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Date: 06 Jul 95 02:56:02 EDT
From: Mike Darwin <>
Subject: Fwd: re: darwin's comments, p...
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DATE: 5/10/95 9:52 AM
RE: Fwd: re: darwin's comments, p...
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Subject: Fwd: re: darwin's comments, p...
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From: (Pichugin)
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Date: 95-05-10 05:47:21 EDT
Date: 10 May 95
The answers on questions 3, 4, 4, 9, 12 could find in our report
(The Immortalist, v. 25, N 8, 1994, p. 5 ) and in our generalized one.
For example, the CI method for freezing sheep heads did not provide for
the use of filters and etc.
5. Our devices are not adapted for automatic registration such very slow
rates, therefore we registered temperature by hand and
only by day. Thus we could only partially determine cooling
curves using periods of measurement. The temperatures of the phase
transitions were ranged from -18 C to -20 C for all three
cases of freezing the glycerolized sheep heads. However, even using the
such method of measurement of rates, it was possible to get the
good reproduction of procedure of freeze-thawing.
6. Glycerol was not in the fixative.
7. The fixative solution contained 2% glutaraldehyde (pure for
"Electron Microscopy"), prepared on phosphate isotonic buffer.
8. In general, the protocol used to prepare the tissue for EM was
reported in The Immortalist, v25, N 8, p5. Slices stained by 1% uranyl
acetate solution, washed out and then stained by 1% lead citrate
solution. The pieces of the tissues were 1x1x3 mm.
10. One of our omissions was that we did not exactly fix and report the
magnifications of each light or electron micrographs and gave only
limits of magnifications on the whole. The magnifications of the light
micrographs were 400 - 600. The magnifications of the electron micrographs
were 7,000 - 12,000.
11. Black and white photos are quite acceptable for registration
of histological preparations stained by Nissl's method.
Certainly, it is better to use colour photos, but we have not
such opportunity.
12. The size of the tissue pieces for light microscopy was 5x5x3. They were
fixed at 20 C. The tissue pieces for electron microscopy were fixed at 4 C.
About Darwin's "General Evaluation of The Research".
About the procedures of fixation. Darwin touched upon the
subject in the several places of his comments. His caveats concerned
fixation glycerolized tissues. So, Darwin's "Caveats on These Comments"
devoted the question completely. The intact non-frozen tissue, the
non-frozen tissue after glycerol perfusion and reperfusion, and the
non-glycerolized tissue after thawing were fixed without osmotic
stresses because they were iso-osmotis with respect to the fixative
solutions. Yes, we made an omission fixing the glycerolized tissues
without addition of glycerol in the fixative solutions. What influence
could this have on the results? The light micrographs (60 - 65) would
look nearly the same only without light-coloured halos around neuron
bodies. We saw micrographs of histological samples fixed by the such
way in articles of another investigators. Any substantial change or
breach of histological picture did not occur. Dr. Fahy have written
that "shrinkage spaces around cells don't worry me" in his comment of
the micrograph N 60. In case of electron microscopy, the injury of fine
structure by osmotic stresses of irregular fixation may be more
significant but on the surface of tissue pieces. Significant changes
may do not happen inside the pieces because as penetrating of molecules
of glutaraldehyde into the glycerolized pieces, concentration of
glycerol maintains still high. Glutaraldehyde quickly fixes biological
structures. Glycerol cannot well interfere with fixation. After
fixation, osmotic stresses can no longer change ultrastructure of the
tissues. Of course, ultrastructure of the glycerolized tissues would
look some better, if we used glycerol in the fixative. The picture of
osmotis stresses of the glycerolized samples does not look far worse
than ones of the non-frozen tissue after glycerol perfusion and
reperfusion. If serious osmotic stresses during fixation of the
glycerolized samples had been, the very strongly injuries would have
been like ones of the thawed and reperfusated tissues. Speed
penetration of water into the glycerolized tissues during fixation
accompanies strongly edema of the tissues, however the glycerolized
tissues looked dehydrated in our case, thus these tissues did not
change at fixation in general.
Yuri Pichugin
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