68
3.
FIXATION
sought by many, but achieved in considerable measure of success by only
a few. The approach has been to start with tissue that has been frozen
with extreme rapidity to minimize ice-crystal formation. Then the
frozen tissue has been dehydrated, either by evaporative drying as
described in Chapter 3.14, or by substitution as indicated in Chapter
3.15.
Freezing tissue fast enough to prevent or at least minimize ice crystal
formation demands that certain precautions be taken. Perhaps the most
important consideration for quick freezing is the size of the specimen.
Hanzon and Hermodsson (1960) dissected fresh tissue on the surface of a
stainless steel box filled with an ice water mixture to cool the tissue
somewhat while the tissue was prepared. They minced tissue, accepting
only those pieces which did not exceed 0.2 mm in one of the dimensions.
Such tiny pieces were put on a thin fiber of nylon which was then
plunged into precooled propane. Elfvin (1963) preferred cotton threads.
In our laboratory we have used ribbons of very thin polyethylene sheet.
We have also found that comparatively stiff, but very thin "needles" can
be drawn from thick nitrocellulose solutions, and serve well for this
purpose. "Duco Cement," as manufactured by the Du Pont Co., and sold
in tubes in stationery stores, is readily available in this country, and is
easy to use. A short length of very fine wire serves as a "handle," and its
tip is dipped into an extruded drop of cement. Then, as the wire is
pulled away, a thin thread of nitrocellulose is created which quickly
hardens. The thickness of the thread can be controlled by the rate at
which it is drawn.
The freezing bath most commonly used is precooled isopentane. This
has the practical advantage of being a liquid at ordinary room temperatures, and also remaining liquid until —160° C. It is important that
tissue not be plunged directly into liquid air or nitrogen. Then a gas
film forms immediately on the surface of the specimen and acts as an
insulating layer, which prevents the rapid transfer of heat.
Liquid propane has a small advantage over liquid isopentane in that it
does not freeze until —190° C, very nearly the temperature of liquid
nitrogen. However, it is of course a gas at room temperature at atmospheric pressure, so its preparation is a problem. One method, described
by Feder and Sidman (1958), connects a 4-5 ft length of
in. plastic
tubing to a gas cylinder, and chills the mid-portion of the tubing with
liquid nitrogen. Slowly escaping gas then is condensed in the tubing, and
the liquid propane is collected in a precooled flask. Rebhun (1961)
3.
FIXATION
sought by many, but achieved in considerable measure of success by only
a few. The approach has been to start with tissue that has been frozen
with extreme rapidity to minimize ice-crystal formation. Then the
frozen tissue has been dehydrated, either by evaporative drying as
described in Chapter 3.14, or by substitution as indicated in Chapter
3.15.
Freezing tissue fast enough to prevent or at least minimize ice crystal
formation demands that certain precautions be taken. Perhaps the most
important consideration for quick freezing is the size of the specimen.
Hanzon and Hermodsson (1960) dissected fresh tissue on the surface of a
stainless steel box filled with an ice water mixture to cool the tissue
somewhat while the tissue was prepared. They minced tissue, accepting
only those pieces which did not exceed 0.2 mm in one of the dimensions.
Such tiny pieces were put on a thin fiber of nylon which was then
plunged into precooled propane. Elfvin (1963) preferred cotton threads.
In our laboratory we have used ribbons of very thin polyethylene sheet.
We have also found that comparatively stiff, but very thin "needles" can
be drawn from thick nitrocellulose solutions, and serve well for this
purpose. "Duco Cement," as manufactured by the Du Pont Co., and sold
in tubes in stationery stores, is readily available in this country, and is
easy to use. A short length of very fine wire serves as a "handle," and its
tip is dipped into an extruded drop of cement. Then, as the wire is
pulled away, a thin thread of nitrocellulose is created which quickly
hardens. The thickness of the thread can be controlled by the rate at
which it is drawn.
The freezing bath most commonly used is precooled isopentane. This
has the practical advantage of being a liquid at ordinary room temperatures, and also remaining liquid until —160° C. It is important that
tissue not be plunged directly into liquid air or nitrogen. Then a gas
film forms immediately on the surface of the specimen and acts as an
insulating layer, which prevents the rapid transfer of heat.
Liquid propane has a small advantage over liquid isopentane in that it
does not freeze until —190° C, very nearly the temperature of liquid
nitrogen. However, it is of course a gas at room temperature at atmospheric pressure, so its preparation is a problem. One method, described
by Feder and Sidman (1958), connects a 4-5 ft length of
in. plastic
tubing to a gas cylinder, and chills the mid-portion of the tubing with
liquid nitrogen. Slowly escaping gas then is condensed in the tubing, and
the liquid propane is collected in a precooled flask. Rebhun (1961)