[Coral-List] Response from separation zooxanthellae - coral
carlazilber at yahoo.com.br
Sat Jun 6 16:57:29 EDT 2009
I have received many requests regarding the answers on how to separate
zooxanthellae from corals. Most people answered attaching some papers that
deal with the issue , while others sent some interesting answers. The
answers are below and I have attached some papers that were sent to me.
Thanks again for all the info!
Best regards to all!
Carla Zilberberg, Ph.D.
Departamento de Zoologia
Universidade Federal do Rio de Janeiro
Ilha do Fundão, Rio de Janeiro, RJ
cep: 21941-590, Brasil
Tel: 21- 2562-6389/ 21-2562-6361
Response from Coral list Separation of zooxanthellae and coral
please let us know if you receive useful protocols for extracting coral DNA
as we are looking for protocols too.
What we found is that you can use a press with 0.1mm barrier that cracks the
coral cells but not the algae, and centrifuge the algae down.
School of Natural Sciences, Medina Lab
University of California, Merced
chris.voolstra at gmail.com
I worked with soft corals
I could not get good DNA from Ethanol-preserved corals but I got a 100%
yeald when I preserved them in DMSO buffer:
20% DMSO, 0.25M EDTA in NaCl saturated water.
I worked with the zooxanthellae and not with the coral tissue, but
separating them from eachother was done by homogenizing the
tisue+zooxanthellae in seawater in 50ml tubes anf then centrifuging
them at 2000 rpm for 30 minutes. The zooxanthellae pellet at the bottom and
the coral tissue floats at the top.
You should repeat this a few times in order to make sure the tissue is left
Hope this helps
Zoology department Tel Aviv University
It is quite easy to separate zooxanthellae from coral tissue, and to check
whether the algae have been removed completely - you can illuminate samples
of the coral tissue with white light and look for the fluorescence of the
algae (red) if you have a suitable microscope
- or even just check microscopically.
It would be very inadvisable to use ethanol before the algae are removed
from the tissue. Ethanol will definitely extract pigments from the algae
and may also damage its wall. I have not done DNA work myself, but I doubt
if you need to fix the coral tissue until after you have extracted the
algae; all you do is remove the tissue from the skeleton and centrifuge.
You can remove tissue with a brush (we use a
toothbrush in our physiological work) of b grinding bits of coral.
Grinding is messier as you get bits of calcium carbonate from the skeleton
into the initial slurry. Getting rid of the algae is no problem but you
will also have a lot of bacteria in with the coral tissue. My colleague Dr
Adrienne Grant has done some work on getting coral cells out of tissue
intact and with minimal bacterial contamination - you could contact her at
agrant at bio.usyd.edu.au.
I am not at my own computer so I haven't got access to references at the
moment. Most people who work on zooxanthellae (including us) have
protocols in their papers for separating coral tissue from algae.
Only Adrienne, as far as I know, has tried to remove bacteria.
Atchison, A.D., P.W. Sammarco, and D.A. Brazeau. 2008. Genetic
connectivity in corals on the Flower Garden Banks and surrounding oil/gas
platforms, Gulf of Mexico. J. Exp. Mar. Biol. Ecol. 365: 1-12.
Brazeau, D., P.W. Sammarco, and D.F. Gleason. 2005. A multi-locus genetic
assignment technique to assess local recruitment of Agaricia agaricites on
coral reefs. Mar. Biol. 147: 1141-1148.
I separate zooxanthellae from coral tissue quite often, and it is rather
simple. I first blast the tissue from the skeleton with a waterpik (the kind
you can buy to clean your teeth) with filtered sea water over a large beaker
or into a plastic bag. This will make a frothy coraltissue/zooxanthellae
mix. I then transfer the froth and water into 50ml tubes and centrifuge for
about 15 minutes (our large centrifugue only goes to about 4000rpm, but if
you can, get up around 10,000rpm and you can go for less time, about 10
min). This should make a dark pellet at the bottom and light colored froth
at the top . In your case, you want to keep the froth and get rid of the
pellet. If the pellet has much light stuff on top of it, I would try to keep
that too, as that is your tissue also. Then put what you want to keep into a
glass homogenizer and homogenize until uniform. Then redistribute into tubes
(I go to smaller tubes here, so I can use a smaller centrifuge in our lab
and get up to 10,000 rpm) and spin for 10 min again. Continue this until it
appears you have completely separated your zoox from your tissue. I am
usually interested in keeping the zoox, but it should work for you to keep
the tissue as well. I do know some corals are less "frothy" than others, so
you may really have to watch the top of your pellet in the bottom of the
tubes for white or light yellowish tissue as that may be where your tissue
ends up at first. You may want to keep the entire water collumn above the
pellet as it might have tissue in it. I'm not quite sure how you can
concentrate your tissue down at the end, maybe put the tissue containing
water through a 40 micron filter basket and it might catch it and
concentrate it. Hope that helps.
Ginnie Lintecum Carter, MS
Smithsonian Institution/ Hawaii Institute of
46-007 Lilipuna Road
Kaneohe, HI 96744
<mailto:ginlintecum at gmail.com> ginlintecum at gmail.com
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