Hi Allen
The best procedure for onion root tip squashes that I have ever used
with freshmen students (they have about a 60 - 70% success rate, and some
of the failures are from things like using the wrong end of the root) is
as follows: (I use fresh roots - not fixed)
Grow onion sets in sand in cool conditions (I think about 10 days, but
do not exactly remember the timing) and cut off the last centimeter or
two of the roots. Refrigerate in a weak sucrose solution for an hour or
so before the lab. (Or you could just have the students harvest a root
tip themselves)
1. Place the root on a slide with a few drops of water. Using a razor
blade and a dissecting microscope if necessary, separate the meristem
region (and root cap), and discard the remainder of the root.
2. Add one drop of ethanolic HCl ( 1 : 1 95% ethylalcohol : conc. HCl)
and allow to remain 45 - 50 seconds. Remove the excess acid with paper
towel and immediately wash with one or two drops of water. Remove excess
water, and add one drop of Orcein stain. (The ethanolic HCl partially
dissolves the cell wall, and makes it possible for the DNA to react with
the stain.) (The preparation can be left for a few minutes as long as it
does not dry out)
3. With the blunt end of a metal probe, gently but thoroughly squash the
root tip in the stain, ensuring that the cells remain on the slide. Add
one drop of 45% acetic acid, and carefully place a cover slip on the
slide.
4. Place the slide between two pieces of paper towelling. With the the
flat end of a metal probe (or the eraser end of a pencil), gently tap the
cover slip vertically through the covering paper towel to spread out the
cells on the slide. Then blot the slide carefully before placing on the
stage of a microscope for observation. (If the cells are still piled on
top of each other, this step can be repeated using a little more force.)
With a little searching, most students can find mitotic stages. Some
problems: If everything is blurry, the ethanolic HCl may have been on
for more than a minute before being washed off, and digested too much of
the cells. A few students cut off the meristem, and are left only with
the root cap, but if they have left too much root, the meristematic
region can be hard for a novice to find. You could direct them to look
for the region where the cells are quite square as opposed to elongated.
One advantage of this method is it is so easy and short, that for those
students who have found a root which does not contain dividing cells, it
is not intimidating to just do another root.
I hope you can use it for your lab!
Lucy Dyer
758 George Street
Fredericton, N.B.
E3B 1K5
