[Histonet] RE: paraformaldehyde crystals v Prills

[David A. Wright]

In reply to:
Histonet Digest, Vol 26, Issue 19 Message: 2

Hello All

It may not help Carl in the UK (I see you are at King's
College, London)  but we have tried various suppliers'
paraformaldehyde and much prefer Fisher Scientific's own brand
(T353-500) precisely because it is a fine prill - particulate
enough not to blow away easily but fine enough to dissolve
"easily".

In my experience though, no suppliers' paraformaldehyde ever
disssolves completely (there is always a fine haze,presumably
of high MW polymer) in a timescale that I find acceptable.  It
will settle out, so if you are happy to decant/pipet off the
clear supernatant the next day, that may be ok, but I use it
for brain perfusions and worry about clogged capillaries.  I
always vacuum filter it through a Buechner Funnel but this is
time consuming and takes a lot of filters unless you want to
watch your formaldehyde de-gas down the vacuum line.  So for
this reason alone (avoiding filtration) it is probably a very
  good idea to follow John Kiernan's formalin suggestion,
especially if you only need a small amount.

This, however raises the question of why you are repeatedly
making small amounts if you don't like messing with the
powder.  Contrary to rumo[u]r, buffered paraformaldehyde keeps
very well (as Dr Kiernan has pointed out - see the archives);
 I have often used it after several months in the cold and
found the fixation to be excellent. Just check the pH first.

A second point is that, from the variations in the protocol he
tried, I'm not sure that Carl understands the desirability of
using NaOH in the prep.  The paraformaldehyde polymer
hydrolyses faster at basic than neutral pH.  If a small amount
of alkali is added simultaneously with or after the PBS
tablet, the pH will not be basic enough to have this effect. 
The NaOH must be added first and the PBS only added once the
PFA is in solution.  I prefer to use dibasic sodium or
potassium phosphate as this directly gives a basic solution
(pH 10-11) in which the PFA dissolves easily (15-20 min at ca.
50°C) and then I neutralize with phosphoric acid once the
solution clears.

I hope this helps
cheers
-David
David A. Wright. Ph.D
The University of Chicago/Neurosurgery
=======================================================
Date: Tue, 17 Jan 2006 12:55:20 -0500
From: "John A. Kiernan" <jkiernan <@t> uwo.ca>
Subject: Re: [Histonet] paraformaldehyde crystals v Prills
To: Carl Hobbs <carl.hobbs <@t> kcl.ac.uk>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <43CD2F88.7CA05654 <@t> uwo.ca>
Content-Type: text/plain; charset=us-ascii

Carl Hobbs wrote:
 
Many of our Groups use paraformaldehyde crystals ( Sigma
P6148) to make up their solution of formalin. Works very well;
4g + 1tab PBS + 6microlitre 10MNaOH.....stir overnight.
Dissolved completely:pH7.2  I am trying to get away from using
this paraformaldehyde: it is very light and prone to spread
around the fume hood when it's weighed out.

     So, I bought PRILLS from Sigma:Problem is, they just do
not dissolve completely, unless I preboil the water.( heating
to 60C is not hot enough to dissolve the Prills.  I have tried
all combinations of the formulation above( eg: no NaOH,
dissolving PBS tablet before adding paraformaldehyde). I want
the procedure with the least risk; boiling may be a higher
risk than the spread of powder.
NB: I cannot turn down/off the fume hoods, for the weighing of
the crystals( which behave more like a very fine powder),
hence my desire to use the heavier Prills.

Be grateful for any insights.
Carl

You can avoid the fine powder and insolubility
problems by making your fixative from formalin
rather than paraformaldehyde. 

A good mixture, shown to be OK even for electron
microscopy, is that of Carson FL, Martin JH & Lynn
JA 1973, Am. J. Clin. Path. 59:365-373. Its
composition is:
Formalin 100 ml
Water 900 ml
Monobasic sodium phosphate, monohydrate 18.6 g
Sodium hydroxide 4.2 g
The pH is 7.2-7.4 and the solution can be kept for
several months.

As always, don't rely on the accuracy of this or
any other email! Consult the original paper before
making and using the fixative.
-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm

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