Sponge sorting

Daryl Webb dwebb at waite.adelaide.edu.au
Wed Feb 26 20:19:27 EST 1997


Jeff Barry (bpijb at picr.cr.man.ac.uk) asks:
> I have been asked to see if anyone out there has had experience sorting
> the symbionts of sponges or more specifically ...
> Has anyone done anything similar?


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 at 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






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