Re: Acidic tol blue

From:"J. A. Kiernan" <>

On Thu, 21 Jun 2001, Erin Ellison wrote:
> I'm trying to stain oesophageal mucosal glands in vivo - the only reference 
> I can find is too cryptic for me and I'd appreciate a translation into a 
> methodology I could use, if this makes sense to anyone.
> "Muller's fixative acidic toluidine blue (Schridde 1904)"

I've not seen Schridde's paper, but your one-liner 
is quite informative.  Here are some facts, and also
some guesswork that I think is probably accurate.

1. Muller's fluid is an old fixative (2.5% potassium
dichromate in 1% sodium sulphate). It's good for
cytoplasmic things such as bits of mucus, and even
mitochondria and the Golgi apparatus, but it's
poor for nuclei, allowing them all to look much
the same, and micro-anatomical preservation
on a larger scale is lousy (meaning things shrink
differently while being dehydrated etc, so that
paraffin sections end up full of artifactual splits, 
spaces and general distortion). By 1904 Muller's
was an old-fashioned fixative, superceded for
nearly all purposes by better mixtures. Perhaps
Schridde was old, or so obscessed with seeing mucus
in cells that he didn't notice the nuclei or the
layers of tissue deep to the mucosa.

2. Acidic toluidine blue could mean many things.
In 1904 it may have meant a solution in 0.1N HCl
(pH close to 1) or in 0.1N acetic acid (pH between
2 and 3 with the dissolved dye). Either solution 
is too acidic to stain nuclei, but it would
stain acid glycoproteins of mucus: at pH 1 only
the sulphated glycoproteins stain, and with
toluidine blue they are metachromatic (red). At
pH 2.5 the sialoglycoproteins also stain, but
in the orthochromatic (blue) colour.
A modern equivalent to Schridde's method (if my
speculations are correct) is to stain with toluidine
blue at pH 2.5 after a better fixative than Muller's.
Neutral buffered formaldehyde or an acetic-alcohol
should be fine. If you are worried about losing
the stain during dehydration, use alcian blue

There is an abundance of published literature to
support all that is in the paragraphs above, and
much of it is summarized and cited in textbooks 
and in the catalogues and leaflets of companies 
that sell stains.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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