3.14.
FREEZE-DRYING
71
been used extensively in "blood bank" work. Fernandez-Moran refers to
some of these results. He explored the technique himself, at times working tissue into buffered salt baths containing as much as 30-60% glycerol.
He has told me of infiltrating living chick embryos with about 20%
glycerol, allowing it to be absorbed through their external membranes
while the temperature was dropped slowly to about 0° C. At about this
point the heart would stop and circulation would cease. Then tissue was
removed from the embryo and was passed through a series of precooled
glycerol solutions until finally it was equilibrated with 60% glycerine
at —25° C. Such tissue still soft, then could be frozen safely, and relatively
slowly, at still lower temperatures without ice crystal formation. To
some extent, at least, this procedure should be applicable even to adult
mammalian tissues, for cold glycerine solutions might be perfused via
circulatory pathways while either the whole animal, or the part to be
studied, was chilled by external packing. It seems, however, that even a
small amount of glycerine, perhaps no more than 10%, provides a great
deal of protection. Thus, any tissue or cell suspension that can withstand
isolation in a salt solution presumably can be glycerinated with modest
amounts of glycerine (10-30%) without producing damage.
Lovelock and Bishop (1959) described the use of dimethyl sulfoxide as
a superior substance to prevent freezing damage to living cells. Subsequent investigation has indicated it to be nearly inert pharmacologically. It can be employed much as glycerine, but has the advantage
of penetrating cells much more rapidly. A technical bulletin and a
medical bibliography are available upon request from the Crown Zellerbach Corp., Chemical Products Div., Camas, Washington.
3.14. Fr eeze-Dry ing
Sjostrand and Baker (1958) were the first investigators who were really
successful in adapting freeze-drying techniques to electron microscopy.
Figure 6 is of one of their preparations. Note that specimens which have
FIG. 6. (a) Frozen-dried mouse pancreas prepared by Sjostrand and Baker (1958).
A phosphotungstic acid "stain" was used to improve contrast. Note that lipids have
been extracted so that the membranes of the nucleus (nuc), the mitochondria (mit.),
and the cisterns of the endoplasmic reticulum (e.r.) appear as pale lines, while proteinaceous material survived the treatment, and subsequently was densely stained. Ice
artifacts (ice) are particularly prevalent in the nucleus, but are not serious in the
cytoplasm, (b) An enlarged view of a mitochondrion.
FREEZE-DRYING
71
been used extensively in "blood bank" work. Fernandez-Moran refers to
some of these results. He explored the technique himself, at times working tissue into buffered salt baths containing as much as 30-60% glycerol.
He has told me of infiltrating living chick embryos with about 20%
glycerol, allowing it to be absorbed through their external membranes
while the temperature was dropped slowly to about 0° C. At about this
point the heart would stop and circulation would cease. Then tissue was
removed from the embryo and was passed through a series of precooled
glycerol solutions until finally it was equilibrated with 60% glycerine
at —25° C. Such tissue still soft, then could be frozen safely, and relatively
slowly, at still lower temperatures without ice crystal formation. To
some extent, at least, this procedure should be applicable even to adult
mammalian tissues, for cold glycerine solutions might be perfused via
circulatory pathways while either the whole animal, or the part to be
studied, was chilled by external packing. It seems, however, that even a
small amount of glycerine, perhaps no more than 10%, provides a great
deal of protection. Thus, any tissue or cell suspension that can withstand
isolation in a salt solution presumably can be glycerinated with modest
amounts of glycerine (10-30%) without producing damage.
Lovelock and Bishop (1959) described the use of dimethyl sulfoxide as
a superior substance to prevent freezing damage to living cells. Subsequent investigation has indicated it to be nearly inert pharmacologically. It can be employed much as glycerine, but has the advantage
of penetrating cells much more rapidly. A technical bulletin and a
medical bibliography are available upon request from the Crown Zellerbach Corp., Chemical Products Div., Camas, Washington.
3.14. Fr eeze-Dry ing
Sjostrand and Baker (1958) were the first investigators who were really
successful in adapting freeze-drying techniques to electron microscopy.
Figure 6 is of one of their preparations. Note that specimens which have
FIG. 6. (a) Frozen-dried mouse pancreas prepared by Sjostrand and Baker (1958).
A phosphotungstic acid "stain" was used to improve contrast. Note that lipids have
been extracted so that the membranes of the nucleus (nuc), the mitochondria (mit.),
and the cisterns of the endoplasmic reticulum (e.r.) appear as pale lines, while proteinaceous material survived the treatment, and subsequently was densely stained. Ice
artifacts (ice) are particularly prevalent in the nucleus, but are not serious in the
cytoplasm, (b) An enlarged view of a mitochondrion.