2/20/97
need it, I can't find the information.
Can anyone recommend some solvents, other than alcohols and acetone,
suitable for freeze-substitution. I am having some difficulty obtaining
adequate substitution in some cells of my material (plant bits) and want to
try solvents which may penetrate more effectively.
Thanks in advance.
Kim Rensing
krensing@uvic.ca
than acetone or ethanol. But I doubt penetration is the problem. Why do
you think you are getting bad penetration?Are you sure your solvents are
totally dry? I recommend using a fresh unopened bottle each time. Avoid
adding molecular sieves which can make matters worse.
Thomas E. Phillips, Ph.D.
Associate Professor of Biological Sciences
Director, Molecular Cytology Core Facility
3 Tucker Hall
University of Missouri
Columbia, MO 65211
(573)-882-4712 (voice)
(573)-882-0123 (fax)
tphillips@biosci.mbp.missouri.edu
Our Hazardous Waste folks don't like to deal with the substitution fluids
we use so check with your waste disposal people before you use the
following method:
Make stock solutions of 4% Osmium tetroxide (1g in 25 ml HPLC grade
Acetone*) and 0.1% Uranyl acetate (0.025g in 25 ml HPLC grade acetone). Mix
stock solutions 1:1 for your substitution fluid.
Note: Uranyl acetate takes several hours to go into solution so I make it
the night before I need it (keep it in the dark at room temp). Then chill
it to -80.
*The acetone must be prechilled to -80 before you add the osmium. Keep
solutions on dry ice or use a cold block when you bring them out of the
-80. Mix the solutions into prechilled sample vials.
References: Hoch HC (1986) Freeze-substitution of fungi. In: Aldrich HC,
Todd WJ (eds) Ultrastructure techniques for microorganisms. Plenum, NY
Howard RJ and O'Donnell KL (1987) Freeze substitution of fungi for
ctyological analysis. Experimental Mycology 11: 250-269
Best regards,
Beth
Beth Richardson
EM Lab Coordinator
Botany Department
University of Georgia
Athens, GA 30602
Phone - (706) 542-1790
FAX - (706) 542-1805
Email - beth@dogwood.botany.uga.edu