236
7.
"STAINING"
the intensity of staining. Huxley and Zubay (1961) slightly alkalized their
solutions with dilute KOH to pH 4.8.
Investigators occasionally have thought of uranyl acetate as having
value as a secondary fixative. Thus people have followed osmium tetroxide
fixation with secondary treatments of uranyl salts. Kellenburger, Ryter,
and Sechaud (1958) employed overnight post-osmic treatment with 0.5%
uranyl acetate in their preservation of bacteria (Chapter 3.4). Their
thought was that it particularly helps in the preservation of nucleoproteins—an opinion that Marinozzi and Gautier (1962) seem to confirm
in their studies of more complex cells.
Uranyl nitrate has been employed as a stain, and certainly it is easier
to handle than the acetate because of its great solubility and superior
stability. It can be dissolved in absolute alcohol and used as a block stain
before embedding. However, there seems to be general agreement that
it is not as effective a stain as the acetate, and it has fallen into disuse.
7.5. Phosphotungstic Acid Stain
Phosphotungstic acid originally was introduced as an effective electron
stain by Hall et al. (1945) in their studies of collagen, and WohlfarthBotterman (1956) employed it as a tissue stain. This is a most curious
compound in that 24 atoms of tungsten are combined in each molecule.
Thus, every molecule that can be attached to a binding site enhances
electron density tremendously. Ordinary compounds, containing 1 or 2
heavy atoms that might be similarly attached, cannot possibly be as
effective.
Phosphotungstic acid remained the most effective and
important
electron stain until Watson (1958) developed his alkaline lead hydroxide
stain, and at the same time emphasized the virtues of uranyl acetate as a
staining agent. These stains have largely superseded phosphotungstic acid
for general purposes when used with cross-linked plastic embedments,
but phosphotungstic acid deserves continuing interest if only because it
is a somewhat selective stain, demonstrating particularly extracellular
connective tissue components including collagen, elastin, and the mucopolysaccharides of basement membranes and other structures (Fig. 29).
The studies of collagen, in which numerous specific transverse bands are
intensified by this stain, demonstrate that phosphotungstic acid has quite
7.
"STAINING"
the intensity of staining. Huxley and Zubay (1961) slightly alkalized their
solutions with dilute KOH to pH 4.8.
Investigators occasionally have thought of uranyl acetate as having
value as a secondary fixative. Thus people have followed osmium tetroxide
fixation with secondary treatments of uranyl salts. Kellenburger, Ryter,
and Sechaud (1958) employed overnight post-osmic treatment with 0.5%
uranyl acetate in their preservation of bacteria (Chapter 3.4). Their
thought was that it particularly helps in the preservation of nucleoproteins—an opinion that Marinozzi and Gautier (1962) seem to confirm
in their studies of more complex cells.
Uranyl nitrate has been employed as a stain, and certainly it is easier
to handle than the acetate because of its great solubility and superior
stability. It can be dissolved in absolute alcohol and used as a block stain
before embedding. However, there seems to be general agreement that
it is not as effective a stain as the acetate, and it has fallen into disuse.
7.5. Phosphotungstic Acid Stain
Phosphotungstic acid originally was introduced as an effective electron
stain by Hall et al. (1945) in their studies of collagen, and WohlfarthBotterman (1956) employed it as a tissue stain. This is a most curious
compound in that 24 atoms of tungsten are combined in each molecule.
Thus, every molecule that can be attached to a binding site enhances
electron density tremendously. Ordinary compounds, containing 1 or 2
heavy atoms that might be similarly attached, cannot possibly be as
effective.
Phosphotungstic acid remained the most effective and
important
electron stain until Watson (1958) developed his alkaline lead hydroxide
stain, and at the same time emphasized the virtues of uranyl acetate as a
staining agent. These stains have largely superseded phosphotungstic acid
for general purposes when used with cross-linked plastic embedments,
but phosphotungstic acid deserves continuing interest if only because it
is a somewhat selective stain, demonstrating particularly extracellular
connective tissue components including collagen, elastin, and the mucopolysaccharides of basement membranes and other structures (Fig. 29).
The studies of collagen, in which numerous specific transverse bands are
intensified by this stain, demonstrate that phosphotungstic acid has quite