352
11. NEGATIVE STAINING
which might be hydrated and penetrated by the stain would remain unobserved.
The nature of negative staining is such that one has to think particularly of concomitant drying artifacts. Insofar as usually a supporting
film is on one side of the specimen, these can be expected to be asymmetric. Huxley has avoided this in some of his elegant studies of fibrous
protein macromolecules by letting the suspension bridge holes in the
supporting film. The holes are made as described earlier (Chapter 6.4).
Apart from symmetrical drying forces, this method has the additional
advantage that the elimination of the supporting film allows a significant
gain in contrast and resolution. It seems evident that the finest micrographs of very small objects will be obtained in this way.
The practical success of negative staining depends upon adjusting the
thickness of the embedding film so that it almost exactly corresponds
with the thickness of the specimen. This can be controlled accurately only
if the specimen can be included in a standardized aerosol preparation
which also includes the stain. Even then, the remains of the dried droplets will have thicker centers than edges and, indeed, one cannot expect
the droplets to be of strictly uniform size or thickness. Thus, it will
only be in limited areas that conditions will be optimal for specimen visualization. If stain is added as a single drop on the surface of the grid,
control becomes a matter of experience and judgment. In general, one
should add more than the desired amount of stain from a fine pipette
or a hyperdermic syringe. Then the excess is removed with filter paper
until the size of the drop seems about correct. Finally, the residual fluid
is allowed to evaporate. It is not likely that the film of stain left behind
will be of strictly uniform thickness and one must expect to have to
search for desirable areas. It can be expected, too, that the film of stain
will tend to be too thick on some grids and too thin on others, so that
there is an inherent inefficiency in this system which only familiarity with
the particular problems can minimize.
It will be realized that it is often possible to prepare well standardized
aerosol samples of suspended particulates. Employing a negative stain
with these may prove to be the simplest and most pertinent means of
production control which can give quick information about the degree
of purity and dispersion. Providing one has made advance preparations,
there is no reason why the specimen cannot be in the microscope within
10 minutes of its collection, if all that is required is its dilution with
stain, and spraying it upon a prepared grid.
11. NEGATIVE STAINING
which might be hydrated and penetrated by the stain would remain unobserved.
The nature of negative staining is such that one has to think particularly of concomitant drying artifacts. Insofar as usually a supporting
film is on one side of the specimen, these can be expected to be asymmetric. Huxley has avoided this in some of his elegant studies of fibrous
protein macromolecules by letting the suspension bridge holes in the
supporting film. The holes are made as described earlier (Chapter 6.4).
Apart from symmetrical drying forces, this method has the additional
advantage that the elimination of the supporting film allows a significant
gain in contrast and resolution. It seems evident that the finest micrographs of very small objects will be obtained in this way.
The practical success of negative staining depends upon adjusting the
thickness of the embedding film so that it almost exactly corresponds
with the thickness of the specimen. This can be controlled accurately only
if the specimen can be included in a standardized aerosol preparation
which also includes the stain. Even then, the remains of the dried droplets will have thicker centers than edges and, indeed, one cannot expect
the droplets to be of strictly uniform size or thickness. Thus, it will
only be in limited areas that conditions will be optimal for specimen visualization. If stain is added as a single drop on the surface of the grid,
control becomes a matter of experience and judgment. In general, one
should add more than the desired amount of stain from a fine pipette
or a hyperdermic syringe. Then the excess is removed with filter paper
until the size of the drop seems about correct. Finally, the residual fluid
is allowed to evaporate. It is not likely that the film of stain left behind
will be of strictly uniform thickness and one must expect to have to
search for desirable areas. It can be expected, too, that the film of stain
will tend to be too thick on some grids and too thin on others, so that
there is an inherent inefficiency in this system which only familiarity with
the particular problems can minimize.
It will be realized that it is often possible to prepare well standardized
aerosol samples of suspended particulates. Employing a negative stain
with these may prove to be the simplest and most pertinent means of
production control which can give quick information about the degree
of purity and dispersion. Providing one has made advance preparations,
there is no reason why the specimen cannot be in the microscope within
10 minutes of its collection, if all that is required is its dilution with
stain, and spraying it upon a prepared grid.