Re: Urea and Tol Blue (or other?) stain
|From:||"J. A. Kiernan" <email@example.com>|
I missed the original posting on this. does anyone have a reference
for this staining method using urea + toluidine blue, or to other
methods in which urea is mixed with a dye? It's interesting
because urea (in quite high concentration) has been used in
experiments on mechanisms of staining. The urea molecule can
form up to 3 hydrogen bonds with suitable N, O or H atoms in
a dye, a tissue, or water. It therefore competitively inhibits
staining due to weak, non-ionic forces (which include hydrogen
bonding and van der Waals forces) but it doesn't prevent staining
due to attraction of oppositely charged ions. The staining of
nuclei and mucus by a cationic thiazine dye like toluidine blue
is mainly ionic, but non-ionic forces are involved in joining
dye molecules to one another to produce the metachromasia (red
colour) seen in some mucus, cartilage, mast cells etc.
When staining is not due principally to simple ionic attraction
(as in the case of collagen in Van Gieson's and similar methods, or
pure nuclear stains such alum-haematoxylin, etc) a high concentration
of urea prevents the staining. It's entirely reasonable to use lower
concentrations of urea to reduce "background" coloration by dyes.
It would be interesting to know who introduced this as a practical
procedure, in conjuction with toluidine blue or any other
dye-based staining method.
On Fri, 29 Sep 2000, Beckers wrote:
> A big thank you to anyone who helped me with the question on using urea
> crystals and 1% toluidine blue for demonstrating basal cell carcinoma
As an afterthought, the only other staining method I can think of
that has urea as a major ingredient is Ungewitter's silver method
for axons in paraffin sections (Stain Technol. 26: 73-76, 1951).
This 2-hour method is very good. I used it a lot in the late 1960s
(when young and in a hurry) but abandoned it soon after I turned 30
(and got a real job) because Holmes' method (takes at least 12 hours,
sometimes 48) was more reliable and needed much less silver nitrate.
The cost of AgNO3 went through the roof in about 1974 because of
some greedy American millionaire buying up all the silver bullion.
The world price of silver soon settled again to a sane level, but
the price of silver nitrate remains exorbitant to this day.
The chemistry of Ungewitter's method was quite thoroughly
studied by British investigators (Rowle, Brain, Gough) in the
early 1970s. It devolves around the equilibrium between urea and
ammonium cyanate and the low (but not too low) solubility of silver
cyanate in water. If anyone has read this far and would like to
know more, I'll be happy to provide references and discuss them,
either privately or (preferably) in the open forum of HistoNet.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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