Re: Urea and Tol Blue (or other dyes?); also etc.

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 seems entirely reasonable to use
   lower concentrations of urea to suppress "background" or even
   certain types of specific 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 
> 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
> -----------------------------------------------
  An irrelevant 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 pretty 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. Growl!
    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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