1/29/97
ectomycorrhizal with a basidiomycete fungus, Rhizopogon vinicolor. I am
trying to localize calcium in the fungal tissue and in the interface area
between the plant and the fungal cells. Tracers for calcium have proven
difficult, i.e. flourescent probes for calcium do not cross fungal
membranes and are probably too large to pass through cell walls as calcium
does. I am interested in references on Alizarin red, or "Dahl's method for
Calcium", whether these are applicable to botanical specimens, and what
about fixing the calcium in place so that it is not displaced by
sectioning, etc., prior to staining? Thanks in advance for any advice.
Janet K. Dye
Ph. D. Graduate Student, Soils
Land, Air, and Water Resources
University of California
Davis, California USA
(916) 752-0199
(916) 668-4217
jkdye@ucdavis.edu
The Alizarin technique for calcium is called "Dawsons" and is quite old. We
don't have the original reference, but for bone the proceedure is: fix in
10% NFS, wash then transfer to 0.5% aq KOH to which enough alizarin red S
has been added to turn solution to deep purple, leave for 24 hours or till
bones are distinctly red, transfer thro KOH-glycerine series,3:1, 1:1, 1:3,
to pure glycerine. Store in pure glycerine plus few crystals of thymol.
Refs for meth blue plus alizarin red staining of bone may help: Bechtol
1948 Stain Technol 23, p3-9; Burdi and Flecker 1968 Stain Technol 43,
p47-48; Lundvall 1927 Anat Anz 62, p353-373. If you still require more
info, contact me via e-mail and we will try to help.
Miss A.J.Wilson
Electron Microscope Unit
St George's Hospital Medical School
Cranmer Terrace
Tooting London SW17 ORE
Tel: 0181 725 5220
1. Fix in 95% ALC for 48-72Hrs. Prolonged fix in alcohol renders tissue
less liable to maceration.
2. Remove fats in acetone for 2-4 days then return specimens to alcohol for
12-24 hrs.
3. Place in 1% KOH until bones or digits appear thro' muscle
Transfer to 0.1% Alizarin Red S in 1% KOH. (Other stains eg alcian blue, or
victoria blue can be used to counter-stain cartilage or bone at this
stage.)
4. Leave till stained or desired bone colour, change to fresh stain if
necessary.
5. CLEARING
Place in solution of 1g KOH, 20mls glycerine, 79mls dist water, leave till
sample clears.
6. When clear, pass thro' increasing conc of glycerine, 50, 70, 90, 100%
Miss A.J.Wilson
Electron Microscope Unit
St George's Hospital Medical School
Cranmer Terrace
Tooting London SW17 ORE
Tel: 0181 725 5220
awilson@s1.sghms.ac.uk
and comments.
Bruce has recommended (pyro?) antimonate to precipitate Ca in place,
localizing it, and I assume followed by TEM and electron probe to confirm
the presence of Ca. I have heard of this technique, especially as applied
to plants which employ sequestering of excess Ca as oxalate crystals in
specialized cells called idioblasts. But I have also heard that your
specimen must have very high levels of Ca present in some form, such as Ca
crystals, in order for this method to work. So I also would assume that
this method won't work for Ca which is held on the ion exchange groups of
the cell wall, or as non-crystalline co-ions in the vacuole?
Anyway, I will read up on the pyroantimonate method and see if it will
track Ca even when it isn't a precipitate. Also will look at Alizarin red
and try to translate to botanicals.
Thanks, Janet.
Janet K. Dye
Ph. D. Graduate Student, Soils
Land, Air, and Water Resources
University of California
Davis, California USA
(916) 752-0199
(916) 668-4217
jkdye@ucdavis.edu
> Bruce has recommended (pyro?) antimonate to precipitate Ca in place,
Yes, pyroantimonate. This can, however, wreck havoc with the
ultrastructure; try it and see.
> localizing it, and I assume followed by TEM and electron probe to confirm
> the presence of Ca.
You can also locate the antimony by EDS.
> I have heard of this technique, especially as applied
> to plants which employ sequestering of excess Ca as oxalate crystals in
> specialized cells called idioblasts.
If you have ~ micron-sized CaC2O4 xtals, you can use EDS directly
without using pyroantimonate--there is plenty of Ca in them.
> But I have also heard that your
> specimen must have very high levels of Ca present in some form, such as Ca
> crystals, in order for this method to work. So I also would assume that
> this method won't work for Ca which is held on the ion exchange groups of
> the cell wall, or as non-crystalline co-ions in the vacuole?
EDS needs a large fraction of a %, or ~mM concentrations (wet) to
detect and quantitate an element. It is irrelevant what the chemical form
of the element is. These amounts must only be present in the analysed
volume; the overall amount can be very much less as long as it is present
in small, concentrated regions.
> Anyway, I will read up on the pyroantimonate method and see if it will
> track Ca even when it isn't a precipitate.
Any Ca++, and other Ca which has a greater affinity for pyroan-
timonate than for its ligands will be precipitated. Good luck.
Yours,
Bill Tivol
tivol@wadsworth.org
measurements with EPMA (=EDS) is about 0.3 mmole/kg dry wt.
Dr. Andrew P. Somlyo
aps2n@elvis.med.virginia.edu
Charley Smalls and I used the pyroantinomate method years ago to localize Ca2+
in developing muscle at sites where Ca2+ is not normally crystaline. However,
it also precipitates Mg2+ if I recall, so you have to do some special
controls. The refs are in our paper:
Smalls, C.M. & Goode,D. (1977) Ca+2 - accumulating components in
developing skeletal muscle. J. Morphol.151: 213-238.
Dr. M. Dennis Goode Phone (301) 405-6917
Department of Zoology Fax (301) 314-9358
University of Maryland e-mail goode@zool.umd.edu
College Park MD 20742
detection limit for Ca by EPMA=EDS, or electron probe, is suprisingly high.
My results with the fungus I work with would indicate a 100 x higher level
of Ca held in exchangeable form on the cell walls, after exposure to
realistic soil solution levels of Ca. And the literature on the host
plant, Douglas Fir, would indicate Ca levels 25-30 x higher than the fungus
(making some assumptions there). So, apparently, electron probe will do
this, although I understand that asking a yes/no tracer question is a lot
easier than asking how much Ca (and how exact?). Ca oxalate crystals have
been observed to form in the walls and possibly in the vacuoles/vesicles of
the structure I work with, so confirming that is not very interesting.
What is interesting is where did the Ca come from is such quantities and
where does it go, if anywhere. (The normal habitat for this symbiosis is
an acidic, leached, relatively low Ca soil.) Maybe using Sr (stable) as a
short term tracer for Ca is more useful. Still reading up and thinking,
Janet.
Janet K. Dye
Ph. D. Graduate Student, Soils
Land, Air, and Water Resources
University of California
Davis, California USA
(916) 752-0199
(916) 668-4217
jkdye@ucdavis.edu
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