6/10/98
she wishes to view their ultrastructure. There are about 50 cells per
colony and about 4 colonies per 3cm polystyrene petri dish. Our problem
is that the colonies are growing between two layers of agar. The bottom
layer is 0.5% agar in PBS and the top layer is 0.33% agar in PBS. The
colonies break up and float away during processing and the agar just
moves around. The colonies are not attached/embedded in the agar.
We have tried (1)cutting around the colonies and sucking the whole lot
up (agar + colony) and treating it as a pellet but the cells are
impossible to find in the resin, and (2)we have tried to just gently
fix the mass and process it as a whole but we end up with no cells as
the colony breaks up and the cells disperse.
Is there anyone out there who could offer a suggestion.
Thanks
Sarah Ellis
Research Division
Peter MacCallum Cancer Institute
Locked Bag #1
A'Beckett Street
Melbourne, Victoria 3000
Australia
Phone 61-3-9656 1244
Fax 61-3-96561411
Email s.ellis@pmci.unimelb.edu.au
ting around the colonies, cooling the dish to 0 C (to harden the agar, but
not freeze the cells or medium), then extract the agar + cells? If this
gives you the intact colony between the agar layers, you could then trim
away some of the agar. Good luck.
Yours,
Bill Tivol
tivol@wadsworth.org
The cells will stick to the agarose during an aldehyde fixation, but if the
concentration of agarose is too weak the binding will also be weak. Try
cutting around the colonies and dropping 2% agaorose over the combined
layers. When you fix this pellet in aldehyde-based fixative, the whole
unit will cross-link. When you postfix in osmium the cells will turn
blacker than the agarose. The contrast between agarose and cells will
allow you to find the cell layer, when you section. If you need further
help, feel free to contact me.
Margaret Springett
e-mail hukee.margaret@mayo.edu
IEM Specialist at Mayo Foundation
1426 Guggenheim
Rochester, Mn. 55905
You might try first infiltrating the cells and agar in situ with a protein
like bovine serum albumin (or possibly gelatin) and then fixing the entire
petri dish contents by gently overlaying some 4% glutaraldehyde. The glut
should not mix with the albumin but should layer on top of it. Allow this
to stand for several hours. The glut will diffuse into the albumin, cross
link it to a firm configuration that you will be able to cut with a razor
blade into cubes. You will probably have to re-fix the blocks that you
initially trim out to firm them up even more. An additional hour in glut
should do this.
You should experiment with this first to get the times and proper
concentrations of protein, but we used it successfuly a number of years
ago. You might even use microwaving to accelerate the fixation.
A second possibility would be to do a freeze substitution fixation. But
this will be more complicated. Try the easier one first.
John J. Bozzola, Ph.D., Director
Center for Electron Microscopy
Neckers Building, Room 146 - B Wing
Southern Illinois University
Carbondale, IL 62901
U.S.A.
Phone: 618-453-3730
Fax: 618-453-2665
Email: bozzola@siu.edu
Keep agar layers as thin as possible (barely covering the plate and
then the inoculated cells. It will be only a couple mm thick.
Infiltrate/embed the whole agar layer in situ as you would
for an adherrent culture.
Use a microscope to locate the cells, circling the colony with a thin
magic marker on the bottom of the plate.
After baking and before you peel up the agar/resin layer, transfer the
circle to the upper surface of the layer. You can even put this circle
of resin back under a microscope to see the cells which will be slightly
brown from the osmium.
THEN cut out the colony and glue onto a blank stub.
We have done this successfully with very small colonies.
Sara E. Miller, Ph. D.
P. O. Box 3020
Duke University Medical Center
Durham, NC 27710
Ph: 919 684-3452
FAX: 919 684-8735