Re: methylene blue/eosin

From:Barry Rittman <>

Methylene blue, azure B, azure IV, azure A, azure C  are compounds formed in the
progressive oxidation of methylene blue to Bernthsen's methylene violet.
Azure I and II are gnerally azure mixtures and vary according to how the dye is
It is difficult to prepare a high purity dye of any of these compounds. Most
powders are mixtures of two or more of these compounds and are prepared by
oxidation in alkaline conditions and heat.
Polychrome methylene blue, the original methylene blue of the Romanowski stains,
was a mixture as it was from a bottle of methylene blue that had been left on a
shelf for a considerable time.

Susan Owens wrote:

> Date: 5 Mar 2001 12:55:14 -0600
> From:
> Subject: thanks re Methylene Blue eosinate
> I have come to the conclusion that my old procedure is probably just a
> Giemsa stain (we're staining for protozoan parasites), so that's what
> I'm going to do.  To date, no one has been able to tell me just what
> methylene blue eosinate is, so I'm assuming it is such an ancient dye
> that it hasn't been produced in 90 years OR it has a completely
> different name and isn't traced back to this older usage.  At any rate,
> this has been a real puzzler and I thank you all for trying your
> collective best to help me.
> Connie M
> ----------------------------------------------------------------------------
> ----
> Connie, just saw your post today....The 7th edition of Biological Stains by
> H.J.Conn(1961) The Williams & Wilkins Company talks about the methylene blue
> eosinate (and others).
> Inpart see below:
> "The first worker to combine eosin and methylene blue was Romanovsky (1891).
> He realized that a mixture of these two dyes had great selective properties
> as a stain, and showed it to be excellent for blood, particularly in bring
> out the malarial parasite..He also appreciated that it was more than a
> mixture of the two dyes and that some new dye having the property of giving
> the nuclei a red color was present.  It was some time later before the
> nature of this new dye was known, although it was subsequently named azure I
> or methylene azure; it's true chemistry has scarcely been understood until
> recently(see pg.96).Methylene violet, and "methylene azure," which probably
> was also present, had already been described by Bernthsen(1885). How these
> new dyes were formed in the Romanovsky stain was not known; although
> Romanovsky stated that different lots of methylene blue solution varied in
> their ability to give a good blood stain, and that old solutions on which a
> scrum had formed were best."
> A great deal more is said, if you can find a copy it makes interesting
> reading.....Azure I(Giemsa) and Azure II(Giemsa)
> There seems to be several more "trade" names for the mixtures,trade names
> for 'secret mixtures'.
> Do find the book.
> Hope this helps.
> Susan

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