Dear All, I am seeding 5*105 cells/well of BHK-21 in 6-well plate for zika virus. The dilution used is 10-2 to 10-6 . I am using 2% Agarose and 2% DMEM with 1% FBS. While during incubation, after 3 day, I am observing plaque in all dilution. But after fixation with 10% formaldehyde and staining with 0.1% crystal violet , I can't see any plaque. What is the reason for that. I am attaching my observation picture and final plate picture.
Dear Dipak,
Your plaques are too small to detect using crystal violet staining. I can see the cells in the plaque, which take up the crystal violate and hence you can not see the plaques. I think you need to wait more time for the plaque to expand and the cells are coming apart from the middle of the plaque. Sometimes, the fixation method also contributes to the visibility of the plaque. Ice-cold methanol is better in this case.
I also suggest you to use Vero cells instead of BHK. BHKs form a rigid layer of cells instead of a monolayer, and it 's hard to judge on the confluency for plaque assay. Vero cells are good in this case, where the cells detach from the plaques forming a visible spot with crystal violate.
I hope this information is helpful to you. Goodluck
Dear Dipak,
I also agree with the previous comment, your plaques are too small at three days and the crystal violet is staining the cells still residing within the small plaques that are forming. I would also recommend using Vero cells instead of BHKs. I incubate my Zika plaque assays 7 days before staining, but I have seen as little as 5 days in other articles. I hope this is helpful and gives you a starting point to optimize the assay for your lab.
thanks to both of you. Now I am getting plaque after incubation for 7 days. But in BHK-21 cells, plaque are more visible as compare to vero cells.
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