From P.Bond@PLYMOUTH.AC.UKWed Apr 30 16:15:52 1997 Date: Wed, 30 Apr 1997 10:03:22 GMT From: Peter Bond To: ALGAE-L@Danann.hea.ie Subject: Fucus fixation 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 thallus tissue. 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 physodes. 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 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: ALANC@GECKO.BIOL.WITS.AC.ZA > 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 (Phaeophyceae). Callow et al (1978). J. Cell Sci. 32:45-54 Fertilization in the brown algae. I. SEM and other observations on Fucus serratus. 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! Gareth. *************************************** Gareth Edwards School of Biological Sciences The University of Birmingham Birmingham B15 2TT Phone: 0121-414-5573 Email: g.o.edwards@bham.ac.uk Dear Peter, You have my utmost respect for wanting to be a phycologist. Are you doing a 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. Cheers, Rob Holland Washington Singer LAbs, University of Exeter, Perry Road, Exeter, Devon, EX$ 4QG. Dear Peter, 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 100% resin. 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. H. Mooney Marine Microbiology Section, Martin Ryan marine Science Institute, University College, Galway, Ireland. My thanks to everybody who wrote. Pete Bond. Dept. Biological Sciences University of Plymouth Room 317,Davy Building Drake Circus Plymouth Devon PL4 8AA