[Nhcoll-l] Formalin transfer

[a.j.van_dam at lumc.nl]

Dear All,


In our museum, all the formaldehyde (4%) preserved anatomical specimens have been transferred to 65% glycerol. The first step in the protocol is to get rid of most of the formaldehyde by flushing the specimen in slowly running tap water. The duration depends on the size and shape of the specimen.


The advantage of running water is that free formaldehyde extraction will go faster and because of the constant water flow, there is also less chance on microbial attack.


In the Netherlands, our occupational health and safety dept. allows us to discard formalin through the drain as long as it is sufficiently diluted by the running tap water.


Kind regards,


Dries

Andries J. van Dam<http://www.linkedin.com/in/andriesvandam> | curator-conservator

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________________________________
Van: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu> namens Dirk Neumann <d.neumann at leibniz-lib.de>
Verzonden: vrijdag 23 september 2022 08:40:57
Aan: nhcoll-l at mailman.yale.edu
Onderwerp: Re: [Nhcoll-l] Formalin transfer

Indeed, Tonya, this developed into a very interesting threat and discussion (again)!

Rob and I chatted a bit offline on concentrations and migration of the 'free' formaldehyde out of tissue matrixes yesterday. Andrew (answering to Mandy's post further down here), raises an important point, i.e. the size and amount of specimens that are stepped, and how long they may be exposed in the water without re-solving formaldehyde out of its covalent bounding with cellular components, proteins and membranes etc. and thus weakening the original fixation or even causing damage to delicate specimens. This could be small specimens, but also iso-osmotic marine invertebrates with weak integumentary systems. The dear colleagues at the MNHN around Marc Herbin presented very interesting details on observed damages on cellular level during the Fluid Preservation Conference in Paris, which has been published in Collection Forum recently (link<https://watermark.silverchair.com/i0831-4985-34-1-157.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAugwggLkBgkqhkiG9w0BBwagggLVMIIC0QIBADCCAsoGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMy4IiE4_JoB4oGq55AgEQgIICmxT6Zc0-neYUT50Ts-qpVBIj1GIiFs5E1hrcGhx9MO9mMPCItwID63bnFzDRlqnL-LNska1cGVtzCBLibf_nkD4z4ANI2evGlMY85RvKwmtqQThgjzkiopMlBKnD4WlALmxaDJViKNvrYqZDpYIy7kGdmTlfJ9AM8zqYZta5ASXPG351chQ5i7ig9MOQizRjozYjYA9OzbLd7Ofginkh5uVtJozvtDmNKhjPaxNUgDp0f87HqmnRk6SOU3OWoB2TYF46WoqD1sUXYztpkWoC1OIdErKhpvP0TZoVosoVCAH8FRoMQOYGh1bsmLMoZuEUFnrJO6XN8EQZ5pXR9EziXiVSUqlg7pTVKRJasMuA_aylkYmM-0IyD4R5xS1lxZPw8wSXKKnEEJJARM-z7MsHeGejttsow_DeyUMlHCVLrGTCMrQb17p_o4OwA5oT8F752zMoG0QLCeSZPPhH4lkcfHCMXw5GeTC3sGYwuNYsd0RoLkmyTCKdviVTB7ce8eEL4gamv9EfIWaPbvC8BCWyHkRt-Rr5Xds14G1SVFyBD4CTHwe1Z9arA7adZdUzSd7XvHEyetRm5bfiNPFemvTnylxgZwKdzhB_ywwAnCL-Qv2R1Cb3PPCIq6WjM4wwSlsmY4nx6dvfzvaxqljYLc9QZq5yh5KhRCeDxInUP98mDm9kAeWsihcXUvyxPlnMDdFAoHNtEA3UjC6cVbeXvbCfljkQ2m8ObkQvEl_ubv_n54OH0ubJ9aRYq69obour-ZrQG6HtPZzYhOVtwPbRUX1tl4515dUVrvWj5o2by4arZCkYjQLvEzbjqmgxvplQCLF0RVmFRS0H7D0k047mDTIInM8LoAsNE5tp3S6dTKO4e5zSRkwQuOKN5jtubOo>).

During the initial watering step, the aim is to mobilise the free, unbound formaldehyde to migrate from the higher concentration inside the organism into the water, which has the initial formaldehyde concentration of 0%. For small specimens, this initial exposure in water should not be longer than one day, or even be avoided at all, i.e. starting with a reasonably low alcohol concentration to avoid damage on the cellular level caused by the osmotic pressure.

When the formaldehyde migrates into the water, its concentration in the water increases, and this increase can be noticed, e.g. by the typical formaldehyde smell, as Rob pointed out. At this point, the formaldehyde concentration in the water has reached a level, where the gas pressure drives it out of the water phase. The easiest way to reduce the concentration (and to avoid the formaldehyde to escape), is to change the water more frequently, i.e. in shorter intervals, or to let the water rinse through the container (preferably from below), as the formaldehyde solution that escapes from the specimen is heavier as the water and will layer at the bottom of the jar or container.

But during the stepping in the alcohol ladder, 'free' formaldehyde is continued to be removed from the organism, as it follows passively with the water that is osmotically removed from the specimens. As Rob pointed out, the concentration difference is an important factor here, and thus it is not surprise, that the amount of formaldehyde that is removed with staging steps 0/20/40/60/70 or 0/30/50/70 may differ, and again, body size, amount of specimens and container size are relevant factors that have an influence.

To answer Rob's question "what is the optimum amount of formaldehyde to carry over into storage solutions": as least as possible, to avoid conservation issues in the preservation fluid, and to during (long-term) storage (e.g. formation of paraformaldehyde).

What is the ideal residual concentration of formaldehyde during staging? It depends - there is no straight answer, as so often when discussing conservation issues.

If your limitation is the capacity of the fume hood when removing 'free' formalin or transferring specimens from formalin into alcohol, you could e.g. use larger containers (with specimens inside being separated e.g. in soft nets net) and change the water more frequently, to avoid that the vapour pressure drives the formaldehyde gas out of the water.

Perhaps this is a way forward, even though it definitely is not a straight forward answer!

With best wishes
Dirk



Am 23.09.2022 um 02:00 schrieb Haff, Tonya (NCMI, Crace):
As usual, what a great thread! I learn a lot from you all regularly, so thanks everyone for the shared knowledge!

Mandy maybe you don’t need another fume hood, if keeping the lids on buckest closed is ok? But FYI and for others who have asked about our ‘portable’ fume hood… we purchased ours from Laftech Technologies last year. I’ve attached the quote (it’s pricey – we’re lucky we can afford it right now), which details which one and which filters we purchased. I know that there are other brands out there as well, and as I haven’t used them I can’t say that the one we have is the best, but so far it seems to function well (and it can be fitted with Formalin specific filters).

Cheers,

Tonya

From: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu><mailto:nhcoll-l-bounces at mailman.yale.edu> On Behalf Of Robert Waller
Sent: Friday, 23 September 2022 9:03 AM
To: Mandy Reid <Mandy.Reid at Australian.Museum><mailto:Mandy.Reid at Australian.Museum>; nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu>
Cc: Rhiannon Stephens <Rhiannon.Stephens at Australian.Museum><mailto:Rhiannon.Stephens at Australian.Museum>; Harry Leung <Harry.Leung at Australian.Museum><mailto:Harry.Leung at Australian.Museum>
Subject: Re: [Nhcoll-l] Formalin transfer

Hi Mandy,
That is a great question – what, if any, is the optimum amount of formaldehyde to carry over into storage solutions?
My credibility, again if any, is with physical chemistry of solutions and ends abruptly at the solution-specimen interface.
Going beyond that, I must defer to John, Dirk, et al.
I do hope they will have some evidence based advice on what would be an ideal residual concentration of formaldehyde, recognizing that would likely depend on taxonomic group being preserved.
Rob


From: Mandy Reid <Mandy.Reid at Australian.Museum<mailto:Mandy.Reid at Australian.Museum>>
Sent: Thursday, September 22, 2022 4:53 PM
To: Robert Waller <rw at protectheritage.com<mailto:rw at protectheritage.com>>; nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu>
Cc: Rhiannon Stephens <Rhiannon.Stephens at Australian.Museum<mailto:Rhiannon.Stephens at Australian.Museum>>; Harry Leung <Harry.Leung at Australian.Museum<mailto:Harry.Leung at Australian.Museum>>
Subject: RE: Formalin transfer

Hi Rob
Thank you very much for that advice. That is really helpful.

I also received the below response. Do you think flushing the formalin from tissues could reverse the cross linking and hence fixation of the specimen? It seems to me that if this is the case, rinsing the surface formalin and then transferring through an ethanol series would have exactly the same effect in the long term as soaking the specimen out in water, so I am not sure of the advantage of only rinsing the surface of the specimen as stated by Andrew below. It assumes that no liquids subsequently enter the specimen when I imagine it would ultimately reach equilibrium with the preservative.

We don’t go through an ethanol series because of the time involved and also because it would require huge volumes of ethanol.

Do you have a recommended procedure for transferring specimens from formalin to ethanol or do you think simply soaking out in water (lids on) then placing straight in 80% ethanol is OK in terms of the longevity and integrity of the tissue?

Cheers
Mandy


Hi Mandy,

Interesting – we only wash the surface formalin off before running through stepping alcohol. The information we’d always worked to was that flushing all formalin out of the tissues was undesirable long term (especially with small fishes), as the cross-linking caused by formalin can be partially reversed. There is an equilibrium reaction between aldehyde & alcohol forming acetal & water. Do you have more recent and improved info on changing this process?

I can’t see

Cheers
Andrew

>><<<)o>
Assistant Curator NE (Fishes)
Museum of New Zealand
04 381 7314
027 7339363


Dr Mandy Reid
Collection Manager | Malacology
Australian Museum  1 William Street Sydney NSW 2010 Australia
T 61 2 9320 6412 M 61 0431 829 842
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From: Robert Waller <rw at protectheritage.com<mailto:rw at protectheritage.com>>
Sent: Thursday, 22 September 2022 11:57 PM
To: Mandy Reid <Mandy.Reid at Australian.Museum<mailto:Mandy.Reid at Australian.Museum>>; nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu>
Cc: Rhiannon Stephens <Rhiannon.Stephens at Australian.Museum<mailto:Rhiannon.Stephens at Australian.Museum>>
Subject: RE: Formalin transfer

Hi Mandy,
The vapor pressure of formaldehyde in aqueous solutions that are less than 40%w/v is real (we can smell it) but considerably lower than the vapor pressure of water in the solution. Therefore, unless the surrounding atmosphere is very close to 100%RH (speculating, perhaps > 99%), then water will be leaving the solution faster than formaldehyde does. Therefore, keeping a lid on makes more sense than leaving it off if the goal is extracting formaldehyde while keeping formaldehyde concentration as low as possible in the solution.
Best,
Rob

From: Nhcoll-l <nhcoll-l-bounces at mailman.yale.edu<mailto:nhcoll-l-bounces at mailman.yale.edu>> On Behalf Of Mandy Reid
Sent: Wednesday, September 21, 2022 9:17 PM
To: nhcoll-l at mailman.yale.edu<mailto:nhcoll-l at mailman.yale.edu>
Cc: Rhiannon Stephens <Rhiannon.Stephens at Australian.Museum<mailto:Rhiannon.Stephens at Australian.Museum>>
Subject: [Nhcoll-l] Formalin transfer

We are currently working on a large project at the Australian Museum to transfer our entire mollusc collection out of formalin (in which it has been stored for historical reasons) and into ethanol for long term preservation.

We normally soak each specimen lot out in 3 changes of water in a fume hood with lids left off the containers/vials etc. to assist with degassing of the formaldehyde in solution. Our problem is a bottleneck in our workflow due to limitations imposed by our fumehood work space. I am wondering whether others in the group have faced similar issues and whether soaking out can possibly be done in water but leaving the lids on the containers (hence could be done on a lab bench after pouring off the formalin in the fume hood)? We don’t have a way to measure residual formalin after the soaking-out process so don’t know whether or not this will be effective.

Any advice would be much appreciated.
Mandy

Dr Mandy Reid
Collection Manager | Malacology
Australian Museum  1 William Street Sydney NSW 2010 Australia
T 61 2 9320 6412 M 61 0431 829 842
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Stiftung Leibniz-Institut zur Analyse des Biodiversitätswandels
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