224
7.
"STAINING"
chamber for performing this operation. The bottom can be fitted with
a piece of filter paper of suitable size which then is dampened, and serves
to maintain a saturated atmosphere when the lid is in place. A small
rectangle cut from a sheet of dental wax is placed on the filter paper, and
drops of stain slightly wider than grid diameters are placed upon this.
Grids then are floated upon these drops (Fig. 26). An important precaution is to use a staining solution free of contamination to begin with.
This can be obtained easily, however, if stain is removed from the center
of an undisturbed bottle with a clean pipette. One also should work
reasonably rapidly in setting up the drops of stain, and placing the grids,
in order to avoid excessive evaporation. If the stain does become too
concentrated as a result of evaporation, precipitates may appear. As an
added precaution the filter paper in the bottom of the Petri dish may be
dampened with a sodium hydroxide solution rather than plain water
to remove carbon dioxide from the contained atmosphere.
The time necessary to achieve staining depends entirely upon the embedment. Methacrylate, as the easiest material to stain, should not require
more than 5 minutes exposure. Epoxy resins generally require the longest
exposures, with Araldite being the most refractive, and requiring perhaps 30 minutes, Epon somewhat less, and Maraglas even less. The
polyester, Vestopal W, in general is more easily stained than either
Epon or Araldite, and may require no more than 10 minutes exposure.
In general, excessively long staining does not accomplish anything, and
increases the risk of contaminations developing. Thus one seeks to use
the minimum time that seems to be required to produce a reliable and
maximal staining effect.
After staining each grid is washed, first in a jet of 0.02 N sodium
hydroxide stored in a plastic wash bottle. Then a final spray of distilled
water is used, and the grids are allowed to dry.
Twice in the year that we have been using the Reynolds' stain in the
manner described, difficulties with contamination developed. In one instance this was traced to a tendency on the part of a particular individual
to breathe heavily on floating grids as he picked them up from their
drops. Being nearsighted, he held his head unusually close to objects he
examined, and apparently overwhelmed the chelating capacity of the
citrate with carbon dioxide. In the second instance, we had trouble with
staining naked sections that were full of holes where collagen had ripped
out. It turned out that staining solution was getting through to the upper
surface of the sections where it presumably concentrated by evaporation,
7.
"STAINING"
chamber for performing this operation. The bottom can be fitted with
a piece of filter paper of suitable size which then is dampened, and serves
to maintain a saturated atmosphere when the lid is in place. A small
rectangle cut from a sheet of dental wax is placed on the filter paper, and
drops of stain slightly wider than grid diameters are placed upon this.
Grids then are floated upon these drops (Fig. 26). An important precaution is to use a staining solution free of contamination to begin with.
This can be obtained easily, however, if stain is removed from the center
of an undisturbed bottle with a clean pipette. One also should work
reasonably rapidly in setting up the drops of stain, and placing the grids,
in order to avoid excessive evaporation. If the stain does become too
concentrated as a result of evaporation, precipitates may appear. As an
added precaution the filter paper in the bottom of the Petri dish may be
dampened with a sodium hydroxide solution rather than plain water
to remove carbon dioxide from the contained atmosphere.
The time necessary to achieve staining depends entirely upon the embedment. Methacrylate, as the easiest material to stain, should not require
more than 5 minutes exposure. Epoxy resins generally require the longest
exposures, with Araldite being the most refractive, and requiring perhaps 30 minutes, Epon somewhat less, and Maraglas even less. The
polyester, Vestopal W, in general is more easily stained than either
Epon or Araldite, and may require no more than 10 minutes exposure.
In general, excessively long staining does not accomplish anything, and
increases the risk of contaminations developing. Thus one seeks to use
the minimum time that seems to be required to produce a reliable and
maximal staining effect.
After staining each grid is washed, first in a jet of 0.02 N sodium
hydroxide stored in a plastic wash bottle. Then a final spray of distilled
water is used, and the grids are allowed to dry.
Twice in the year that we have been using the Reynolds' stain in the
manner described, difficulties with contamination developed. In one instance this was traced to a tendency on the part of a particular individual
to breathe heavily on floating grids as he picked them up from their
drops. Being nearsighted, he held his head unusually close to objects he
examined, and apparently overwhelmed the chelating capacity of the
citrate with carbon dioxide. In the second instance, we had trouble with
staining naked sections that were full of holes where collagen had ripped
out. It turned out that staining solution was getting through to the upper
surface of the sections where it presumably concentrated by evaporation,