Preservative for environmental SEM

Bruno Pernet pernet at
Fri Sep 8 16:51:42 EST 2000

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 

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

>>> Nicola Diane Chapman <N.D.Chapman at> 09/08/00 07:29 AM >>>


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 


Nicola Diane Chapman
Heriot-Watt University
<N.D.Chapman at>

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