X-Message-Number: 7733
Date: Sun, 23 Feb 1997 01:52:46 -0800 (PST)
From: Doug Skrecky <>
Subject: Is Permafrost Burial a Viable Option?
IS PERMAFROST BURIAL A VIABLE OPTION?
(From Vol.32 August 1996 Canadian Cryonics News)
By Doug Skrecky
Preserved tissue requires a high degree of stability if
resuscitation after a prolonged period of storage is contemplated. Can
permafrost burial in combination with either chemical fixation or
freeze-drying stabilize tissue DNA, RNA and proteins adequately for
resuscitation to be even a theoretical possibility?
The most effective fixative for the preservation of high molecular
weight DNA is ethanol. This is the only fixative that was able to yield
1327 bp DNA amplication products after 30 days of fixation at room
temperature. *1 Diluting to 50% ethanol greatly harms DNA preservation.
*2 Some intriguing work has found that either adding at least 25 mM
sodium chloride or 30 mM potassium chloride to formaldehyde solutions
halts DNA deterioration at room temperature at least temporarily (over 24
hours). There is also a strong temperature dependance with no DNA
deterioration being observed over a 24 hour period if the temperature was
less than 10 C. *3 These results are not surprising as both salt addition
and lowering of temperature are known to stabilize the hydrogen bonds
between DNA strands. Unfortunately the effect of either temperature or
salt on long term DNA preservation in ethanol has not yet been
investigated.
The most effective fixative for the preservation of high molecular
weight RNA appears once again to be ethanol. *4
The most effective fixative for the preservation of protein antigens
are zinc based fixatives such as B5. *5 Nonetheless ethanol is still a
large improvement over most other fixatives and since B5 destroys both
DNA and RNA it at least is out of the running.
Overall, ethanol with possibly a dash of salt would seem to be the
best overall fixative. However to be effective virtually all water would
have to be removed from tissue for ethanol to be an effective stabilizer
in the long term. Inclusion of sufficient desiccant inside the casket to
mummify an ethanol preserved corpse would thus be an absolute
requirement. No desiccant - no resuscitation.
Which leads us to freeze-drying as a possible alternative. Storage
of freeze-dried tissue (no cryoprotectant used) with desiccant under
nitrogen at 37 C showed extensive DNA, RNA and protein degradation. At
room temperature both DNA and protein were perfectly stable over a 6
month period, though some slight RNA deterioration was still detected. *6
As with formaldehyde fixation, the stability of freeze dried tissue
appears to be very sensitive to temperature.
My recommendations for permafrost burial are as follows:
1. Freeze-drying is to be preferred to fixation.
2. If fixation is attempted ethanol is the first choice. If a
formaldehyde solution is used add salt to it.
3. Add enough desiccant to the casket to insure mummification.
*1 "Effect of Fixatives and Fixation Times on Tissues: Authors' Reply"
American Journal of Clinical Pathology 96(1): 144-145 1991
*2 "Ethanol Fixation of Bladder Irrigation Specimens for Flow Cytometric
Analysis" Cancer 63: 1780-1783 1989
*3 "The Effect of Formalin Fixation on DNA and the Extraction of High
Molecular Weight DNA From Fixed and Embedded Tissues" Pathology Research
Practice 189: 66-72 1993
*4 "Effect of Fixation on the Amplification of Nucleic Acids From
Paraffin Embedded Material by the Polymerase Chain Reaction" Journal of
Histochemistry Cytochemistry 39: 351-354 1991
*5 "A Simple Technique for Preservation of Fixation Sensitive Antigens in
Paraffin Embedded Tissues" Journal of Histochemistry Cytochemistry 42:
1127-1134 1994
*6 "Degradation of Macromolecules During Preservation of Lyophilized
Pathological Tissues" Pathology Research Practice 191: 420-426 1995
Postscript to "Is Permafrost Burial a Viable Option?":
After looking for any research on the effect of salt on ethanol
solutions I found the following article from the journal Nucleic Acids
Research (Vol.6 No.6 1979 2089-2107), which was entitled "The Circular
Dichroism and X-ray Diffraction of DNA Condensed From Ethanolic
Solutions". This article made mention of the fact that DNA did not
precipitate in 60% ethanol unless 0.025 M sodium chloride was added.
Since concentrated ethanol solutions preserve DNA by precipitating it it
does seem that adding salt would likely be of some benefit in alcohol
fixation. This precipitation is reversible by lowering the ethanol
concentration to 30%.
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