46
3.
FIXATION
3.6. Duration of Osmium Tetroxide Fixation
In the early years of tissue work with osmium tetroxide solutions it was
common practice to fix for relatively long periods of time, perhaps overnight or even longer. After 1952, however, it became apparent that not
more than a very few hours gave much superior results in most instances.
At the Third International Conference on Electron Microscopy in London in 1954 (published 1956), Palade clearly and dramatically demonstrated that prolonged exposure to the fixative allowed proteinaceous
substances to leach out of muscle tissue. Membranous structures, predominantly lipid in nature, were less affected than purely proteinaceous
material. This suggests the deliberate use of long fixation to differentiate
membranous structures under conditions when these tend to be masked.
Ordinarily, however, it is obvious that a short fixation period is to be
preferred when the "total" contents of a cell are to be preserved. It has
since become evident that 30-60 minutes ordinarily is ample time for
optimal preservation. Millonig (1962), however, advocates 2-4 hours.
In this writer's laboratory an effort was made to determine the minimum time that might yield successful preservation. Exposure for as little
as 3 minutes produced as satisfactory fixation of kidney as any longer
period, although, perhaps the depth of fixation was not as great. It must
be realized, therefore, that "complete" fixation occurs very quickly, within
minutes or even seconds of the time when the fixative reaches the tissue.
Little or nothing is gained by prolonging exposure excessively, and deleterious processes start.
3.7. Criteria of Good Osmium Tetroxide Fixation
Criteria of good osmium tetroxide fixation should mainly relate to
what this fixative can accomplish when used alone as the primary preservative. Admittedly, there may be some organelles and/or inclusions
that are not fixed adequately by such treatment. The most conspicuous
deficiency seems to be in the preservation of chromosomes, but we have
every confidence that proteins in general can be delicately preserved, and
the cytomembrane systems of cells retained in place. Unfortunately, there
may be great discrepancies between what is possible and what actually
is accomplished. It is hoped that what follows will at least serve to in-
3.
FIXATION
3.6. Duration of Osmium Tetroxide Fixation
In the early years of tissue work with osmium tetroxide solutions it was
common practice to fix for relatively long periods of time, perhaps overnight or even longer. After 1952, however, it became apparent that not
more than a very few hours gave much superior results in most instances.
At the Third International Conference on Electron Microscopy in London in 1954 (published 1956), Palade clearly and dramatically demonstrated that prolonged exposure to the fixative allowed proteinaceous
substances to leach out of muscle tissue. Membranous structures, predominantly lipid in nature, were less affected than purely proteinaceous
material. This suggests the deliberate use of long fixation to differentiate
membranous structures under conditions when these tend to be masked.
Ordinarily, however, it is obvious that a short fixation period is to be
preferred when the "total" contents of a cell are to be preserved. It has
since become evident that 30-60 minutes ordinarily is ample time for
optimal preservation. Millonig (1962), however, advocates 2-4 hours.
In this writer's laboratory an effort was made to determine the minimum time that might yield successful preservation. Exposure for as little
as 3 minutes produced as satisfactory fixation of kidney as any longer
period, although, perhaps the depth of fixation was not as great. It must
be realized, therefore, that "complete" fixation occurs very quickly, within
minutes or even seconds of the time when the fixative reaches the tissue.
Little or nothing is gained by prolonging exposure excessively, and deleterious processes start.
3.7. Criteria of Good Osmium Tetroxide Fixation
Criteria of good osmium tetroxide fixation should mainly relate to
what this fixative can accomplish when used alone as the primary preservative. Admittedly, there may be some organelles and/or inclusions
that are not fixed adequately by such treatment. The most conspicuous
deficiency seems to be in the preservation of chromosomes, but we have
every confidence that proteins in general can be delicately preserved, and
the cytomembrane systems of cells retained in place. Unfortunately, there
may be great discrepancies between what is possible and what actually
is accomplished. It is hoped that what follows will at least serve to in-