At last, a very late follow-up to my plea for help on TEM Fucus
fixation as requested by several list members.
My delayed response is due to waiting to ensure my results were
consistent, which they seem to be for both germlings and mature
For those interested, my procedure was fairly straightforward:
2.5% glutaraldehyde in phosphate buffer (0.1M, pH7.2) with caffeine
added at 1.0% and NaCl at 3.0%. Fix for 2-3hrs.
Post fix in buffered 1.0% osmium tetroxide.
Dehydrate in alcohol.
Embed in Spurrs resin.
I infiltrated my tissue for 2 weeks, changing the resin every three
days, and got pretty good results, even with cells packed with
Many thanks for all the helpful comments.
Here are the responses to my initial request for ideas:
We have used SEM EDAX and XRF...very difficult to standardise, then
for super subcellular localization we used a neutron probe at the
National Accelerator Centre...very interestng but very expensive (had
to be done on a collaboration basis). Tried the AA spec for specific
metals but our equipoment is shot so gave that up as a bad job....any
suggestions from your end. I would appreciate a summary of the
replies, to find out who else is looking at metals.
We are supposed to have a relatively pristine coast...but found large
amounts of arsenic in Osmundaria.
Look for ward to hearing from you. say hi to murray
Alan T Critchley
Botany Department, University of the Witwatersrand,
Johannesburg, P Bag 3, WITS 2050, South Africa.
Tel: +27 11 716 2305 (off.), +27 11 716 2201 (seccy)
Fax: +27 11 403 1429 (Dept.), +27 11 716 8030 (Univ.)
E-Mail: [log in to unmask]
> I am working with with heavy metal uptake in Fucus, and would
> greatly appreciate any information on preparatory techniques for TEM
> and SEM, with and without X-ray analysis to supplement my collection
> of potential protocols. There must be a definifitive method out
> there somewhere.
I expect that you probably already know of the following references,
but here goes...
Motomura (1994). Protoplasma 178:97-110
Electron and immunofluorescence microscopy on the fertilization of
Fucus distichus (Fucales, Phaeophyceae).
Manton & Clarke (1955). J. Exp. Bot. 7:416-432
Observations with the electron microscope on the internal structure of
the spermatozoid of Fucus.
Maier (1995). Phycologia 34:441-443
On the fine structure of flagellar hairs in the brown algae
Callow et al (1978). J. Cell Sci. 32:45-54
Fertilization in the brown algae. I. SEM and other observations on
Brawley et al (1976). J. Cell Sci. 20:233-254
Fine-structural studies of the gametes and embryo of Fucus
vesiculosus L. (Phaeophyta). I. Fertilization and pronuclear fusion.
Hope that there may be something helpful there!
School of Biological Sciences
The University of Birmingham
Birmingham B15 2TT
Email: [log in to unmask]
You have my utmost respect for wanting to be a phycologist. Are you
PhD or post-doc? Who's your supervisor? I know a few of the names
there through John Wedderburn. I hope I can be of some help in your EM
endeavours. The SEM and TEM protocols I used were fairly standard with
minor mods. I will look up some refs plus my own tips for you in the
next few days. All I can say for now is that you have to use a
modified primary fixative that contains caffeine. This will stabilise
phenolics. Clayton & Ashburner 1994 European Journal of Phycology
29:1-9 has EM on eggs and zygotes of D.potatorum. Nice paper too. I
have obtained equally as good micrographs as they have. As far as
simplyfying techniques I didn't really need to. One SEM advantage that
we now have up here is a cryo-stage . I know there is one at Plymouth
but so do you probably. That will help save a lot of time.
Washington Singer LAbs,
University of Exeter,
Exeter, Devon, EX$ 4QG.
in case you did not get many replies to your query on TEM fixation in
particular, I could send you my protocol which I used to fix
phytoplankton i was exposing to triphenyltin (antifouling agent). I
don't know if it will work with macroalgae but realise there may not
be many people working in the field so decided to reply anyway.
I think my protocol is pretty standard anyway as it involves
gluteraldehyde fixation (3% glut buffered but made up in seawater***)
Osmium tetroxide fixation (also made up in s/w) Dehydration with
acetone series slow infiltration with resin: acetone solutions up to
I have submitted a paper detailing this protocol along with the tips
on using sea water based fixatives etc. as I feel it really improved
my fixation, and have not seen it in papers. Hope thisw helps, Helen.
Marine Microbiology Section,
Martin Ryan marine Science Institute,
My thanks to everybody who wrote.
Dept. Biological Sciences
University of Plymouth
Room 317,Davy Building
Devon PL4 8AA