[Histonet] Question concerning H. pylori staining

jkiernan at uwo.ca jkiernan at uwo.ca
Mon Apr 13 01:34:29 CDT 2020

In the method of Leung et al. (1996 J. Histotechnol. 19:131), the periodic acid oxidizes the neutral gastric mucus to generate aldehyde groups, as in the first part of the PAS method. Treatment with an acidified bisulphite solution then converts the aldehyde groups to aldehyde bifulphite groups, which are strong anions. Alcian yellow (a cationic azo dye) binds strongly to these anionic sites (but not to nucleic acids) and may then change into a permanently insoluble pigment. (The chemistry is similar to that of staining with alcian blue 8G; this isn't the place to discuss all that!)

The next step (mildly alkaline aqueous toluidine blue) imparts blue to nearly everything not already coloured yellow; it cannot displace alcian yellow from the already stained neutral gastric mucus, making for strong contrast. The bacteria stain blue by virtue of their anionic components (nucleic acids and materials in their cell walls) so do nuclei of cells. Toluidine blue, like most dyes with small molecules, stains quickly and is also easy to remove. Alcohol-water mixtures remove such dyes faster than either water or 100% alcohol. The original Leung method recommended removing most of the water by careful blotting, and then taking the slide(s) directly to the first of 3 changes of 100% alcohol.

Real alcian yellow (CI 12840, Ingrain yellow 1) has not been manufactured for many years. Other methods for showing blue bacilli in yellow mucus are available, but the chemistry for getting the yellow is not the same.

John Kiernan
Anatomy, UWO, London, Canada
= = =
From: Gudrun Lang via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 12 April 2020 03:38
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] Question concerning H. pylori staining

I found this instruction for a Hp- stain, that sounds similiar to yours.
They want the slides to be airdried after water-rinsing and before xylen.
But the result should be blue bacteria, not purple.
I would try to let the slides air-dry for about half a minute, then rinse
very-very short in absolute ethanol (one dip). The colour should turn blue
and some dye will be extracted to give a clearer result. Then fast into
xylen, dip a few times, then coverslip.

I assume, that the periodic acid should render some mucins stainable with
Alcian yellow, to give a more contrasted result.

Gudrun Lang

-----Ursprüngliche Nachricht-----
Von: Bob Richmond via Histonet [mailto:histonet at lists.utsouthwestern.edu]
Gesendet: Donnerstag, 9. April 2020 20:05
An: Histonet at lists.utsouthwestern.edu
Betreff: Re: [Histonet] Question concerning H. pylori staining

Michelle (where) asksi: >>We have a question about staining for H-Pylori
Using Quick Stain (Periodic acid 1%, Alician Yellow, Sodium Metabisulfate,
Toluidine Blue stock, Sodium Hydroxide) we notice what clearly  looks like
the H-Pylori purple stained clusters, but after dehydration in 100% alcohol
the purple clusters seem to disappear. Should we just dehydrate using a
slide oven instead of the alcohol? For how long at what temp? Could the
alcohol be affecting the purple color making it too light to see?<<

You identify Helicobacter pylori by its morphology - curved, angled, or
"gull wing" bacilli. Is that what you're seeing? If you do this stain, you
should know how it's interpreted.

The Alcian yellow (correct spelling) method is needlessly complex. What
does the periodic acid do? - A solution of toluidine blue (Diff-Quik 2 or a
generic equivalent) suffices - don't use Diff-Quik 1 with it - dehydrate
through alcohols rapidly.

Bob Richmond
Samurai Pathologist
Maryville TN
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