Re: Lillies fixative
From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
(This message is rather long and is about wrongly
naming things after people. It contains some information
but no direct, practical advice. Read on or not, as
you see fit. There is a significant suggestion at the end.)
I think kkdulany@unmc.edu's mixture is not one of Lillie's.
Fourteen fixatives based mainly on formaldehyde are described
and discussed on pp 30-34 in Lillie'ss big book (4th & last edn,
1976, published less than 3 years before his death at age 83), and
this is not one of them. It would have been quite unlike Lillie
to have invented such a mixture - adding acetic acid to neutral
buffered formalin, and adding alcohol to a phosphate buffer, if
that is what's used in the NBF. (Sodium and potassium phosphates
are insoluble in alcohol and can be expelled from solution in
alcohol-water mixtures. Lillie knew all this stuff.)
Lillie himself described 3 formaldehyde fixatives: a phosphate-
buffered formalin pH 7.0; calcium acetate-formalin; and Lillie's
(1949) AAF. The last of these is: Formalin 10, Acetic acid 5,
alcohol (95-100%): 85 parts by volume (Anatomical Record 103: 611.
Ref from Lillie's book; I haven't checked the original).
Lillie's AAF has the same ingredients as the mixture below. I
haven't done the sums to compare concentrations, but with the mixture
below you may lose the benefits of having acetic acid in the
mixture. Otherwise, if the buffer doesn't hold the pH near neutrality,
you lose the (questionable) advantage of an alcoholic formaldehyde
fixative being neutral. You can't have it both ways! pH about 3 is
good for nuclei, chromosomes etc. pH 7 to 7.6 is poor for chromatin
in light microscopy but good for cytoplasmic details (mitochondria,
some granules, Golgi complex, etc). Alcohol is bad for some of these
cytoplasmic features (but good for glycogen) and is generally better
in conjunction with paraffin embedding than an entirely non-coagulant
formaldehyde solution.
(If a fixative has a coagulant ingredient There is less differential
shrinkage in the mounted and stained sections. By differential
shrinkage I mean such things as empty spaces around cells in the CNS,
separation of epithelium from underlying layers in gut, and many
departures from ideal preservation of glomeruli and tubules in the
kidney. Differential shrinkage is usually present after fixation
in aqueous formaldehyde and paraffin embedding. These artifacts are
not seen in frozen or plastic-embedded sections. They indicate
failure of the fixative to protect the tissue against processing
into paraffin. J.R. Baker surmised that cross-linking of proteins
by formaldehyde impaired penetration of large wax molecules. He may
be right, but I don't know of any experimental work that tests this
hypothesis. Do you?)
---------------------------------
On Thu, 12 Oct 2000 kkdulany@unmc.edu wrote:
> Lillie's Fixative
> Stock solution"
> 200 ml. glacial acetic acid
> 800 ml. 95% alcolhol
>
> Working solution:
> 1 part stock solution
> 3 parts 10% neutral buffered formalin
---------------------------------
That was rather long, but I'm grateful to kkdulany@unmc.edu for
making me sit up and think and pull Lillie's book down off the
shelf. I'm not saying there's anything wrong with the mixture
described above. If it works well with surgical specimens of
suspected mammary tumours, that's good. I don't think it was
really invented by Lillie, who was one of the founding fathers
of histochemistry, especially in the context of pathology. The
credit for mixing acetic acid, alcohol and neutral buffered
formalin is due to someone else, and if this mixture is really
useful it should be critically investigated.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
Phone: (519) 661-2111
FAX (Department): (519) 661-3936
E-mail: kiernan@uwo.ca
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