254
7.
"STAINING"
uniform emulsion film on top of a Parlodion film which might have
usefulness in particular situations. A somewhat crude variant of this
method is to create the film simply by blowing a bubble.
Caro and van Tubergen (1962) determine the necessary exposure time
for ultrathin sections by comparison with what is necessary for a satisfactory autoradiograph of a relatively thick section observed by conventional
microscopy. For the latter, they routinely cut 0.4 [i sections of all material
being used for electron microscopy. The thick sections are picked up on
glass microscope slides and dipped into a 1:1 dilution of the emulsion
(the emulsion must be melted first at 45°C for 15 minutes, but is returned to room temperature before use). Excess emulsion is drained, and
the slides dried in a vertical position. The rule of thumb relationship
between the exposure time for such a conventional preparation, and
ultrathin preparations for electron microscopy, is that the latter require about ten times the exposure of the former. Thus, if there is a
suitable photographic response in a light microscope preparation in one
week, a useful preparation for electron microscopy will require 2-4
months exposure.
Storage during exposure time of course must be in total darkness. The
black plastic slide-boxes which are available nowadays serve very well
for this purpose, and their lids are sealed on with black plastic electrical
tape. The temperature of storage during exposure is not of great importance. However, Caro and van Tubergen (1962) present experimental
evidence that higher grain counts can be expected from storage at room
temperature than if low temperatures are used. Thus, the higher temperatures improve sensitivity, apparently without significantly altering
the background count.
Caro and van Tubergen (1962) have made a somewhat extended study
of developers. If Kodak D-19 is used, each grain consists of a highly coiled,
long filament. The whole mass is apt to have a diameter of 0.3-0.4 \i.
Kodak Microdol-X produces a single strand with minimal coiling and
can be regarded as a superior grain for autoradiographic work. Unfortunately there seems to be no way of knowing which ends of such
filaments were the starting points of their growth. Thus one-half their
length represents the resolution error that must be accepted. This
developer, however, seems to be the most practical one for most purposes. These investigators did explore p-phenylenediamine developers
which can be made to produce smaller grains with asymmetrical ends so
7.
"STAINING"
uniform emulsion film on top of a Parlodion film which might have
usefulness in particular situations. A somewhat crude variant of this
method is to create the film simply by blowing a bubble.
Caro and van Tubergen (1962) determine the necessary exposure time
for ultrathin sections by comparison with what is necessary for a satisfactory autoradiograph of a relatively thick section observed by conventional
microscopy. For the latter, they routinely cut 0.4 [i sections of all material
being used for electron microscopy. The thick sections are picked up on
glass microscope slides and dipped into a 1:1 dilution of the emulsion
(the emulsion must be melted first at 45°C for 15 minutes, but is returned to room temperature before use). Excess emulsion is drained, and
the slides dried in a vertical position. The rule of thumb relationship
between the exposure time for such a conventional preparation, and
ultrathin preparations for electron microscopy, is that the latter require about ten times the exposure of the former. Thus, if there is a
suitable photographic response in a light microscope preparation in one
week, a useful preparation for electron microscopy will require 2-4
months exposure.
Storage during exposure time of course must be in total darkness. The
black plastic slide-boxes which are available nowadays serve very well
for this purpose, and their lids are sealed on with black plastic electrical
tape. The temperature of storage during exposure is not of great importance. However, Caro and van Tubergen (1962) present experimental
evidence that higher grain counts can be expected from storage at room
temperature than if low temperatures are used. Thus, the higher temperatures improve sensitivity, apparently without significantly altering
the background count.
Caro and van Tubergen (1962) have made a somewhat extended study
of developers. If Kodak D-19 is used, each grain consists of a highly coiled,
long filament. The whole mass is apt to have a diameter of 0.3-0.4 \i.
Kodak Microdol-X produces a single strand with minimal coiling and
can be regarded as a superior grain for autoradiographic work. Unfortunately there seems to be no way of knowing which ends of such
filaments were the starting points of their growth. Thus one-half their
length represents the resolution error that must be accepted. This
developer, however, seems to be the most practical one for most purposes. These investigators did explore p-phenylenediamine developers
which can be made to produce smaller grains with asymmetrical ends so