CuteChlamyFixation Method for Mitotic Cells

From Peter Luykx

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 supernatant 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.