Re: Urea and Tol Blue (or other?) stain

From:"J. A. Kiernan" <>

  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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