260
7.
"STAINING"
blue to stain Epon sections. They prepared a 0.1% toluidine blue solution in 2.5% sodium carbonate at pH 11. They stained for 30 minutes
to 2 hours. This was followed by a wash in water, and then very brief
dips in 90 and 100% alcohol which served to differentiate the stain. The
sections then were passed quickly through xylol and mounted with Permount. In a symposium discussion of a paper by Mercer (1963), Charles
and Meek separately emphasized the usefulness of alkaline toluidine blue.
If a 1% solution dissolved in 1% borax is used at 85 °C, staining occurs in
Araldite in 1-2 minutes. Actually a convenient and usually effective way
of staining with alkaline toluidine blue is to place a slide with sections
upon it on a hotplate, and flood the slide with stain. When a little steam
or a few bubbles are seen it can be assumed that staining is complete.
Charles indicated that strongly alkaline toluidine blue solutions are not
stable, and thus should be made up freshly before use. We have not found
this to be strictly true and always necessary.
Crystal violet is a dye ordinarily regarded as having many of the same
properties as toluidine blue, and this has been used, with basic fuchsin as
a counterstain, by Moore and his co-workers (1960) to stain methacrylate
sections. Thionine also belongs in this category, and has been used with
fuchsin and azure as counterstains with Vestopal W sections by Tzitsikas
and his co-workers (1961). The latter investigators used only a 3 minute
exposure, but at a temperature of 60°-80°C.
Richardson and his co-workers (1960) generalized that most basic dyes
will stain sections embedded in Araldite if they are made sufficiently
alkaline. They recommend pH 11, and they often use brief heat. Their
particular recommendation was an adaptation of Mallory's azure IImethylene blue stain, in which equal parts of 1% azure II dissolved in
water, and 1% methylene blue dissolved in 1% borax solution were mixed
directly on the slide, and heated for 5 minutes. This staining followed a
5 minute pretreatment with 1% periodic acid or saturated potassium
persulfate. It was the borax which gave a pH of approximately 11 when
the stains were dissolved in it.
Mercer (1963), in the course of a symposium discussion, has indicated
that Sudan dyes work quite well in Araldite, and that a number of other
dyes are effective too if the solution is warmed. He also mentions an
intriguing method ascribed to Birbeck which involves heating a section
with a drop of permanganate solution which is said to result in a
beautiful definition of the membranes. As a part of the discussion of this
7.
"STAINING"
blue to stain Epon sections. They prepared a 0.1% toluidine blue solution in 2.5% sodium carbonate at pH 11. They stained for 30 minutes
to 2 hours. This was followed by a wash in water, and then very brief
dips in 90 and 100% alcohol which served to differentiate the stain. The
sections then were passed quickly through xylol and mounted with Permount. In a symposium discussion of a paper by Mercer (1963), Charles
and Meek separately emphasized the usefulness of alkaline toluidine blue.
If a 1% solution dissolved in 1% borax is used at 85 °C, staining occurs in
Araldite in 1-2 minutes. Actually a convenient and usually effective way
of staining with alkaline toluidine blue is to place a slide with sections
upon it on a hotplate, and flood the slide with stain. When a little steam
or a few bubbles are seen it can be assumed that staining is complete.
Charles indicated that strongly alkaline toluidine blue solutions are not
stable, and thus should be made up freshly before use. We have not found
this to be strictly true and always necessary.
Crystal violet is a dye ordinarily regarded as having many of the same
properties as toluidine blue, and this has been used, with basic fuchsin as
a counterstain, by Moore and his co-workers (1960) to stain methacrylate
sections. Thionine also belongs in this category, and has been used with
fuchsin and azure as counterstains with Vestopal W sections by Tzitsikas
and his co-workers (1961). The latter investigators used only a 3 minute
exposure, but at a temperature of 60°-80°C.
Richardson and his co-workers (1960) generalized that most basic dyes
will stain sections embedded in Araldite if they are made sufficiently
alkaline. They recommend pH 11, and they often use brief heat. Their
particular recommendation was an adaptation of Mallory's azure IImethylene blue stain, in which equal parts of 1% azure II dissolved in
water, and 1% methylene blue dissolved in 1% borax solution were mixed
directly on the slide, and heated for 5 minutes. This staining followed a
5 minute pretreatment with 1% periodic acid or saturated potassium
persulfate. It was the borax which gave a pH of approximately 11 when
the stains were dissolved in it.
Mercer (1963), in the course of a symposium discussion, has indicated
that Sudan dyes work quite well in Araldite, and that a number of other
dyes are effective too if the solution is warmed. He also mentions an
intriguing method ascribed to Birbeck which involves heating a section
with a drop of permanganate solution which is said to result in a
beautiful definition of the membranes. As a part of the discussion of this