[Nhcoll-l] [EXTERN] Re: Preserving frozen fish

Tue Aug 19 15:09:27 EDT 2025

just briefly adding to John's thoughtful recommendations:

the size of the specimens is a relevant factor, also for the decision to inject formaldehyde or (just) ethanol. As it can be assumed that freezing has damaged the cell membranes, I would personally always prefer formalin injection, because weak thawed tissues will not get better for the reasons John mentioned if treated with ethanol. This might be less of a problem in fresh fish, but it sounds like a lovely frezzer-job with a lot of 'history' waiting to be dealt with.

When I had to fix large frozen Whitefish, I thawed them (rinsing) in cold water for few hours, and as soon as the outer 2-3 centimetres where thawed and I could stretch the fins and body, I moved them into 4-7 % formalin for complete thawing. If the specimens looked to be dead for quite some time before hitting the freezer, I would inject the belly with formalin as well. As the gut contents still can be frozen, pressure from the injected formalin might build up, therefore the needle/syringe should be removed carefully and PSA should be in place. It might be useful to cover the injection hole with a finger when removing the needle.

Hope this helps
Dirk

Am 19.08.2025 um 20:51 schrieb John E Simmons:
I suggest you separate this into two issues, (1) preserving tissues for DNA and (2) preserving the whole fish as a fluid preparation.

For DNA preservation, I think that the best procedure will be to remove a tissue sample from the frozen specimen and store the sample at -80C (for example, in liquid nitrogen). Ultracold temperature is a much better long-term preservative than 100% ETOH.

The problems with preserving directly in 100% alcohol are that:
(1) 100% ETOH is chemically dehydrated and may have traces of the dehydrant chemicals in it that may interfere with future molecular work (not to mention that 100% ETOH is more expensive than 95.6% ETOH).
(2) As the alcohol penetrates the specimen it dehydrates the tissues, which impedes the penetration of alcohol to deeper tissues. Using 70% alcohol means less dehydration of the tissues (allowing deeper penetration) while still providing a good biocide to protect the specimen during the preservation process.
(3) Once the specimen is preserved you will need to change the alcohol anyway because it will have become diluted with water extracted from the specimen. The chance of diluting the 100% ETOH to 70% by extraction of water from the specimen is slim, so there is no real gain in starting with 100%.

Preservation in 70% ETOH without using a formaldehyde fixative works fine as long as the alcohol can get deep enough into the specimen. This is why I recommend thawing the specimen in 70% ETOH and injecting the alcohol or cutting into the specimen as it thaws to get the alcohol deeper into the specimen, and after 24 hr to change the specimen to fresh alcohol. Using 100% or even 95.6% alcohol will produce a more dehydrated specimen, not a better preserved one, and is not likely to produce very good DNA.

If you thaw the specimen in a 1:9 buffered formaldehyde and water solution, the recommendation to inject the fixative into (or cut into) the specimen as it thaws still holds, but you can still get good results even if the formaldehyde mixture becomes diluted (formaldehyde penetrates better than alcohol).

Formaldehyde has only been available since the mid 1890s, so specimens preserved prior to that were preserved directly in alcohol. It is instructive that almost all of the pre-formaldehyde instructions for preservation call for the use of alcohol at a concentration below full strength and to change to fresh alcohol preservative after 24 hr.

I hope this information is helpful.

--John

John E. Simmons
Writer and Museum Consultant
Museologica
and
Investigador Asociado, Departamento de Ornitologia
Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima

On Tue, Aug 19, 2025 at 2:13 PM Rebecca Abrams <ridabrams at berkeley.edu<mailto:ridabrams at berkeley.edu>> wrote:
Hello all,
I have an ever increasing number of frozen fishes coming to me from a local aquarium that I will be preserving in either ethanol or formalin then ethanol. Our usual protocol is to use 100% ethanol for reasons of DNA preservation (I know this is an unusual protocol) if the specimen will fit into one of our jars, otherwise it goes into formalin then into 70% ethanol.
Both ASIH's online resources and John Simmons's Fluid Preservation say to avoid preserving frozen specimens and if one has to then to thaw them in the preservative and inject with preservative as they thaw.

If anyone has tips and tricks for this in particular for fish that would be very helpful. I have some rather large friends waiting for me to prep that I would like to not ruin by turning them to mush first. I know that I can skeletonize them, but I would very much like to have at least one in fluid.

Thank you,
Rebecca Abrams
MA, M.Ed
Collections Manager- Fishes
Museum of Vertebrate Zoology
3101 Valley Life Science Building
University of California, Berkeley, CA, 94720
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Stiftung Leibniz-Institut zur Analyse des Biodiversitätswandels
Postanschrift: Adenauerallee 127, 53113 Bonn, Germany

Stiftung des öffentlichen Rechts;
Generaldirektion: Prof. Dr. Bernhard Misof (Generaldirektor), Adrian Grüter (Kaufm. Geschäftsführer)
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