242
7.
"STAINING"
weeks when green crystals of vanadium oxide make their appearance as
a result of hydrolysis. The solution can be expected to have a pH of
about 3.6 which cannot be altered greatly without precipitates forming.
Vanadatomolybdate is a more vigorous stain which is stable for months.
It is made by mixing 20 ml of 1% vanadyl sulfate with 80 ml of 1%
ammonium heptamolybdate (ammonium paramolybdate, commercial
"molybdic acid"). A purple solution is first formed, which slowly oxidizes
to a clear yellow, which then is the staining solution. Oxidation can be
speeded up by bubbling oxygen through the solution. This stain also
will have a low pH, about 3.2. This can be modified over a considerable
range, but without improving the staining properties.
Grids with sections are floated face down on the surface of drops of
these solutions. No doubt the optimal staining time must vary depending
upon the embedment, but 15-30 minutes of exposure is indicated as
appropriate for epoxy-embedded tissue. After staining, grids simply are
rinsed with distilled water and dried.
7.8. Chromyl Chloride Stain
Bullivant and Hotchin (1960) have suggested using vaporous chromyl
chloride as an electron stain. This is the one effective gaseous stain so far
proposed other than the vapors of osmium tetroxide. Chromyl chloride
contains hexavalent chromium. This is an extremely reactive material
which probably must be regarded as highly toxic. The fact that chromyl
chloride exists as a gas presumably makes it particularly dangerous to
handle. Fortunately it is destroyed by water, which makes its disposal
relatively easy.
Chromyl chloride is produced by the reaction of 1 mole of K 2 Cr 2 0 7
and 4 moles of KC1 with 3 moles of H 2 S0 4 . The reaction is best carried
out in a simple distillation apparatus equipped with a dropping funnel.
Approximately appropriate proportions of K 2 Cr 2 0 7 and KC1 are ground
together in a mortar, and this blended powder is placed in the flask. Then
an excess of concentrated sulfuric acid is added slowly, which liberates
heat, and generates a heavy red gas that is the chromyl chloride. The
latter condenses as a red liquid with a boiling point of 117°C.
Since chromyl chloride vapor is extremely reactive with water, it is
essential to use it as a stain under circumstances where there is a low
humidity. It may be necessary to use a desiccator; otherwise a Petri dish
7.
"STAINING"
weeks when green crystals of vanadium oxide make their appearance as
a result of hydrolysis. The solution can be expected to have a pH of
about 3.6 which cannot be altered greatly without precipitates forming.
Vanadatomolybdate is a more vigorous stain which is stable for months.
It is made by mixing 20 ml of 1% vanadyl sulfate with 80 ml of 1%
ammonium heptamolybdate (ammonium paramolybdate, commercial
"molybdic acid"). A purple solution is first formed, which slowly oxidizes
to a clear yellow, which then is the staining solution. Oxidation can be
speeded up by bubbling oxygen through the solution. This stain also
will have a low pH, about 3.2. This can be modified over a considerable
range, but without improving the staining properties.
Grids with sections are floated face down on the surface of drops of
these solutions. No doubt the optimal staining time must vary depending
upon the embedment, but 15-30 minutes of exposure is indicated as
appropriate for epoxy-embedded tissue. After staining, grids simply are
rinsed with distilled water and dried.
7.8. Chromyl Chloride Stain
Bullivant and Hotchin (1960) have suggested using vaporous chromyl
chloride as an electron stain. This is the one effective gaseous stain so far
proposed other than the vapors of osmium tetroxide. Chromyl chloride
contains hexavalent chromium. This is an extremely reactive material
which probably must be regarded as highly toxic. The fact that chromyl
chloride exists as a gas presumably makes it particularly dangerous to
handle. Fortunately it is destroyed by water, which makes its disposal
relatively easy.
Chromyl chloride is produced by the reaction of 1 mole of K 2 Cr 2 0 7
and 4 moles of KC1 with 3 moles of H 2 S0 4 . The reaction is best carried
out in a simple distillation apparatus equipped with a dropping funnel.
Approximately appropriate proportions of K 2 Cr 2 0 7 and KC1 are ground
together in a mortar, and this blended powder is placed in the flask. Then
an excess of concentrated sulfuric acid is added slowly, which liberates
heat, and generates a heavy red gas that is the chromyl chloride. The
latter condenses as a red liquid with a boiling point of 117°C.
Since chromyl chloride vapor is extremely reactive with water, it is
essential to use it as a stain under circumstances where there is a low
humidity. It may be necessary to use a desiccator; otherwise a Petri dish