RE: [Histonet] RE: formalin [Longish]

From:Kemlo Rogerson

WOW! Give up, you're the man!!!!

-----Original Message-----
From: John A. Kiernan [] 
Sent: 16 May 2005 19:11
To: Kemlo Rogerson
Cc: Glencross Hedley (RW3) Central Manchester & Manchester Children's;
Subject: Re: [Histonet] RE: formalin [Longish]

Kemlo, I think your atmospheric pressure ideas
would be true only if our atmosphere consisted
largely of gaseous formaldehyde, which fortunately
is not the case. (Formaldehyde molecules are said
to be present in interstellar space; perhaps that's
why there isn't any life out there among the fixed 

The gas dissolves in water because it's very soluble 
and also because, as you point out, there is a chemical 
reaction that forms methylene hydrate. In 37% 
formaldehyde (neat formalin) 37% of the weight is 
derived from formaldehyde gas and 63% from water. 
40% is the wt/vol  expression (There are 40 g of 
formaldehyde in 100 ml of the 37% wt/wt solution.) 

Despite the smell, not much formaldehyde escapes
into the air. When formalin evaporates, most of the 
formaldehyde ends up as the solid polymer 
paraformaldehyde. Evaporation might occur more
quickly on top of Mt Everest than at sea level,
especially on a sunny day with a brisk wind; 
I'm sure the YETIS (Yogic Engineering and Technical
Institute for Sherpas) up there know this, and fix 
their specimens in capped jars and vials.  It is 
possible to distil aqueous solutions of formaldehyde, 
and Walker's book ("Formaldehyde", 3rd ed 1964) gives 
tabulated data for the composition of distillates 
obtained under different conditions. 

Regarding the quotation, "It has been suggested that 
the hydrated form, methylene glycol, is the reactive 
component ..." this was certainly once believed (see
for example, Walker's book and the first 3 editions
of Pearse's Histochemistry). The more recent view
is that the methylene glycol constitutes a reserve,
always in equilibrium with a very small concentration
of dissolved formaldehyde molecules and water. When
a formaldehyde molecule is pulled out of the solution
(by combining with part of a protein molecule, for 
example), it is immediately replaced from the
reservoir of methylene glycol. I do not know enough
chemistry to fully understand the experimental 
evidence, but it was published in chemical journals,
presumably with knowledgeable chemists doing the
peer-reviewing. For a recent review of fixation, see
D. Hopwood's chapter in Bancroft & Gamble's "Theory 
and Practice of histological Techniques" (2002). The
fixation chapter in the 4th edition of Pearse (Vol 1, 
1980) is older but seems still to be up to date. The
short review "Formaldehyde fixation" by Fox et al
(1985) J Histochem Cytochem 33:845-853 is worth
reading because it covers the earliest applications
as well as the current understanding of th chemistry
and its practical implications.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Kemlo Rogerson wrote:
> "Formaldehyde, as 4% buffered formaldehyde (10% buffered formalin), is the
> most widely employed universal fixative particularly for routine paraffin
> embedded sections. It is a gas with a very pungent odour, soluble in water
> to a maximum extent of 40% by weight and is sold as such under the name of
> formaldehyde (40%) or formalin (a colourless liquid). Formaldehyde is also
> obtainable in a stable solid form composed of high molecular weight
> known as paraformaldehyde. Heated paraformaldehyde generates pure gaseous
> formaldehyde which, when dissolved in water, reverts mostly to the
> form. Aqueous formaldehyde exists principally in the form of its
> monohydrate, methylene glycol, CH2(OH)2, and as low molecular weight
> polymeric hydrates or polyoxymethylene glycols. It has been suggested that
> the hydrated form, methylene glycol, is the reactive component of
> formaldehyde but this has been disputed2"
> Fixation and fixatives
> Anthony S-Y Leong
> So as it is a gas it must be held in solution by atmospheric pressure? If
> that atmospheric is raised or lowered then lesser or greater amount of gas
> can be held in solution; the same with increasing or decreasing
> temperatures. So is formalin 40% formaldehyde gas up Everest or is it much
> less? Atmospheric pressure is reduced so the Saturated Vapour Pressure of
> the solution may exceed barometric pressure and the gas is driven off
> the pressures equilibrate; plus it is very cold. Do you have to make a
> stronger dilution up Everest to end up with a 4% buffered formaldehyde?
> there any Histo Labs up there? Who cares?
> Kemlo Rogerson
> Cellular Pathology Manager
> East Lancashire Hospitals NHS Trust
> DD. 01254-294162
> Mobile 0774-9754194
> -----Original Message-----
> From: Glencross Hedley (RW3) CM&MC Manchester
> []
> Sent: 16 May 2005 14:02
> To:
> Subject: [Histonet] RE: formalin
> Hi everyone
> It has always been my understanding that "formalin" like "Hoover" &
> "Sellotape" (and countless other words) is just a trade name. This
> describes a 37% solution of formaldehyde gas in water, and we should be
> talking about 3.7% (4%) formaldehyde and not 10% formalin.
> Regards
> Hedley Glencross
> Manchester Cytology Centre UK
> For all my years I have believed that formaldehyde was the 37%
> concentrate without buffers, and that formalin was 10% buffered... learn
> something new every day!
> Thanks John!
> -----Original Message-----
> From: John A. Kiernan []
> Sent: Friday, May 13, 2005 11:31 AM
> To: Weems, Joyce
> Cc:
> Subject: Re: [Histonet] quick question about formaldehyde
> Dear Joyce,
> Formalin = 37% formaldehyde; so no, it doesn't
> make a difference. Formalin is not buffered; it
> does contain about 10% methanol, which is put
> in to retard polymerization. When diluted to
> make a 4% formaldehyde fixative, the methanol
> concentration is 1%. Buffering of the dilute
> solution offsets pH changes due to the
> Cannizzaro reaction. It also inhibits the
> formation of blood-derived "formalin pigment"
> which forms after fixation in an acidic
> formaldehyde solution.
> Tim Morken is correct in saying we don't know
> the extent of chemical change in 12 year-old
> formalin. The fact that there's no expiry date
> sugggests that it's not much. For what it's
> worth, I've used formalin that's more than 5
> years old and fixation has been OK.
>                       John Kiernan
>                       london, Canada

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