Mitotic squashes

Lucy Dyer ldyer at unb.ca
Wed Sep 9 14:43:29 EST 1998


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




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