From: GABLES::PLUYKX 3-NOV-1994 11:02:22.43
To: IN%"amacdona at cs-acad-lan.Lakeheadu.Ca"
CC: PLUYKX
Subj: RE: fixative wanted
On a fixative for mitotic cells in Chlamydomonas:
See E. Harris, 1989. THE CHLAMYDOMONAS SOURCEBOOK. Academic Press, pp.
109-112, for a list of chromosome numbers, along with references to the methods
that people have used in the past. None of them are 100% satisfactory, as far
as getting a good chromosome spread is concerned. Some of the methods are
rather elaborate.
A simple method that I have used, simply for determining if cells are
actually in mitosis or not, is the following.
1. Put 1-2 ml of a liquid culture in a 12- or 15-ml conical glass
centrifuge tube (actually any kind of test tube will do), centrifuge to bring
the cells to the bottom.
2. Aspirate off or pour off the supernatent, add 2-3 ml of freshly made up
fixative (i.e, within the last 3-4 hours): ethanol:glacial acetic acid in a 3:1
ratio, swirl to suspend cells.
3. Sometime within the next hour or so, recentrifuge, remove the supernatent
again, add fresh fixative. (This is to remove the traces of water.)
(If the tube is tightly capped at this point to prevent
evaporation, the material can be stored almost
indefinitely -- months, at least.)
4. Let the fixed cells sit in the fixative for a day (many days, weeks, or
months is okay), then recentrifuge, remove supernatent, and resuspend in 45%
acetic acid (1-2 ml) -- e.g., 45 ml acetic acid + 55 ml water.
5. Let the cells sit in the 45% acetic acid for at least 20 min. (several
hours is okay), recentrifuge if necessary to concentrate the cells at the
bottom of the tube, remove one drop of cells with a Pasteur pipette, put it on
a slide, add a coverslip that has been rimmed with Vaseline, to prevent
evaporation and to keep from being overcome with acetic acid fumes while
looking through the microscope. It is best to use a SMALL drop, which doesn't
spread all the way to the edges of the coverslip.
(A convenient way to rim a coverslip with Vaseline is to spread a small dab
on the heel of the left hand, then scrape the (clean) coverslip across the heel
of the hand, building up a little ridge of vaseline along one edge in this way.
Then turn the coverslip 90 degrees, repeat, etc., until all four edges are
rimmed.)
6. Observe with phase contrast. The 40x objective is high enough to see
whether a cell is in mitosis or not (although chromosomes can't be counted
reliably).
Additional note to (5) above: It is best not to SQUASH the cells under the
coverslip, just compress them very slightly with gently tapping on the edges of
the coverlip (which also gives a good Vaseline-seal).
Because mitosis is rapid, in asynchronous log-phase cultures only about 1%
of the cells will be in mitosis at any given time, so you may have to do a lot
of looking to find the mitoses.
---- Peter Luykx
Dept. of Biology
Univ. of Miami
Coral Gables, FL 33124
pluykx at umiami.ir.miami.edu
