Re: 4% paraformaldehyde vs 10% bnffered formalin

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From:"J. A. Kiernan" <>
To:Colleen Forster <>
Date:Wed, 13 Oct 1999 10:42:57 -0400 (EDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

(I took the liberty of expanding the Subject line)
On Tue, 12 Oct 1999, Colleen Forster wrote:

> I have an educational test question I need help with. What
> is the difference in how para works compared to BNF ??

   They both work exactly the same way.

   The only difference in composition of the two
   fixatives is that 10% BNF (or NBF) contains
   about 1% methanol. If made from ancient formalin
   it will contain more methanol and also some
   formate ions, and less than the official 3.7 to 4%
   of formaldehyde. Probably the methanol and formate
   make no difference whatever to the properties of
   the solution as a fixative, if the pH is in the
   range 7 to 7.6.
> I use 4% para for all my research perfusion and wonder why
> that is recommended over 10% BNF? 

    1. Tradition, dating from about 1960.
    2. When you use paraformaldehyde you have complete control
       over the composition of the fixative, and it will be the
       same every time you make it up. If something goes
       unexpectedly wrong, you won't wonder, "Was it because
       of using a 5 year-old stock bottle of formalin?"
         This is reassuring, but within reasonable limits it  
       is doubtful if "old formalin" makes bad fixatives.
       An exception may be formalin that has stood for a long
       time in the cold and contains a considerable precipitate
       (which is paraformaldehyde) in the bottom of the bottle.
       Fortunately the concentration of dissolved formaldehyde
       has little effect on its fixative properties.

> Also what is the actual chemistry going on that para needs to 
> be at 4 C when fixing vs room temp like BNF??

    The chemistry is just the same, but the reactions of fixation
    occur more slowly at the lower temperature. To achieve the
    same amount of cross-linking of protein molecules you might
    need 10 hours at 24C or 40 hours at 4C. For some research
    purposes, especially preservation of enzymatic activity in
    a tissue, it is desirable to have minimal chemical fixation,
    often only a few minutes, to avoid enzyme inhibition while
    getting just enough structural stabilization to allow the
    cutting and handling of frozen sections. The quality of the
    final preparation is often surprisingly good, probably
    because further fixation occurs after doing the histochemistry,
    when the sections are dehydrated in alcohol. A short time in
    formaldehyde makes it more difficult to cut frozen sections
    and gives poor results when it precedes paraffin embedding.
      To get the best out of a fixative that has formaldehyde 
    as the only active ingredient, you need to fix for at least
    one week. Some authorities say 2 or more weeks. There are
    plenty of fixative mixtures that do a better job than
    formaldehyde alone, and in much shorter times, but there
    is always some trade-off: nearly always destruction of
    enzymatic activity, and sometimes also interference with
    immunohistochemical stainability. Formaldehyde is one
    of the worst fixatives when it comes to clogging up antigenic
    sites, but its slow reactivity allows you to get away with
    incomplete fixation of structural proteins. Furthermore,
    the cross-linking can be partly undone by heating in
    water (antigen retrieval).
> Anyone know the answers to these trivia I would appreciate it.

    Your questions were not trivial, Colleen. Queries along these
    lines appear regularly on the HistoNet listserver, and they
    serve to emphasize that if you are doing histotechnology you
    can't know too much about formaldehyde. 

    Here are two trivia that are too true: 
    1. There is no such animal as "4% paraformaldehyde." When the
       powder has gone into solution it has depolymerized, and the
       solution does not contain any paraformaldehyde (which is,
       by definition, an insoluble polymer of formaldehyde).
    2. There is no formaldehyde in any solution that claims to
       contain the compound. Concentrated solutions (formalin)
       consist almost entirely of low polymers. In a dilute aqueous
       solution the solute is HOCH2OH (methylene glycol), not
       HCHO (formaldehyde, which is a gas). Alcoholic fixatives
       that contain "formaldehyde" actually contain compounds
       analogous to methylene glycol (methylal, ethylal etc).
       Their chemical reactions are essentially the same as those
       of formaldehyde:
          That's why if you'll pardon the pun,
            Formality isn't much fun.
          Though no-one says "methylene glycol"
            It's a chemical version of "formol-,"
          Which word was despised by Baker the Wise,
            Who correctly insisted on "formal-."

       That's why formal-saline is OK but formol-saline is non-U.

    If you want to do some serious reading about matters of
    formaldehyde, fixation, life, the universe and everything
    just ask, and I'll send you a reading list about formaldehyde
    and fixation. I'm told that the answer to the larger question
    is 42, but the question itself is unknown.  

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

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