Dear Nikki,
I don't know enough about environmental SEM to recommend fixation
protocols specifically for it. For normal SEM I usually fix larvae
according to the following protocol, which might be adapted for use for
ESEM:
1. concentrate larvae in a small volume of filtered seawater.
2. relax them in 1:1 mixture of 7.5% magnesium chloride and seawater
(5-30 minutes).
3. OPTIONAL STEP. In order to concentrate small freely swimming
larvae into a very small volume of seawater I often kill them with a drop of
dilute formalin (final concentration of 1% or so, but the exact
concentration doesn't matter much) and let them settle to the bottom of
the vial. After they've settled I wash them twice with filtered seawater,
letting them settle between washes, and resuspend them in less than a
milliliter of seawater to conserve osmium tetroxide in the next step.
4. add enough 4% osmium tetroxide to make a final concentration of
about 1%. Osmium tetroxide is an unpleasant and dangerous chemical,
so read about it before you use it and definitely do this in a fume hood
with appropriate disposal containers ready. Leave larvae in osmium for 1-
2 hrs.
5. wash three times in distilled water.
At this point I normally dehydrate larvae in an ascending concentration
series of ethanol solutions to 100%, then critical point dry them. For
ESEM, I suppose you have the option of somehow mounting wet larvae,
though I assume you would need to get rid of most of the water
surrounding them without completely destroying cilia and other surface
structures, which seems to me like it might be rather challenging.
Hope that's of use. Is there a particular advantage of ESEM over normal
SEM for small, delicate specimens like annelid larvae? I imply don't know
enough about it.
Bruno Pernet
Smithsonian Marine Station
701 Seaway Drive
Fort Pierce, FL 34949
pernet at sms.si.edu
>>> Nicola Diane Chapman <N.D.Chapman at hw.ac.uk> 09/08/00 07:29 AM >>>
Hi,
Sorry to ask yet another question. However I was wondering whether
anyone knows the best preservative recipe in order to preserve
trochophores for an environmental SEM I.D. We are aware that
glutaldehyde (?) is a possible preservative but do not know the
proportions.
Thanks
Nikki
--
Nicola Diane Chapman
Heriot-Watt University
<N.D.Chapman at hw.ac.uk>
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