228
7.
"STAINING"
eluded. After staining is complete, the staining solution is removed by
dilution with quantities of gas-free water before removing and drying
the grids.
First a stock solution of uncontaminated stain is collected under a layer
of paraffin oil in a separatory cylinder equipped with a Teflon stopcock
[Fig. 27(a)]. It can be stored thus for weeks or months. A film of lead
salts may slowly develop on the inner surface of the glass, but the interior
fluid can be expected to remain free of contaminating suspended crystals.
Drops of clean staining solution then can be released at will through the
stopcock. There will be minimum contact with air if the stem of the
cylinder is cut off close to the stopcock as illustrated. After use, residual
staining solution can be removed from the lip of the stem with absorbent
paper. However, it has proved best not to try to clean this region particularly thoroughly. Solid deposits of lead salts will accumulate on the lip,
and it is a mistake to try to remove these immediately preceding the
withdrawal of stain. Rather, discard the first few drops of stain with each
new withdrawal, which will wash away any detachable debris. Then
clean solution will be delivered.
Our staining containers are made essentially as Feldman (1962) suggested [Fig. 27(b)]. A strip of heavy Lucite (Plexiglas) sheet, or a bar of
this material, is drilled so that small (diameter, 8 mm; depth, 4 mm) cups
are made in it which will accommodate 2-3 grids, and which serve as the
staining containers. The drill holes are fire-polished so that their smooth
walls and bottoms can be kept clean. Grids with sections upon them are
put in the cups and submerged under drops of uncontaminated stain which
then fills each hole so that there is a slightly bulging meniscus. A rectangle
of clean, heavy glass is added as a cover without unnecessary delay after
filling each container, and without allowing air bubbles to be trapped.
Before the staining operation is begun, a generous quantity (a liter or
more) of de-gassed water should have been prepared by boiling de-ionized
or distilled water for a few minutes in an Erlenmeyer flask. When staining is finished this is used as the dilutant and wash water. Before removing the specimens, the entire plastic bar with its loaded and covered
cups is placed in a suitably proportioned pan, and is totally submerged in
the de-gassed wash water. The water can be used while it is still uncomfortably hot. The heavy glass covers are not removed from the specimen
chambers except under the wash water when the staining solution then is
free to diffuse freely and be greatly diluted in the gas-free water. It is
best to aid this by stirring appropriately with a glass paddle. Most of the
7.
"STAINING"
eluded. After staining is complete, the staining solution is removed by
dilution with quantities of gas-free water before removing and drying
the grids.
First a stock solution of uncontaminated stain is collected under a layer
of paraffin oil in a separatory cylinder equipped with a Teflon stopcock
[Fig. 27(a)]. It can be stored thus for weeks or months. A film of lead
salts may slowly develop on the inner surface of the glass, but the interior
fluid can be expected to remain free of contaminating suspended crystals.
Drops of clean staining solution then can be released at will through the
stopcock. There will be minimum contact with air if the stem of the
cylinder is cut off close to the stopcock as illustrated. After use, residual
staining solution can be removed from the lip of the stem with absorbent
paper. However, it has proved best not to try to clean this region particularly thoroughly. Solid deposits of lead salts will accumulate on the lip,
and it is a mistake to try to remove these immediately preceding the
withdrawal of stain. Rather, discard the first few drops of stain with each
new withdrawal, which will wash away any detachable debris. Then
clean solution will be delivered.
Our staining containers are made essentially as Feldman (1962) suggested [Fig. 27(b)]. A strip of heavy Lucite (Plexiglas) sheet, or a bar of
this material, is drilled so that small (diameter, 8 mm; depth, 4 mm) cups
are made in it which will accommodate 2-3 grids, and which serve as the
staining containers. The drill holes are fire-polished so that their smooth
walls and bottoms can be kept clean. Grids with sections upon them are
put in the cups and submerged under drops of uncontaminated stain which
then fills each hole so that there is a slightly bulging meniscus. A rectangle
of clean, heavy glass is added as a cover without unnecessary delay after
filling each container, and without allowing air bubbles to be trapped.
Before the staining operation is begun, a generous quantity (a liter or
more) of de-gassed water should have been prepared by boiling de-ionized
or distilled water for a few minutes in an Erlenmeyer flask. When staining is finished this is used as the dilutant and wash water. Before removing the specimens, the entire plastic bar with its loaded and covered
cups is placed in a suitably proportioned pan, and is totally submerged in
the de-gassed wash water. The water can be used while it is still uncomfortably hot. The heavy glass covers are not removed from the specimen
chambers except under the wash water when the staining solution then is
free to diffuse freely and be greatly diluted in the gas-free water. It is
best to aid this by stirring appropriately with a glass paddle. Most of the