Re: Methylene Blue-Schiffs (also S. spicer)

From:"J. A. Kiernan" <>

My comments follow the quoted lines from Rena, which
may be horribly fragmented and mangled by Winders
email software. 

On Tue, 19 Jun 2001, Rena Fail wrote:
> A Schiff's methylene blue will stain DNA purple to red and RNA blue. Ref: 

> A.J.Garvin,B.J.Hall, R.M.Brissie, and S.S. Spicer, Cytochemical 
> differentiation of nucleic acids with a Schiff's Methylene Blue 
> Sequence.  J. of Histochem & Cytochem., Vol. 24 No4, April, 1976, 
> pg587  This one uses Bouin's  as a mordant , 2% schiff's for 30 
> min.,running water for 5, 0.5% periodic acid  for 5, stain in 1% Schiff's 
> for 10 min, wash in running water for 5-10min. stain in methylene blue pH 6 
> for 1-3 min. dif in 95% deh. clear and mount.
> An older method of Dr. Spicer's is in the 3rd edition of the AFIP manual on 
> pg 133

I haven't checked the JHC paper, but are you sure it includes a 
periodic acid step?  This would make glycoproteins etc Schiff-
positive in addition to DNA, and why should one want to do that?

The way the method works is:  Bouin fixation for 24 hrs causes 
INCOMPLETE Feulgen hydrolysis of DNA. The resulting aldehydes
are detected with Schiff's reagent, and the RNA is then stained 
with methylene blue (or azure A in the "Spicer's Feulgen-azure A"
method in Lillie & Fullmer, p.175; toluidine blue would also be

The INCOMPLETENESS of the nucleic acid hydrolysis is the key
feature of this technique. A complete Feulgen hydrolysis, usually
done these days with 5N HCl (pH about -0.3) at 20C, converts all 
the DNA to aldehydes but it also changes all the RNA to small, 
soluble fragments, so that no RNA remains to be stained with a 
basic dye like methylene blue. Bouin's fluid (pH about +1) for
24 hrs at 20C is a less ferocious acid than either this or the 
traditional Feulgen reagent, which was 1.0N HCl (pH zero) at
60C for about an hour. That was the regular method when Spicer 
wrote up this now venerable method for DNA + RNA in 2 colours. 

Sam Spicer's histochemical publications span more than 40 years
and include clever and insightful contributions to lectin
histochemistry in the late 1990s. He also gave us the generally
accepted classification and nomenclature of histochemically 
discernable mucosubstances, long before the advent of lectins 
as useful staining reagents.

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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