[Histonet] Malmberg's fixative

J. A. Kiernan jkiernan <@t> uwo.ca
Wed Sep 29 09:44:34 CDT 2004

Is this for preserving vital staining with
methylene blue? That's the only use of
ammonium picrate that I've come across.
Sorry but I cannot find a reference to 
Malmberg. Someone will!

To make a saturated ammonium picrate
According to Peter Gray (1954: The
Microtomist's Formulary and Guide, 
p.396-397), add picric acid to a
saturated aqueous solution of same,
so that plenty of undissolved picric 
acid is present. Add ammonia, with
shaking, drop by drop until the
surplus picric acid starts to dissolve.
Then add more ammonia until the
solution smells of free ammonia.

Gray warns of the explosive properties
of ammonium picrate; the solution
must not be allowed to evaporate to
dryness. This is a much more dangerous
compound than picric acid (see Merck
Index, for example).

The compound usually used to immobilize
methylene blue in vitally stained tissue
is ammonium molybdate, often 6% or so, 
acidified or in a phosphate-citrate
buffer, pH 5.5. This can be followed
by a real fixative such as formaldehyde
or glutaraldehyde prior to dehydration,
clearing etc.

A prominent user of vital methylene blue
in recent decades has been T. Muller, who
has introduced various innovations to
the staining method and the fixation
that follows, especially for central
nervous tissue. Here is a list of some 
of his papers.

Muller T (1989) Paraffin sections of nervous tissue supravitally
stained with methylene blue: a new, reliable and simple fixation
technique. Stain Technol. 64: 93-96.

Muller T (1990) Supravital staining of murine brain with
methylene blue according to the Cajal method: a simple and
reliable preparation technique. J. Neurosci. Methods 32: 223-226.

Muller T (1992) Light-microscopic demonstration of methylene blue
accumulation sites in mouse brain after supravital staining. Acta
Anat. 144: 39-44.

Muller T (1994) Large nerve cells with long axons in the granular
layer and white matter of the murine cerebellum. J. Anat. 184:

Muller T (1994) Supravital uptake of cationic dyes by mast cell
granules: a light and electron microscope study. Biotech.
Histochem. 69: 171-176.

Muller T (1996) The innervation of taste buds in the soft palate
of the rat as revealed by methylene blue staining. Arch. Histol.
Cytol. 59: 47-54.

Muller T (1998) Methylene blue supravital staining: an evaluation
of its applicability to the mammalian brain and pineal gland.
Histol. Histopathol. 13: 1019-1026.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
"McCollough, Carol" wrote:
> Greetings Histonetters:
> I have been asked for a recipe for Malmberg's fixative (1:1 ammonium picrate and glycerol).  I cannot find a method for the preparation of ammonium picrate.  Does anyone have a recipe or a commercial source for this nasty?
> Thanks very much.
> Regards -
> Carol
> **********************
> Carol B. McCollough, HT/HTL(ASCP)
> Diagnostics & Histology Laboratory Manager
> Oyster Disease Research Project
> Fisheries Service
> Maryland Department of Natural Resources
> Cooperative Oxford Laboratory
> 904 S. Morris Street
> Oxford, Maryland 21654
> 410-226-5193
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