7.5.
PHOSPHOTUNGSTIC ACID STAIN
237
specific binding sites in proteins. However, presumably all proteins have
some such sites so that in effect this substance serves in some degree as a
"universal" stain for proteinaceous material. Cytomembranes are poorly
revealed, without the exaggerated contrast that can be demonstrated
with the alkaline lead stains. The net effect of staining protein-rich cells
with phosphotungstic acid is a picture that lacks the dramatic contrast
variations of one stained with lead salts for almost everything is stained
to the same degree, and the image tends to look flat (Fig. 29).
Phosphotungstic acid can be used very easily as a stain. It can be
dissolved in either aqueous or alcoholic solutions. The latter are often
much more effective in penetrating sections than the former.
The concentration of phosphotungstic acid used as a stain is not very
important. With time, even very dilute solutions (less than 0.1%) will
produce as intense a staining reaction as much stronger solutions. There
probably is never any reason to exceed 5%.
Most salts of phosphotungstic acid are insoluble so that distilled or
de-ionized water must be used in making solutions. We often have had
the impression that phosphotungstic acid either had contaminants or
reacted with contaminants while being dissolved, and that these reaction
products formed surface films. Such films are completely invisible to the
naked eye, but sometimes dirty sections disastrously. It is very easy to
eliminate such a film simply by pouring the freshly mixed solution from
one container to another. Apparently the contaminating film stays behind
in the mixing container, and the solution can be expected to remain
clean from then on.
Sections mounted on grids most simply can be floated on the surface
of aqueous solutions of phosphotungstic acid. When alcoholic solutions
(40-50%) are used, the grids are apt to sink, but this should not be a
matter of concern. Total immersion ordinarily will do no harm.
Mild heat often can be used to help phosphotungstic acid penetrate
cross-linked plastics. Long exposure times (1-4 hours) may also be required under some circumstances. One should proceed cautiously with
such treatments, however, for we think we have seen interactions between
the phosphotungstic acid and the copper of the supporting screens which
deposited "dirt" on the specimens. If considerable heat or prolonged
staining is required, it is possible to stain unmounted sections by floating
them on the surface of a staining solution. To do this, the sections are
transferred from the trough to the staining solution in a manner indicated
PHOSPHOTUNGSTIC ACID STAIN
237
specific binding sites in proteins. However, presumably all proteins have
some such sites so that in effect this substance serves in some degree as a
"universal" stain for proteinaceous material. Cytomembranes are poorly
revealed, without the exaggerated contrast that can be demonstrated
with the alkaline lead stains. The net effect of staining protein-rich cells
with phosphotungstic acid is a picture that lacks the dramatic contrast
variations of one stained with lead salts for almost everything is stained
to the same degree, and the image tends to look flat (Fig. 29).
Phosphotungstic acid can be used very easily as a stain. It can be
dissolved in either aqueous or alcoholic solutions. The latter are often
much more effective in penetrating sections than the former.
The concentration of phosphotungstic acid used as a stain is not very
important. With time, even very dilute solutions (less than 0.1%) will
produce as intense a staining reaction as much stronger solutions. There
probably is never any reason to exceed 5%.
Most salts of phosphotungstic acid are insoluble so that distilled or
de-ionized water must be used in making solutions. We often have had
the impression that phosphotungstic acid either had contaminants or
reacted with contaminants while being dissolved, and that these reaction
products formed surface films. Such films are completely invisible to the
naked eye, but sometimes dirty sections disastrously. It is very easy to
eliminate such a film simply by pouring the freshly mixed solution from
one container to another. Apparently the contaminating film stays behind
in the mixing container, and the solution can be expected to remain
clean from then on.
Sections mounted on grids most simply can be floated on the surface
of aqueous solutions of phosphotungstic acid. When alcoholic solutions
(40-50%) are used, the grids are apt to sink, but this should not be a
matter of concern. Total immersion ordinarily will do no harm.
Mild heat often can be used to help phosphotungstic acid penetrate
cross-linked plastics. Long exposure times (1-4 hours) may also be required under some circumstances. One should proceed cautiously with
such treatments, however, for we think we have seen interactions between
the phosphotungstic acid and the copper of the supporting screens which
deposited "dirt" on the specimens. If considerable heat or prolonged
staining is required, it is possible to stain unmounted sections by floating
them on the surface of a staining solution. To do this, the sections are
transferred from the trough to the staining solution in a manner indicated