[Histonet] paraformaldehyde

[Monson, Frederick]

My preference is to prepare 20% formaldehyde in 200-400ml lots (but a
liter can also be made!) from paraformaldehyde and keep it in the
refrigerator.  In my experience, 20% is about as high as I can go, if I
want to keep it for a time. 

What I have copied between the lines is the protocol I use for my own
purposes.  It can be altered, but none of the safety concerns are
changed.
------------------------------------------------------------------------
----
ALL WORK WITH THESE CHEMICALS SHOULD BE PERFORMED IN A FUNCTIONING
CHEMICAL HOOD! 
------------------------------------------------------------------------
---
Careful Preparation of Formaldehyde from Paraformaldehyde (PFA), and the

Safe Use of pHCHO Thereafter  

!!!!!DO IT ALL IN A CHEMICAL FUME HOOD!!!!!
		
1.	Weigh 80g paraformaldehyde - (NOTE:  this powder must be handled
with great care to prevent 'powder splashing' during weighing.)
2.	Suspend in 400ml HOH with constant stirring and  raise
temperature to 50-60oC
3.	Add 5  OR 10N NaOH dropwise at 2min intervals until solution
begins to clear.  Then dropwise at 5min intervals until clearing is
maximized.  DO NOT BOIL!!!!!
4.	Filter on a Beutner funnel with light suction using Whatman #2
paper and store at 4oC in a screw cap bottle.  This solution keeps for 1
or more months at 4oC.
5.	Portions of aliquots can then be diluted to working solutions as
required with buffered saline or other solutions. (NOTE: Working
solutions are used and stored at 4oC in appropriate containers.)
Dilution of the stock solution is performed quickly and dispensing of
the working solution may be accomplished by pouring or pipetting the
working solution into vials. 

Safety concerns:	Formaldehyde is a gas which saturates water at
about 40%(w/v) at ambient temperature and pressure.  Evolution of the
gas occurs most readily when concentrated solutions are diluted, and
when temperatures are high.   IT IS DANGEROUS TO DISPOSE OF 10% OR
HIGHER SOLUTIONS OF FORMALDEHYDE IN A SINK!  Paraformaldehyde is a
polymer of HCHO that depolymerizes under conditions of mild heat
(optimum is 60oC with constant stirring)) and alkalinity.  Solutions
>20%(w/v) tend to repolymerize on standing.  Disposal is only permitted
in accordance with local, state and federal guidelines.  Find out what
they are for YOU!  



Formaldehyde and Paraformaldehyde are NOT synonyms.

When in any doubt, ASK questions.  Obey LOCAL Regulations!!!!
------------------------------------------------------------------------
--
ALL WORK WITH THESE CHEMICALS SHOULD BE PERFORMED IN A FUNCTIONING
CHEMICAL HOOD! 
------------------------------------------------------------------------
---

Hope this helps,
	  

Frederick C. Monson, PhD
Technical Director
Light, Electron, X-Ray and Scanning Probe Imaging and Analysis Center
(LEXSPIAC)
Large Scientific Instrument Core
Geology, West Chester University
S. Church St. and W. Rosedale Ave.
West Chester, PA, 19320
610-738-0437
fmonson <@t> wcupa.edu
URL:  http://darwin.wcupa.edu/cgi-bin/casireserve.cgi

============================================
Knowledge is the key to happiness.  Ignorance might be the key to
contentment.
============================================




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Hawkins,
Hal K.
Sent: Monday, July 24, 2006 3:44 PM
To: histonet
Subject: [Histonet] paraformaldehyde

 

We need to prepare 4% phosphate-buffered paraformaldehyde for a
cooperative study in fairly large volumes, about 2 L at a time.  

 

My only experience in preparing this fixative involved heating to over
60 C, adding sodium hydroxide chips until all was dissolved, then adding
the buffer and adjusting the pH.

 

There seems to be a simpler method in which pre-mixed Dulbecco PBS is
warmed and the paraformaldehyde is dissolved in the buffer.  Has anyone
used this method and does it work without further adjustment of the
final pH?

 

Thanks in advance,

 

 

Hal Hawkins

UTMB, Galveston, Texas

 

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