262
7.
"STAINING"
avoid softening of the plastic embedment, and the consequent eventual
wrinkling of the sections. In essence the staining reaction intensifies the
original distribution of the osmium salts, and is a uniform dark brown,
almost black color. The effect is almost like looking at an electron micrograph or, for that matter, a line drawing. The stain provides additional
contrast to phase contrast images, and permits the use of thinner sections
than would be otherwise possible. The stain seems to be so reliable, and
can be so easily applied, that it should prove to be quite useful.
McGee-Russell, in commenting upon a symposium paper by Mercer
(1963), described the use of Sudan black B as a "negative stain," specifically and only with Epon-embedded sections. A standard saturated
solution of this dye, refluxed in 70% alcohol, is used at room temperature
for 1-2 hours. The Epon plastic embedment is darkly stained, but most
parts of the specimen do not stain at all, and reveal very fine detail as
light images against a dark background. In this way, structures not easily
given a positive stain are revealed in negative contrast.
7.14. Removal of Cured Epoxy Resins
It is reasonable to think that the staining of thick sections for conventional microscopy might be improved by removing the embedding
medium. Certainly this is true with methacrylate sections where there is
no particular problem in dissolving the embedment. Cross-linked plastics
are essentially insoluble, however, in all ordinary solvents even though
powerful organic solvents such as trichloroethylene and ketones may
extract some components, or at least swell the plastic. This may help
staining in particular cases (Chapter 7.12), but it also would be desirable
to completely remove the embedment.
This writer knows of no way to dissolve cured Vestopal or any other
polyester resin. However, Mayor et al. (1961) have indicated that Araldite,
and presumably other epoxy resin formulations which use dodecenyl
succinic anhydride as a hardener, can be dissolved in a mixture of sodium
methoxide, benzene and methyl alcohol.
It is necessary to make one's own sodium methoxide by dividing about
2.5 gm of metallic sodium into cubes ^ in. or less on a side, and dropping
these piece by piece into 25 ml of methyl alcohol in a hood or wellventilated area. Considerable heat will be generated, and much bubbling
will ensue so this process should be done cautiously in an adequately
7.
"STAINING"
avoid softening of the plastic embedment, and the consequent eventual
wrinkling of the sections. In essence the staining reaction intensifies the
original distribution of the osmium salts, and is a uniform dark brown,
almost black color. The effect is almost like looking at an electron micrograph or, for that matter, a line drawing. The stain provides additional
contrast to phase contrast images, and permits the use of thinner sections
than would be otherwise possible. The stain seems to be so reliable, and
can be so easily applied, that it should prove to be quite useful.
McGee-Russell, in commenting upon a symposium paper by Mercer
(1963), described the use of Sudan black B as a "negative stain," specifically and only with Epon-embedded sections. A standard saturated
solution of this dye, refluxed in 70% alcohol, is used at room temperature
for 1-2 hours. The Epon plastic embedment is darkly stained, but most
parts of the specimen do not stain at all, and reveal very fine detail as
light images against a dark background. In this way, structures not easily
given a positive stain are revealed in negative contrast.
7.14. Removal of Cured Epoxy Resins
It is reasonable to think that the staining of thick sections for conventional microscopy might be improved by removing the embedding
medium. Certainly this is true with methacrylate sections where there is
no particular problem in dissolving the embedment. Cross-linked plastics
are essentially insoluble, however, in all ordinary solvents even though
powerful organic solvents such as trichloroethylene and ketones may
extract some components, or at least swell the plastic. This may help
staining in particular cases (Chapter 7.12), but it also would be desirable
to completely remove the embedment.
This writer knows of no way to dissolve cured Vestopal or any other
polyester resin. However, Mayor et al. (1961) have indicated that Araldite,
and presumably other epoxy resin formulations which use dodecenyl
succinic anhydride as a hardener, can be dissolved in a mixture of sodium
methoxide, benzene and methyl alcohol.
It is necessary to make one's own sodium methoxide by dividing about
2.5 gm of metallic sodium into cubes ^ in. or less on a side, and dropping
these piece by piece into 25 ml of methyl alcohol in a hood or wellventilated area. Considerable heat will be generated, and much bubbling
will ensue so this process should be done cautiously in an adequately