Flow on Nematode eggs

Scott Tighe stighe at zoo.uvm.edu
Fri Mar 1 10:15:19 EST 2002


Dear Janet:

I have worked with nematode eggs a lot in the past but not on flow.
However, I would strongly recommend examining the prep under the scope
to determine size of the ova and the amount of actual mature nematodes.
For the most part ova range from 50-80um and are oval shaped depending
on the genus. Also, many preps have mature nematode presents are they
are often up to 700um x 50um or so. If you have mature nematodes
present, they can be filtered out with 150um filter mesh. Sorry to say I
have no data regarding DNA staining, but you may need to treat the ova
with an agent to create spearoplast condition to remove the outer wall
of the ova, usually extremely low [conc] of sodium hypochlorite (believe
it or not). I hope someone else can add to this from the list serve. I'd
like to hear their responses.

Sincerely

Scott Tighe
Vermont Cancer Center

janet dow wrote:
>
> I have a client who wish to run flow on nematode eggs (Meloidigyne
> incognita and Meloidigyne hapla) to stain to look at genome size.  We have
> tried twice to run them and the first time saw no florescence so we change
> protocols to PI but the second time we clogged both my machines with the
> samples( I am using a BD FACScan and a FACSCalibur).   Can anyone help
> us-she has found a paper from 1984 which used flow on a machine made at Los
> Alamos but it doesn't specify what size nozzle they used.  She also does
> not know how big the eggs are but you can visualize them in the solution
> and they settle out of solution very quickly.
>
> thank you in advance for all your help-I have always had such great luck
> with questions on this group.
>
> Sincerely
>
> Janet Dow
>
> Janet Dow
> Research Technician and Manager
> Flow Cytometry Facility
> North Carolina State College of Veterinary Medicine
> Room C-314
> Raleigh, NC 27606
> (919)513-6364



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