58
3.
FIXATION
then is removed from the fixative and washed or stored in a 0.2 M sucrose
solution buffered at pH 7.4 with 0.1 M phosphate or cacodylate. They do
not hesitate to store tissue fixed in this way for days, weeks, and even
months before using it, either for histochemical work or for delayed
post-fixation with osmium tetroxide. Their histochemical reactions have
been carried out in fixed tissue blocks before embedding. It has been
general experience that absolute quantities of enzymes are too low in
ultrathin sections to leave visible reaction byproducts, even though their
functional presence sometimes can be demonstrated in thick sections at
the level of conventional microscopy (Chapter 4.16).
This writer has not yet had sufficient experience with glutaraldehyde
to be able to assess all of the difficulties that may be involved in its use.
A pure glutaraldehyde solution should be water-clear. However, it
obviously is a rather unstable compound which decomposes to produce
straw-colored byproducts that are highly acidic. Presumably this can be
minimized by storing stock solutions in a refrigerator. It also has been
proposed that thin layers of barium carbonate be added to the bottoms
of stock bottles of glutaraldehyde. This nearly insoluble salt then serves at
least to remove acid as it may be formed, and thus extends the useful life
of the solution. After discoloration has occurred, glutaraldehyde can be
distilled. As it is originally supplied, by the Dow Chemical Co., it is in
the form of a 25% solution. It distills at just about 100°C, so that aldehyde and water are recovered simultaneously. It can be observed that
a residue will remain that has a much higher boiling point. Our own
distillations, however, have resulted in solutions which were still quite
acidic, although they easily could be made alkaline by buffering.
Although we have not titrated such distillates, and cannot say how much
acid was present, we suspect that the amount was insignificant and did
not affect its subsequent use as a fixative. Other laboratories apparently
have been satisfied to purify glutaraldehyde by mixing it with activated
charcoal, and then filtering.
In our own laboratory we have not used glutaraldehyde exactly as
Sabatini et al. have, but rather have substituted it for formaldehyde in
the Millonig buffer, as indicated in the previous section. Admitting our
experience with glutaraldehyde is limited, we have nonetheless been
unable to detect any morphological
differences which seem to be particularly significant between glutaraldehyde and formaldehyde irrespective of
whether or not post-fixation with osmium tetroxide was employed. Our
comparisons have been made with rat liver slabs given brief (10-15
3.
FIXATION
then is removed from the fixative and washed or stored in a 0.2 M sucrose
solution buffered at pH 7.4 with 0.1 M phosphate or cacodylate. They do
not hesitate to store tissue fixed in this way for days, weeks, and even
months before using it, either for histochemical work or for delayed
post-fixation with osmium tetroxide. Their histochemical reactions have
been carried out in fixed tissue blocks before embedding. It has been
general experience that absolute quantities of enzymes are too low in
ultrathin sections to leave visible reaction byproducts, even though their
functional presence sometimes can be demonstrated in thick sections at
the level of conventional microscopy (Chapter 4.16).
This writer has not yet had sufficient experience with glutaraldehyde
to be able to assess all of the difficulties that may be involved in its use.
A pure glutaraldehyde solution should be water-clear. However, it
obviously is a rather unstable compound which decomposes to produce
straw-colored byproducts that are highly acidic. Presumably this can be
minimized by storing stock solutions in a refrigerator. It also has been
proposed that thin layers of barium carbonate be added to the bottoms
of stock bottles of glutaraldehyde. This nearly insoluble salt then serves at
least to remove acid as it may be formed, and thus extends the useful life
of the solution. After discoloration has occurred, glutaraldehyde can be
distilled. As it is originally supplied, by the Dow Chemical Co., it is in
the form of a 25% solution. It distills at just about 100°C, so that aldehyde and water are recovered simultaneously. It can be observed that
a residue will remain that has a much higher boiling point. Our own
distillations, however, have resulted in solutions which were still quite
acidic, although they easily could be made alkaline by buffering.
Although we have not titrated such distillates, and cannot say how much
acid was present, we suspect that the amount was insignificant and did
not affect its subsequent use as a fixative. Other laboratories apparently
have been satisfied to purify glutaraldehyde by mixing it with activated
charcoal, and then filtering.
In our own laboratory we have not used glutaraldehyde exactly as
Sabatini et al. have, but rather have substituted it for formaldehyde in
the Millonig buffer, as indicated in the previous section. Admitting our
experience with glutaraldehyde is limited, we have nonetheless been
unable to detect any morphological
differences which seem to be particularly significant between glutaraldehyde and formaldehyde irrespective of
whether or not post-fixation with osmium tetroxide was employed. Our
comparisons have been made with rat liver slabs given brief (10-15