10. Particulate Specimens, Mounting,
Shadowing, and Replication
10.1. Introductory Remarks
There are, of course, some circumstances where it is desirable to
examine as directly as possible dispersed or fragmented biological
material. This is the common problem of the bacteriologist and the
virologist who often have no need for ultrathin sections. Analytical work
with cells and tissues sometimes yields particulate suspensions which
may consist of macromolecules or whole organelles for direct examination. There is also an increasing awareness that even cytomembrane
systems may withstand cellular disintegration, and so be studied in
isolation. Of course, sometimes it is easy to isolate fibrous components
of tissue. Furthermore, there are hard tissue elements that are difficult
to section at best, but their natural, cleaved, or etched surfaces can be
studied without serious problems. The older techniques which are useful for these operations include refined methods of fragmentation and
suspension, shadowing with heavy metals to enhance surface contrast,
and the very delicate replication of surfaces.
These methods had already been considerably developed before ultrathin sectioning proved successful. Replication techniques in general
were not developed by biologists, but rather by metallurgists who were
concerned with visualizing crystal structure in hard materials. The first
successful replication usually is credited to Mahl in 1940. Substantial
improvement, which gave much impetus to replication, followed the introduction of the Formvar technique by Schaefer and Harker (1942).
With the introduction of shadowing methods by Williams and Wyckoff
(1944), it was immediately obvious that contrast in replicas could be
greatly enhanced by combining replication with shadowing. This has
become almost universal practice. Shadowing, of course, also has been a
boon to those who would study dispersed or fragmented material. This
particularly includes the preparations of bacteriologists and virologists.
More recently, people also have come to realize that "negative staining"
326
Shadowing, and Replication
10.1. Introductory Remarks
There are, of course, some circumstances where it is desirable to
examine as directly as possible dispersed or fragmented biological
material. This is the common problem of the bacteriologist and the
virologist who often have no need for ultrathin sections. Analytical work
with cells and tissues sometimes yields particulate suspensions which
may consist of macromolecules or whole organelles for direct examination. There is also an increasing awareness that even cytomembrane
systems may withstand cellular disintegration, and so be studied in
isolation. Of course, sometimes it is easy to isolate fibrous components
of tissue. Furthermore, there are hard tissue elements that are difficult
to section at best, but their natural, cleaved, or etched surfaces can be
studied without serious problems. The older techniques which are useful for these operations include refined methods of fragmentation and
suspension, shadowing with heavy metals to enhance surface contrast,
and the very delicate replication of surfaces.
These methods had already been considerably developed before ultrathin sectioning proved successful. Replication techniques in general
were not developed by biologists, but rather by metallurgists who were
concerned with visualizing crystal structure in hard materials. The first
successful replication usually is credited to Mahl in 1940. Substantial
improvement, which gave much impetus to replication, followed the introduction of the Formvar technique by Schaefer and Harker (1942).
With the introduction of shadowing methods by Williams and Wyckoff
(1944), it was immediately obvious that contrast in replicas could be
greatly enhanced by combining replication with shadowing. This has
become almost universal practice. Shadowing, of course, also has been a
boon to those who would study dispersed or fragmented material. This
particularly includes the preparations of bacteriologists and virologists.
More recently, people also have come to realize that "negative staining"
326