3.5.
TEMPERATURE CONTROL OF FIXATION
45
washing with several changes of carbon tetrachloride, followed by a
number of changes of absolute alcohol. The specimens are then ready
for embedding.
3.5. Temperature Control of Osmium Tetroxide
Fixation
There are theoretical reasons for thinking that chilling osmium tetroxide fixatives might produce a more delicate preservation of tissues than
would otherwise be obtained. At least one might expect that cytolytic
activity would be minimized. A number of laboratories including those
of the Rockefeller and Karolinska Institutes have at times advocated
precooling the fixative to near 0°C temperatures. Palade has indicated
his belief that thicker masses of tissue can be well preserved at low
temperatures than would otherwise be possible. Rhodin has thought that
there were qualitative differences as well as quantitative ones. The present writer confirms these opinions.
In our own laboratory we have on occasion compared tissue preserved
at near 0°C with samples fixed at 21°, 37°, and 50°C. There were subtle
qualitative differences among the four samples, although we did not
push the investigation far enough to analyze these in detail. Clearly the
higher temperatures should be avoided. We have the subjective impression that the best results follow a short initial fixation at 0° C, after which
the vial with its tissue is removed from the ice bath and allowed to equilibrate at room temperature where it remains until finished.
Millonig (1962) states that he does not regard temperature as of great
importance. This writer is willing to agree that perhaps it is not important under conditions that are truly optimal in other respects, but still
advocates it as an easy form of insurance.
In talking of low temperature fixation one must bear in mind that no
doubt osmium tetroxide begins to act within seconds after the tissue
is immersed, so that if tissue is to be minced in a drop of fixative, even
this must be precooled, or the tissue may be minced "dry" and subsequently added to chilled fixative. If a tissue is to be fixed in situ, relatively large quantities of chilled fixative must be flushed over the surface
to cool the tissue as rapidly as possible. Even then effective cooling certainly is limited.
TEMPERATURE CONTROL OF FIXATION
45
washing with several changes of carbon tetrachloride, followed by a
number of changes of absolute alcohol. The specimens are then ready
for embedding.
3.5. Temperature Control of Osmium Tetroxide
Fixation
There are theoretical reasons for thinking that chilling osmium tetroxide fixatives might produce a more delicate preservation of tissues than
would otherwise be obtained. At least one might expect that cytolytic
activity would be minimized. A number of laboratories including those
of the Rockefeller and Karolinska Institutes have at times advocated
precooling the fixative to near 0°C temperatures. Palade has indicated
his belief that thicker masses of tissue can be well preserved at low
temperatures than would otherwise be possible. Rhodin has thought that
there were qualitative differences as well as quantitative ones. The present writer confirms these opinions.
In our own laboratory we have on occasion compared tissue preserved
at near 0°C with samples fixed at 21°, 37°, and 50°C. There were subtle
qualitative differences among the four samples, although we did not
push the investigation far enough to analyze these in detail. Clearly the
higher temperatures should be avoided. We have the subjective impression that the best results follow a short initial fixation at 0° C, after which
the vial with its tissue is removed from the ice bath and allowed to equilibrate at room temperature where it remains until finished.
Millonig (1962) states that he does not regard temperature as of great
importance. This writer is willing to agree that perhaps it is not important under conditions that are truly optimal in other respects, but still
advocates it as an easy form of insurance.
In talking of low temperature fixation one must bear in mind that no
doubt osmium tetroxide begins to act within seconds after the tissue
is immersed, so that if tissue is to be minced in a drop of fixative, even
this must be precooled, or the tissue may be minced "dry" and subsequently added to chilled fixative. If a tissue is to be fixed in situ, relatively large quantities of chilled fixative must be flushed over the surface
to cool the tissue as rapidly as possible. Even then effective cooling certainly is limited.