[Nhcoll-l] [EXTERN] Re: Best practices for ethanol-fixed specimens

Fri Apr 24 11:03:39 EDT 2026

I agree with John, the term fixation doesn't just mean a chemical change, though it often does. I think more broadly one could argue that fixation = any of several irreversible processes that largely arrest biological activity, both in and on tissues. Chemical and physical processes can both fix tissues. For example, one also refers to "heat fixation" in microbiological contexts, which just means "killing and drying out the cells on a slide by passing it over a flame".

The main difference between fixed and unfixed tissues is that the former have been irrevocably altered by some means, and that the normal biological process of decomposition has been arrested (not just slowed down). Usually this is accomplished by dehydration (physical or chemical) but also by cross-linking in the case of fixatives like formalin.

-N

--

[The Ohio State University]
Nathaniel F. Shoobs
Curator of Mollusks
College of Arts & Sciences Dept. of Evolution, Ecology, and Organismal Biology
Museum of Biological Diversity, 1315 Kinnear Rd, Columbus, OH 43212
614-688-1342 (Office)
mbd.osu.edu/collections/invertebrates<https://mbd.osu.edu/collections/invertebrates>

From: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu> on behalf of John E Simmons <simmons.johne at gmail.com>
Date: Friday, April 24, 2026 at 10:43 AM
To: Dirk Neumann <d.neumann at leibniz-lib.de>
Cc: NHCOLL-new <nhcoll-l at mailman.yale.edu>
Subject: Re: [Nhcoll-l] [EXTERN] Re: Best practices for ethanol-fixed specimens

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A correction needs to be made concerning the use of the term “pseudo-fixative.”

Fixation has many meanings, but in general it refers to a chemical or physical process that is used to prevent postmortem changes in tissue. From a histological point of view, fixation also prepares tissues to be receptive to staining, sectioning, or other treatments. There are dozens of definitions of fixation in the literature, and dozens of recipes for fixatives to produce particular results. Tissues can be fixed by dehydration, heat, salts, acids, and other chemicals. Fixation has been practiced for a very long time―histologists cite the year 1833 as the date of the origin of modern fixation because that was when chromic acid came into use as a hardening agent, well before formaldehyde had even been discovered, but specimens were being fixed by various means well before the 1833. The choice of fixative or fixation process depends on what organism you are working with and what results you want.

As discussed in detail in the second edition of Fluid Preservation: A Comprehensive Reference (2025) the term pseudo-fixative is both incorrect and misleading. It seems to have been coined by Stoddard to differentiate between fixatives that form covalent bonds or crosslinks (e.g., formaldehyde and gluteraldehyde) and fixatives that do not form crosslinks. In fact, there is no such thing as a pseudo-fixative because fixation cannot be defined by the formation of crosslinks. There are many ways to fix tissue that do not form crosslinks.

The specimens in question that were put into 95% ethanol have no doubt been fixed, and as Dirk has correctly pointed out, that process has no doubt resulted in osmotic damage to the tissues. Other treatments can still be done on the specimens, including other fixation techniques, but to refer to the use of 95% ETOH as a pseudo-fixative is incorrect. Fixation in alcohol does, in fact, result in changes in the tissues in that it unwinds and disorders proteins and alters the patterns of hydrogen bonding as the tissue is dehydrated.

--John

John E. Simmons
US Fulbright Senior Scholar
Writer and Museum Consultant, Museologica
and
Research Associate, Earth and Mineral Sciences Museum & Art Gallery, Penn State University
and
Investigador Asociado, Departamento de Ornitología, Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima

On Fri, Apr 24, 2026 at 1:43 AM Dirk Neumann <d.neumann at leibniz-lib.de<mailto:d.neumann at leibniz-lib.de>> wrote:
... and that "once they are stable in 95%", but osmotic damage is done (which usually is irreversible and includes cell rupture, which makes the specimens less suited for histology and analysis of fine cell structures, embryos tend to be used for).

With best wishes
Dirk

Am 23.04.2026 um 23:35 schrieb Simon Moore:
ACHTUNG/ATTENTION: Diese E-Mail stammt von einem externen Absender. / This e-mail comes from an external sender.

Hi Beth,

Remember that EtOH is only a pseudo-fixative as it doesn’t really bring about a chemical change.
Sorry to be such a pedant!

With all good wishes, Simon

Simon Moore MIScT, RSci, FLS, ACR
Conservator of Natural Sciences and Cutlery Historian.

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[cid:ii_19dbfeac7c39ba1137c1]

> On 23 Apr 2026, at 21:30, Elizabeth Wommack <ewommack at uwyo.edu><mailto:ewommack at uwyo.edu> wrote:
>
> Hi Verity,
>
> One thing you could do is check to see if they have finished fixing in the 95% ETOH. Check to see if the concentration of the ETOH they are in is 95%. If it is less then they may still be exchanging water with the preservative, and you might want to put them in some fresh 95% ETOH for a bit longer. Once they are stable at 95% ETOH, I switch ETOH fixed material to 70% ETOH for long-term storage.
>
> cheers,
> Beth
>
> Elizabeth Wommack, PhD
> Curator and Collections Manager of Vertebrates
> University of Wyoming Museum of Vertebrates
> Berry Biodiversity Conservation Center
> University of Wyoming,
> Laramie, WY 82071
> ewommack at uwyo.edu<mailto:ewommack at uwyo.edu>
> pronouns: she, her, herself
> www.uwymv.org<https://urldefense.com/v3/__http://www.uwymv.org__;!!KGKeukY!08nloWV5SAQc_MHA2Kf7_856FI9GEc-cCxs7dF0xxzmBdyL5-xEueNeHeCbseH0GL8aWvuvVLHLjyXO43yJ2_5I$>
> UWYMV Collection Use PolicyFrom: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu><mailto:nhcoll-l-bounces at mailman.yale.edu> on behalf of Dirk Neumann <d.neumann at leibniz-lib.de><mailto:d.neumann at leibniz-lib.de>
> Sent: Thursday, April 23, 2026 00:09
> To: nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu> <nhcoll-l at mailman.yale.edu><mailto:nhcoll-l at mailman.yale.edu>
> Subject: Re: [Nhcoll-l] [EXTERN] Re: Best practices for ethanol-fixed specimens
>  ◆ This message was sent from a non-UWYO address. Please exercise caution when clicking links or opening attachments from external sources.
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> Hi Verity,
>
> I would opt for 70%; 95% not only strongly dehydrates the specimens, but also causes a lot of osmotic pressure on tissue membranes, which might effect the usefulness of the specimens for morphological analysis. Tissue subsampling and subsequent storage of tissues in 95% for later molecular investigation might be better.
>
> Hope this helps
> Dirk
>
>
> Am 22.04.2026 um 22:50 schrieb Shoobs, Nate:
>>
>>
>> Depends on what you want to use them for!
>> Formalin is not a great long-term preservative, even though it's a great fixative. Since the tissue is fixed already, unless you want to post-fix for some specific purpose like histology, I'd leave them in ethanol.
>> It's fine to keep things in 95% indefinitely, but if you think they will be used mainly for anatomical study, swapping the 95% for 70-75%  is better for long term storage and will keep the tissues flexible. Some institutions use 80% as a compromise between genetic and anatomical preservation. If you typically voucher frozen material, I'd recommend taking your tissue samples and freezing them before stepping the lot(s) down in concentration. You could even chuck a whole individual in 95% into the freezer.
>>
>> Personally, I rarely if ever use graded ethanol series for going from high to lower concentration, especially for fresher material, I only bother when going from low-high, but I'm unsure how vertebrates typically handle.
>> My 2 cents!
>> -Nate
>> --<image001[26].png>
>>
>> Nathaniel F. Shoobs
>> Curator of Mollusks
>> College of Arts & Sciences Dept. of Evolution, Ecology, and Organismal Biology
>> Museum of Biological Diversity, 1315 Kinnear Rd, Columbus, OH 43212
>> 614-688-1342 (Office)
>> mbd.osu.edu/collections/invertebrates<http://mbd.osu.edu/collections/invertebrates>
>> From: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu><mailto:nhcoll-l-bounces at mailman.yale.edu> on behalf of Mathis,Verity L <vmathis at flmnh.ufl.edu><mailto:vmathis at flmnh.ufl.edu>
>> Date: Wednesday, April 22, 2026 at 4:38 PM
>> To: nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu> <nhcoll-l at mailman.yale.edu><mailto:nhcoll-l at mailman.yale.edu>
>> Subject: [Nhcoll-l] Best practices for ethanol-fixed specimens
>>
>> This Message Is From an External Sender
>> This message came from outside your organization.
>> Report Suspicious
>>  Hello all
>> I am hoping to get feedback on the best practices for some recently fixed fluid specimens.  During some recent field work, our lab fixed some rodent fetuses in 95% ethanol while prepping.  I’m hoping for some advice on how best store them long-term now that they are back in the lab/collections.  Do I keep them in 95% ETOH, re-fix them in formalin, or step them down to 70% ETOH for long-term storage?  Or something else altogether?
>>  Thanks for any insight!
>> Verity
>> ******************************
>> Verity L. Mathis, Ph.D.
>> Mammal Collections Manager
>> Florida Museum of Natural History
>> University of Florida
>> 1659 Museum Road
>> Gainesville FL 32611
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Postanschrift: Adenauerallee 127, 53113 Bonn, Germany

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