Re: [Histonet] Bringing pH up

From:"John A. Kiernan"

Sodium carbonate is an alkali. (The Merck index
gives 11.6 as the pH of an aqueous solution of
unspecified strength.) A 1.0M solution of ammonia 
has pH 11.6; this is equivalent to concentrated
ammonium hydroxide diluted to about 16
volumes with water. The pH will be more stable
with sodium carbonate because it isn't losing
ammonia (gas) or absorbing carbon dioxide from
the air. 

Neither compound constitutes a buffer. Buffers
based on carbonate-bicarbonate combinations are
effective in the range pH 9.2-11.0, and 
ammonia/ammonium chloride mixtures are for
pH 8.3-10.8

A simple buffer that's effective in the range 
of pH 11-12 is the Na2HPO4-NaOH system. For
pH 11.6 take 50 ml 0.05M Na2HPO4 and add
13.5 ml of 0.1M NaOH (Perrin & Dempsey 1974
Buffers for pH and Metal Ion Control. London:
Chapman & Hall Science Paperbacks, p.151)

An alkaline solution of toluidine blue does not
need to be buffered. Above pH 9 it will stain
everything. The higher pH may help the dye
penetrate into epoxy-embedded semi-thin sections.
Penetration can also be enhanced by exposing
the section (before staining) to sodium ethoxide,
which is made by dissolving NaOH pellets in
100% ethanol. This "etches" (partly depolymerizes)
the resin.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
"Johnson, Teri" wrote:
> I've been handed a request to make 1% toluidine blue "buffered to pH
> 11.6 with sodium carbonate."  I always thought sodium carbonate was a
> neutral pH.  Is it possible to bring the pH of this dye solution up with
> sodium carbonate, and if so, what %, N, or M solution would you
> recommend using to do this?
> Interestingly in this procedure, a "1% ammonia" solution is used as a
> rinse.  I tend to think the dye solution is buffered with sodium
> carbonate and the pH is brought up to 11.6 with ammonium hydroxide,
> although I have no reference other than the quoted text for
> clarification.  Thanks for any advice you can offer!
> Teri Johnson
> Managing Director Histology Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, Missouri  64110
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