X-Message-Number: 7733
Date: Sun, 23 Feb 1997 01:52:46 -0800 (PST)
From: Doug Skrecky <>
Subject: 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
      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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