X-Message-Number: 7665
Date: Fri, 7 Feb 1997 11:04:07 -0800 (PST)
From: Doug Skrecky <>
Subject: Another Visser Technique Suggestion

   According to Mike Darwin cracking is not a problem with the Visser
 technique. I suppose this may be due Visser's clever choice of
 cryoprotectant, which has an exceptionally low melting point of -61 C.
 This may lower the glass transition temperature and prevent cracking.
   Here's yet another possible explanation for the variability of Visser's
 results, again with a simple solution. When the techinque works, it may
 be due to partial vitrification of the rat heart. To achieve this a
 period of slow cooling by exposure to nitrogen gas must preceed the fast
 cooling that accompanies immersion in liquid nitrogen. Variations in the
 length of exposure to nitrogen gas before immersion could then account
 for the variations in the results of the Visser technique.
   For partial vitrification to happen the heart would have to be frozen
 slowly at least initially, so that the cryoprotectant has time to become
 what is called freeze concentrated as ice forms between cells. Rat hearts
 are able to survive partial ice formation. (Cryobiology 29: 470-477 1992)
 Once this ice has formed the remaining extracellular cryoprotectant is so
 concentrated that it vitrifies when the heart is quickly cooled.
 Intracellular freezing is avoided because cells have time to dehydrate
 during the slow cooling phase so that they too are vitrified. This works
 well for mouse embryos for example. (Cryobiology 31: 423-433 1994)
   This suggestion could be tested by immersing the rat heart for
 different periods of time in (for example) pure dimethylformamide cooled
 to various temperatures. After the time period for slow cooling is over
 remove the heart from the dimethylformamide cooling bath and immerse it
 directly in liquid nitrogen.

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