62
3.
FIXATION
preservation of DNA with aldehyde fixation than is usually possible with
osmium tetroxide.
Specific granules sometimes appear with unexpected contrast and
staining properties. We frequently have had the impression that organelles stand out against the cytoplasmic background with much better than
usual contrast. We have worried somewhat that this may have been
indicative of an extraction of cytoplasmic proteins, but one usually has
the opposite impression, that unexpected quantities of some materials
were preserved. Sabatini et al. (1963) have observed the same phenomenon: "The mitochondrial matrix, the ground substance of the hyaloplasm, and the structural contents of the endoplasmic reticulum, as well
as the contents of intercellular and pericapillary spaces, appeared denser
after the double fixation than after osmium tetroxide alone. This suggested that in those places a greater proportion of material is retained by
the use of the former procedure."
Sabatini et al. (1963) have encountered a problem in post-fixation with
osmium tetroxide, particularly after glutaraldehyde. If the glutaraldehyde
is not rather thoroughly washed out, the lipids of the cytomembranes
may not be adequately osmicated unless excessive fixation times are
employed. In practice, they recommend washing for at least several hours,
or even overnight, in a buffered sucrose solution. In private conversation,
Dr. Barrnett has suggested to this writer that the explanation of this
phenomenon may be that glutaraldehyde molecules become quite firmly
attached at practically all potential binding sites associated with the
phospholipids. Then they are not easily displaced by osmium complexes.
This writer, however, has not observed this phenomenon when using
formaldehyde as described earlier, or even glutaraldehyde in the manner
in which he has employed it. The explanation may be that we have been
interested in aldehydes mainly as "killing agents," and generally our
tissue exposure to the aldehydes has been a matter of minutes rather than
hours. Also, we have used a different and stronger buffer, and these salts
may have modified the reaction. Of course our experience has been
principally with formaldehyde, and we routinely have gone directly from
the aldehyde fixative into the osmium tetroxide with no washing whatsoever.
In our own considerable experience with formaldehyde as a primary
fixative, we have noted only two consistent "deleterious" effects. The
first of these is a slight apparent coarsening of the preservation of hyalo-
3.
FIXATION
preservation of DNA with aldehyde fixation than is usually possible with
osmium tetroxide.
Specific granules sometimes appear with unexpected contrast and
staining properties. We frequently have had the impression that organelles stand out against the cytoplasmic background with much better than
usual contrast. We have worried somewhat that this may have been
indicative of an extraction of cytoplasmic proteins, but one usually has
the opposite impression, that unexpected quantities of some materials
were preserved. Sabatini et al. (1963) have observed the same phenomenon: "The mitochondrial matrix, the ground substance of the hyaloplasm, and the structural contents of the endoplasmic reticulum, as well
as the contents of intercellular and pericapillary spaces, appeared denser
after the double fixation than after osmium tetroxide alone. This suggested that in those places a greater proportion of material is retained by
the use of the former procedure."
Sabatini et al. (1963) have encountered a problem in post-fixation with
osmium tetroxide, particularly after glutaraldehyde. If the glutaraldehyde
is not rather thoroughly washed out, the lipids of the cytomembranes
may not be adequately osmicated unless excessive fixation times are
employed. In practice, they recommend washing for at least several hours,
or even overnight, in a buffered sucrose solution. In private conversation,
Dr. Barrnett has suggested to this writer that the explanation of this
phenomenon may be that glutaraldehyde molecules become quite firmly
attached at practically all potential binding sites associated with the
phospholipids. Then they are not easily displaced by osmium complexes.
This writer, however, has not observed this phenomenon when using
formaldehyde as described earlier, or even glutaraldehyde in the manner
in which he has employed it. The explanation may be that we have been
interested in aldehydes mainly as "killing agents," and generally our
tissue exposure to the aldehydes has been a matter of minutes rather than
hours. Also, we have used a different and stronger buffer, and these salts
may have modified the reaction. Of course our experience has been
principally with formaldehyde, and we routinely have gone directly from
the aldehyde fixative into the osmium tetroxide with no washing whatsoever.
In our own considerable experience with formaldehyde as a primary
fixative, we have noted only two consistent "deleterious" effects. The
first of these is a slight apparent coarsening of the preservation of hyalo-