10/29/97

I have been having particular difficulty with a batch of fish

eggs I have tried to prepare for SEM. They were fixed in glut.

followed by osmium and then dehydrated to 100% EtOH. I have

then dried them from liquid CO2 in a CPD. A few eggs have survived

the drying intact, the rest either exploding or collapsing.



I have extended the periods in EtOH, and soaking in the CO2 (with

lots of flushing), and have slowly vented the gas to minimize

any 'shock'. I have tried taking the eggs from 100% EtOH through

a graded series of Freon before drying, but the eggs shrivel up

by the time I get to 50% Freon. I also tried drying from Peldri,

going in a graded series from 100% EtOH to 100% Peldri and letting

them soak in the liquid Peldri overnight. By that stage, they

had again collapsed.



I guess freeze-drying might be my next

move, but thought I'd ask if others may have suggestions on how

to get these specimens through the CPD stage. I have processed

fish eggs before, but these seem to be quite robustly encapsulated.



Thank you.





Carolyn J. Emerson

email: cemerson@plato.ucs.mun.ca



Biology Department

Memorial University

St. John's, NF A1B 3X9

Tel: (709) 737-7515

Fax: (709) 737-3018

Hi Carolyn,

You might try some of the mordant techniques using glutaraldehyde, tannic

acid, guanidine HCl and osmium by:

Gamliel, Scanning Elec. Microsc. 1985: IV; pp1649-1662, 1985.

or

Osmium, tannic acid, uranyl acetate as per:

Shroeter, etal., J. Elect. Microsc. Techn. v1 pp219-225, 1984.



Judy Murphy published two nice reviews covering non-coating techniques

which may also help to strengthen cells against collapse:

Scanning Electron Microsc. 1978, vol II, pp 175-194

Scanning Electron Microsc. 1980 , vol I, pp 209-



Klaus Peters also published a paper titled "Improved handling of structural

fragile cell-biological specimens by the exchange method." J. of

Microscopy v 118, pp 429-441.



I could dig them out and FAX to you if you do not have access to these journals.



good luck



cheers



Edward J. Basgall, PhD

The Pennsylvania State University

Surface Chemistry Group ejb11@psu.edu

Materials Research Institute Building Ph: 814-865-0493

University Park, PA 16802-7003 FAX: 814-863-0618

http://www.personal.psu.edu/ejb11/

Privilege does not absolve one of ecological responsibility.

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