Hi, I am working on measuring the activity of a certain virus.
From last week, when I do plaque assay, a big hole is formed in the center of the well.
I've never seen this before, but the same result is repeated again and again.
What's wrong with my plaque assay?
I am using the methylcellulose as an overlay medium.
Please let me know what I did wrong.
I attached my result
From your picture it seems that it is only on the leftmost wells, and only in the samples with less viruses. Is it always like that? If so, these wells contains more cells, and the hole appears because somehow these cells are detaching.
If it happens more on the plate border wells, there is some sort of evaporation on your system, and the outer wells suffers more from it (loss of liquid), Thus it concentrates the contents and affects the cell growth. If it happens more in the controls or in the more diluted virus samples, it means that the higher cell concentration are making the monolayers unstable to a point where your staining process makes it detach. If the problem is more widespread (not directly related to border wells or control samples), then it may be a matter of uneven growth in the wells. In this case when you seed the cells they may be concentrating on the well center instead of being evenly distributed, and that leads to monolayer detaching.
Anyway, I would try to solve it by avoiding evaporation (sealing the plates with parafilm or using a more appropriated incubator), by adjusting the cell concentration to avoid overgrowth, and by making sure that the cells are evenly distributed at the time of seeding (it can be easily verified by optical microscopy).
I think Gabriel is right.
It is probably due to excessive cell growth or dehydration in the central area of the well.
And you can also try the following variants if you do not want to change the solidifying agent:
Slightly reduce the number of cells per well.
Slightly increase the volume of medium in each well.
Reduce the incubation time of the experiment.
Reduce the percentage of supplemental serum in the experiments.
Hi Jong,
These big holes as you have presented in pictures are most likely due to the cells lifting off. Which cells are you using for plaquing? what virus is this?
1. Your cells might have got dried when you remove your RPMI 1640+FCS and add virus in wells. One plate at a time/ make the timing as short as possible between media removal and adding viruses.
2. Try with low viscous CMC.
3. Try adding crystal violet directly on CMC, or you could add PBS to make CMC less viscous while removing from the media before adding CV.
If you could write your protocol of plaque assay here, I could hopefully give some more solutions for the problem that is presented here.
Narayan
The remarks quoted above are interesting. The observation of all the plates shows that the central area of wells is a clear, so, when the cellular concentration is less dense which results in an attenuated coloration. Also, it is possible that in the center the bottom of the boxes is convex which leads to a lower level of medium with reduction of the cells ... Are you using agar or methylcellulose ?
Ria Bedi
I think both Narayan and Gabriel is right. The center cells might have got dried and died before adding agar or medium.
Hi Jong Won Yoon how did you solve your problem with your Plaque assays?? I am having also problems with my plaque assays, I cannot see any good plaques to count and to titrate my Influenza virus propagated in MDCK cells, could you please give me some pieces of advice for my experiments? Many thanks in advance :)
Yes, these holes from excessive growth of cells try to use 90% confluent cells and use less cell count. Additionally, handle cells gently during washing and adding the media.
I used to have a similar problem while doing focal forming assay. I have tailed spots. Did anyone face the same kind of problem?
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