[Nhcoll-l] [EXTERN] Re: [EXTERNAL] Best practices for transferring specimens from RNAlater to ethanol + transferring specimens from -80 to room temp

Thu Jul 2 10:44:18 EDT 2026

Hi Nate,

yes, slugs are ... are challenge. We once had a PhD that had to fix them alive to preserve the DNA (back in the older days) .... no fun.

Frankly, if you notice that the mucus starts swelling I would brush it off gently. The skin should be sturdy enough, and usually, the chances that you loose scales or delicate appendices in slugs is minor :-)

If you inject, it might be worth aiming for the lung (assuming it is a pulmonate) and/or gut to build up a reservoir inside the specimen.

With best wishes
Dirk

Am 02.07.2026 um 16:32 schrieb Shoobs, Nate:
Thanks Dirk and John,
I will definitely pay very close attention when doing a test with DI water to ensure nothing drastic happens.

I think injections might be a bit overkill for these specimens, as they're small (less than 2 inches in length) and are already in multiple parts. But just to clarify what you mean John, were you suggesting injecting water during the initial rinsing step? Or only suggesting injecting fixatives at the later steps?
The specimens will have thawed already if I rinse them in DI water before transferring them to the graded ethanol series. I understand you may be talking about general procedure rather than our specific case.

Tangentially I will also note that in general, at least among most malacologists, excess surface mucus is not considered an important part of the specimen especially in slugs, and is usually discarded during the initial preservation process when doing the first transfer from fixative ethanol to the permanent storage ethanol. In the past this served the purpose of ensuring the concentration of the storage ethanol was actually the desired concentration (because the water in the specimen can dilute ETOH, but also because water in solution can get absorbed by mucus as Dirk noted). It also makes for an aesthetically nicer specimen that is better for examination/photography/etc.
Nowadays this has additional importance because mucus is a major inhibitor<https://royalsocietypublishing.org/rstb/article/376/1825/20200162/31520/Sticky-problems-extraction-of-nucleic-acids-from> of various molecular biological techniques like DNA extraction/purification and enzymatic processes like PCR. I don't think there's ever a shortage of mucopolysaccharides in and on the tissues of preserved molluscan specimens, especially slugs, even if  the surface mucus is wiped or rinsed off!

Best,
Nate

--
[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><mailto:nhcoll-l-bounces at mailman.yale.edu> on behalf of John E Simmons <simmons.johne at gmail.com><mailto:simmons.johne at gmail.com>
Date: Thursday, July 2, 2026 at 10:02 AM
To: Dirk Neumann <d.neumann at leibniz-lib.de><mailto:d.neumann at leibniz-lib.de>
Cc: 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: [EXTERNAL] Best practices for transferring specimens from RNAlater to ethanol + transferring specimens from -80 to room temp

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Both to avoid the problems resulting from fixatives or preservatives not penetrating thick skin, skin covered with scales, skin coated in mucous, aor frozen tissues, the specimens should be injected with small amounts of the fluid as the tissue thaws. Particularly for larger specimens, the injection sites should be marked on a drawing to avoid over-injecting some sites or failing to inject others. With frozen specimens take extra care not to over-inject because fluids cannot penetrate frozen tissues until they thaw.

A natural mucous coating is part of the specimen and should be preserved if possible--a series of coordinated injections of small amounts of fluid over time as the specimen thaws will keep the specimen intact.

--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 Thu, Jul 2, 2026 at 3:53 AM Dirk Neumann <d.neumann at leibniz-lib.de<mailto:d.neumann at leibniz-lib.de>> wrote:
Hi Nate,

monitor the specimens closely when transferring them into DI water; the mucopolysaccharids might attract water and swell (again). The mucus can absorb up to 98% water, covers the entire body of slugs and shield them against dessication - and impairs fluid transfer. Probably the reason for the reported ineffective penetration rate of tissues, so be alert.

Can also happen with fish - we once attempted to fix a frozen sturgeon. "Rinsing and cleaning it quickly first after thawing" turned out into a complete disaster and left us with a slime-choked sink.

With best wishes
Dirk

Am 02.07.2026 um 05:05 schrieb Shoobs, Nate:
Hey all,
Thanks for the replies. To clarify — The specimens are frozen at -80 currently in the collector’s lab. Once they get shipped here, I could keep them in at -80 or transfer to room temp storage. Or I could even transfer to ethanol and put back in -80.

My main desire is that the specimens remain stable morphologically  in perpetuity.

To John’s point RE preservation of larger specimens — The collector indicated that RNALater was not very effective in penetrating the tissues of the specimens (small terrestrial slugs) so the specimens were dissected in the field to increase penetration into different organ systems. And some have apparently degraded a bit while being stored in -80 in RNAlater.

So there are two questions — one is a procedural question which I think is answered: “how do I best transfer from RNAlater to 70% ETOH” answer: “soak in DI water and then step up through a graded ETOH series”.

But the other question is, is this the best practice / thing one should do if one has important vouchers like this that need to be kept in perpetuity.

The storage medium/space is not an issue (I have plenty of space in our -80) and in our room temp wet collection.
-Nate
-
Nathaniel F. Shoobs, Curator of Mollusks
College of Arts & Sciences Dept. of Evolution, Ecology, and Organismal Biology, The Ohio State University
Museum of Biological Diversity
1315 Kinnear Rd, Columbus, OH 43212
614-688-1342 (Office)
mbd.osu.edu<http://mbd.osu.edu/>
________________________________
From: John E Simmons <simmons.johne at gmail.com><mailto:simmons.johne at gmail.com>
Sent: Wednesday, 01 July 2026 21:36:50
To: Kevin Kocot <kmkocot at ua.edu><mailto:kmkocot at ua.edu>
Cc: Shoobs, Nate <shoobs.1 at osu.edu><mailto:shoobs.1 at osu.edu>; Natural History Collections Listserv <nhcoll-l at mailman.yale.edu><mailto:nhcoll-l at mailman.yale.edu>
Subject: Re: [Nhcoll-l] [EXTERNAL] Best practices for transferring specimens from RNAlater to ethanol + transferring specimens from -80 to room temp

RNA later is marketed as a preservative for RNA and DNA and histological samples of 0.5 cm or thinner, not for larger, whole specimens. It contains sodium citrate tribasic dehydrate (as a buffer) and ammonium sulfate (a protein precipitator), but as far as I have been able to determine, it does not contain any chemicals known to be successful long-term preservatives. The few published reports of it as a successful fixative or preservative are all based on relatively short time periods. Assuming the specimens are not frozen, I would transfer the specimens to deionized water (not distilled water or tap water), the step then up through 20% concentration stages to 70% ethyl alcohol.

--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 Wed, Jul 1, 2026 at 7:48 PM Kevin Kocot <kmkocot at ua.edu<mailto:kmkocot at ua.edu>> wrote:
Hi Nate and all,

What temperature are the specimens currently at? If already frozen solid at -80, I would leave them as-is as they are stable. Otherwise, I would not directly transfer to ethanol but stepwise hydrate/rinse them and then dehydrate them in ethanol.

Best,
Kevin

________________________________
From: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu<mailto:nhcoll-l-bounces at mailman.yale.edu>> on behalf of Shoobs, Nate <shoobs.1 at osu.edu<mailto:shoobs.1 at osu.edu>>
Sent: Wednesday, July 1, 2026 5:41 PM
To: Natural History Collections Listserv <nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu>>
Subject: [EXTERNAL] [Nhcoll-l] Best practices for transferring specimens from RNAlater to ethanol + transferring specimens from -80 to room temp

Hey all,
I have some specimens coming in to our collection soon that have been preserved in RNAlater, but have  been successfully sequenced and are vouchers for a forthcoming publication.
The specimens will mainly serve as morphological vouchers now, as they have been sequenced to the extent that they likely ever will be (transcriptomes have been successfully sequenced).
The RNAlater seems like it might not be a good long-term storage solution for morphological vouchers.

Does any other collection have experience with:

  1.
transferring RNAlater specimens to ethanol (70%)
  2.
deciding whether to store a specimen at -80 long term, vs transitioning it to room temp storage

Best,
Nate

--
[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>
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Stiftung des öffentlichen Rechts;
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Collection Manager, Hamburg

Postal address:
Museum of Nature Hamburg
Leibniz Institute for the Analysis
of Biodiversity Change
Dirk Neumann
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20146 Hamburg
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Sitz der Stiftung: Adenauerallee 160 in Bonn
Vorsitzender des Stiftungsrates: Dr. Michael Wappelhorst

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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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