7.3.
ALKALINE LEAD HYDROXIDE STAINS
227
than aqueous stains, and also because we had less trouble with contamination. Our eventual method of avoiding contamination (see below) almost
precluded their use, however, and we returned to aqueous stains.
The great problem with Watson's stain always has been the contamination which develops as lead carbonates are formed at interfaces
and within solutions. If allowed to form, these highly hydrophobic
crystals attach themselves to the plastic of sections with such great
avidity that often it is difficult to find even a high magnification field
that is not contaminated with a sprinkling of these crystals (Fig. 10).
Much ingenuity has gone into the design of procedures and equipment
to prevent this.
It is not practical to consider in detail all of the practices and devices
which have been advocated to eliminate contamination. In general, they
include (1) efforts to form clean, de novo surfaces just before commencing
staining; (2) the protection of surfaces from atmospheric carbon dioxide
by staining in nitrogen atmospheres, either within large containers such
as glove boxes or plastic bags, or in streams of gas continuously flowing
over drops of stain; (3) by avoiding passing the grids through an interface of stain, by first submerging the grids in water, then replacing this
with stain by an exchange of solutions, and finally returning to water
before removing the grids from their bath; and (4) by minimizing the
gaseous atmosphere as by placing the grids in an empty syringe with its
plunger in, and then sucking up the stain. Also, obvious precautions
have been taken to de-gas all water used with the lead stain, as well, of
course, as using only distilled or de-ionized water. To varying extents
these different procedures surely have been helpful. However, some were
costly to set up and others costly to run. In some cases one could stain
only a single grid at a time, and all too often a good deal of contamination appeared in spite of the efforts. In our own laboratory we finally
settled on a compromise method, mainly inspired by the work of Feldman
(1962), which has the advantages of considerable reliability, ease of
handling, and simple equipment. A description of this method follows,
which will be the only technique detailed here.
Our procedure depends upon covering the grids to be stained with an
uncontaminated staining solution from which air subsequently is expartly through Lucite bars (b), and flooded to overflowing with staining solution.
Heavy glass squares serve as lids (b). Stain subsequently is removed by great dilution
after sinking the covered chambers in freshly boiled distilled water, and only then
removing their lids (Chapter 7.3).
ALKALINE LEAD HYDROXIDE STAINS
227
than aqueous stains, and also because we had less trouble with contamination. Our eventual method of avoiding contamination (see below) almost
precluded their use, however, and we returned to aqueous stains.
The great problem with Watson's stain always has been the contamination which develops as lead carbonates are formed at interfaces
and within solutions. If allowed to form, these highly hydrophobic
crystals attach themselves to the plastic of sections with such great
avidity that often it is difficult to find even a high magnification field
that is not contaminated with a sprinkling of these crystals (Fig. 10).
Much ingenuity has gone into the design of procedures and equipment
to prevent this.
It is not practical to consider in detail all of the practices and devices
which have been advocated to eliminate contamination. In general, they
include (1) efforts to form clean, de novo surfaces just before commencing
staining; (2) the protection of surfaces from atmospheric carbon dioxide
by staining in nitrogen atmospheres, either within large containers such
as glove boxes or plastic bags, or in streams of gas continuously flowing
over drops of stain; (3) by avoiding passing the grids through an interface of stain, by first submerging the grids in water, then replacing this
with stain by an exchange of solutions, and finally returning to water
before removing the grids from their bath; and (4) by minimizing the
gaseous atmosphere as by placing the grids in an empty syringe with its
plunger in, and then sucking up the stain. Also, obvious precautions
have been taken to de-gas all water used with the lead stain, as well, of
course, as using only distilled or de-ionized water. To varying extents
these different procedures surely have been helpful. However, some were
costly to set up and others costly to run. In some cases one could stain
only a single grid at a time, and all too often a good deal of contamination appeared in spite of the efforts. In our own laboratory we finally
settled on a compromise method, mainly inspired by the work of Feldman
(1962), which has the advantages of considerable reliability, ease of
handling, and simple equipment. A description of this method follows,
which will be the only technique detailed here.
Our procedure depends upon covering the grids to be stained with an
uncontaminated staining solution from which air subsequently is expartly through Lucite bars (b), and flooded to overflowing with staining solution.
Heavy glass squares serve as lids (b). Stain subsequently is removed by great dilution
after sinking the covered chambers in freshly boiled distilled water, and only then
removing their lids (Chapter 7.3).