11.4.
SPECIMEN
PREPARATION
351
present in the specimen with the stain material. It is also almost mandatory if one is using highly hydrophobic carbon support films. Dilute
serum albumin makes a good wetting agent for these purposes. Only
traces of albumin, perhaps as little as 0.01%, are enough. The grids with
their supporting films can be deliberately coated with protein in advance
of mounting the specimen, or, often, protein can be added to the specimen
suspension along with the stain and deposited with it. Of course, when
cell particulates are being studied, it ordinarily can be assumed that
some soluble protein will be present, and it may not be necessary to add
more. When the supporting film is adequately wet, the negative stain
spreads out as in Fig. 42(c) and (d). In this instance, the sodium phosphotungstate also penetrated the lumens of the fibrils so that their cores
appear dark. Another preparation which lacked good wetting properties
is illustrated in Fig. 42(a) and (b). In this instance, the sodium phosphotungstate did not spread out well over the supporting film, and neither
did it fill the lumens of the fibrils. In this comparison it can be seen,
however, that the revealed detail differed markedly in the two specimens,
so that the one type of preparation complemented the other. It was quite
advantageous to be able to study both images. Thus, one cannot categorically say what should be done in every situation.
Curiously, albumin and some other proteins that might be added as
wetting agents are not visualized by the negative stains currently being
commonly employed. The "invisible" proteins presumably have such
an open structure that they are penetrated by the stain. Smaller organic
molecules, for instance sucrose, ordinarily also are invisible. Even modest
amounts of salt may not hamper the final viewing of the specimen.
Thus, one often can use quite complex suspending media without their
components interfering with the final imaging of the specimens. This
allows a flexibility which is not permissible in shadowed specimens. However, what surely is an advantage under most circumstances might lead
to important interpretive errors in other situations. A negatively stained
image may not show all components of the specimen, even those substances which might be layered on the surface. Component structures
FIG. 42. Frayed fibrils from within sperm flagella, "negatively stained" with sodium
phosphotungstate. Great detail can be revealed as in (b), an enlarged portion of (a).
The character of the "stain" deposit depends much upon physical forces at work as
the preparation is made, particularly upon surface tension interactions. In (c) and (d)
the stain wet the supporting film and the filaments, even penetrating the latter to
demonstrate a hollow core. In (a) and (b) the supporting film and apparently also the
specimen filaments were effectively hydrophobic, and different features of the specimen were revealed. Dilute protein serves to increase wettability.
SPECIMEN
PREPARATION
351
present in the specimen with the stain material. It is also almost mandatory if one is using highly hydrophobic carbon support films. Dilute
serum albumin makes a good wetting agent for these purposes. Only
traces of albumin, perhaps as little as 0.01%, are enough. The grids with
their supporting films can be deliberately coated with protein in advance
of mounting the specimen, or, often, protein can be added to the specimen
suspension along with the stain and deposited with it. Of course, when
cell particulates are being studied, it ordinarily can be assumed that
some soluble protein will be present, and it may not be necessary to add
more. When the supporting film is adequately wet, the negative stain
spreads out as in Fig. 42(c) and (d). In this instance, the sodium phosphotungstate also penetrated the lumens of the fibrils so that their cores
appear dark. Another preparation which lacked good wetting properties
is illustrated in Fig. 42(a) and (b). In this instance, the sodium phosphotungstate did not spread out well over the supporting film, and neither
did it fill the lumens of the fibrils. In this comparison it can be seen,
however, that the revealed detail differed markedly in the two specimens,
so that the one type of preparation complemented the other. It was quite
advantageous to be able to study both images. Thus, one cannot categorically say what should be done in every situation.
Curiously, albumin and some other proteins that might be added as
wetting agents are not visualized by the negative stains currently being
commonly employed. The "invisible" proteins presumably have such
an open structure that they are penetrated by the stain. Smaller organic
molecules, for instance sucrose, ordinarily also are invisible. Even modest
amounts of salt may not hamper the final viewing of the specimen.
Thus, one often can use quite complex suspending media without their
components interfering with the final imaging of the specimens. This
allows a flexibility which is not permissible in shadowed specimens. However, what surely is an advantage under most circumstances might lead
to important interpretive errors in other situations. A negatively stained
image may not show all components of the specimen, even those substances which might be layered on the surface. Component structures
FIG. 42. Frayed fibrils from within sperm flagella, "negatively stained" with sodium
phosphotungstate. Great detail can be revealed as in (b), an enlarged portion of (a).
The character of the "stain" deposit depends much upon physical forces at work as
the preparation is made, particularly upon surface tension interactions. In (c) and (d)
the stain wet the supporting film and the filaments, even penetrating the latter to
demonstrate a hollow core. In (a) and (b) the supporting film and apparently also the
specimen filaments were effectively hydrophobic, and different features of the specimen were revealed. Dilute protein serves to increase wettability.