Re: Methyl green staining

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From:"J. A. Kiernan" <> (by way of Marvin Hanna)
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On Fri, 28 Jan 2000, Robert G. Russell wrote:

> 3. The texts recommmend 1% methyl green in distilled water - is this the
> general recommendation out there?.

    For a simple counterstain it doesn't matter much. Slight
    acidification (e.g. pH 4) prevents staining of collagen
    and of most cytoplasms. It's really just like any other
    basic dye.

>                             ... I presume chlorofrom extraction of MG
> is not necessary for routine counterstain use, only for use in DNA,RNA
> staining.

   If the methyl green was manufactured less than about 20 years
   ago it shouldn't contain crystal violet, and chloroform extraction
   shouldn't be needed for any purpose. The dye sold as "methyl
   green" has in fact been ethyl green (CI 42590) for many years,
   and this is noted in tiny print in chemical catalogues. True
   methyl green (CI 42585), which was always contaminated with
   crystal violet, is now a curiosity found only in old bottles
   at the backs of shelves.

   It is high time this stain was routinely called ethyl green
   because that's what it is, and it's better than the old
   methyl green on account of not containing crystal violet
   as an impurity. The combination with pyronine G should be
   called EGP, not MGP!

   The colour of ethyl green is about half-way between green
   and blue. The same was true of methyl green, after extraction
   with chloroform. (The Colour Index name for CI 42585 is
   Basic blue 20.)

   The reason for dehydrating in n-butanol is that this does not
   extract much of either the ethyl green or the pyronine from
   stained sections. Extraction of ethyl green by water and of
   pyronine by lower alcohols made the traditional Unna-
   Pappenheim method difficult to do. n-butanol is only partly
   miscible with water, so you have to blot the slides with
   filter paper before going into the first change. This solvent
   also has an unpleasant cough-making smell. There are also
   published EGP methods using t-butanol (quite a nice smell),
   but this solvent is more expensive.

   When ethyl green is used alone, as a nuclear counterstain that
   contrasts with the brown of oxidized DAB, the dehydration is
   less critical. After a brief rinse in water (tap water, if
   neutral extracts less dye than pure water, which is always
   rather acidic) go into the first of 3 changes of 100% ethanol.
   Avoid alcohol-water mixtures, which extract this dye (and many
   others) from stained sections much more aggressively than
   either water or 100% ethanol.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1
   Phone: (519) 661-2111
   FAX (Department): (519) 661-3936

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