[Nhcoll-l] Alcohol concentration for terrestrial vertebrates

Fri May 7 03:48:42 EDT 2021

Dear Tonya, John, Simon, Dirk - well all,

Also I agree. Since I will soon retire I want to share some experiences:
When we started to take care of the collection of wet vertebrates in Stockholm in 1975 there was no overlap in time (well 1 week) with the previous staff (the previous curator was employed 1934-1974). So we had to invent the wheel. The initial ambition was to keep a concentration between 70 and 80% ethanol. (We also tested the new suggested conservation fluid Phenoxetol, which after some years showed to be a disaster). To compensate for evaporation, we tried to stick to 80%. New material was fixed in formalin for at least a week, washing in water, 20% ethanol for two days or more, 50% for two days or more, and final storage in 80%. Also we removed all bad jars from the collection – and a bad jar was a jar that needed topping. Expedition material was sorted and identified etc after this stage with the result that many specimens was changed to 80% once more. It took more than 10 years to realize that 80% was to strong. But also that every change of alcohol, or topping, resulted in a higher concentration ethanol since the lowering effect of the alcohol concentration through remnants of the previus stage fluid inside the specimens was removed. Also the small amounts of formalin in the specimen was reduced for each change of fluid. Especially for tiny fish we could find obvious shrinking. Today we are careful

1.       To keep the specimens in 70% (not more, not less)

2.       Not to rinse to much in water. Rather remove the formalin from the surface of the specimen only.

3.       Don´t change the fluid if it is not necessary.

4.       If you have to remove all fluid, add maybe 80-90% of fresh (70%) ethanol and the rest used ethanol from another specimen.
All formalin fixed specimens has a small amount of formalin left - that is good.
Some substances in the specimen dissolve in the alcohol (just look at an alcohol preserved Anguilla…). Every change of alcohol add to the removing of lipids etc - that is bad.

As far as we know, formalin was used for the first time at the NRM in 1904, but only occasionally! Still in the 1940s ethanol was commonly used for fixation in the field. When the museum moved from downtown Stockholm to north of the city in 1916, the economy for alcohol was reduced due to world war I (otherwise Sweden was not involved). This led to the invention to use a diluted formalin solution for the exhibition jars (for specimens fixed in ethanol!). The research collection continued to be stored in ethanol. Our collection is old. We estimate that our oldest specimens in ethanol are from the 1720s (from the Seba collection). Still many specimens from before 1758 are in remarkable good condition. In some specimens it is even possible to get small pieces of DNA with ancient DNA technic – but usually not. This sounds contradicting to some statements above. We don’t know too much about the preservation history of these specimens, but what we know might be of general interest. The initial fixation and preservation was in distilled wine (=“spiritus vini”). We don’t know the concentration, and probably it was not pure ethanol, but also contained small amount of other fractions from the wine, more like strong cognac. The Royal collection (of king Adolf Fredrik with many Linnaean types) was donated to the Royal Swedish Academy of Sciences in 1801. NRM was founded in 1819, but immediately in practice fused with the Academy. In 1848 the collections of the Academy was formally donated to the Museum. From the 1740s to 1970 this collection of  vertebrates in alcohol was moved four times. Jars and fluid was probably changed twice. But most of the time the collection was stored cool and dark. Glasses and fluids was expensive so the ratio: specimen volume / conservation fluid volume was high up to 1900.  From 1801-1898 the major part seems to have been almost untouched, except that the whole collection was moved 1500 meters in 1829.

I was once asked how long a specimen could be stored in alcohol. With the reservation that our old specimens will be stored like today, no sudden disasters etc (and no climate change), I decided that to 2220 = 500 years would be possible, maybe 1000 years.

Erik Åhlander
vertebrate zoology and museum history

ZOO
Swedish Museum of Natural History
PO Box 50007
SE-10405 Stockholm
Sweden
+46 0 8 5195 4118
+46 0 70 225 2716
erik.ahlander at nrm.se

Från: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu> För Dirk Neumann
Skickat: den 7 maj 2021 08:36
Till: nhcoll-l at mailman.yale.edu
Ämne: Re: [Nhcoll-l] Alcohol concentration for terrestrial vertebrates

Hi Tonya (and John and Simon ;-)

concur with John and Simon, specimens should be kept in 70%; Simon pointed to the diluting effects and the image below nicely illustrates this: even if you use more steps for transferring specimens (0/20/40/60/80 vs. 20/30/50/70), tissues are still soaked with 60% or less high concentrated EtOH.

Depending on size, body mass and number of specimens (i.e. amount of tissue in the jar), the effect can be considerable (see "staining" in the images below; in the left one, body fluids released from these tall whitefish are indicated by the reddish haemoglobin stain at the bottom of the jar, the overall greenish colour in the right comes from chlorophyll released from the guts of these herbivorous distichodus fish).

I do the initial filling usually with 73-75% EtOH to reach 70%; aside from vertebrates high EtOH concentrations can be an issue in malaise traps because there the specimens usually are collected over several days or weeks in 96-80% EtOH. As Simon pointed out this quickly dehydrates specimens and weakens the joints holding all the antennae, appendices, bristles of invertebrates. Another issue is that in unsorted malaise trap samples there often is a thick deposit of specimens at the bottom of the container. Because the diluted less high concentrated ethanol is heavier, it layers at the bottom of the jar (cf. whitefish jar). Inside malaise trap containers, this diluted EtOH may get trapped in the thick specimen deposit.

Usually, I leave jars for few day to see if there are any unwanted effects before moving them into the collection.

Hope this is useful, with best wishes
Dirk

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Am 07.05.2021 um 00:17 schrieb Simon Moore:
Thanks John and Tonya,

What John says is true about the staging of alcohols and the final concentrations.  80% was what I was advised at the NHM in London when I worked there and by the time larger terrestrial vertebrates ‘end up’ in 80%, you will often find that with the mix of lower grade alcohols from the staging process, once things have settled down / equilibrated, then the net result is around 70% anyway.  Higher grade alcohols  can lead to embrittlement of certain tissues as well as evaporation issues.

I have also found the staging process necessary for the more fragile specimens as they undergo changes in Osmotic pressure during this process which can cause syneresis or shrinkage in softer tissues.

With all good wishes, Simon

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

www.natural-history-conservation.com<https://url11.mailanyone.net/v1/?m=1leu5X-0006gs-5g&i=57e1b682&c=P_JTOxhc00RtGTsMjTrryrRBThMnzI5k1ol2aDVqPITm0G_xR0drpuNIhh-krJ6ihFhOLJnXYNjI5fJDeS7rag0t-LwIYs0jmRWXIk2uN2sYVvoo4O9RHPsKEAKiAK-LvbrlH-pnEJMM5dJlJOlNvswIXfaiFJxHBKwsoJX5uQ31zmivYbvBNJdb61ZNkqDKGinISZmQBmu6t6VBola0IT4zHh0nqkiHmWvI7KCXEbWncO8-owQTcerGpMed6sP9>

[cid:3543D570-FCD3-4F5F-B04C-2A6D3A8D9E27 at home][cid:image002.jpg at 01D7431E.C9D5A260]

On 6 May 2021, at 22:50, John E Simmons <simmons.johne at gmail.com<mailto:simmons.johne at gmail.com>> wrote:

Tonya,
Thank you for your kind words about my book. The recommendation for staging up to 80% concentration was by made by my friend Simon Moore, who I cited in that sentence. In general, I do not recommend using 80% ETOH as a preservative for terrestrial vertebrates, but rather 70%. Preservation is alcohol is a trade-off between dehydration of the specimens and providing them suitable protection against biological deterioration. At 70%, ETOH is a very good biocide; below that, not so good, and above 70%, too strong for most specimens (note that there are some instances in which 80% might be preferred).

I do not recommend using stronger alcohol as a hedge against evaporation--that leads to uneven concentrations of preservatives and can be a real mess to work with in a collection.

For how-to instructions on preserving, transferring specimens, and managing a fluid preserved collection, you might want to check Herpetological Collecting and Collections Management (3rd edition, 2015). The instructions for preserving and managing fluid preserved animals will work for most other specimens as well as for reptiles and amphibians.

Hope this helps,
--John

John E. Simmons
Writer and Museum Consultant
Museologica
and
Associate Curator of Collections
Earth and Mineral Science Museum & Art Gallery
Penn State University
and
Investigador Asociado, Departamento de Ornitologia
Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima

On Thu, May 6, 2021 at 4:05 PM Haff, Tonya (NCMI, Crace) <Tonya.Haff at csiro.au<mailto:Tonya.Haff at csiro.au>> wrote:
Hello all,

I am enjoying reading John Simmon's fantastic book on fluid preservation. In it I read one suggestion for stepping specimens up out of formalin fixative into preservation alcohol as follows: from 20% ETOH to 40% to 60% and finally to 80%. We typically place our specimens in 70% ETOH, and I know higher concentrations can cause some problems with specimen dehydration. All our specimens are terrestrial vertebrates. I presume the final 80% provides a buffer against ETOH evaporation or leaching of water from the specimen into the fluid in the jar, to ensure that the alcohol concentration in the preservation fluid stays sufficiently high? But to me this is not quite clear. I wonder if any of you have thoughts on this, or if you would be willing to share how you step your specimens up in ETOH?

Thank you!

Tonya

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