[Histonet] mordanting in Bouin's, storage in 70%, processing mouse tissues

Bryan Hewlett bhewlett <@t> cogeco.ca
Wed Jun 21 20:39:37 CDT 2006

Elizabeth, Paul and Gayle,

With respect to the use of  "mordant's" for trichrome and triacid connective 
tissue stains.

To quote John Kiernan:
"A mordant is a substance that serves to bind a dye to a substrate.
Thus cotton can be impregnated with tannic acid, with which cationic dyes 
form insoluble salts.
The tannic acid serves as a mordant for the dye, which would not adhere to 
the uncharged molecules of the cotton.
In histological parlance, however, the term is restricted to metal ions that 
are able to bind covalently to suitable dye molecules,
forming complexes (AKA 'coordination compounds' or 'dye-metal complexes')."
(See also; Baker, J.R. Principles of biological microtechnique: 1958)

Zenker and Helly fluids (as Gayle correctly mentions) were traditionally 
favoured as the fixatives of choice for connective tissue stains.
These fixatives contain mercuric chloride and chromium salts, both of which 
qualify as mordants in the strict sense of the term.
As Gayle mentions, it is possible to fix tissue in formaldehyde and then 
apply the mordant(s) as a pretreatment prior to staining with connective 
tissue stains.
However, it is doubtful if any true mordant action is occurring ( see 
Horobin: Histochemistry.1982),
since these fluids also re-align the reactive side chains on the proteins 
and favour acidophilia.

Bouin's fluid is also a favoured fixative for connective tissue stains, 
however, its mode of action is completely different.
It is, correctly speaking, an accentuator. i.e. a substance that improves 
the binding of a dye to a substrate.
Bouin's fluid works equally well as a staining pre-treatment on 
formaldehyde-fixed tissue prior to performing the connective tissue stain.
Bouin's fluid as a purported 'mordant'(sic) for these stains is a complete 
NO metal ions are present in Bouin's fluid. The principle active ingredient 
is picric acid.
The anionic dyes WILL bind to the fixed proteins in connective tissue, how 
well depends on the nature of the fixative!
Formaldehyde fixation leaves the proteins with a preponderance of acidic 
carboxyl side chains and hence slight overall basophilia.
Treatment in picric acid re-aligns the reactive side chains on the proteins, 
so that there is now a
predominance of basic amino groups and hence maximal binding of anionic dyes 
Mercury fixation is simply not necessary! Nor is any other metal mordant. 
Troubleshooting Histology stains: Horobin and Bancroft 1998, page 129).

Best regards,


----- Original Message ----- 
From: "Gayle Callis" <gcallis <@t> montana.edu>
To: "Monfils, Paul" <PMonfils <@t> Lifespan.org>; 
<Histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, June 21, 2006 5:41 PM
Subject: RE: [Histonet] mordanting in Bouin's, storage in 70%,processing 
mouse tissues

> Mallory's triple stain (I really had to dig into my histopast from 1962!) 
> The reason mercuric chloride is used as a mordant for this stain is that 
> Zenkers (which contains mercuric chloride) was always a fixative of choice 
> for this special stain.  One could actually fix in NBF, and mordant in 
> Zenkers before staining , when we did this stain in the first 
> histotechnics class.
> I do not recall ever using Bouins as a mordant for the Triple, but John 
> Kiernan may set us straight on this whole subject. We only used Bouins 
> either as a fixative or mordant for Massons Trichrome, but if it works for 
> Mallory's, I would do it instead of toxic mercuric chloride.
> Overprocessing of your mouse tissue sounds like the culprit for your dry, 
> brittle tissues.  Mouse tissues are generally very small and can be 
> processed 30 to 45 min per change.  Liver and brain  must be totally fixed 
> before processing due to their homogenous nature or processing finished 
> the fixation and generally dries out the tissues even more.
> 70%, 80%, 95% x 2, 100% x 2, xylene X 2 with 3 changes of paraffin at 30 
> min each as heat will dry the tissues out even more.
> After trimming a block, set it on cold ice water block, but return it to 
> the microtome and DO NOT TRIM OFF WHAT YOU HAVE JUST SOAKED!!!!  You want 
> the first sections that come off the sharp blade.   If your room is really 
> cold, you can even use a RT water soak, or try a gauze soaked in water 
> over the just trimmed block.
> We simply wash Bouins fixed or mordanted sections in running tap water 
> until the color is gone, approx 5 min or so.
> Gayle Callis
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University - Bozeman
> PO Box 173610
> Bozeman MT 59717-3610
> 406 994-6367
> 406 994-4303 (FAX)
> _______________________________________________
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