4/16/97
Fellow microscopists
I am doing some TEM of cultured lymphocytes. All membranes seem to
be absent. There are halos where membranes should be but no
membranes.
So far I have tried
2.5 glut in 0.1M cacodylate
2.5 glut in 0.2M cacodylate (caused shrinkage)
4 paraformaldehyde 1 glut in 0.1M cacodylate.
Post fixing in Osmium dehydrating in ethanol and embedding in
Spurr
I am going to try making up the fix in the culture medium next.
Has anyone any thoughts of anything else to try. I would be
particularly interested in the use of Ca+ Mg+ and sucrose.
Chris Gilpin
Biological Sciences Electron Microscope Unit
G452 Stopford Building
Oxford Road
Manchester
M13 9PT
phone +44 161 275 5170
fax +44 161 275 5171
CGILPIN@fs1.sem.man.ac.uk
For cultured cells I routinely fix in 2% glut/0.1M. cacod
and post fix with 1% OsO4/0.1M. cacod, embed in Araldite, then stain
sections with Ua/Pb with no problems.
What are your fixation conditions - monolayer, pellet of cells.
>From your description sounds like the OsO4/Pb side of things is at fault. I
wouldn't add fixatives to the culture medium, depending on its constituents
you might have a Canazarro type reaction and fix the medium as well as the
cells.
Ian.
imontgom@pop-server.biomed.gla.ac.uk
only about 5 minutes in each fixative at 20 degrees C or at the most for 15
minutes in ice, unless you use much lower concentrations.
Overfixing or storage in con. ethanol removes lipids and hence membranes.
Badly Os overfixed tissues show dark cytoplasms surrounded by "white" cell
membranes.
Regards
Jim Darley
ProSciTech Microscopy PLUS
PO Box 111, Thuringowa QLD 4817 Australia
Phone +61 77 740 370 Fax: +61 77 892 313
jim@proscitech.com.au
I had the same problem once when I was processing some cultured
cells for EM. Morphology was bad at all, but there was no contrast on all
membrane. Then I processed the same cell culture with the same batch of
osmium solution and fresh osmium side by side. It turned out fresh osmium
solved problem.
Hong Yi
Emory, Neurology
hyi@emory.edu
We use 3% glut in 0.1M cacodylate for hematology samples. The membranes are
not very well preserved. They are often blurred, partial and sometimes
absent but nothing like what you have described. The cells still appear
bounded.
We have used cryofixation-freeze substitution and have found excellent
membrane preservation in cell suspensions right down to the trilaminar
plasmalemma.
Next best is a dimethyl sulfoxide pretreatment before fixing in glut. We
use 10% in RPMI-1640 culture media for 10 min. You could probably use
whatever media your cells are growing in. This has given us better membrane
boundaries than glut alone. They show up unilaminar.
Caveat -- DMSO has some interesting properties and effects on cells. It's
best to study the literature especially the safety requirements. It's
probably not good to just dump it down the sink.
Name: Charles Gilbert VOC:(704)355-5261
Carolinas Medical Center FAX:(704)355-8424
Dept of Pediatric Research
PO Box 32861
Charlotte, NC 28232-2861
E-mail: cgilbert@mail.carolinas.org
indicator to check the activity of an osmium solution, ideally without
processing tissue and viewing in the microscope?
I 'm sure at some time we have all picked up a bottle of expensive osmium
and thought do I use it or make fresh up. This happened to me recently and I
tried soaking some osmium into a piece of cocktail stick, which appeared to
darken, but I was wrong.
Malcolm Haswell
University of Sunderland
UK
es0mhs@environment.sunderland.ac.uk
I would put a drop of corn oil (maize oil on your side of the
puddle) on a slide and some of the osmium solution in a small dish (size so
that the slide acts as a lid for the dish). If the osmium is good, vapors
from it will blacken the oil droplet. (Any polyunsaturated oil will do.)
Phil
&&& Illigitimi non carborundum &&&&&&&&
Philip Oshel
Station A
PO Box 5037
Champaign, IL 61825-5037
(217) 355-1143
oshel@ux1.cso.uiuc.edu