[Histonet] Re: Hematoxylin stability

From:"Robert Richmond"

The name associated with an alum hematoxylin usually corresponds to
the oxidant employed. You can find citations of the original
publications in Lillie 3rd ed. or in Bolles-Lee's The Microtomist's
Vade-Mecum if you're fortunate enough to get your hands on a copy of

Harris hematoxylin was oxidized with mercuric oxide. You really can't
handle mercury in a present day hospital laboratory in the USA. Dr.
Harris's name should not be invoked if you aren't oxidizing with HgO.

Delafield's hematoxylin has no added oxidant - you put the bottle
under the counter with a loose stopper and let the ambient oxygen
oxidize it. (Phosphotungstic acid hematoxylin - PTAH, if anyone
remembers that - was usually air-oxidized.)

Mayer hematoxylins are oxidized with sodium iodate, favored because
the oxidation occurs instantly at room temperature. The amount of
iodate should be great enough to produce an immediately usable stain,
but small enough to leave much of the hematoxylin unoxidized in order
to improve the shelf life of the mixture. - The Gill hematoxylins are
Mayer hematoxylin formulations with the molar concentrations of
hematoxylin and iodate rationally calculated, as set out by Baker in
his Cytologic Technique.

I think that commercial hematoxylins today are usually oxidized by
bubbling oxygen through them, but I'm not sure of that. Other oxidants
such as permanganate have been employed on occasion.

Acid, usually acetic, is added in a quantity determined by one's
desire to have a progressive (not further differentiated) or
regressive (differentiated in a subsequent acid bath) stain. The wise
pathologist leaves this choice to the histotechnologist. I do not want
a regressive step in my frozen section stain sequence, however.

A preservative is usually added - ethanol, ethylene glycol, glycerol,
or several others. Chloral hydrate and paraldehyde, which are
controlled substances (like sodium barbital, they are abusable
sedatives) were formerly used as preservatives, but they're not worth
the administrative hassle of keeping a controlled substance in the

Bob Richmond
Samurai Pathologist and long-ago occasional hematoxylin brewer
Knoxville TN

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