Plaque Assay - Plaque Assay (Jun/15/2008 )

Hello,

I have been doing couple of plaque assays and trying to get it standardized, and my having problems. I am attaching two plates of the plaque assays. One is worked fine and the other one did not. The Crystal violet used could be one of the reasons, but I have used the same stain again and it did not work.

This is a rotavirus Plaque assay. The Cells are MA104

thekid

hi

would you pleaaaaase give your protocol to me too?
I use neutral red for visualization......but it fails all the time, can't get the plaques in the end. I work with a paramyxovirus.

you dissolve cystal violet in what? water, PBS or alcohol?

All I notice is that the second picture is much more purple. Is the pH right? Presence of any reducing agents? Expired solutions?

The crystal violet is at 0.05% solution in alcohol and then made up with water. The pH of the solution is ok, the CPE shown by the virus is really fast and the staining as you can see is totally different between the two plates.

I am not sure why?


thekid

It kind of looks like you washed the stain out in the lighter coloured plates, maybe the cells didn't attach well and were removed when you took off the media.

Did you wash one plate but not the other before adding crystal violet.

BTW, I usually make my crystal violet in 6% glutaraldehyde, ethanol solutions shrink the cells causing an over estimation of the area infected in plaque assays.