How to titer adenovirus stock - OD 260 vs. Plaque assay (Sep/24/2005 )
I was wondering what is the best method to titer my amplified adenoviral stock. My lab protocol says to take the O.D. 260nm to determine particles/ml. I'm pretty sure though that this shouldn't be used with an unpurified stock of virus. I'm infecting 293A cells with my crude adenoviral stock and then collecting the amplified virus. Therefore, this virus is unpurified. My understanding also was that the O.D. 260nm reading only tells you the number of particles of virus and what we're looking for is the number of plaque forming units (pfu). My lab for some reason equates O.D. 260 as pfu which I think is incorrect, correct me if I'm wrong.
I've been looking at the ViraPower Adenovirus Expression Kit protocol from Invitrogen and their method to titer adenoviral stock is to do a plaque assay by infecting 293A cells with different dilutions of virus and then after overlaying with an agarose media and staining with MTT, to count the number of plaques and determine pfu/ml. My feeling is that this is more accurate for what I need.
The main reason I ask is because the first method (OD) takes about 5 minutes to get the data. The other method takes about a week to a week and a half. I want to make sure the latter method is the most correct method before I embark on that path. Does anyone know which method I need? Thanks
If your lab has a good reasoning for their linking an OD260 value to pfu then you could possibly use it. If they've done this multiple times taking an OD260 and then running a plaque assay they should have a pretty good idea of the relationship between their OD values and pfu/ml. (Basically like using OD600 values to relate to cfu/ml for bacteria)
Saying that, I would personally do a plaque assay so I have my own assurances that my pfu/ml will be correct and not based on the "lab accepted" conversion from OD260. To me this seems like an issue of how much do you need to trust your data.
But, not having done virology lately, see what other people say too.