Re: [Histonet] Long term storage of tissue

From:Robert Krug

Peggy:  Thanks for sharing your expertise.  I would also like to thank the 
other people who have responded to my query.    I have a copy of Bancroft 
and Gamble.   I also have the 2nd and 4th Editions of Bancroft and 
Stevens.  The 2nd Edition has a nice section on preparing Museum Quality 
tissues. The chapter implies the tissues seemed to be used only for 
viewing and  the tissues are processed and stored in various Kaiserling 
solutions.  No mention is ever made to the tissues being utilized for 
anything except viewing. 

What I am really trying to locate is a study or a textbook which states at 
what intervals the formalin should  be changed out - or does it  ever need 
to be changed.  For every reference I find documenting the formation of 
formic acid, I find another piece of information which simply states 
tissues may be stored in formalin forever.  From the time I first started 
working in Histology, I was always told that tissues stored in formalin 
had to have the formalin changed on a yearly basis.  I can't remember that 
we ever changed formalin solutions.  There were no OSHA requirements for 
formaldehyde monitoring at that time. Changing the solutions or disposing 
of formalin fixed tissues was always a dreaded experience in the days 
before OSHA reclassified formaldehyde.   The bottles of formaldehyde 
simply advised the user to "seek fresh air if you feel light headed." If 
we needed to archive tissue, the tissue was always fixed in formalin and 
placed in 70% ethanol before storing.   Why?  Because that's the way the 
lab had always handled tissue.     I do have some experience with 
spirochette controls which were fixed in formalin for around 1 week and 
then stored in 70% alcohol.  This tissue was eventually embedded into 
paraffin blocks, but the tissue was probably 5 years old before the last 
of the tissue was embedded.  The staining with the Steiner Steiner 
procedure remained unchanged from the time the tissue was first processed 
to the time the last  wet tissue was section and processed. 

When I was in the Army, the entire block of tissue from the visceration 
was stored in a large pan and submersed in formalin.  The bodies went to 
the funeral directors without the tissues being returned.  The volume of 
10% NBF was never really sufficient to properly fix the large volume of 
tissue retained.  Eventually the formalin evaporated and fungus would 
sometimes be noted on the tissues. If the reaction were unreversible, why 
the formation of fungus?   Although the center of the tissues were 
unquestionably poorly fixed, it seems the tissue surface should have been 
fixed.   By that time  the fungal growth was noted, the cases had been 
signed out and the tissues were incincerated.  I seriously doubt if many 
labs today would consider handling human tissues the way we did in the 
 Many thanks

Bob Krug
St John, Missouri

09/23/2004 06:06 PM

        To:     "Robert Krug" 
        Subject:        Re: [Histonet] Long term storage of tissue

To build on what van der Loos and Pierce stated -

Yes, formic acid is created when formaldehyde reacts with oxygen. Long 
storage in formic acid, even low levels, will cause destruction of 
in the tissue.

Yes, additional cross-links are formed, which can interfere with staining,
by blocking epitopes or distorting the proteins so they are not stainable.

>From personal experience. One year, we got a great case of lung TB from 
autopsy, and a wonderful kidney with amyloid from another autopsy. We
grossed a lot of each for controls, and stored the rest in formalin. About 
years later, we had run out of the blocks of controls, and went back to 
stored wet stock, and grossed a bunch more blocks.

Neither would now stain positive for the AFB or the amyloid. They could
still be "seen" on the H&E, but the Kinyoun would no longer stain the
mycobacterium, nor would the Congo red stain the amyloid. No matter how we
altered the stain (increase time, heat, less differentiation, etc.).

Now, I don't know if the proteins in the TB cell wall and the amyloid
proteins were ruined by acid destruction or excess formalin cross-links or
by protein distortion. It really didn't matter the root reason. Basic fact 
the tissue would no longer stain. Lost were two cases of great control

As for a source - How about "Theory and Practice of Histological 
5th ed., John D. Bancroft and Marilyn Gamble, 2002, Churchill-Livingstone.
In the chapter on "fixation", under the topic "duration":

"Long fixation in these aldehydes (fixatives) is known to severely inhibit
enzyme activity and immunological reactions. This has been attributed to
limitations in substrate diffusion and cross-link formation between 

Most histotechnique textbooks discuss the formation of formic acid with 
term storage of tissue in formalin.

Hope this helps.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

----- Original Message -----
From: "Robert Krug" 
Sent: Wednesday, September 22, 2004 2:57 PM
Subject: [Histonet] Long term storage of tissue

> I have a question concerning the long term storage of tissue which is 
> addressed in any of the histology textbooks I have on hand.  After many
> hours of searching, I was unable to find an  answer through a websearch.
> Can tissue be preserved in 10% neutral buffered formalin for decades
> without changing the 10% NBF (provided the container is sealed and does
> not leak)?
> A former coworker of mine once worked at the CDC and she stated the CDC
> used 70% alcohol  for the long term storage of tissue.  A review of the
> Internet shows that the Australian Museum Fish site states they have 
> in "formaldehyde and then stored in alcohol"  for the last 80 years. 
> site claims to have thousands of specimens on hand.    Another site I
> found recommended storage in formalin as a way to keep the original 
> of the tissue and recommended against long term storage of tissue in
> alcohol for various reasons.
> The unused 10% neutral buffered formalin solution does decompose over 
> and is typically assigned an expiration period.  Once the tissue has 
> fixed in 10% neutral buffered formalin, is the reaction stable, meaning
> the bonds are stable and no decomposition will occur?  I was always told
> the formalin solution degrades with time and may form formic acid and
> other by products.   Given enough time the remaining formalin in 
> might  form formic acid, drop out of solution as paraformaldehyde or
> escape as formaldehyde gas.   Another coworker feels that once fixation
> has occurred, the fixation is permanent and there is no need to change 
> solution.  I realize the fixation bonds can be broken through washing in
> running water and with select chemicals.  But given a sealed container,
> will the bonds remain permanent and the tissue free of decomposition or
> fungal growth?
> Any jounal articles or textbook references you have to support your view
> point would be greatly appreciated.
> Many thanks
> Bob Krug
> St John, Missouri
> _______________________________________________
> Histonet mailing list

Histonet mailing list

<< Previous Message | Next Message >>