3.13.
RAPID FREEZING
69
indicates that liquid Freon 12, 13, or 14 can be collected in the same
way, and will also serve as good quenching baths. They offer the advantage of not being explosive in the presence of oxygen as are liquid isopentane and propane. Freon 12 can be liquified most easily since its boiling point is only — 22°C, while the boiling points of the other Freons are
much below that. The freezing point of Freon 12 is — 160°C, and that of
the other Freons is —181 and — 184°C, respectively.
It is evident that a dry ice temperature (—72° C) also would be adequate
sometimes for freezing small bits of tissue. Then a precooled container
of ethyl ether or heptane can be used. Under some circumstances,
absolute alcohol might be used as the quenching bath, but it tends to
absorb water so readily that the formation of an annoying sludge of ice
crystals is almost inevitable.
Fernandez-Moran (1960) has explored the possibilities of using liquid
helium II at —272° C to freeze tissues extremely rapidly. Although this
may prove to be an essential procedure for particular projects, its great
cost and usual unavailability will preclude its being used except when
required for special purposes.
For some elegant freeze-substitution work with nervous tissue, van
Haarveld and Cowell (1964) found that their tissue could be frozen more
reliably and effectively by contact with precooled metal than by a fluid
bath. They chose silver for this purpose because of its well-known
property as an excellent thermal conductor. Slabs of tissue (cerebellar
folia) were laid upon its highly polished surface so that good contact was
established. Only the cortical layer in immediate contact with the freezing-plate was studied finally.
It was of course necessary to protect the silver freezing plate from
moist air so that ice films did not form upon its cold surface, which then
would insulate the contact with the specimen. Therefore an apparatus
of some complexity was required, and the freezing plate was located at
the bottom of a well immersed in liquid nitrogen. A flow of dry helium
gas was passed first through a coil in the liquid nitrogen bath to cool it,
and then escaped into the bottom of the well, just above the silver
freezing plate. Thus no appreciable quantity of air reached this area,
and ice and oxidative films were avoided.
The possibility of inhibiting ice crystal formation by glycerinating
tissue in advance of freezing deserves much further study. Agriculturalists
interested in preserving viable spermatozoa for artificial insemination
have known of its protective action for more than a decade. It has also
RAPID FREEZING
69
indicates that liquid Freon 12, 13, or 14 can be collected in the same
way, and will also serve as good quenching baths. They offer the advantage of not being explosive in the presence of oxygen as are liquid isopentane and propane. Freon 12 can be liquified most easily since its boiling point is only — 22°C, while the boiling points of the other Freons are
much below that. The freezing point of Freon 12 is — 160°C, and that of
the other Freons is —181 and — 184°C, respectively.
It is evident that a dry ice temperature (—72° C) also would be adequate
sometimes for freezing small bits of tissue. Then a precooled container
of ethyl ether or heptane can be used. Under some circumstances,
absolute alcohol might be used as the quenching bath, but it tends to
absorb water so readily that the formation of an annoying sludge of ice
crystals is almost inevitable.
Fernandez-Moran (1960) has explored the possibilities of using liquid
helium II at —272° C to freeze tissues extremely rapidly. Although this
may prove to be an essential procedure for particular projects, its great
cost and usual unavailability will preclude its being used except when
required for special purposes.
For some elegant freeze-substitution work with nervous tissue, van
Haarveld and Cowell (1964) found that their tissue could be frozen more
reliably and effectively by contact with precooled metal than by a fluid
bath. They chose silver for this purpose because of its well-known
property as an excellent thermal conductor. Slabs of tissue (cerebellar
folia) were laid upon its highly polished surface so that good contact was
established. Only the cortical layer in immediate contact with the freezing-plate was studied finally.
It was of course necessary to protect the silver freezing plate from
moist air so that ice films did not form upon its cold surface, which then
would insulate the contact with the specimen. Therefore an apparatus
of some complexity was required, and the freezing plate was located at
the bottom of a well immersed in liquid nitrogen. A flow of dry helium
gas was passed first through a coil in the liquid nitrogen bath to cool it,
and then escaped into the bottom of the well, just above the silver
freezing plate. Thus no appreciable quantity of air reached this area,
and ice and oxidative films were avoided.
The possibility of inhibiting ice crystal formation by glycerinating
tissue in advance of freezing deserves much further study. Agriculturalists
interested in preserving viable spermatozoa for artificial insemination
have known of its protective action for more than a decade. It has also