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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">Dear Andrew Charles and Simon,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">Thank you for your informative answers. I will try and see how it goes then.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">Sincerely,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US">Joosep Sarapuu<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D;mso-fareast-language:EN-US"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">From:</span></b><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"> Bentley, Andrew Charles [mailto:abentley@ku.edu]
<br>
<b>Sent:</b> Friday, May 21, 2021 8:25 PM<br>
<b>To:</b> Joosep Sarapuu <Joosep.Sarapuu@loodusmuuseum.ee>; Julian Carter <Julian.Carter@museumwales.ac.uk>; Simon Moore <couteaufin@btinternet.com><br>
<b>Cc:</b> NHCOLL-new <nhcoll-l@mailman.yale.edu><br>
<b>Subject:</b> Re: [Nhcoll-l] Fluid preservation correction<o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><a name="_GoBack"></a><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif">Joosep<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif">Yes, standard practice for fixing amphibians, reptiles, fish and anything else that will be ending up in alcohol, is to inject with formalin into all body cavities
to ensure goof formalin uptake throughout the body and thereafter submerge in formalin for 2-4 days depending on the size of the specimens. Obviously, it is advantageous to first remove tissues and preserve however appropriate (liquid nitrogen, 95% ethanol,
buffer) before fixation. Specimens can then be lightly rinsed to remove any excess formalin from the surface of the specimens after which they can be stepped into ever increasing concentrations of ethanol – 20, 40, 60, 70 or similar.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif">John Simmons book on herpetological collecting is a great resource here for outlining these techniques:
<a href="https://www.amazon.com/Herpetological-Collecting-Collections-Management-3rd/dp/0916984907">
https://www.amazon.com/Herpetological-Collecting-Collections-Management-3rd/dp/0916984907</a><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif">Andy<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"> A : A : A :<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"> }<(((_°>.,.,.,.}<(((_°>.,.,.,.}<)))_°><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"> V V V<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">Andy Bentley<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">Ichthyology Collection Manager<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">University of Kansas<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">USA<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">Tel: (785) 864-3863<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">Email: <a href="mailto:abentley@ku.edu">abentley@ku.edu</a> <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"><a href="http://ichthyology.biodiversity.ku.edu/">http://ichthyology.biodiversity.ku.edu</a><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"> <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"> A : A : A :<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"> }<(((_°>.,.,.,.}<(((_°>.,.,.,.}<)))_°><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"> V V V<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="color:black">From: </span></b><span lang="EN-US" style="color:black">Joosep Sarapuu <<a href="mailto:Joosep.Sarapuu@loodusmuuseum.ee">Joosep.Sarapuu@loodusmuuseum.ee</a>><br>
<b>Date: </b>Friday, May 21, 2021 at 2:15 AM<br>
<b>To: </b>"Julian uk>" <<a href="mailto:Julian.Carter@museumwales.ac.uk">Julian.Carter@museumwales.ac.uk</a>>, Simon Moore <<a href="mailto:couteaufin@btinternet.com">couteaufin@btinternet.com</a>>, Andrew Bentley <<a href="mailto:abentley@ku.edu">abentley@ku.edu</a>><br>
<b>Cc: </b>"<a href="mailto:nhcoll-l@mailman.yale.edu">nhcoll-l@mailman.yale.edu</a>" <<a href="mailto:nhcoll-l@mailman.yale.edu">nhcoll-l@mailman.yale.edu</a>><br>
<b>Subject: </b>RE: [Nhcoll-l] Fluid preservation correction<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Dear Julian, Simon and Andrew Charles,</span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D"> </span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Thanks for all your answers, they were very helpful.</span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D"> </span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D">One more question appeared when i read your answers: How to collect specimens for fluid preservation? (For example frog). Do i first inject formaline
to the body and then put it also to formaline and then when i return to lab I put it in EtOH? Or some better solutions?</span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D"> </span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Sincerely,</span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Joosep Sarapuu</span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D">Estonian Museum of Natural History</span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#1F497D"> </span><span lang="EN-US"><o:p></o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif">From:</span></b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif"> Julian Carter [<a href="mailto:Julian.Carter@museumwales.ac.uk">mailto:Julian.Carter@museumwales.ac.uk</a>]
<br>
<b>Sent:</b> Friday, May 21, 2021 1:56 AM<br>
<b>To:</b> Simon Moore <<a href="mailto:couteaufin@btinternet.com">couteaufin@btinternet.com</a>>; Bentley, Andrew Charles <<a href="mailto:abentley@ku.edu">abentley@ku.edu</a>><br>
<b>Cc:</b> NHCOLL-new <<a href="mailto:nhcoll-l@mailman.yale.edu">nhcoll-l@mailman.yale.edu</a>>; Joosep Sarapuu <<a href="mailto:Joosep.Sarapuu@loodusmuuseum.ee">Joosep.Sarapuu@loodusmuuseum.ee</a>><br>
<b>Subject:</b> Re: [Nhcoll-l] Fluid preservation correction</span><span lang="EN-US"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US"> <o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-family:"Calibri",sans-serif;color:black">Any fixation in formalin will have a notable effect on the viability of the DNA for later research, even for a few days. There are a complex mix of factors occurring
such as crosslinking effects, hydrolytic degradation, the acidity of a solution altering conformations and functional group arrangements etc. Most damage to the DNA will occur at point of death of the organism, how quickly it is preserved, and how it is preserved.
Thus Andy's comment rightly points out that subsequent fixation and preservation treatments may have already subsequently effected the quality of the preserved DNA.</span><span lang="EN-US"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-family:"Calibri",sans-serif;color:black"> </span><span lang="EN-US"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-family:"Calibri",sans-serif;color:black">Cheers</span><span lang="EN-US"><o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-family:"Calibri",sans-serif;color:black">Jules</span><span lang="EN-US"><o:p></o:p></span></p>
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<p class="MsoNormal" style="background:white"><span lang="CY" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#212121">Prif Gadwraethydd, Gwyddorau Naturiol / Principal Conservator Natural Sciences</span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span lang="EN-US" style="color:black"><a href="mailto:julian.carter@amgueddfacymru.ac.uk" target="_blank"><span lang="CY" style="font-size:11.0pt;font-family:"Calibri",sans-serif">julian.carter@amgueddfacymru.ac.uk</span></a></span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span lang="EN-US" style="color:black"><a href="mailto:julian.carter@museumwales.ac.uk" target="_blank"><span lang="CY" style="font-size:11.0pt;font-family:"Calibri",sans-serif">julian.carter@museumwales.ac.uk</span></a></span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span lang="EN-US" style="color:black"><a href="https://nam10.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.researchgate.net%2Fprofile%2FJulian_Carter&data=04%7C01%7Cabentley%40ku.edu%7C0b741d987b5d47eeb11808d91c282f3f%7C3c176536afe643f5b96636feabbe3c1a%7C0%7C0%7C637571781207469734%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C1000&sdata=dZ92zYc04DPsBM65c8Z8%2BwptUZ1XV6XzZ4TrBpNuWaQ%3D&reserved=0" target="_blank"><span lang="CY" style="font-size:11.0pt;font-family:"Calibri",sans-serif">https://www.researchgate.net/profile/Julian_Carter</span></a></span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#212121">Twitter: @NatHistConserve</span><span lang="EN-US"><o:p></o:p></span></p>
<p class="MsoNormal" style="background:white"><span lang="CY" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:#212121">Ffon/Tel: (+44) 029 20573230 / (+44)07870448074</span><span lang="EN-US"><o:p></o:p></span></p>
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<p class="MsoNormal"><b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">From:</span></b><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black"> Nhcoll-l <</span><span lang="EN-US"><a href="mailto:nhcoll-l-bounces@mailman.yale.edu"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">nhcoll-l-bounces@mailman.yale.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">>
on behalf of Simon Moore <</span><span lang="EN-US"><a href="mailto:couteaufin@btinternet.com"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">couteaufin@btinternet.com</span></a></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">><br>
<b>Sent:</b> 20 May 2021 23:09<br>
<b>To:</b> Bentley, Andrew Charles <</span><span lang="EN-US"><a href="mailto:abentley@ku.edu"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">abentley@ku.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">><br>
<b>Cc:</b> NHCOLL-new <</span><span lang="EN-US"><a href="mailto:nhcoll-l@mailman.yale.edu"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">nhcoll-l@mailman.yale.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">>;
Joosep Sarapuu <</span><span lang="EN-US"><a href="mailto:Joosep.Sarapuu@loodusmuuseum.ee"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif">Joosep.Sarapuu@loodusmuuseum.ee</span></a></span><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;color:black">><br>
<b>Subject:</b> Re: [Nhcoll-l] Fluid preservation correction</span><span lang="EN-US">
<o:p></o:p></span></p>
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<p class="MsoNormal"><span lang="EN-US" style="font-size:11.0pt">I don’t think that a fixation in formalin can alter DNA for a few days provided the samples is transferred into 96% EtOH as soon as possible after fixation and that no formaldehyde is still present
(via the sample) in the preservative fluid. I am unsure of there is a critical level for formalin presence that might compromise DNA.<br>
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With all good wishes, Simon<br>
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Simon Moore MIScT, RSci, FLS, ACR<br>
Conservator of Natural Sciences and Cutlery Historian,<br>
<br>
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> On 20 May 2021, at 19:52, Bentley, Andrew Charles <</span><span lang="EN-US"><a href="mailto:abentley@ku.edu"><span style="font-size:11.0pt">abentley@ku.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt">> wrote:<br>
> <br>
> Julian<br>
> <br>
> That is presuming that the specimens were not first fixed in formalin before being placed in 95% ethanol.<br>
> <br>
> Andy<br>
> <br>
> A : A : A :<br>
> }<(((_°>.,.,.,.}<(((_°>.,.,.,.}<)))_°><br>
> V V V<br>
> Andy Bentley<br>
> Ichthyology Collection Manager<br>
> University of Kansas<br>
> Biodiversity Institute<br>
> Dyche Hall<br>
> 1345 Jayhawk Boulevard<br>
> Lawrence, KS, 66045-7561<br>
> USA<br>
> <br>
> Tel: (785) 864-3863<br>
> Fax: (785) 864-5335 <br>
> Email: </span><span lang="EN-US"><a href="mailto:abentley@ku.edu"><span style="font-size:11.0pt">abentley@ku.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt">
<br>
> </span><span lang="EN-US"><a href="http://ichthyology.biodiversity.ku.edu"><span style="font-size:11.0pt">http://ichthyology.biodiversity.ku.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt"><br>
> <br>
> A : A : A :<br>
> }<(((_°>.,.,.,.}<(((_°>.,.,.,.}<)))_°><br>
> V V V<br>
> <br>
> <br>
> From: Nhcoll-l <</span><span lang="EN-US"><a href="mailto:nhcoll-l-bounces@mailman.yale.edu"><span style="font-size:11.0pt">nhcoll-l-bounces@mailman.yale.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt">> on behalf of "Julian uk>" <</span><span lang="EN-US"><a href="mailto:Julian.Carter@museumwales.ac.uk"><span style="font-size:11.0pt">Julian.Carter@museumwales.ac.uk</span></a></span><span lang="EN-US" style="font-size:11.0pt">><br>
> Date: Thursday, May 20, 2021 at 1:51 PM<br>
> To: Joosep Sarapuu <</span><span lang="EN-US"><a href="mailto:Joosep.Sarapuu@loodusmuuseum.ee"><span style="font-size:11.0pt">Joosep.Sarapuu@loodusmuuseum.ee</span></a></span><span lang="EN-US" style="font-size:11.0pt">>, "</span><span lang="EN-US"><a href="mailto:nhcoll-l@mailman.yale.edu"><span style="font-size:11.0pt">nhcoll-l@mailman.yale.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt">"
<</span><span lang="EN-US"><a href="mailto:nhcoll-l@mailman.yale.edu"><span style="font-size:11.0pt">nhcoll-l@mailman.yale.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt">><br>
> Subject: Re: [Nhcoll-l] Fluid preservation correction<br>
> <br>
> Hi Joosep,<br>
> <br>
> 96% Ethanol (with no additives) will actually be better for DNA preservation as it effectively dehydrates the specimen and reduces hydrolytic effects. So I’d keep them in the 96% EtOH. ‘Cold and dry’ tends to be best for biomolecule preservation.<br>
> <br>
> As to the denatured EtOH – then probably best to transfer into equivalent absolute ethanol. The ‘denaturant’ used can be very variable though usually something like MEK or Methanol, both of which are better avoided with DNA samples.<br>
> <br>
> Hope that helps.<br>
> <br>
> All the best,<br>
> <br>
> Jules<br>
> <br>
> <br>
> Julian Carter (he/him)<br>
> Prif Gadwraethydd, Gwyddorau Naturiol / Principal Conservator Natural Sciences<br>
> </span><span lang="EN-US"><a href="mailto:julian.carter@amgueddfacymru.ac.uk"><span style="font-size:11.0pt">julian.carter@amgueddfacymru.ac.uk</span></a></span><span lang="EN-US" style="font-size:11.0pt"><br>
> </span><span lang="EN-US"><a href="mailto:julian.carter@museumwales.ac.uk"><span style="font-size:11.0pt">julian.carter@museumwales.ac.uk</span></a></span><span lang="EN-US" style="font-size:11.0pt"><br>
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> Twitter: @NatHistConserve<br>
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> <br>
> <br>
> <br>
> <br>
> From: Nhcoll-l <</span><span lang="EN-US"><a href="mailto:nhcoll-l-bounces@mailman.yale.edu"><span style="font-size:11.0pt">nhcoll-l-bounces@mailman.yale.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt">> On Behalf Of Joosep Sarapuu<br>
> Sent: 20 May 2021 14:12<br>
> To: </span><span lang="EN-US"><a href="mailto:nhcoll-l@mailman.yale.edu"><span style="font-size:11.0pt">nhcoll-l@mailman.yale.edu</span></a></span><span lang="EN-US" style="font-size:11.0pt"><br>
> Subject: [Nhcoll-l] Fluid preservation correction<br>
> <br>
> Dear all,<br>
> <br>
> I have a question about fluid preservation correction.<br>
> • I have three samples of animals in 96% EtOH right now, but i know that the best for DNA would be 75% EtOH. Can i just dilute this to the right amount or i have to wash it in destilled water and then step by step to 75% EtOH?<br>
> • I have one animal in denaturated EtOH. This one i have to wash and then also step by step to 75% normal EtOH?<br>
> <br>
> <br>
> Sincerely,<br>
> Joosep Sarapuu<br>
> <br>
> <br>
> <br>
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