[Nhcoll-l] [External] Re: [EXTERN] Removing specimens from formaldehyde

Wed Oct 2 10:08:58 EDT 2024

I've been watching this thread, because I also have received some specimens in formalin which I'd like to transfer to ethanol. I have not seen any mention of buffering. Is the best-practice process simply to rinse in deionized water and then start up the ethanol ladder to the desired concentration, or should one insert a K2HPO4 buffering step after the water rinse (as suggested in the National Park Service Conserve O Gram 11/1)?

Cindy Opitz (she/her)
Director of Research Collections
Museum of Natural History and Old Capitol Museum
Instructor, Museum Studies Certificate Program
The University of Iowa
11 Macbride Hall, Iowa City, Iowa 52242
Office: 319.335.0481
cindy-opitz at uiowa.edu
mnh.uiowa.edu, oldcap.uiowa.edu

-----Original Message-----
From: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu> On Behalf Of Simon Moore
Sent: Tuesday, October 1, 2024 5:23 PM
To: a.j.van_dam at lumc.nl
Cc: NHCOLL-new <nhcoll-l at mailman.yale.edu>
Subject: [External] Re: [Nhcoll-l] [EXTERN] Removing specimens from formaldehyde

Thanks everyone, this is a most interesting string.

When rehydrating specimens from dried out I take them up the dehydration ladder of alcohols, I tend to use 20% increases for most, 10% for fragiles and 30% stages for more robust specimens.  Smaller specimens tend to average about 2 hours in each change and I have not noted any osmotic shrinking / syneresis.  By smaller, I mean those that fit into a c. 1 litre jar.

With all good wishes, Simon

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

http://www.natural-history-conservation.com/

> On 1 Oct 2024, at 09:53, <a.j.van_dam at lumc.nl> <a.j.van_dam at lumc.nl> wrote:
>
> Dear all,
>
> Just to be correct, I suggested four steps (30-50-70-75) instead of the three (30-50-75) John is referring to, of which the last step (above 70%) being very small (70-75) to prevent osmotic shock since the 'ethanol conc. versus osmotic pressure curve' is exponential (see John's reference: figure 7 in Waller and Strang, 1996), which also implies that the first step can be a bit higher being 30%, then 2 times steps by 20%, and when reaching 70% a much smaller step by 5% because above 70% the exponential curve start to rise significantly. Nowadays, in histology the standard is to start even with a higher first step of 50%. To be on the save side, I suggest to start with an intermediate step of 30%.
>
> Kind regards,
>
> Dries
> Andries J. van Dam | curator-conservator  Anatomical Museum |
> Directorate of education | Leiden University Medical Center | Building
> 3 (V3-32) P.O.Box 9600 | 2300 RC Leiden | Netherlands Visiting
> address: Hippocratespad 21 | Tel: +31 (0)71 52 68356 | E-mail:
> A.J.van_Dam at lumc.nl  Scientific associate | Natural History Museum
> London
>
> Van: John E Simmons <simmons.johne at gmail.com>
> Verzonden: maandag 30 september 2024 22:24
> Aan: Dirk Neumann <d.neumann at leibniz-lib.de>
> CC: Dam, A.J. van (DOO) <a.j.van_dam at lumc.nl>;
> nhcoll-l at mailman.yale.edu <nhcoll-l at mailman.yale.edu>
> Onderwerp: Re: [Nhcoll-l] [EXTERN] Removing specimens from
> formaldehyde  There are several drawbacks to using the suggested steps of 30-50-75.
>
> According to Waller and Strang (1996), transfer in concentration steps greater than 20% risks causing cellular rupture to specimens because of the effect of ethanol concentration on osmotic pressure (see figure 7 in Waller and Strang 1996). As stated in their paper, “…it is clear that the osmotic pressure rises steadily with ethanol concentrations for solutions below about 75% v/v and begins to rise more rapidly at concentrations above about 80% v/v. These facts suggest that, from considerations of osmotic pressure, solutions with approximately equal concentrations are appropriate for stepping specimens up to higher ethanol concentrations, up to about 75%v/v.” This means that the abrupt change from water to 30% ETOH should be avoided, and as the concentration of ethanol nears 75% it is very close to the osmotic pressure shift, which should also be avoided by not going above 70%.
>
> In addition, starting with the abrupt change to 30% ETOH will cause more rapid dehydration than a 20% step, and rapid dehydration is potentially destructive to tissues.
>
> Lastly, the assumption that using 75% ethanol may result in a 70% concentration after stepping up may well be incorrect (depending on the volume of fluid used and the specimen). The recommendation for using 70% ethanol is based on the fact that 70%, ETOH is a very good biocide, but ethanol preservation is balance between providing an antiseptic environment and excessive dehydration of the specimens, so there is no reason, but some risk, in using ETOH at concentrations greater than 70% (see figures 12 and 13 in Waller and Strang 1996).
>  Referernce:
> Waller and Strang. 1996. Physical chemical properties of preservative
> solutions—I. Ethanol-water solutions. Collection Forum 13(2):70-85
>
> 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 Mon, Sep 30, 2024 at 3:53 PM Dirk Neumann <d.neumann at leibniz-lib.de> wrote:
> Hi Dries and all,
>
> also added offline that especially with these large specimens it would be worth keeping an eye on layering. The final concentration should be at 70%, and you are correct that it might be worth filling the jar up with 75% instead of 70% to end up at 70% (and not 65%).
>
> But I am not sure that the steep steps 30/50/70 are necessary for achieving this; if specimens have been sitting in formalin for long time, it might be worth considering lees steep steps (0/20/40/60/70-75) and allow for more time.
>
> Pragmatically, when I had to handle a lot of large specimens (often large whitefish), there was the risk of what where cautioning, i.e. that specimens would arrive too fast at "70%" and the residual water could dilute the ethanol concentration to well below 70%.
>
> Therefore, I delayed labelling of specimens usually for some time and "added" the time it took to rearrange shelves to free space to monitor the freshly filled 70% jars if I could spot any layering. You could also exchange the 70% after half a year to make sure that the concentration has not dropped significantly below 70%.
>
> Both concentrations surely work, but I always preferred the slower option.
>
> All the best
> Dirk
>
>
> Am 30.09.2024 um 20:28 schrieb a.j.van_dam at lumc.nl:
> Dear Vanessa,
>  There is a very simple way to monitor the progress of fluid exchange when transferring specimens from an aqueous solution of 4% formaldehyde to 70% ethanol.
>  Since the density of ethanol (d=0.79) is much lower than the density of water (d=1.00) and the density of buffered formaldehyde 4% (d=1.02), during the time the specimen is in one of the transfer baths (ethanol 20-40-60-70), the density of the surrounding fluid will slowly rise due its exchange with the heavier fluid inside the specimen.
>  When measuring the surrounding fluid periodically (e.g. once a week) with a densimeter you will see a logarithmic decrease in density until there is hardly a significant change anymore, which indicates that the transfer step has been fully completed and the specimen can be placed in the next bath. This way of monitoring will ensure correct transfer times without having to worry about the variables of type of specimen, shape, size, etc.
>  Like Dirk and John, I would also recommend four baths, but with slightly different concentrations: 30-50-70-75, which gives after complete exchange an end concentration a little over 70%.
>  Kind regards,
>  Dries
>  Andries J. van Dam | curator-conservator  Anatomical Museum |
> Directorate of education | Leiden University Medical Center | Building
> 3 (V3-32) P.O.Box 9600 | 2300 RC Leiden | Netherlands Visiting
> address: Hippocratespad 21 | Tel: +31 (0)71 52 68356 | E-mail:
> A.J.van_Dam at lumc.nl  Scientific associate | Natural History Museum
> London
>
> Van: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu> namens John E
> Simmons <simmons.johne at gmail.com>
> Verzonden: maandag 30 september 2024 17:55
> Aan: Dirk Neumann <d.neumann at leibniz-lib.de>
> CC: nhcoll-l at mailman.yale.edu <nhcoll-l at mailman.yale.edu>
> Onderwerp: Re: [Nhcoll-l] [EXTERN] Removing specimens from
> formaldehyde  Vanessa, Dirk's advice is correct.
>
> The reason we lack reliable recommendations for soaking time for each step is that there are too many variables to consider, such as the surface-to-volume ratio of the specimen, thickess of the specimen, whether the specimen has thin skin, scales, fur, a shell, and so forth, and the density and structure of the dermal layers and internal tissues.
>
> There are several papers that give penetration times for formaldehyde or ethanol, but these rates should not be extrapolated for whole specimens. All of the published penetration rates   that I have reviewed  are based on small samples (often no more than 1 cubic cm in volume) of gels or agars, etc., so the penetration rates are not transferable to whole organisms. For example, the penetration rates of formaldehyde published by Steedman (1976) are based on gelatin and casein gels, Medawar (1941) used plasma clots, and Baker (1958) used gelatin/albumin gels.
>
> The rate of penetration of fixatives and preservatives is complicated by the fact that the chemicals modify the tissues as they penetrate them, which greatly impedes the rate of penetration of more of the fluid, and quickly limits the depth of penetration of the fluid (this is why it is recommended to inject formaldehyde or other fixatives into specimens). In addition, penetration rates of preservative fluids are temperature dependent.
>
> I hope that someday we will have enough research on penetration, fixation, and preservation rates that we can come up with some general guidelines for time required for each soaking step, but until that day comes, Dirk's advice is the best we have.
>
> --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 Mon, Sep 30, 2024 at 11:30 AM Dirk Neumann <d.neumann at leibniz-lib.de> wrote:
> Dear Vanessa,
>
> rising and staging times depends on the size of the specimens and how readily superfluous the formaldehydes is diluted from them. The specimens shown may require 2-3 days of rinsing, and then slowly going up 20/40/60/70. Each of these steps may take 1 week or longer, it depends how much formaldehyde comes out of them.
>
> All together you should assume at least a month, but it can take you longer.
>
> With all best wishes
> Dirk
>
>
> Am 30.09.2024 um 17:14 schrieb Vanessa Pitusi:
> Dear all,
>  Recently, I have discovered that most of our larger specimens kept in the large collection jars, are kept in formalin (photo for reference).
>  I have looked into removing the specimens from formalin and placing them into ethanol. I understand the steps that have to be taken, but I was wondering if anyone has advice on the soaking time for each step. That is the only thing that is kept vague in the texts that I have read. One reference mentioned that tortoises and racoons take two to three days.
>  Most the specimens that I will work with a large fish, cephlapods, and birds.
>  In case any of you have done this, any advice on this or the process is appreciated!
>  I am also open to having a quick chat via Teams or Zoom.
>  Kind regards,
> Vanessa
>
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> ****
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> Collection Manager, Hamburg
>  Postal address:
> Museum of Nature Hamburg
> Leibniz Institute for the Analysis
> of Biodiversity Change
> Dirk Neumann
> Martin-Luther-King-Platz 3
> 20146 Hamburg
> +49 40 238 317 – 628
> d.neumann at leibniz-lib.de
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>
> Stiftung des öffentlichen Rechts;
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> Grüter (Kaufm. Geschäftsführer) Sitz der Stiftung: Adenauerallee 160
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>  --
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>
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