348
11. NEGATIVE STAINING
conservative comparison, principally because the weight density of most
biological material probably cannot even be doubled by coupling with
any presently known positive stain.
11.3. Negative Stains
Sodium phosphotungstate has proved to be by far the most useful
negative stain. It generally is used in the form of a 1% aqueous solution,
although sometimes 0.5 or 2% is employed. This is prepared by starting
with a solution of phosphotungstic acid and adding drops of concentrated sodium hydroxide until the desired pH is achieved. In many circumstances, particularly when dealing with isolated proteins and virus
particles, a pH 5-6 seems to give optimal results. With such systems it
often has seemed undesirable to pass neutrality into the alkaline range.
Solutions are easily prepared freshly before use.
Sodium tungstate is a highly soluble compound that can be used just
about as the phosphotungstate. Its anhydrous density is slightly higher
(4.2 as compared with 3.8).
Uranium salts have been the other materials fairly commonly employed. Although Valentine and Home (1962) emphasize the use of
uranium nitrate because of its great solubility, a number of investigators
have used the acetate successfully. Huxley (1962) continues to use uranium acetate for studying proteins at low pH. Its unadjusted pH is 4.3,
but it can be acidified further, and thus used with materials that require
the maintenance of a very low pH. The anhydrous density of uranium
nitrate is 3.7.
Valentine and Home (1962) are much interested in cadmium iodide as
a negative stain. It has the virtue of good solubility coupled with a substantially higher density (5.7) than the tungstate compounds. However,
the gain in contrast will not prove very significant under most circumstances.
Valentine and Home (1962) also suggest that silver nitrate has a considerable potentiality as a negative stain. It of course has a high solubility.
Also its anhydrous density (4.4) is slightly greater than that of sodium
tungstate, and substantially better than uranium nitrate.
Valentine also has reported that he has begun experiments with thallium fluoride, which has the extraordinarily high anhydrous density of
11. NEGATIVE STAINING
conservative comparison, principally because the weight density of most
biological material probably cannot even be doubled by coupling with
any presently known positive stain.
11.3. Negative Stains
Sodium phosphotungstate has proved to be by far the most useful
negative stain. It generally is used in the form of a 1% aqueous solution,
although sometimes 0.5 or 2% is employed. This is prepared by starting
with a solution of phosphotungstic acid and adding drops of concentrated sodium hydroxide until the desired pH is achieved. In many circumstances, particularly when dealing with isolated proteins and virus
particles, a pH 5-6 seems to give optimal results. With such systems it
often has seemed undesirable to pass neutrality into the alkaline range.
Solutions are easily prepared freshly before use.
Sodium tungstate is a highly soluble compound that can be used just
about as the phosphotungstate. Its anhydrous density is slightly higher
(4.2 as compared with 3.8).
Uranium salts have been the other materials fairly commonly employed. Although Valentine and Home (1962) emphasize the use of
uranium nitrate because of its great solubility, a number of investigators
have used the acetate successfully. Huxley (1962) continues to use uranium acetate for studying proteins at low pH. Its unadjusted pH is 4.3,
but it can be acidified further, and thus used with materials that require
the maintenance of a very low pH. The anhydrous density of uranium
nitrate is 3.7.
Valentine and Home (1962) are much interested in cadmium iodide as
a negative stain. It has the virtue of good solubility coupled with a substantially higher density (5.7) than the tungstate compounds. However,
the gain in contrast will not prove very significant under most circumstances.
Valentine and Home (1962) also suggest that silver nitrate has a considerable potentiality as a negative stain. It of course has a high solubility.
Also its anhydrous density (4.4) is slightly greater than that of sodium
tungstate, and substantially better than uranium nitrate.
Valentine also has reported that he has begun experiments with thallium fluoride, which has the extraordinarily high anhydrous density of