Re: [Histonet] Taft's Method For Nucleic Acids

From:John Kiernan

You enquired about
"Taft's method" for green DNA and red RNA.
Taft studied and possibly improved the method. 
Brachet had recognized that it stained the nucleic 
acids in different colours, about 10 years earlier. 
The original technique dated from about 1910 (Unna
& Pappenheim); it helped to define the plasma cell. 

If you mean the technique described and investigated by
E.B.Taft in 1951 (Stain Technol 26,205; Exp Cell Res 2,312),
it is a complicated procedure, developed at a time when
methyl green was always contaminated with crystal violet
and pyronine dyes were frequently unreliable. Things are
easier these days. ALSO: Please note that the correct
spelling is pyronine, not pyronin. Organic bases have
-ine endings. 

Methyl green (CI 42585) has probably not been manufactured 
for 30 or 40 years. Dye powders and solutions are still
labelled "methyl green" even by the most trustworthy
suppliers, but the dye they contain is ethyl green
(CI 42590). The catalogue of at least one huge company
indexes ethyl and methyl green, both as CI 42590. That's
almost honest.  

This may seem a trivial point, but it is not. Methyl green 
spontaneously loses a methyl group from its quaternary 
nitrogen atom. The product is crystal violet, which must 
be removed from the aqueous dye solution by repeated 
extractions with chloroform in a separating
funnel. (I did this in the 1960s and early 1970s, and it 
could take up half a working day.) 
Ethyl green is much more stable than methyl green, and the 
chloroform extraction is not needed. The staining method 
should be renamed ethyl green-pyronine

Suppliers should proudly sell ethyl green rather than the 
old, troublesome methyl green. The Biological Stain
certifies ethyl green, pyronine B and pyronine Y. The tests
and requirements have been published by Penney et al (2002)
in Biotech Histochem 77: 237-275. 

Staining with ethyl green & pyronine always need careful
experimentation to get critical separation of the DNA and
RNA colours. 

John Kiernan
London, Canada.
"Farley, Sunni R" wrote: 
> I am looking for information regarding Methyl Green-Pyronin staining for
> nucleic acids.  I have contacted Rowley Biochemical Inc (Danvers, MA) about
> their Methyl Green Pyronin stain and Differentiating Solution.  They
> provided me with the chemical composition of these solutions and also the
> protocol
> for Taft's Method  for Nucleic Acids.  Does anybody routinely use these
> items and this method?  Also, have you used them on B5-fixed tissue?
> Any information and/or suggestions would be much appreciated.  Thank you for
> your time!
> Sunni R. Farley
> Histotechnician
> Pathology Department
> Seattle Cancer Care Alliance
> 825 Eastlake Ave East
> Seattle, Washington 98109
> (206) 288-1312

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