Comments on: Detecting viruses: the plaque assay

By: Diarra

Diarra — Thu, 19 Jan 2012 09:14:00 +0000

Hi, i’m a master student,
My problem is that, at the end of my plaque i have some damage on the cells. Not all the bottom of the plate is intact, almost 10-30% of the bottom of my plate are damaged.
Please what are the possible causes of that?
Thank you

By: vinay

vinay — Fri, 06 Jan 2012 05:35:00 +0000

how to calculate virus copy number?

By: Cadaver Hibrido

Cadaver Hibrido — Tue, 20 Dec 2011 14:34:00 +0000

Rodrigo
To determinate the presence of virus, you should determinate the E2 and E6 genes. So, you can determinate if HPV is in integrated form or episomal status.
The load viral is determinated by the quantification of E6 copy number

By: Bosy

Bosy — Mon, 19 Dec 2011 21:02:00 +0000

بس ياجماعه حد ممكن يجاوبني علي سؤال ده اذا سمحتوا احنا عارفين ان الفيروس مش بيعيش الا داخل خلايا العائل طيب ازاي بيعيش علي ميديا عاديه احنا اخدنا التجارب ف المعمل بس الفيروس مش بيعيش علي ميديا لكن بيعيش ف عائل ياريت حد يجاوبني

By: Hjilrefavj

Hjilrefavj — Mon, 19 Dec 2011 20:56:00 +0000

شكرا علي الكلام العلمي الجميل ده جزاكم الله خير

By: profvrr

profvrr — Thu, 08 Dec 2011 21:52:00 +0000

You should count between 10-100 plaques (that is for a 10 cm plate).

By: Rajendharpadma

Rajendharpadma — Wed, 07 Dec 2011 15:08:00 +0000

hello sir,
I am testing chickungya virus titre by plaque assay method could you please tell me that how to minimize the variation between replicates and shall i consider 100 plaques for calculation or shall i consider in between 10-100 please help me in this aspect

By: Guest

Guest — Wed, 16 Nov 2011 18:37:00 +0000

Hi Sir,
Nice. Can you please also upload protocol for calculating viral titers after isolating from mouse organs (like Spleen or Lung)? I want to know about how to isolate viruses from organs and what solutions can I use for that? Thanks

By: Aaron Olea

Aaron Olea — Sat, 12 Nov 2011 03:18:00 +0000

w0w its so amazing !!!

By: Sonal lad

Sonal lad — Fri, 07 Oct 2011 13:29:00 +0000

hi sir,i am bsc microbiology student. and my problem is that ,now in msc we have done the practical of isolating the phagefor about 10 times but still we failed and still could not isolate the phage .so i want standard procedure and guidance.thank u for this post.

By: Abdul kapur

Abdul kapur — Thu, 22 Sep 2011 11:27:00 +0000

hi sir please explain the quantification,chaion virusracterization,and purificat

By: Saeed ahmed

Saeed ahmed — Sun, 11 Sep 2011 10:09:00 +0000

i have one question if you can help me i shall be very thankfull to you for this act my question is
the
bacterial OD value is 0.4 which is equivalent to 1*10^8 cfu/ml and the
bacteriophage valuse is 1*10^9 and i want the MOI valuse 0.1 then how i
can calculate it i am waiting for your reply.

By: Happychronicles

Happychronicles — Sun, 04 Sep 2011 22:14:00 +0000

Actually isn’t the value 1.7x10e-9?

By: Xenobio

Xenobio — Mon, 22 Aug 2011 10:13:00 +0000

If you can find some chikungunya-specific antiserum or monoclonal antibodies you could do an immunostaining procedure to detect foci of virus infection, instead of simply staining with crystal violet to see live vs. dead cells.

You can also try using an overlay of carboxymethylcellulose or Avicel (it’s a brand name for a mixture of cmc + insoluble cellulose) because they form suspensions that are viscous enough to stop the virus from moving around, but still liquid and can be aspirated. As opposed to solid agarose overlays which can be a bit tricky to peel off without messing up your cells.

By: OLUROPO2OLAJIDE

OLUROPO2OLAJIDE — Sun, 19 Jun 2011 18:27:00 +0000

The number of plate form represent the number of virus OLAJIDE OLUROPO

By: Curits

Curits — Fri, 13 May 2011 16:50:00 +0000

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.

By: profvrr

profvrr — Mon, 18 Apr 2011 16:43:00 +0000

Try 10% methanol.

By: Vinniethepolar

Vinniethepolar — Mon, 18 Apr 2011 16:37:00 +0000

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

By: profvrr

profvrr — Wed, 13 Apr 2011 18:50:00 +0000

I would proceed…sometimes CPE isn’t a predictor of good plaquing.

By: Drgvmd

Drgvmd — Mon, 11 Apr 2011 09:38:00 +0000

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

By: Are all virus particles infectious?

Are all virus particles infectious? — Fri, 21 Jan 2011 17:31:05 +0000

[...] we take the titer of a virus preparation (in plaque forming-units per milliliter) and divide it by into the number of virus particles in the sample, we obtain a [...]

By: Multiplicity of infection

Multiplicity of infection — Thu, 13 Jan 2011 14:58:30 +0000

[...] it is not possible to calculate the MOI the virus titer can be determined – for example by plaque assay or any other method of quantifying infectivity. Share this [...]

By: Sujit pujhari

Sujit pujhari — Tue, 11 Jan 2011 18:19:00 +0000

May I know in which cell line you are using to titrate JEV.

By: D_diston

D_diston — Sat, 04 Dec 2010 19:51:00 +0000

Hi,

Do you have a reference for this detection limit?

Thanks,
Dave

By: profvrr

profvrr — Tue, 30 Nov 2010 21:35:00 +0000

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.

By: Shalini

Shalini — Tue, 23 Nov 2010 08:19:00 +0000

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??

By: Tlawson

Tlawson — Mon, 01 Nov 2010 21:21:00 +0000

Thank you! We ordered TCA today.

By: profvrr

profvrr — Mon, 01 Nov 2010 15:32:00 +0000

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.

By: Tlawson

Tlawson — Sat, 30 Oct 2010 20:36:00 +0000

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?

By: Saad Saleem

Saad Saleem — Tue, 19 Oct 2010 16:24:00 +0000

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

By: profvrr

profvrr — Thu, 14 Oct 2010 20:00:12 +0000

If the plaques overlap after 3 days incubation then you should

certainly stain after two days.

By: Cassie

Cassie — Wed, 13 Oct 2010 15:57:49 +0000

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.

By: dracolll

dracolll — Sat, 04 Sep 2010 04:00:10 +0000

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.

By: profvrr

profvrr — Thu, 29 Jul 2010 22:13:47 +0000

In a plaque assay, a two-fold variability is routine due to errors in
pipetting, dilution, etc. Five-fold would not be acceptable.

By: Juan Carlos

Juan Carlos — Thu, 29 Jul 2010 21:23:50 +0000

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).

By: profvrr

profvrr — Thu, 08 Jul 2010 19:23:45 +0000

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.

By: T4phage

T4phage — Mon, 05 Jul 2010 15:43:14 +0000

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

By: profvrr

profvrr — Thu, 01 Jul 2010 23:34:08 +0000

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?

By: T4phage

T4phage — Thu, 01 Jul 2010 18:21:23 +0000

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

By: Lyndsinz_89

Lyndsinz_89 — Sat, 19 Jun 2010 01:03:40 +0000

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.

By: profvrr

profvrr — Thu, 06 May 2010 19:41:17 +0000

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.

By: profvrr

profvrr — Thu, 06 May 2010 19:40:47 +0000

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.

By: shirod

shirod — Thu, 06 May 2010 07:25:59 +0000

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?

By: megha

megha — Mon, 05 Apr 2010 15:24:05 +0000

how to stain the plaques, I mean do we need to add agar containing stain in nutrient medium to the plate?

By: megha

megha — Mon, 05 Apr 2010 08:24:05 +0000

how to stain the plaques, I mean do we need to add agar containing stain in nutrient medium to the plate?

By: profvrr

profvrr — Thu, 25 Mar 2010 09:54:53 +0000

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
http://www.virology.ws/2009/07/13/measurement-o....

By: murine

murine — Thu, 25 Mar 2010 04:05:51 +0000

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.

By: Claudia

Claudia — Fri, 19 Feb 2010 15:39:04 +0000

Get help!!! Thanks

By: Claudia

Claudia — Fri, 19 Feb 2010 15:38:48 +0000

Get help!! Thanks

By: Cenef

Cenef — Tue, 16 Feb 2010 12:24:23 +0000

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?