In article <4sdr2l$2fr at panix.com>, iayork at panix.com (Ian A. York) wrote:
> Flu isn't one of the viruses I've worked with, but our lab is going to be
> playing with some for a while. We received a large volume of frozen
> virus, which we tossed into our -80 oC freezer while we looked dubiously
> at each other. We need to thaw and aliquot this. Can anyone tell me:
> -how much reduction in titre we can expect from the freeze-thaw?
> -how to accurately titrate the stocks, if we need to repeat the titre?
> -anything else a novice flu person should know?
>> We're just going to be using this as targets in cytotoxic T cell assays,
> so we don't need to get fancy with it.
I can't give you figures on how much infectivity you can expect to lose,
but I wouldn't be _too_ worried about it. If the virus has been egg-grown
and is still in allantoic fluid then it should be fine (there is plenty of
protein in there to act as a cryoprotectant) - if it has been purified and
put out into (say) PBS then you could have a bit more trouble, but the
sort of concentrations of virus you get (up to 10~9 EID50/ml in allantoic
fluid: more if it has been purified) should make this pretty irrelevant.
As for infectivity assay, you could try plaquing in MDCK cells but you
need to worry about things like trypsin concentration in the overlay (or
it probably won't plaque at all). Personally I'd go for 50% egg infectious
dose assays (EID50) provided you have the facilities to handle this. You
can use eggs which have been incubated at 37oC for 10 days for this.
Typically we make up a decimal dilution series and put 0.2ml of each
dilution into 4 replicate fertile eggs for each dilution - locate the
embryo and mark the position (you need a dark room and a light source for
this) then pierce the shell over the air sac and at the injection site
(1/2 way down the egg away from the embryo, yolk sac and major blood
vessels). Inject at a 45o angle downwards using a syringe with 25g needle.
Seal the injection sites with a (hot) 1:1 mix of paraffin wax and
vaseline, then incubate at 35 oC for 48 hours, chill for 2-4 hours minimum
(overnight is best) and test for heamagglutinating ability in the
allantoic fluid by adding an equal volume of 1% erthrocytes (we use human,
but chicken or guinea pig are also commonly used). A nice reference for
all this old fashioned stuff is:
Barrett, T. & Inglis, S.C. (1985) Growth, purification and titration of
influenza viruses. pp119-130 In Mahy, B.W.J.(ed) Virology: A practical
approach IRL press, Oxford (ISBN 0-904147-78-9)
If you do decide to use eggs, remember to turn them 3 time a day or so
(we've got a huge incubator with turning shelves that does it for us!).
Hope this helps some, now I'd better get back to finishing my Phd.! Good luck.
Microbial Molecular Genetics and Cell Biology Group,
School of Biological Sciences, Biology West Building,
University of Birmingham,
West Midlands, B15 2TT.
Tel. (+44) (0)121 414 6555
Fax. (+44) (0)121 414 6557
E-mail g.e.price at bham.ac.uk