[Nhcoll-l] Information about fluid collections

Mon Aug 21 12:39:42 EDT 2017

Hi all

Whilst I appreciate the approach is to reduce use of what is perceived hazardous or flammable, a move into KIII is likely to be damaging to the other criteria of preserving surviving DNA in the specimens. Thus if genetic research is a feature of the collection then best to keep it in an ethanol solution, especially if that is what the specimen is currently preserved in. Also it is going to be a lot of work to move a significant number of specimens into new solutionsâ€¦ However, like Erik, I do not fully understand the constraints you are working with to require considering such a task.

Rob Waller also raises a very important point over what osmotic pressure is â€“ you may not require a â€˜stepping approachâ€™ at all as it is related to the molar concentration of all the dissolved species in the solution and thus it will be worth having a working idea of the osmotic pressures of the solutions you are planning to move from and to before making these decisions e.g. KIII has a dissolved salt and glycerol so the osmotic pressure is potentially quite high and may not be very different to an ethanol solution. The classic â€˜stepping upâ€™ approach is usually to move a specimen from formaldehyde to alcohol and serves as a wash step as well as a means of reducing the effects of tissue shrinkage.

It is difficult to know what to suggest as an alternative â€“ glycerol on its own will potentially work with most specimens but I think best to get guidance from those who use it regularly in collections such as the anatomical ones. Others potential low hazard alternatives such as propylene glycol (Te Papa in NZ are preserving their big squid in this) have not been used widely enough to know their value as long term preservatives.

Thus not sure this discussion is making your decision any easierâ€¦ However all the best with the challenges of this project!

Best wishes

Jules

Prif Gadwraethydd, Gwyddorau Naturiol
Principal Conservator Natural Sciences
julian.carter at amgueddfacymru.ac.uk<mailto:julian.carter at amgueddfacymru.ac.uk>
julian.carter at museumwales.ac.uk<mailto:julian.carter at museumwales.ac.uk>
029 20573230
07870448074

From: nhcoll-l-bounces at mailman.yale.edu [mailto:nhcoll-l-bounces at mailman.yale.edu] On Behalf Of Erik Ã…hlander
Sent: 21 August 2017 16:07
To: 'nhcoll-l at mailman.yale.edu' (nhcoll-l at mailman.yale.edu) <nhcoll-l at mailman.yale.edu>
Subject: Re: [Nhcoll-l] Information about fluid collections

Hi Juliette, hi Simon,

We were one of those museums who read about the transfer to 2-phenoxyethnol and tested it on a few accessions in 1976. It seemed to work excellent in the first place and was also cheaper than ethanol! A few years later we had to transfer all specimens to 70% ethanol to avoid a catastrophe! They were getting softer and softer. After the transfer to ethanol they showed minor signs of dessication, but they were safe, and many are now type specimens.

Since that experience we avoid anything but ethanol. We have a few specimens kept in ethanol for almost 300 years. The melanin colour pattern is still visible.

We use glycerol for clear and stained specimens but we avoid thymol because of its toxicity. We are aware of the risk for fungus,  but it is less than 1000 specimens, and quite easy to monitor.

My immediate reaction to Juliettes first mail, was that discussion on changing fluid is the wrong way, important is to get localities were you can keep ethanol and formalin, but I know nothing of the planned use of the collection, its history, size etc, so I will not argue!

I can only wish you good luck!

[nrm_logo]

Erik Ã…hlander
Collection Manager, fish and herptiles
Swedish Museum of Natural History
Department of Zoology
Box 50007
SE-104 05 STOCKHOLM
Sweden
erik.ahlander at nrm.se<mailto:erik.ahlander at nrm.se>
+46-8-51954118
+46-70-2252716

FrÃ¥n: nhcoll-l-bounces at mailman.yale.edu<mailto:nhcoll-l-bounces at mailman.yale.edu> [mailto:nhcoll-l-bounces at mailman.yale.edu] FÃ¶r Simon Moore
Skickat: den 21 augusti 2017 16:17
Till: Galpin Juliette <juliette.galpin at lehavre.fr<mailto:juliette.galpin at lehavre.fr>>
Kopia: nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu>; Baglione Gabrielle <gabrielle.baglione at lehavre.fr<mailto:gabrielle.baglione at lehavre.fr>>; Cremiere CÃ©dric <cedric.cremiere at lehavre.fr<mailto:cedric.cremiere at lehavre.fr>>
Ã„mne: Re: [Nhcoll-l] Information about fluid collections

Hi Juliette,

Generally I have found that stages in a ladder take about 2 hours immersion but this is averaging; larger more muscled specimens obviously take a bit longer while the specimen composition can adjust to the specific gravity of the new fluid.
The problem with what Rob has said is that generally, most specimens can accommodate the change from ethanol to Kaiserling without all the in-between stages, however, I have found in practise, that this is risky, leading to osmotic shrivelling in some tissues, so I make it good practice to make the stages, even though Rob suggests that itâ€™s unnecessary.

I have often found that wherever these changes are proposed on paper, or for health and safety reasons, that they are new ideas that present a Utopian view on how collections should be managed.  In reality, this is quite different and I could quote at least one example where a major museum proposed in the 1970s, that all of its fluid specimens should be moved into a propylene glycol and 2-phenoxyethanol preservative (originally designed for zooplankton) and with disastrous and expensive results!  This is one good reason why I dislike people changing fluids for specimens in which they have safely resided for years.  As for Health & Safety or flammability issues, I have not heard of any (yet) problems with using the formalin and alcohol as preservatives; we are adults after all and understand the risks and precautions involved. For areas of the world where there is geological instability or where Summer temperatures frequently rise to 40 deg. C, this might be different.

Anyway, this is my opinion and although it may be beneficial to transfer some of the collection to KIII, I would not advise it as a blanket preservative.  Instead and by all means, take fresh material through the Kaiserling preservation technique.

Iâ€™m sorry if this has been a difficult enquiry.

With all good wishes, Simon.

Simon Moore MIScT, RSci, FLS, ACR
Conservator of Natural Sciences and Cutlery Historian,
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On 21 Aug 2017, at 14:21, Galpin Juliette <juliette.galpin at lehavre.fr<mailto:juliette.galpin at lehavre.fr>> wrote:

Hi all,

Thanks again for all these advices.
I understand better all the steps of the process I have to follow to make it right â€¦
Do you have any idea of how long the process (successive baths) takes for a specimen like a fish (salmon for example) ?

But, with the last message of Robert Waller, I donâ€™t know what I should do any more â€¦ Is it best to follow the ladder, or to transfer directly the specimens into Kaiserling, maybe with just a bath of water or ethanol to make the specimen release the formol, just for 1 day ?
And actually, Iâ€™ve never done measurements of osmotic pressure â€¦ So it scares me a little to do it 3 times for each specimen.
The collection is not really used nor studied scientifically for the moment. We donâ€™t think it will be more studied in the future, but its reconditioning will have to allow scientific studies, even if we donâ€™t know in which purposes yet. Actually, the principal goal is to make the collection safer for its move and storage.

In fact, if Iâ€™m understanding all that you said right, you advise us to change our mind and use other solutions which will be easier to put in place and will be less at risk for the specimens â€¦ Am I right ?

Thanks again !
Best wishes.

Juliette Galpin

     De : rw at protectheritage.com<mailto:rw at protectheritage.com> [mailto:rw at protectheritage.com]
EnvoyÃ© : dimanche 20 aoÃ»t 2017 19:26
Ã€ : Galpin Juliette; 'John E Simmons'; 'Dirk Neumann'
Cc : nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu>; Baglione Gabrielle; Cremiere CÃ©dric
Objet : RE: [Nhcoll-l] Information about fluid collections

Hi all,
Much of this discussion is beyond my area of expertise but I do want to point out some apparent misunderstandings about osmotic pressure.
Osmotic pressure is a product of water activity and is independent of what substances are causing the reduction in water activity, that is, whether ethanol, glycerol, potassium acetate or whatever. It would seem unwise to step through dilutions of ethanol then progressions of concentration to another preservative solely for the purposes of reducing osmotic pressure differences. The least osmotic pressure difference may be through direct transfer or some other path. I do want to recognize that there may be other reasons for wanting to go through a stepped procedure but we should not think that is the only, or the best, way to complete a transfer while avoiding osmotic pressure differences. Measurements of osmotic pressure differences between initial, final, and intermediate solutions will be required. For ethanol water solutions the data available in: Waller and Strang, Physical-chemical properties of preservative solutions I: Ethanol-water solutions. Collection Forum 12(2), 70-85, 1996.. I suspect for Kaiserling solution new measurements will be required. These can be easily made through freezing point depression measurements â€“ an experiment many of us may have conducted in grade school.

Finally, I hope options other than Kaiserling solution will be identified and evaluated before a course of action is decided upon. Most notably, what are the current and anticipated uses of the collection, and how will collection utility for those purposes be affected by whatever preservative solution is chosen, from among several options considered.
Best,
Rob

Robert Waller, PhD, CAPC, FIIC
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De : Simon Moore [mailto:couteaufin at btinternet.com]
EnvoyÃ© : vendredi 18 aoÃ»t 2017 11:59
Ã€ : Galpin Juliette
Cc : nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu>
Objet : Re: [Nhcoll-l] Information about fluid collections

Hi Juliette,

Firstly, it is wise to make up the Kaiserling III preservative well in advance before use, this will help to avoid some of the air bubble issues.  Make sure that the glycerine is thoroughly mixed in, there should be no Schleren optic effects!
Ideally to move specimens from alcohol to K III does involve a large osmotic pressure change and I am always advising against it.  I have never preserved medusae or other jelly forms in it, however some jewel anemones (Corynactis viridis) survived well but these were collected fresh, narcotised and taken through the Kaiserling process!

I would suggest, that if this process really must go ahead that you try a few experimental runs first.  It will take up much of your time, bearing in mind that you will have to take each specimen down a hydration ladder to water and then into a KIII solution with only half the glycerine content so that the specimens can accommodate to this stage - they will float at first and then gradually sink, then they can be transferred to the next stage - this will happened all the way down the ladder of alcohols - 60%, 40%, 20%, 10%, water, half-strength KIII then normal KIII.  You will need a small tank for each stage - the clip-top plastic â€˜really usefulâ€™ boxes are good for this and you will need one of those kitchen serving/straining ladles with holes in it to transfer the specimens gently through each stage!

I have had no experience of preserving DNA in KIII, I have always used 96% ethanol.  For the formula I have always used: Kaiserling III (preservative): Potassium acetate (200g), glycerol (300), distilled water (900).  There are many later variations and modifications including Lunquistâ€™s.

Thymol is a useful additive to prevent fungal growth and the camphor is good although I have never tried this modification before.  Normally the pot. acetate will be enough to prevent germination of fungal spores but itâ€™s always best to take precautions, especially with so many specimens.

Feel free to come back to me and the List if you have any issues, further questions.

With all good wishes, Simon.

Simon Moore MIScT, RSci, FLS, ACR
Conservator of Natural Sciences and Cutlery Historian,
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On 18 Aug 2017, at 08:50, Galpin Juliette <juliette.galpin at lehavre.fr<mailto:juliette.galpin at lehavre.fr>> wrote:

Hi Everybody,

Thank you all for answering me so quickly !

Actually, the collection needs to be moved into safer fluids, for its own conservation (the fluids are evaporating), and because it will be moved to a new storage, that need to match new standards (no flammable or toxic products allowed, no evaporation of products, keeping the possibility of studying DNA, etc.). It is compulsory from our guardianship and funders of our new storage location. And Kaiserling seemed like a good idea, because it matches all these constraints. But I completely understand all the reservations you have regarding this operation and the harm it could do to specimens. That is why I need all the help possible to do this operation, keeping the risks to a minimum.

Simon, you assumed well, we have many marine organisms like fishes, shellfishes, cnidarians, sponges, ascidians, and so on... But we also have mammals, reptilians and amphibians for instance. Will Kaiserling be fine for these specimens too ? Do they need more precautions ?

How could I do to avoid having these bubbles due to changing in osmotic pressure ? I donâ€™t know how to measure this osmotic pressure and watch out for its changeoverâ€¦ And we donâ€™t know in which fluid the collections are currently preserved, neither the way they were fixed.

Could you help me set the better process to do this changeover with all precautions needed please ? For a changeover into ethanol, Iâ€™ve seen that the specimens must have been adapted to their new fluid by successive baths of ethanol at different concentrations. Do I have to follow the same process with Kaiserling ?

I will only use the preservative fluid, because all the specimens have already been fixed (I guess).
For the formula of the preservative solution, Iâ€™ve found that it is 900g Potassium acetate ; 6L Water ; 3,6L glycerol ; and some thymol. What are the good proportions for the mixture then ?! Thank for the recipe and the way to do it, but is it the same with thymol than with camphor ?

Again, thank you very much to all of you for helping me to implement this delicate operation.
Kind regards.

Juliette Galpin

Juliette,
You have asked a very interesting question. I agree with the responses from my colleagues concerning the caution about osmotic pressure and the kinds of specimens involved. Could you please tell us (1) what kinds of specimens are in the collection (marine invertebrates or vertebrates, fresh water or terrestrial invertebrates or vertebrates?) and (2) the reason why you are considering changing the preserving to Kaiserling? Kaiserling has been mostly used for anatomical specimens in an attempt to preserve the color of the tissues, and as far as I know it has not been used on a very wide variety of natural history specimens.
Sincerely,
John

John E. Simmons
Museologica
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On Wed, Aug 16, 2017 at 9:09 AM, Dirk Neumann <dirk.neumann at zsm.mwn.de<mailto:dirk.neumann at zsm.mwn.de>> wrote:
Dear Simon,
dear Galpin,

the baseline for changing the holding fluid is: Do not change, unless there is a good reason to do so.

>From a collection care point of view, fire protection or toxicity problems - for me - would not be a proper reason if in the long run the new condition would harm the integrity of the specimen.

Also, because of the acidity, I would refrain of changing the fluid unless there is a real good reason to do so (maybe part of the collection, or specific specimens from a lot containing several specimens for a specific research purpose).

Btw:
Mahoney (1973) Laboratory Techniques in Zoology gives the following recipes (Pick-Judah formula)
(Simon, please comment if this wouldn't make sense)

KI (fixative)
Potassium acetate      42.5g
Potassium nitrate        22.5g
Distilled water               1.6 L
40% formalin solution 400ml (full strength)

Salt are dissolved in hot water, formalin is added to the cooled solution

KII (colour reviver)
EtOH      96 %

KIII (perservative)
Sodium      acetate      36.0g
Distilled water          120.0ml
Camphor                      5.0ml (4% EtOH solution)
Glycerol                        72ml

(The solution is made by adding the ingredients in the order given. At first, the camphor is thrown out of solution but redissolves on allowing the solution to stand)

All the best
Dirk

Am 16.08.2017 um 13:19 schrieb Simon Moore:
Hi Juliette,

No problem about getting in touch.

You mention re-packing a collection.  Is this collection to be moved or stored or did you mean that is needs to be moved into safer fluids?  The latter is indicative of a move for museums with such collections to be rid of fluids that are flammable and/or toxic.

There are various concerns about this as the condition of the specimens is what should be the real issue here.  As you are based at LeHavre I am assuming that you should have many marine organisms in fluids?  For many such organisms a move to Kaiserling III will be fine but you have to bear in mind that the osmotic pressure of KIII is considerably different to that of ethanol, even formalin.  For delicate organisms (medusae, siphonophores) this changeover has to be carried out very carefully or you will end up with air bubbles inside them that appear when binary/tertiary azeotropes are used - miscible fluids of different specific gravities.  You also need to bear in mind that Kaiserlingâ€™s technique was devised mainly for anatomy specimens as the preservative is good for maintaining the colour of blood and organs that filter it (liver, kidney).  I have successfully colour-preserved marine organisms that were freshly collected and using the 3 stage technique - fixation, colour enhancement and preservation.
All of this may sound rather negative but I am just warning you to be careful with the changeover.  I would also post the enquiry onto the conservation forum: nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu> to see if anyone else is doing this and to see if they have any concerns.

The formula for the Kaiserling method is: Kaiserling I (fixative): 40% formaldehyde (400) [tlv 3], potassium acetate (50 g), potasÂsium nitrate (30 g), distilled water (1000).
Kaiserling II (enhancer/colour restorer): 80-90% ethanol. Use until colour returns if it has chemically â€˜fadedâ€™ in fixative â€“ may be minutes for smaller specimens. Some like to add some potassium hydrosulphite if being stored for more than a few days.
Kaiserling III (preservative): Potassium acetate (200g), glycerol (300), distilled water (900).

With all good wishes, Simon.

Simon Moore MIScT, RSci, FLS, ACR
Conservator of Natural Sciences and Cutlery Historian,
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On 16 Aug 2017, at 10:37, Galpin Juliette <juliette.galpin at lehavre.fr<mailto:juliette.galpin at lehavre.fr>> wrote:

Hello,

My name is Juliette Galpin and Iâ€™m in charge of all the scientific collection in the Natural History Museum of Le Havre, in France.
Iâ€™m following the advice of Judith White, in Natural History Museum, who told me to get in touch with you. Hope you wonâ€™t mind.

We have a collection which is maintained in fluids, and we want to repack it. The specimens are currently in liquid as Alcool, Ethanol and a bit of Formol. And we wish to recondition it in Kaiserling III, which is a mixture of glycerin and acetate of potassium without any formol. I donâ€™t know if you know, or use, this liquid.
Iâ€™ve found some of your papers about collections in fluids, but there is few literature about Kaiserling precisely, and I donâ€™t really know how to create this liquid correctly.
Do you know this product, or someone who might know and use it, or maybe know the process to adapt the specimens to their new conservation fluid ?

Thank you very much for your answer and all the help you can provide me,

Regards.
Juliette Galpin
<image001.jpg>
Juliette GALPIN
In charge of Natural history Collections
Natural history Museum
Ville du Havre
juliette.galpin at lehavre.fr<mailto:juliette.galpin at lehavre.fr>
TÃ©l. : 02 35 54 75 89 / 02.32.74.79.90

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---------

Dirk Neumann

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postal address:

Bavarian Natural History Collections

The Bavarian State Collection of Zoology

Dirk Neumann, Section Ichthyology / DNA-Storage

Muenchhausenstr. 21

81247 Munich (Germany)

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