2 replies to this topic

[kba]

Hello,

I performed a plaque assay with Bovine Enterovirus and understand how to calculate the PFU, but am unsure which wells to use to calculate it. We only used one well for each dilution in the series, so I can't average the wells. Generally, the examples that I have found on the internet and in textbooks have from 20-50 plaques, but the largest number I have is 16. For one dilution, for example, from the 10^-2 to the 10^-7 dilutions there were 16, 10, 5, 0, 0 plaques produced. For the other results (it was a time-series) I have used the wells where there were more than 10 plaques, but I am not sure for this one as the dilutions were lower (I think something must have gone wrong during the experiment, since the pfu is so much lower at this time-point). e.g. for later time-point, I used 10^-4 to 10^-7 dilutions and got 0, 12 and 2 plaques and have used the plaque number and dilution from the well with 12 plaques.

Sorry if this is confusing, can anyone suggest the best well to use?
Thanks.

Edited by kba, 15 January 2011 - 08:53 AM.

[bob1]

The following page from Clontech has a good description of a protocol...

PFU protocol

[protolder]

Hola, I work with baculovirus and i had the same problem that you have, There isn´t relation between the dilution and the found plaques. So I prefer use the agarose method to isolate clones and titering in a 96 well plate by end point dilution. I seed 100ul of 10e5 cells/ml and infect  each colum  10 ul of diluted virus.At the first days of incubation,infection is clear in low dilutions virus wells, and high diluted needs more time.So you count the infected and uninfected wells in each dilution and there is an excell method to calculate the exact titer(Reed and Muench calculator)Look internet. Buena suerte