3.4.
SPECIAL PURPOSE OSMIUM TETROXIDE FIXATIVES
41
pyridines which destroyed the proper action of the fixative and seriously
damaged tissue. These contaminants caused cloudy solutions, whereas if
the collidine was of sufficient purity, the resulting solutions were absolutely clear. Thus the investigator who would use this buffer must be
prepared to purify it himself. Bennett and Luft devised a somewhat
laborious way of doing this to which the interested investigator is referred
for details. Holt and Hicks (1961) simply redistilled collidine at 170172°C, and stabilized it by the addition of about 0.5% chromatographic
grade alumina, finally storing it at 0-2°C.
If the beginner is bewildered by this discussion he should rest assured
that any one of the above formulations is quite capable of giving satisfactory results in most situations. If his first efforts are disappointing he
should not blame the fixative, or jump to a new formula; rather he
should consider the problems of reaching the tissue with fixative as discussed in the previous chapter. The real difficulty in obtaining good
material for electron microscopy is almost always a difficulty of getting
the fixative to the tissue under optimal conditions.
3.4. Special Purpose Osmium Tetroxide Fixatives
A number of variant osmium tetroxide fixatives have been designed
for special purposes. It is not practical to include all of these, but the
selected group which follows will indicate something of their range.
In 1955 Dalton proposed a fixative that has theoretical interest and
has enjoyed some popularity. This fixative is based on osmium tetroxide,
but uses a chrome vehicle at pH 7.2 which has some buffering properties,
for dichromate ions form equilibrium mixtures with chromate ions.
Dalton hoped that the chromate and dichromate ions would be fixatives
in their own right, and thus might be expected to preserve cell components that were not fixed by the osmium tetroxide. There was also the
likelihood that chromium atoms would become attached to cell structures
and thus produce the effect of an "electron stain." He thought it might
be particularly effective when fixing complex lipids.
The formulation is as follows:
Stock buffer:
4% potassium dichromate, brought to pH 7.2 with KOH
Stock salt solution:
NaCl
3.4%
SPECIAL PURPOSE OSMIUM TETROXIDE FIXATIVES
41
pyridines which destroyed the proper action of the fixative and seriously
damaged tissue. These contaminants caused cloudy solutions, whereas if
the collidine was of sufficient purity, the resulting solutions were absolutely clear. Thus the investigator who would use this buffer must be
prepared to purify it himself. Bennett and Luft devised a somewhat
laborious way of doing this to which the interested investigator is referred
for details. Holt and Hicks (1961) simply redistilled collidine at 170172°C, and stabilized it by the addition of about 0.5% chromatographic
grade alumina, finally storing it at 0-2°C.
If the beginner is bewildered by this discussion he should rest assured
that any one of the above formulations is quite capable of giving satisfactory results in most situations. If his first efforts are disappointing he
should not blame the fixative, or jump to a new formula; rather he
should consider the problems of reaching the tissue with fixative as discussed in the previous chapter. The real difficulty in obtaining good
material for electron microscopy is almost always a difficulty of getting
the fixative to the tissue under optimal conditions.
3.4. Special Purpose Osmium Tetroxide Fixatives
A number of variant osmium tetroxide fixatives have been designed
for special purposes. It is not practical to include all of these, but the
selected group which follows will indicate something of their range.
In 1955 Dalton proposed a fixative that has theoretical interest and
has enjoyed some popularity. This fixative is based on osmium tetroxide,
but uses a chrome vehicle at pH 7.2 which has some buffering properties,
for dichromate ions form equilibrium mixtures with chromate ions.
Dalton hoped that the chromate and dichromate ions would be fixatives
in their own right, and thus might be expected to preserve cell components that were not fixed by the osmium tetroxide. There was also the
likelihood that chromium atoms would become attached to cell structures
and thus produce the effect of an "electron stain." He thought it might
be particularly effective when fixing complex lipids.
The formulation is as follows:
Stock buffer:
4% potassium dichromate, brought to pH 7.2 with KOH
Stock salt solution:
NaCl
3.4%