could you specify your problem for optimizing plague assay? I did influenza plague assay to titer the virus using 6-well plates with 2 replicates.

plaque assays are very common in virology and yes they are widely used in mammalian cells to measure titer mammalian viruses. I agree with Changhui Zhao, it would be great if you would specify the problem: which cells & virus are you using? Is it a mutant virus? Is it replicating in the cells you are using? how do you fix/stain? do you want to use agar overlay? etc.

Like Zhao and Yakimovich said, your question is vague. Please give us the specifics to enable us help you.

Sorry to take time to respond, I was away for sometime. So I have 4 human viruses (Herpers simplex 1, Adenovirus, Coxsackie B1 as well as Semliki forest I bought from ATCC). The ampules are from infected mammalian cultures. They all have recommended cell line (Veros , Hek 293, BHK-21, Fibroblast, respectively) for propagation which I followed. The first step is to allow the virus to absorb into its respective cell line for 1-2 hrs after which I removed the supernatant and added (1% of agar and culture media (2% FBS) and grow plates (6 wells plates (seeded at 8 x 105 cells/well) for 3 to 5days. Now the problem I encounter is that even in control well (no virus) I see plaques) this has been happening for some time now. And I’m not sure if what I think it is plaques is or not. I would like further clarity on this and perhaps use of another quantifying method for these viruses. I’m more concerned about the propagation of these viruses whether they were well propagated since I’m only using plaque assay to quantify them. Your help on this regard will be high appreciated.

I think overgrown cells can detach. I suggest you use media without FBS to do plague, but you should make sure the cells are at least 90% confluent. Another possibility is the agar temperature may be too high. I used agarose instead before and make sure the temperature below 42 Celsius degree before covering the cells.

Is this an Osmolarity problem?

How did you constitute/mix your overlay medium? I ask this because if you dilute the medium significantly when adding agar, it may become hypotonic and kill/shrink the cells resulting in holes that may look like plaques in the monolayer. I usually mix 1 volume of double strength (2X) with 1 volume of 2% agar in water. The final concentration of the overlay is 1X medium and 1% agar. Also of note is that I do not use FBS even when I have to incubate the plaque assay for 7 days. Note also, the temperature issue that has been raised above by Changhui Zao.

The WORST scenario is that you have contaminated your media with an adventitious virus but I doubt this is the case.

Best

Jhn

Thanks so much for all responses sent so far, much appreciated.

Changhui, thanks for you input on removing FBS, will do so, about the temperature I use 42 degrees. John, I mixed my overlay by taking half of culture media (cell-type specific) warmed up to 42 degrees and mix it with half of 2% Agar to make it 1% overlay (also warmed to 42 degrees). What I don't follow is about double strength media. How do you make a double strength media? I think there is a point on your story:).

Thanks again

Lindiwe

Lindiwe,

I think your problem is now clear. You need to use 2X media! How do you make a double strength media? A double strength medium means that everything in the medium is double the normal (recommended) amount. You have two options: If you prefer a ready-made (premixed) liquid medium, you can buy a 2X medium from the same source you bought the normal (1X) medium. If you prefer to make a double strength (2X) medium from a premixed powder, then dissolve two times the amount of powder that is recommended for a normal (1X) medium without changing the recommended final volume of reconstituted medium. This also applies to antibiotics and other supplements such as L-glutamine if they are required to culture your cells but not included in the initial powder.

Please let me know if you need more clarifications.

Best

John

Thanks John will do as recommended!

HI,

Plaque assay, TCID50 or LD50 are commonly used methods for virus titration.  but  keep in your mind, before you choose plaque assay as your method of viral titration. you should first figure out if your virus generates plaques in your target mammalian cell. otherwise, you have to switch to another cell line in which your virus is able to generate plaque, alternatively,  you need to do called "foci formation" assay to titrate your virus through Immunofluorescence staining to visualize the viral replication. For some of engineering viruses in which they can express GFP protein, so you can use GFP microscopy to determine your virus titre.

I have titrate a couples of HSV viruses, this virus has several mutant strains, the wild type virus can replicate in VERO cells, but the mutants do not , you have to use U2OS cell to titrate or propagate them. Also one thing needs to pay attention, this virus needs serum while it is growing, so every time, before absorbing virus you need to remove serum from cells, but afterwards, when you overlay agar/ agarose on the cells,  normal cell culture medium(containing serum) always needs to be added and mixed with agar/agarose. 

good luck with your experiment