There is no doubt that widely-used Kaerber or Spearman or other methods based on virus back titration using 10- fold dilutions have shown acceptable results. Anyway, if we need to estimate virus titer more properly does it make any sense to use lower fold for back titration (e.g. 2-fold or 5-fold). Is this more accurate or an absolute waste of the time?
In this page there is some information about MOI: http://atcc.custhelp.com/app/answers/detail/a_id/410/~/converting-tcid%5B50%5D-to-moi.
Kind regards
As long as you modify your calculations to account for 2 or 5-fold dilutions, you will be fine. 10-fold dilutions are typically used because you hit your ID50 within less serial dilutions that way. If you have a low titer to begin with, it could be more accurate. Especially if your titer is low to begin with. For example, if you're hitting your Infectious Dose 50% after one 10 fold dilution (or even loosing all signs of infection entirely), that would throw off your accuracy completely. You would NEED to calculate your virus titer with a lower serial dilution method in this case (like the 2-fold you suggested). Hope this helps!
What virus are you wanting to titrate? I prefer plaque assays or infectious foci assays. In the latter, you infect cells similar to a plaque assay but stain the cells with a pan antibody to the virus and count the number of infected cell clusters. I have used this for RSV and it works well.
Thanx Christopher. I assume the virus titer by plaque assay is different from titer estimated by CPE of LD. One of our confuse is when you conduct the infection experiment with certain MOI based on TCID50 or LD50 do you really accurate with this result?
In this page there is some information about MOI: http://atcc.custhelp.com/app/answers/detail/a_id/410/~/converting-tcid%5B50%5D-to-moi.
Kind regards
A 2-fold serial dilution would provide you with a more accurate estimation of the virus titer but will take more time and resources. You could titer with 10-fold dilutions first. Then repeat the experiment with 2 fold dilutions in the range of your initial estimation, but once again this takes more time. My experience is that ID50 is more sensitive than plaque assays, as not every infectious virus forms a successful plaque, but if the virus can productively infect a cell, then the progeny will continue to replicate in an ID50 assay. I think the big question is, what do you need the data for and is the increased sensitivity and accuracy required to answer the question you have?
In our lab i am using Plaque assay and CCID50 for virus titration. For plaque assay and CCID 50 , using 10 fold dilution. But CCID50 is more accurate and sensitive if we compare with plaque assay.
plaque assay is better or you can use plate HA if you are working in haemeaggultinating virus like influenza A
Hi Alexander,
I'd say it depends on what virus you want to titer. I do titration pretty much every week in my lab for several viruses like dengue or HCV. For dengue, I use only plaque assay with agar overlay and staining with crystal violet because this is the only way to do it. When you know if your infection works, you just play with the dilutions to determine the best titer. For HCV, I use the focus formation assay that allows me to determine the TCID50 and the FFU/mL. It's very accurate and much easier and faster than the plaque assay.
Good luck.
Thanks Gregory! Does focus formation and plaque counting can make similar results. I'll explain. For instance, we have nice plaques in primary cell culture, but in established cell lines we haven't. Nevertheless, we can count fuorescent foci using IFA. In this particular case can we compare virus titer between cell systems or it is inaccurate?
In my experience, lesion assay (pock, plaque or focus) is easier and more accurate than a dilution method, once you have the system set up. A vital stain that does not fade e.g. Tetrazolium can be more useful than say crystal violet, you need to experiment. Of course, the fewer systems you have to optimise, the easier life becomes.
I would like mention that
the best method to count a virus is by a plaque assay, but not all viruses form plaques and then various strategies have been developed to quantify viruses
here there is a list for different viruses , often you have no choise on what to do
http://en.wikipedia.org/wiki/Virus_quantification
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology