On Wed, 21 Apr 1999, Masayuki Miyagishima, MD asked 2 questions:

> 1) What is the difference between fresh made paraformaldehyde and
> formalin, chemically?

   Almost none. As the paraformaldehyde "dissolves" it depolymerizes
   and becomes formaldehyde. A formaldehyde solution made by diluting
   a stock liquid (37-40%), typically tenfold, also contains a little
   methanol (which is included as a stabilizer in the concentrated
   solution). An older solution, however made, acquires increasing
   concentrations of methanol and formic acid, largely from formaldehyde
   molecules reacting with one another. The acidification is
   largely taken care of by the buffer or other neutralizing additive
   such as calcium acetate, or marble chips. Probably the small
   amounts of methanol and formate ions in a working formaldehyde
   solution make no difference for ordinary light or electron
   microscopy. Conceivably certain enzymes might be adversely
   affected. Perhaps someone knows an example.

> 2) Why does the red bible recommend to use 4% paraformaldehyde at 4 C.
> Is it to make the light fixation?

    The chemical reactions of formaldehyde fixation are slower than 
    those of any other fixative agent. Full cross-linking and
    structural stabilization take 2 weeks, and you need 4 or 5
    days to protect against alcohol, hot wax etc. With brief
    fixation and paraffin embedding you see the consequences of
    only partial chemical cross-linking of proteins, combined with
    partial coagulation by the dehydrating agent. A lower temperature
    will slow down the chemical reactions: a rough & ready rule is
    half the speed for every 10 deg C drop.

    Inadequate formalin fixation can be valuable for several purposes,
    including immunohistochemistry when you want plenty of unreacted
    stretches in the protein chains. For enzyme histochemistry,
    minimal fixation can prevent a cryostat section of an unfixed
    object from disintegrating in an enzyme histochemical incubation
    medium. In fact you should give as much fixation as you can get
    away with. The microanatomy and cytoplasmic details in minimally
    fixed cryosections are rather poor by comparison with a plastic
    section of something thoroughly fixed in a glutaraldehyde-containing
    mixture (glutaraldehyde reacts quickly but penetrates slowly), or
    a thin paraffin section following a classical fixative mixture
    like Helly's fluid.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1