Re: [Histonet] Long term storage of tissue
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
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
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