[Histonet] ammonium bromide in fixative

John Kiernan jkiernan at uwo.ca
Fri Jun 12 23:57:35 CDT 2015

Formalin with ammonium bromide is the fixative prescribed for traditional silver and gold techniques for staining neuroglial</x> cells in free-floating frozen sections. Adding ammonium bromide lowers the pH of the fixative because of a chemical reaction with formaldehyde. An Argentinian histologist</x> called Lascano showed in the 1940s that any sufficiently acidified formaldehyde solution (pH 1.5) was just as good as FAB for Cajal's "gold-sublimate" (astrocytes) and for silver carbonate methods (oligodendrocytes, microglia). The reaction that lowers the pH is

4NH4+ + 6HCHO ---> C6H12N4 + 6H2O + 4H+ 
(Subscripts and superscripts in this equation will not be honoured in this email!)

The 4H+ lowers the pH. The other product, C6H12N4 is hexamethylenetetramine, also known as hexamine in Britain and methenamine in the USA, and used in various histochemical staining methods, notably Grocott's method for fungal cell-walls in sections of animal (including human) tissues. The bromide (Br-) ion of NH4Br is irrelevant, in the FAB fixative and also in Globus's pretreatments (dilute ammonia followed by dilute hydrobromic acid). Globus's "bromuration" treatment was applied to frozen sections of pieces of CNS that had been fixed in non-acidified formalin. 

The references for Lascano's work are:

Lascano, E.F. (1946a). Influencia del pH en la impregnacion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:105-114.

Lascano, E.F. (1946b). Importancia del pH en la fijacion del tejido nervioso. Creacion artificial de fijadores</x> tipo formol-bromuro y formol-nitrato de urano de Cajal. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:185-194.

Lascano, E.F. (1946c). Influencia del pH del fijador en la coloracion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 8:272-276.

Not everyone agreed with Lascano! 

Polak, M. (1948). Sobre la importancia del bromuro de amonio de la solucion fijadora de Cajal en la impregnacion argentica del tejido nervioso. Archivos de la Sociedad Argentina de Anatomia Normal y Patologica 10:224-234.

Do you really need this information to pass your HTL qualifying exam? It isn't easily found in books or with Google Scholar. I came across these papers quite by chance when looking over some old journals that UWO's library had set aside for throwing out (Gasp!), about 1980. Yes, they had hit a new low! Their services have, however, been exceptionally good for the last 10-15 years.

Does anyone still use either Cajal's method for astrocytes or traditional silver carbonate methods to stain oligodendrocytes and microglia? They are difficult for several reasons, take up time, and require an old-fashioned freezing microtome to cut and collect the rather thick sections that are needed. Reliable antibodies for immunostaining glial cell-types have been available for many years, and they work on any kind of section. You need some thickness to appreciate the 3D shapes of astrocytes and oligodendrocytes, however they are stained. 

There's plenty of histo-history in the traditional neuroglia stains, which defined the cell-types for identification by electron microscopy and immunohistochemistry. 

John Kiernan
Anatomy & Cell Biology
University of Western Ontario
London, Canada
= = =
On 11/06/15, "Walters, Katherine S"  <katherine-walters at uiowa.edu> wrote:
> Hi all,
> I am studying to take the HTL certification test and ran across a reference to Formalin Ammonium Bromide. I see that it is very good for central nervous tissue fixation, it must be made fresh and that its pH is 1.5. Does anyone happen to know the reason for ammonium bromide in this fixative? I have been looking online and this has not been explained.
> Also, has anyone taken this test lately? I am curious as to how much old techniques, such as mercuric fixatives will be included?
> Thank you,
> Katherine S Walters
> Histology Director
> Central Microscopy Research Facility
> University of Iowa
> 76 Eckstein Medical Research Building
> 431 Newton Road
> Iowa City, Iowa 52242
> 319-335-8142
> Facility Website:
> http://cmrf.research.uiowa.edu/
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