218
7.
"STAINING"
stain, while its relatives are less so—Epon will be easier, and Maraglas
will be the easiest of all. The polyester, Vestopal W, usually seems to
stain more easily than Epon, and perhaps Selectron is even easier.
A pretreatment of sections with organic solvents, or even their vapors,
often helps staining. Perhaps the most gentle treatment is simply to place
grids with mounted sections on a platform in a closed container with a
little ethyl or amyl acetate in the bottom. A Petri dish with a piece of
filter paper covering its bottom, and with a microscope slide for a platform, is a convenient apparatus. We would expect a 30 minute exposure
to the fumes to accomplish all that could be expected with this treatment,
and would then proceed directly to staining after removing the grids from
the solvent atmosphere. Although we have mainly used ketones for this
purpose, other organic solvents with high vapor pressures might be
equally or more effective under some circumstances. I have recently heard
that trichloroethylene is particularly effective with Epon, and perhaps
with other epoxys, but I have no experience with it. The value of this
sort of treatment may not depend so much on the solvent action of the
chemical used as upon its propensity for swelling the plastic embedment.
We also frequently have exposed mounted sections to dilute (20-50%)
alcohol or acetone for 30 minute periods before starting to stain them.
This works to a degree, perhaps mainly because the specimen becomes as
uniformly wet as possible in advance of applying the stain. Pathways are
opened through the hydrophobic embedment for the unimpeded diffusion of the stain. Pure organic solvents can be used effectively for these
purposes, too, but of course tend to be more hazardous to the sections.
The above methods are "pretreatments" which may facilitate staining,
but it is also possible to affect the staining directly. One way is to use
dilute alcoholic stains (20-50%) rather than stains made up in pure water.
The alcohol then apparently serves as a wetting agent, and often helps
stain penetration. Probably all of the alkaline lead hydroxide stains, and
certainly phosphotungstic acid and uranyl acetate, can be used in this
way. Alcoholic uranyl acetate will not keep, however.
Finally, heat frequently has been used to help stain penetration, and
is often quite effective, although also often hazardous. When heat is
being used the specimens ordinarily must be submerged in a considerable
volume of stain in a closed container. Commonly, a 37°C oven will prove
adequate, although sometimes it may be desirable to go to 60°C. Heat
has been used mainly with uranyl acetate stains which otherwise often
7.
"STAINING"
stain, while its relatives are less so—Epon will be easier, and Maraglas
will be the easiest of all. The polyester, Vestopal W, usually seems to
stain more easily than Epon, and perhaps Selectron is even easier.
A pretreatment of sections with organic solvents, or even their vapors,
often helps staining. Perhaps the most gentle treatment is simply to place
grids with mounted sections on a platform in a closed container with a
little ethyl or amyl acetate in the bottom. A Petri dish with a piece of
filter paper covering its bottom, and with a microscope slide for a platform, is a convenient apparatus. We would expect a 30 minute exposure
to the fumes to accomplish all that could be expected with this treatment,
and would then proceed directly to staining after removing the grids from
the solvent atmosphere. Although we have mainly used ketones for this
purpose, other organic solvents with high vapor pressures might be
equally or more effective under some circumstances. I have recently heard
that trichloroethylene is particularly effective with Epon, and perhaps
with other epoxys, but I have no experience with it. The value of this
sort of treatment may not depend so much on the solvent action of the
chemical used as upon its propensity for swelling the plastic embedment.
We also frequently have exposed mounted sections to dilute (20-50%)
alcohol or acetone for 30 minute periods before starting to stain them.
This works to a degree, perhaps mainly because the specimen becomes as
uniformly wet as possible in advance of applying the stain. Pathways are
opened through the hydrophobic embedment for the unimpeded diffusion of the stain. Pure organic solvents can be used effectively for these
purposes, too, but of course tend to be more hazardous to the sections.
The above methods are "pretreatments" which may facilitate staining,
but it is also possible to affect the staining directly. One way is to use
dilute alcoholic stains (20-50%) rather than stains made up in pure water.
The alcohol then apparently serves as a wetting agent, and often helps
stain penetration. Probably all of the alkaline lead hydroxide stains, and
certainly phosphotungstic acid and uranyl acetate, can be used in this
way. Alcoholic uranyl acetate will not keep, however.
Finally, heat frequently has been used to help stain penetration, and
is often quite effective, although also often hazardous. When heat is
being used the specimens ordinarily must be submerged in a considerable
volume of stain in a closed container. Commonly, a 37°C oven will prove
adequate, although sometimes it may be desirable to go to 60°C. Heat
has been used mainly with uranyl acetate stains which otherwise often