 |
 |
 |
 |
 |
Jeff in a previous life I played a little with sorting animal from
vegetable from mineral with some corals. the investigator wanted to
seperate the symbiotic algae from the animal.
the biggest problem we had was keeping the algae alive for any extended
period after sorting, they just couldnt find a media like the insides of
coral. The best sorting survival we got was just using filtered sea
water.
Technical considerations: (from memory this was a long time ago...)
Instrument was an old EPICS 741 with a few mods and fiddles.
The algae were fairly big, and even after a gradient enrichment step
there was a fair bit of lumpy crud around. (sponge spicules would be
ugly....) I think we ended up using a 100um tip and *low* pressure as
the 741 could barely deflect the big drops. consequently the sort speed
was fairly slow...
Detecting the algae was easy. Fluorescence was awsome, like the
proverbial traffic light. Gating was on fluor only for them and they
ended up quite pure 95% +.
The animal component was another matter. I think we ended up using a 4
way gate with 2 fl's + forward and 90 scatter. even then ISTR that purity
was only around mid - high 80's, with the balance similar sized lumps of
crud.
Basically i found that it could be done, but took quite a while to work up
optimal machine parameters. Be prepared to start from scratch basically.
As for the biological side, well its plenty frustrating to spend all day
to get nice populations only to be told that they all lysed by next
morning... hope you have better luck.
Cheers
--Daryl Webb (dwebb@waite.adelaide.edu.au) Dept. of Plant Science, Waite Institute University of Adelaide, Glen Osmond S.A. 5064 Australia. Voice:61_8 303 7426 Fax:61_8 303 7102
|
|
|
|
|
|
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web