Re: Bielchowsky stain

From:"J. A. Kiernan"

> "Nymeyer, Heather" wrote:
>  ... The method being used is a 20 % silver nitrate 
> solution for 20 mins followed by a 15 min incubation
> (in the dark) of ammoniacal silver. ... A few drops of the reducer
> is added to the ammoniacal silver and checked microscopically
> until fibers are seen.  At the same time the fibers are being
> developed the excess background precipitate is forming.

This is what to expect if you add formaldehyde to an
ammoniacal silver solution; it's one way of making
mirrors. In all the variants I've encountered (and most
bear little resemblance to the methods published by
Max Bielschowsky from 1904-1910), the sections are put
in the reducing solution after the ammoniacal silver.
There may or mmay not be a distilled water rinse
between these steps, and this is a critical feature
affecting the result. A small amount of ammonia-silver
complex must be carried over, either as free liquid or
not washed out of the tissue. 

The principle of the method is that silver precipitation
occurs fastest in the axons because these already
contain small amounts of colloidal silver, formed 
during the immersion in silver nitrate. Eventually
there will be silver precipitation everywhere, hence
the need for microscopic control of the reduction
step. The rate of reduction is affected by a variety
of factors such as the concentrations of silver and
ammonia and the pH. (These are explained by Nauta WJH
& Gygax PA (1951) Stain Technol. 26:5-11 in a paper
about their Bielschowsky-like method for degenerating

If you're staining normal axons there are easier
silver methods than those based on Bielschowsky's.

John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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