42
3.
FIXATION
Stock osmium tetroxide solution:
Osmium tetroxide
2.0%
Fixative:
Buffer
Salt
Osmium tetroxide solution
1 part
1 part
2 parts
Unfortunately the small ions of the vehicle undoubtedly
penetrate
tissue blocks most rapidly, and thus are apt to reach cells and their
organelles in advance of the osmium complexes. Insofar as the former
get there first, they probably do more harm than good so that, often,
decidedly inferior fixation results from the use of this fixative. However,
when cells are immediately exposed to the fixative as in tissue cultures,
there is less likelihood of this happening, and excellent results may be
obtained without particular difficulty.
There is probably less leaching out of cytoplasmic proteins after this
fixative than after the standard mixtures. Thus, there may be a greater
retention of background material, which in turn means a reduced contrast in some situations.
Although Dalton and Palade fixatives preserve the usual cytoplasmic
organelles in almost exactly the same manner, there are sometimes
important differences in the preservation of secretory products, which
may make a comparison worthwhile.
Thorough mixing of the chrome solution, and a careful check of the
pH after titration is important. Failure to do this may have disastrous
consequences.
Although bacteriological techniques in general are outside the scope of
this book, a preparation method developed by Kellenberger, Ryter, and
Sechaud (1958) for fixing and embedding bacteria is quite generally useful for studying cell suspensions and homogenates as well as
finding
general application in bacteriology. These investigators use essentially
the Veronal-buffered osmium tetroxide fixative of Palade with sodium
chloride added, and a lower pH (6.1) than is customary for tissue work
(see below). Also, when using suspensions of organisms, they use what
might be termed a "prefixation." Additionally, they ordinarily use uranyl
acetate as a secondary fixative in the wash.
They begin with a bacterial suspension by mixing 30 ml of the culture
with 3 ml of the fixative, and centrifuge immediately for 5 minutes at
3.
FIXATION
Stock osmium tetroxide solution:
Osmium tetroxide
2.0%
Fixative:
Buffer
Salt
Osmium tetroxide solution
1 part
1 part
2 parts
Unfortunately the small ions of the vehicle undoubtedly
penetrate
tissue blocks most rapidly, and thus are apt to reach cells and their
organelles in advance of the osmium complexes. Insofar as the former
get there first, they probably do more harm than good so that, often,
decidedly inferior fixation results from the use of this fixative. However,
when cells are immediately exposed to the fixative as in tissue cultures,
there is less likelihood of this happening, and excellent results may be
obtained without particular difficulty.
There is probably less leaching out of cytoplasmic proteins after this
fixative than after the standard mixtures. Thus, there may be a greater
retention of background material, which in turn means a reduced contrast in some situations.
Although Dalton and Palade fixatives preserve the usual cytoplasmic
organelles in almost exactly the same manner, there are sometimes
important differences in the preservation of secretory products, which
may make a comparison worthwhile.
Thorough mixing of the chrome solution, and a careful check of the
pH after titration is important. Failure to do this may have disastrous
consequences.
Although bacteriological techniques in general are outside the scope of
this book, a preparation method developed by Kellenberger, Ryter, and
Sechaud (1958) for fixing and embedding bacteria is quite generally useful for studying cell suspensions and homogenates as well as
finding
general application in bacteriology. These investigators use essentially
the Veronal-buffered osmium tetroxide fixative of Palade with sodium
chloride added, and a lower pH (6.1) than is customary for tissue work
(see below). Also, when using suspensions of organisms, they use what
might be termed a "prefixation." Additionally, they ordinarily use uranyl
acetate as a secondary fixative in the wash.
They begin with a bacterial suspension by mixing 30 ml of the culture
with 3 ml of the fixative, and centrifuge immediately for 5 minutes at