[Histonet] (reply) silly questions.---PFA

Tue Dec 16 16:16:07 CST 2008

Kathy,

Here is a copy of the email I sent Danielle

Danielle,

Here is the method we use:

Test for validity of formalin concentrations

Schiff’s reagent (as used in the PAS stain) is used to detect aldehydes and in this technique it is used to titrate a solution of formalin. The concentration of which is unknown (Jaspers 1987).

Solutions:

1.      1 ml of formalin fixative to be tested.
2.      2.4% aqueous sodium bisulphite (sodium metabisulphite) – prepare fresh.
3.      Schiff’s reagent.

Procedure:

1.      Add 1 ml of formalin to 5 ml of sodium bisulphite.
2.      Mix and react for 15 minutes at room temperature stirring from time to time.
3.      Add 100µl of Schiff reagent.

Results:
If solution turns a deep violet than initial concentration of formalin is in excess of 4% (i.e. 1.6% formaldehyde).

By only using 0.6ml of 2.4% sodium metabisulphite, you can see whether it is greater than 0.5% formalin

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

-----Original Message-----
From: Walters, Katherine S [mailto:katherine-walters <@t> uiowa.edu]
Sent: Saturday, 13 December 2008 12:48 AM
To: tifei <@t> foxmail.com; Tony Henwood; Pat Flannery; histonet <@t> lists.utsouthwestern.ed
Subject: RE: [Histonet] (reply) silly questions.---PFA

This may be another silly question, but how does one test the concentration of formaldehyde in solution?

Thanks,
Kathy

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of tf
Sent: Friday, December 12, 2008 2:35 AM
To: Tony Henwood; Pat Flannery; histonet <@t> lists.utsouthwestern.ed
Subject: [Histonet] (reply) silly questions.---PFA

"I looked at the sections and the cell shrinkage (and prominent spaces between cells and connective tissue) indicated that most of the "fixation" seemed to have occured in the processing ethanols. I asked him for some of the fixative he used, tested the formaldehyde concentration and found it to be less than 0.5%!!"

Tony: Do you think this is because of inproper preparation of PFA in his lab, or the common problem in all researchers using PFA?
         I do think most biomedical labs currently are using PFA to prepare the fixatives!

So, anyone has the idea on a correction preparation procedure of 4% PFA? I noticed some of you dissolve PFA powder in NaOH-conditioned alkaline water, then add concentrated PB solution. We here dissolve PFA in concentrated PB solution directly (heat & stir for 2-3 hours), then adjust pH to 7.4.

We dont have big problem in tissue quaility....except when one want to cut the brain in a cryostat rather sliding microtome. Many times the brain sections from the cryostat have "cheese" like holes/cavities, which almost never appear on sliding microtome-prepared sections.

2008-12-12

tf

发件人： Tony Henwood
发送时间： 2008-12-12  06:18:47
收件人： Pat Flannery; histonet <@t> lists.utsouthwestern.edu
抄送：
主题： RE: [Histonet] Silly Question?

Pat,
I agree with you.
In a routine diagnostic histopathology laboratory, it makes little difference what you use. Around the world for over 100 years most labs use 10% neutral buffered formalin made from concentrated 38%(or there
abouts) formalin (or formaldehyde).
Researchers, though, are a different kettle of fish. They will tend to hang on to misinformed, "mystical" methods believing they are being scientific. Funny, you would think that they, as a group, would be the ones pushing the boundaries and critically assessing each step of their research, ensuring that they understand what and why they are doing it. (Disclaimer - not all researchers are like this, thank heavens!!) Using a formaldehyde solution made from polyformaldehyde can cause problems. One researcher used it and wondered why their morphology was sub-optimal and their p53 immunohistochemistry was negative. He assured me that he had fixed small samples of tissue for 6 hours in 4% formaldehyde and then processed them using ethanol, xylene and wax. I looked at the sections and the cell shrinkage (and prominent spaces between cells and connective tissue) indicated that most of the "fixation" seemed to have occured in the processing ethanols. I asked him for some of the fixative he used, tested the formaldehyde concentration and found it to be less than 0.5%!! This also explains the loss of p53 staining. I gave him some of our routine 10% phosphate buffered fomalin, asked him to fix overnight, and try agin. Low and behold problem solved. How's that for a Friday Flamming!!! Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Pat Flannery
Sent: Friday, 12 December 2008 3:59 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Silly Question?
Please humor me on this if it's obvious (to everyone but me):  why do 
we use paraformaldehyde (which is so inconvenient to make up) rather 
than buffered formalin or just diluted formaldehyde itself?
It seems that around here, some folks prefer paraformaldehyde (either 
2% or 4%) and others use formalin, while some others stick to diluted 
formaldehyde (I see all 4 on labels for specimens submitted for 
histology).  Is it mostly a matter of personal preference or where you 
were trained (i.e. force of habit) or is there a valid reason to use 
each solution (basically the same chemical once in solution, merely 
buffered or not)?  The only answer I've gotten when I've asked is, 
"That's what we always use."
Thanks.
-Pat Flannery (not a "real" histologist - I just play one in the lab) _______________________________________________
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Histonet <@t> lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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