66
3.
FIXATION
his tissue. He admitted that there was not much difference but felt that
the sodium salt was in fact slightly better. More recently, Afzelius (1962)
has indicated that he has tried still other permanganates, including ones
with bivalent cations, and has found the calcium salt particularly interesting in that the triple-layered structure of "unit membranes" is easily
seen everywhere. He also found that the widths of the membranes varied
somewhat depending upon which permanganate salt was used. He also
suggested dissolving permanganate salts in organic media, such as
acetone. So far these various possibilities have not been widely explored.
When people first started using permanganate fixation its action was
regarded as capricious. Time and experience have demonstrated that
much of the difficulty resulted from the common practice of embedding
such tissue in methacrylate. As soon as cross-linked plastics became
commonly used as embedments, these difficulties largely vanished. However, there remain problems. In a cube of tissue, ordinarily it will only be
one narrow zone that will be well preserved.
In this writer's own rather limited experience, permanganate-fixed
animal tissue seems unduly sensitive to washing and dehydration. Often
minced cubes of tissues can be observed to swell grossly as they are
washed, and when placed in the lower alcohols. Also they may tend to
crumble and fall apart at this time. This can be entirely avoided by
omitting aqueous washes altogether, by moving the small cubes or thin
slabs of tissue directly into an organic solvent that is miscible with water.
We have particularly used for this purpose anhydrous acetone, or propylene oxide to which 5% absolute alcohol is added. Such ultra-rapid
dehydration followed by epoxy embedding yields perfectly reproducible
and consistent results. Indeed, these observations lead to the thought that
unsatisfactory results with permanganate fixatives in many laboratories
may be due more to differences in methods of dehydration rather than
anything else. Perhaps the most successful laboratories have wittingly or
unwittingly used the most rapid dehydration.
Most animal tissues show some swelling artifacts, particularly in the
mitochondria. Afzelius (1962) believes that some membrane systems often
are altered during preservation. Thus, if one critically compares tissues as
nearly identical as possible, fixed on the one hand with osmium tetroxide,
and on the other with permanganate, there may be significant differences
in the cytomembrane patterns. Most investigators would be inclined to
trust the osmium tetroxide picture more than the permanganate one. The
former agrees well with aldehyde preservation, and in most respects with
3.
FIXATION
his tissue. He admitted that there was not much difference but felt that
the sodium salt was in fact slightly better. More recently, Afzelius (1962)
has indicated that he has tried still other permanganates, including ones
with bivalent cations, and has found the calcium salt particularly interesting in that the triple-layered structure of "unit membranes" is easily
seen everywhere. He also found that the widths of the membranes varied
somewhat depending upon which permanganate salt was used. He also
suggested dissolving permanganate salts in organic media, such as
acetone. So far these various possibilities have not been widely explored.
When people first started using permanganate fixation its action was
regarded as capricious. Time and experience have demonstrated that
much of the difficulty resulted from the common practice of embedding
such tissue in methacrylate. As soon as cross-linked plastics became
commonly used as embedments, these difficulties largely vanished. However, there remain problems. In a cube of tissue, ordinarily it will only be
one narrow zone that will be well preserved.
In this writer's own rather limited experience, permanganate-fixed
animal tissue seems unduly sensitive to washing and dehydration. Often
minced cubes of tissues can be observed to swell grossly as they are
washed, and when placed in the lower alcohols. Also they may tend to
crumble and fall apart at this time. This can be entirely avoided by
omitting aqueous washes altogether, by moving the small cubes or thin
slabs of tissue directly into an organic solvent that is miscible with water.
We have particularly used for this purpose anhydrous acetone, or propylene oxide to which 5% absolute alcohol is added. Such ultra-rapid
dehydration followed by epoxy embedding yields perfectly reproducible
and consistent results. Indeed, these observations lead to the thought that
unsatisfactory results with permanganate fixatives in many laboratories
may be due more to differences in methods of dehydration rather than
anything else. Perhaps the most successful laboratories have wittingly or
unwittingly used the most rapid dehydration.
Most animal tissues show some swelling artifacts, particularly in the
mitochondria. Afzelius (1962) believes that some membrane systems often
are altered during preservation. Thus, if one critically compares tissues as
nearly identical as possible, fixed on the one hand with osmium tetroxide,
and on the other with permanganate, there may be significant differences
in the cytomembrane patterns. Most investigators would be inclined to
trust the osmium tetroxide picture more than the permanganate one. The
former agrees well with aldehyde preservation, and in most respects with