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From:"J. A. Kiernan" <>
To:Jim Manavis <>
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Tue, 21 Mar 2000, Jim Manavis wrote:

> Can anyone help with the technique MGP (Methyl green pyronin). It assists in
> differentiation between DNA and RNA. One of the pathologists has asked for
> it.

  No replies have appeared on the HistoNet listserver yet, so here's quite
  a long one to give you some encouragement.

  First-off, the method should no longer be called MGP. It should be
  EGP because nowadays the DNA stain is not methyl green but ethyl
  This is a _superb_ staining method. It was splendid even in the old
  days, but it became technically easier with the advent of dyes that
  were pure enough to behave reliably. This happened in the 1970s but
  knowledge about the better dyes was slow to spread, and it's only
  in the last decade or so that the technique has shed some of its
  reputation for being troublesome, difficult, fickle etc. 

  The important thing is not to use dyes that have been hanging round
  on some shelf for more than 20 years. It's not that they change with
  keeping. The newer ones with the same names on the labels have higher
  dye contents and don't contain undesirable impurities. 

  There are many variants of the technique. With modern dyes and a
  suitable procedure the only skill that's needed is in avoiding
  undue loss of colours (greenish DNA, red RNA, orange metachromasia
  of mast cell granules & cartilage). It's generally agreed (I think)
  that water extracts the green component and that water-alcohol
  mixtures, ethanol and acetone extract too much of both dyes. 
  n-butanol is the dehydrant of choice, after thoroughly shaking and
  blotting the slides to remove most of the dye solution. 

  Use a procedure taken from a book or paper published less than 10
  years ago. If the instructions tell you to purify your methyl green 
  solution by chloroform extraction, they are out of date. True methyl
  green (CI 42585) always needed this treatment, but it hasn't been
  manufactured for many years. The dye sold under this name is really
  ethyl green (CI 42590), and it doesn't contain crystal violet as an
  inevitable chloroform-soluble contaminant. The dye content on the
  label of a bottle of ethyl green should be close to 90%. NEVER use
  a sample of "methyl green" that is not clearly identified by its
  true CI number (42590) and preferably also its true name of ethyl 
  green.  As to pyronine Y, the traditional Biological Stain Commission
  requirement was a 45% dye content. Since the mid-1980s it has been
  easy to obtain pyronine Y of higher purity and dye content. Only
  more recently published techniques take this into account. The
  instructions for a modern method will allow for the availability
  of high quality ethyl green and pyronine Y.  
  For an EGP method that may meet the exacting standards of the 
  Eurocrats, see Hoyer et al (1986) Histochem J 18: 90-94. This is
  perfectly straightforward, but it needs high-purity dyes. Older
  batches, even if Certified, may not work. 

  The ethyl green-pyronine method is excellent for showing cells that
  are rich in ribosomal RNA (red cytoplasm) in contrast to other
  cells (bluish-green nuclei). In lymphoid tissue, plasma cells
  stand out as occasional great red wheels among the greenish-blue
  sea of lymphocyte nuclei. In the nervous system neurons have red
  cytoplasmic Nissl substance and nucleoli, whereas glial cells
  are seen generally as greenish nuclei - but with oil immersion
  you can see red rings of RNA around the tight little green 
  nuclei of the oligodendrocytes. As with other Nissl stains, it
  is difficult (perhaps impossible) to distinguish some small 
  neurons, as in the granular layer of the cerebellum for example,
  from oligodendrocytes. 

  The only reason not to use the EGP method for everything is that
  it does not impart any colour to collagen or to the cytoplasms
  of cells that are not rich in rRNA. 

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

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