328
10.
PARTICULATE SPECIMENS
benzoate is said to act similarly. If one is to have a clear view of a
shadowed specimen, it is also necessary to eliminate other contaminants,
particularly those which might tend to form amorphous films which
would envelope and bury the particulate matter in which one is interested. This is usually less of a problem when material is to be negatively
stained.
Some fibrous tissue components can withstand considerable mechanical
agitation and, therefore, can be cleaned as well as fragmented by the
judicious use of blending equipment. This technique was developed by
Schmitt and Hall and their co-workers for studying collagen (1942), and
later striated muscle and other fibrous proteins. They started with commercially available electric blenders, but also used microblending equipment of their own design. Frictional heat develops quite rapidly within
the suspension being blended so that running times must be kept short.
There is no standard way of preparing a suspension. Usually, however,
cellular material is liberated either by mechanical agitation (including
supersonic vibrations) or by osmotic shock. The separation of components
then commonly involves differential centrifugation which may be expected to purify and "clean" a preparation which finally will be concentrated.
The simplest way of mounting suspended material is to place a drop
on a grid previously prepared with a supporting film and allow this to
evaporate. However, if the supporting film is hydrophobic, as it is apt
to be, there may be some difficulties. Parlodion and Formvar films are
mildly hydrophobic, and carbon films decidedly so. Carbon stabilized
plastic films will prove most generally convenient and adequate for this
type of work, but it sometimes is desirable to have the plastic on top of
the carbon so that this is the exposed surface. To accomplish this, carbon
must be evaporated on top of the plastic before stripping it from the glass
slide. However, even traces of surface active material or wetting agents
may be enough to allow drops of suspension to spread out over even
hydrophobic supporting films so that difficulties will not be serious. In
this connection, a thin layer of serum albumin deposited in advance
from 0.1% solution can be used. Or traces of albumin or other protein
(even saliva) can be included in the suspending medium which will
reduce its surface tension tremendously.
If a drop of a suspension is simply allowed to evaporate on a supporting film, the suspension is apt to become concentrated as evaporation
proceeds, and there may be much coalescence of material in the final
10.
PARTICULATE SPECIMENS
benzoate is said to act similarly. If one is to have a clear view of a
shadowed specimen, it is also necessary to eliminate other contaminants,
particularly those which might tend to form amorphous films which
would envelope and bury the particulate matter in which one is interested. This is usually less of a problem when material is to be negatively
stained.
Some fibrous tissue components can withstand considerable mechanical
agitation and, therefore, can be cleaned as well as fragmented by the
judicious use of blending equipment. This technique was developed by
Schmitt and Hall and their co-workers for studying collagen (1942), and
later striated muscle and other fibrous proteins. They started with commercially available electric blenders, but also used microblending equipment of their own design. Frictional heat develops quite rapidly within
the suspension being blended so that running times must be kept short.
There is no standard way of preparing a suspension. Usually, however,
cellular material is liberated either by mechanical agitation (including
supersonic vibrations) or by osmotic shock. The separation of components
then commonly involves differential centrifugation which may be expected to purify and "clean" a preparation which finally will be concentrated.
The simplest way of mounting suspended material is to place a drop
on a grid previously prepared with a supporting film and allow this to
evaporate. However, if the supporting film is hydrophobic, as it is apt
to be, there may be some difficulties. Parlodion and Formvar films are
mildly hydrophobic, and carbon films decidedly so. Carbon stabilized
plastic films will prove most generally convenient and adequate for this
type of work, but it sometimes is desirable to have the plastic on top of
the carbon so that this is the exposed surface. To accomplish this, carbon
must be evaporated on top of the plastic before stripping it from the glass
slide. However, even traces of surface active material or wetting agents
may be enough to allow drops of suspension to spread out over even
hydrophobic supporting films so that difficulties will not be serious. In
this connection, a thin layer of serum albumin deposited in advance
from 0.1% solution can be used. Or traces of albumin or other protein
(even saliva) can be included in the suspending medium which will
reduce its surface tension tremendously.
If a drop of a suspension is simply allowed to evaporate on a supporting film, the suspension is apt to become concentrated as evaporation
proceeds, and there may be much coalescence of material in the final