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<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif"">John, thanks for the appreciation. Again some additions to consider with regard to either readjust the buffer or to replace the fluid in case of acidification of formalin:<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif""><o:p> </o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-family:"Calibri","sans-serif"">Formaldehyde degrades by oxidation to formic acid and to the insoluble paraformaldehyde below room temperature. Adjusting the pH by adding more salts won’t compensate for the formaldehyde
loss and does not have a reversal effect on the further degradation of formaldehyde.</span><span style="font-family:"Calibri","sans-serif""><o:p></o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-family:"Calibri","sans-serif"">Dissolved minerals and their ions (like Ca<sup>2+</sup>) can affect the buffer capacity.
</span><span style="font-family:"Calibri","sans-serif"">For example calcium precipitates as calcium phosphate in phosphate buffers.
<o:p></o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-family:"Calibri","sans-serif"">In acidified fluids it is likely that high concentrations of all kinds of dissolved minerals are present (depending on the preserved specimen/tissues), which can precipitate when
the fluid properties are changed. A well-known example is adding ethanol to acidified formalin forming immediately “white clouds” of precipitating salts.
<o:p></o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-family:"Calibri","sans-serif"">When adjusting the pH the chance of precipitation and efflorescence of salts dissolved in the acid formalin increases and might have a negative impact on the structural integrity
of the specimens. Replacing the fluid with freshly buffered formalin seems therefore more preferable than to adjust the pH of the degraded fluid by adding more salts.<o:p></o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-family:Symbol"><span style="mso-list:Ignore">·<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-family:"Calibri","sans-serif"">The use of containers and seals with low oxygen permeability significantly slows down acidification of formalin. Store formalin preserved collections at CONSTANT temperature above
18 degrees Celsius (to prevent polymerisation to paraformaldehyde).<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif""><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif"">Anyway, this discussion shows that there are different views among the specialists how to tackle the problem of acidity and that with regard to this topic and I agree with Julian that there
is a need for additional research that backs up some of our recommendations.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif""><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif"">Regards,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif""><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif"">Dries<o:p></o:p></span></p>
<p class="MsoNormal" style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:black">Andries J. van Dam,
</span><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:#FF8000">conservator</span><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:black"><br>
<br>
Museum of Anatomy<br>
Leiden University Medical Center <br>
Building 3 (V3-32)<br>
P.O.Box 9600 <br>
2300 RC Leiden <br>
The Netherlands <br>
tel: +31 (0)71 52 68356<br>
E-mail: </span><u><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:#0000FE"><a href="mailto:A.J.van_Dam@lumc.nl">A.J.van_Dam@lumc.nl</a><br>
</span></u><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:black">Visiting address: Hippocratespad 21 </span><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:black">
</span><span style="color:black"><o:p></o:p></span></p>
<p class="MsoNormal" style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto"><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:#FF8000">Scientific associate</span><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:black">
Natural History Museum London<br>
<a href="http://www.nhm.ac.uk/" target="_blank">http://www.nhm.ac.uk</a></span><span 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:8.0pt;font-family:"Verdana","sans-serif";color:#FF8000">Directory Board member</span><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:black"> ICOM-CC<br>
<u><a href="http://www.icom-cc.org/" target="_blank" title="http://www.icom-cc.org/">http://www.icom-cc.org</a>
<br>
<br>
</u></span><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:#FF8000">Director</span><span lang="EN-US" style="font-size:8.0pt;font-family:"Verdana","sans-serif";color:black"> Alcomon Company<br>
<u><a href="http://www.alcomon.com/" target="_blank" title="http://www.alcomon.com/">http://www.alcomon.com</a></u></span><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black">
</span><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-family:"Calibri","sans-serif""><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">From:</span></b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> John E Simmons [mailto:simmons.johne@gmail.com]
<br>
<b>Sent:</b> donderdag 3 juli 2014 14:59<br>
<b>To:</b> Dam, A.J. van (DOO)<br>
<b>Cc:</b> Greg Watkins-Colwell; Carola Haas; nhcoll-l@mailman.yale.edu<br>
<b>Subject:</b> Re: [Nhcoll-l] long-term storage of amphibian larvae in formalin<o:p></o:p></span></p>
<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">Dries,<br>
As always I appreciate your observations based on your experience. A few comments:<br>
<br>
On Thu, Jul 3, 2014 at 1:46 AM, <<a href="mailto:A.J.van_Dam@lumc.nl" target="_blank">A.J.van_Dam@lumc.nl</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black">Formalin is not stable!
<o:p></o:p></span></p>
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<p class="MsoNormal">Of course formalin is not stable, that is why it has to be buffered. If it was stable, we could merely neutralize it and be done. In any case, I said the buffering system was stable, not the formaldehyde.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black">We noticed in our fetus collection stored on phosphate buffered formalin (pH 7.3) that after ten years pH is around 6, after 20 years around 5, and after 30 years
around 4 (close to unbuffered formalin). <o:p></o:p></span></p>
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<p class="MsoNormal">What you are describing is acidification of a formaldehyde solution; what Greg described as a solution becoming sufficiently alkaline to cause clearing of tissues. One has to be cautious about extrapolating from one sort of preserved specimen
to another. A human fetus has a very different surface-to-volume ratio than do amphibian larvae, more lipids (your formaldehyde may be acidifying due to the breakdown of lipids into fatty acids), and the overall specimen volume to preservative volume is likely
to be very different for a human fetus and a clutch of amphibian larvae--all of these can be factors in pH change in formaldehyde solutions.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:10.0pt;font-family:"Tahoma","sans-serif";color:black">Therefore, when using buffered formalin as a preservation fluid, I recommend to change the fluid every 10 years.<o:p></o:p></span></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"> I advise monitoring the pH of the formaldehyde solution as well as visually watching for signs of clearing (which would indicate a shift to alkalinity) or evidence of acidification. If there are problems, then
either adjust the buffer or, in worse case scenarios, change the formaldehyde solution. I do not recommend changing preservatives unless you have identified some problem that a fresh preservative solution is likely to resolve. Changing to fresh preservative
solutions (whether formaldehyde-based or alcohol-based) can create other problems.<o:p></o:p></p>
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<p class="MsoNormal">--John<o:p></o:p></p>
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