6/22/97
After dehydration did you Critical point dry (CPD) your cells?
If you did
not then that's your problem.
Looking at your protocol, I would like to suggest the following
:-
1. Fix Marine phytoplankton cells in 2,5% glutaraldehyde in
0,1M
sodium cacodylate buffer pH 7.2 for 1hour
2. Wash in 0.1M sodium cacodylate buffer pH 7.2 for 2 X 5
minutes
3. Post fixation - 2% osmium in 0.1M sodium cacodylate
buffer for 1
hour
4. Wash as in (2)
:
Before dehydration transfer pelleted cells into a specimen
processing
capsule. They are 13mm diameter and 18mm high and have very small
holes in each end, permitting liquid exchange, but which tend to
retain a
small amount of solution thereby reducing surface tension
effects.
Specimen can therefore be retained in the "wet" state up to
dehydration
and the CPD process. The capsule are very useful for CPD small
specimens like Marine cells etc. (available from Agar - G 3314
catalogue
no.). Place the closed capsules into no. 2 pill vials and change
ethanol
solutions in the pill vials using pasteur pipettes.
5. Dehydration: Ethanol
10% 30% 50% 70% 90% - 2 x 5 minutes each
6. 100% 3 x 5 minutes.
7. The capsules containing cells are than transferred into
the CPD
chamber and critical point dried with liquid carbon dioxide.
After CPD mount specimen, coat and view.
Please note: do not wash with distilled water. Your salts will
wash off
during fixation and buffer rinses
all the best.
Vijay H Bandu
Centre for Electron Microscopy
University of Natal
Private Bag X01
Scottsville
3209
South Africa
e- mail bandu@emu.unp.ac.za
telephone : 0331 2605157
fax 0331 2605776
It sounds like these creatures may be better observed without
fixation in their own environment. Would the use of an environmental
microscope be better than a low-vacuum system? In an environmental SEM
or ESEM, 100% relative humidity can be maintained allowing observation
of living creatures and water... The Philips/Electroscan ESEM is the
only system that can do this.
Cheers, Jan