1953). Some problems of animal virology as studied by the plaque technique. Cold Spring Harbor Symp. Quant. Biol., 18, 273-279
Thank you for the post. Plaque assays can be done to determine infectious virus titer for those viruses whose susceptible cells grow in monolayers however some viruses are grown in suspension because of the nature of the host cell e.g. HIV in H9 cells, HHV-8 from induced KS-1 cells. For these viruses, infectious virus titer cannot is determined by plaque assay rather by TCID50. Now, TCID50 is not absolute quantitation of infectious virus unlike plaque assay which is absolute. TCID50 titer is calculated by monitoring viral CPE which is not always easy because some viruses do not produce CPE. In these cases, how can one calculate infectious virus titer?
Thank you.
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TCID 50 is not an assay per se but a calculation method. It can be used if CPE is present but it's usefulness is more evident when no CPE is present, thus the need for an alternative method to plaque assays (a plaque is a CPE). In the case of human coronaviruses (HCoV) an immunoperoxydase assay can be done in 96-well plaque with dilutions of the virus and the TCID calculated based on wich well contains infected (positive) cells (http://www.jove.com/index/details.stp?ID=751). No CPE is detected, only the presence of viral antigens on the surface of the infected cells.
A further note, the mouse coronaviruses (MHV) can be detected by plaque assay despite the fact that they are not “lysogenic”. They wont kill the infected cells per se but induce the fusion of neighbouring cells. The scincytia they form can either be seen using a microscope or by the plaque assay (the scincytia are fragaile and when pouring the fixating agent they get dislodgedm, leaving a plaque).
Raj. About your question, for quantifying infectious viruses you can use immunofluorescence assays and count the focus of infection or currently there are new works to improve the values obtained by qPCR to determinate the infectious viruses, for example with a pre-treatment to eliminate viruses with structural damages, quantifying only infectious viruses.
Vince, do you think that quantitative PCR is an accurate means of comparing influenza virus between different samples, say in a controlled experiment? It seems that most papers stick with plaque assays.
Q-PCR measures viral RNA, not infectivity – a plaque assay is the best
measure for infectivity. So it depends on what you want to measure.
Personally I find plaque assays far easier to carry out than Q-PCR.
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Hi, I just want to know the formula used for the determination of the PFU? Additionally, isn't the stock concentration relevant to the dilution? What stock concentration are you assuming was present there?
Thanks!
The formula for calculating plaque titer is: (number of plaques)
divided by (dilution of the stock virus on the plate that was
counted). In the example in the figure: (17 plaques) divided by
(10e-8) is 1.7 x 10e8 pfu/ml. If you plate a wide range of dilutions
you don't need to know the concentration of the stock – you only need
to know how much to dilute which is related to the maximum virus titer
expected. In this example, the virus is known not to exceed 10e9
pfu/ml so the highest dilution need only be 10e-8.
Actually i would to detect the cyanophages in Singapore. I found that, I cant get plaque but I get clear or lawn. Is it also because of cyanophage present. Thanks.
If the lawn is cleared then you should be able to get plaques. Have
you tried many dilutions? Different agar?
Thanks for your comment. Is is the whole agar (clear) as a plaque? I have not tried on many dilutions, I will try on it. But, why the different agar can give different result? I was trying on the filamentous cyanobacteria phages, do you think this type of bacteria phages able to give small plaque?? Thanks.
DIfferent types of agar (oxoid, noble, bacto, agarose) have different
components that may inhibit plaque formation. For example, certain
rhinoviruses will form plaques only in overlays containing type VII
agarose, not any other type of agar. Yes, there is no reason why a
filamentous phage cannot form a plaque. If the phage clears a lawn of
bacteria then on higher dilution you should see plaques.
Great explanation !! I like it.
DIfferent types of agar (oxoid, noble, bacto, agarose) have different
components that may inhibit plaque formation. For example, certain
rhinoviruses will form plaques only in overlays containing type VII
agarose, not any other type of agar. Yes, there is no reason why a
filamentous phage cannot form a plaque. If the phage clears a lawn of
bacteria then on higher dilution you should see plaques
I'm trying to get information on the optimum volume (or thickness) of the infecting aliquot in the plaque assay. In the above example you use 0.1 ml. As the thickness of this liquid layer affects the probability of virus particle attaching to cells during the adsorption period, it should be matched with the surface area of the monolayer, adsorption time, and temperature. What literature is out there on these factors, and is this virus specific?
Get help!! Thanks
Get help!!! Thanks
Hi Dr Vince,
In case of emerging Picornaviruses, the virus with less adaptation to cell culture (which improves upon 2-3 passages), which method could be more suitable for quantifying the infectious viral particles?
And Thank you for the Virology Infection. Which appears to be like that of HIV.
If the virus causes cytopathic effect in cells it can be titrated
using an endpoint assay, such as 50% tissue culture infectious doses
(TCID50). This is the amount of virus needed to cause cytopathic
effect in 50% of infected cultures. See
https://virology.ws/2009/07/13/measurement-o….
how to stain the plaques, I mean do we need to add agar containing stain in nutrient medium to the plate?
how to stain the plaques, I mean do we need to add agar containing stain in nutrient medium to the plate?
it seems like the procedure is too complicated i am a new micro student , is there a simpler way to do plaque assay? or ways to improve the procedure to get a more readable and countable result?
Depending on the goal, the stain can be either added to the overlay,
or added to the cells after the overlay has been removed. For example,
neutral red dye can be added to the agar overlay to visualize plaques
more easily for isolation. If the goal is not to isolate plaques, but
merely to count them, then the monolayer can be stained after the agar
is removed with a dye such as crystal violet.
The plaque assay is not complicated; it is one of the simplest, and
most informative procedures in virology. I'm making a video of the
entire process which will show how easy it is.
Hi Sir, good day! im a 4th year BS Chemistry student. For us to graduate, we are required to have an undergraduate thesis. The topic that I want to study is entitled “The Antiwart Activity of Ficus septica”. My problem is, how can I culture this kind of virus which is human papillomavirus (HPV) in a simplest yet best method? What is the best assay that I can do to detect the presence of this virus? I will deeply appreciate your help Sir? Thank you very much. God bless and more power.
I'm currently trying to enumerate very low levels of T4 bacteriophage using the plaque assay and I've found that the limit of detection may not be high enough. WHat is the limit of detection of the plaque assay and is there another assay method I could use with a lower limit of detection?
Thanks
The detection limit is roughly 10 PFU per ml, if you are plating out
0.1 ml per plate. You could use a limit dilution method (such as 50%
infectious doses) which involves more assay plates and can therefore
detect fewer than 10 PFU per ml, but the readout is cell killing, not
plaquing. Have you considered nucleic acid detection methods such as
PCR?
Thank you, my next step was to look at other detection methods such as PCR but I was unsure of the detection limits for this assay also, should PCR be able to detect even 1 viable phage? Thanks
PCR will not give information on viable phages, only on the presence
of phage DNA. It should be possible to refine the PCR to detect one
genome.
Hi Dr Vincent,
I’m actually having problems with the variability of the plaque assay. I have repeated the assay several times to determine the concentration of my viral stock and in occasions the variability is two-fold and even greater.
Is there an inherent variability of the technique or is it me to blame?
Thank you in advance (great informative website by the way).
In a plaque assay, a two-fold variability is routine due to errors in
pipetting, dilution, etc. Five-fold would not be acceptable.
Hi Sir, I'd like to know is there any reason, other than preventing contamination, that when we perform plaque assay to titer influenza viruses, we need to invert the plates? Some say it's due to the polarity of influenza virus growth. But that's not quite convincing.
Dear Dr Vincent,
I'm currently doing plaque assay using Vero cells to quantify my Chikungunya virus. I'm now optimizing the incubation period prior to staining. I can see plaque formation 2 days PI via naked eyes. My question is, is it too soon for me to stain the plaques? Or should I incubate longer? Because when I tried incubating them for 3 days but the plaques formed are large and start to overlap each other. I appreciate your help. Thanks in advance.
If the plaques overlap after 3 days incubation then you should
certainly stain after two days.
Hi Dr. Vincent,
I am looking for ways of optimizing plaque assay. In terms of time, or the quality of results. Do you have any suggestions
Hello. I am a biochemist on sabbatical leave in a picornavirus laboratory learning some basic virology techniques. I am working with EMCV but am having trouble with the plaque assay procedure. The lab uses agarose overlays, removes the overlays after incubation, and then fixes/stains with crystal violet/MeOH/H2O solution. This seems to work fine with HeLa cells. When I use this procedure with EMCV-infected L929 cells, however, the cells come off the plate with the overlay. I can see some clear plaques in the wells, and I can see the intact monolayers of cells under a ‘scope before I remove the agarose plugs. Are there any suggestions for keeping the cells attached to the well surface instead of peeling off with the overlay?
Before peeling off the agarose overlay, add enough 10%
tricholoroacetic acid to cover the overlay and incubate 10 minutes.
This will fix the cells to the plastic so they do not come off. We use
this step routinely for all of our plaque assays.
Thank you! We ordered TCA today.
hi
I am trying to do a plaque assay for JEV but I could not get any plaques.
Can you please suggest me some reasons as to why this is happening??
can JEV be inactivated due to some reasons??
There are many factors that determine whether or not a virus will form
plaques in a cell line. It’s important to know that the virus is
cytopathic in the cells, but that alone is not sufficient. The easiest
variable to change is the overlay: there are many different types
(agar, agarose, methylcellulose) and they should be evaluated
individually.
Hi,
Do you have a reference for this detection limit?
Thanks,
Dave
May I know in which cell line you are using to titrate JEV.
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I am trying to do a plaque assay with chikungunya but this virus does not produce a clear cut CPE. Kindly let me know if I can proceed with the plaque assay
I would proceed…sometimes CPE isn’t a predictor of good plaquing.
I have the same problem of having cells come off the plate with the overlay. Besides TCA, any other chemical you can recommend? Thanks very much
Try 10% methanol.
You will only be able to assess the antiwart activity on DNA replication by assaying for viral copy number. You cannot reasonably assess effect of any compound on production of virions because virion production of HPV has no cell model and is not cytolytic.Â