Re: Lillies fixative

From:"J. A. Kiernan" <>

   (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'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 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 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


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