I have performed plaque assay for FMDV. Agarose concentration was 0.8% and it was not low-melting agarose. The cell was perfectly monolayered. Under the microscope, plaque-like "big dot" in monolayered cells was observed. But after staining, no plaque was seen. We used crystal violet for the staining with 10% formaldehyde.
Thanks
Hi, Rubayet.
Plaque assays are really tricky...
What I understand is that your monolayer seems ok, without any plaques, right?
Maybe you doesn't washed properly your plate, or the crystal violet concentration is too high that you can't saw the dot. Maybe you can try to wash again to remove completely the dye/agarose...
If any agarose remains on the surface of the well, you won't be able to see the plaque formation.
I hope this could help you!
I haven't had much success with using agarose with plaque assays for various reasons: if its too hot, you can scald your cells. If its too hard it will take your monolayer with you when you aspirate it off.
In addition, when we do plaque assays to pick plaques, the final concentration of our agarose is 0.5% not 0.8%: you may not be sufficient room for your plaque to grow to be visible to the eye.
For a traditional plaque assay for titering we usually use methyl cellulose: its vicious but allows your plaques to spread and can be easily aspirated. I have included the protocol so that you can see it.
Hi Rubayet,
for a simple PFU /ml titration, you can perform it with 1.5% methylcellulose (remember that when you prepare it after sterilizing it you should keep it in the fridge in order it gets completely disolved) or you can also use Karaya gum. I have used the last one for FMDV and have had good results.
when you wash the plates, try doing it with cold water it helps preventing detachment.
if cristal violet with 10% of formaldehyde does not work you can fix with metanol (kept at -20C) as a first step during 25 minutes at 4 degrees, wash and then dye with cv.
cheers!
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology