2 . M E T H O D S
43
from the cells which remain in the central explant. Activity per cell is
perhaps the most useful manner of expressing the data, and in a steadystate population this is probably not very different from activity per
unit DNA, so long as the culture is reasonably uniform, not multiplying
too rapidly, and when there are no dead cells. Cultures using solid
pieces of tissue, however, are seldom uniform.
For the maintenance of an adequate oxygen supply, for the regulation
of pH, especially in a bicarbonate-buffered medium, or for other purposes, Carrel flasks and various modifications of them have a great
advantage over hanging-drop cultures in that they can readily be
filled with known gas mixtures (e.g. 5°/o C 0 2 + 9 5 % 0 2 , or 5% C 0 2
in air) instead of normal air, and the mixture can be renewed as and
when necessary. Sterile hypodermic needles passed through the rubber
cap which is usually used to seal the flask provide ready means of ingress
and egress.
Many tissues cannot readily be grown when fragments of them are
explanted in a fluid medium directly on to the bottom of Carrel flasks,
since they are either too easily displaced or are damaged by the
relatively large volume of fluid. However, if such tissues are covered
with a layer of well-washed and leached Cellophane then a copious
growth may occur between the Cellophane and the glass and particularly on the Cellophane (Evans and Earle, 1947; Shannon and
Earle, 1951). Opinions differ as to whether the Cellophane should or
should not be perforated so as to form a lattice, as it was in the earlier
experiments along these lines. The use of Cellophane in this way
eliminates the necessity for the capricious and indeterminate plasma
clot and must be regarded as having been a major advance, even though
it has now, in its turn, been largely replaced by the newer methods for
obtaining mass cultures of cells (see p. 60).
If the bottom of a Carrel flask is made optically flat, or has a large hole
drilled in it which is then sealed with a coverslip, the outgrowing cells
can be satisfactorily observed and photographed, especially with the
help of that highly useful instrument for Tissue Culture purposes, the
inverted microscope.
D. R O L L E R TUBES
In order to offset any effects caused by the stagnation of the supernatant fluid and to assist in establishing greater uniformity in the activity
of the tissues than is possible in Carrel flasks, the roller tube was
developed (Gey, 1933). This technique is based on the same principles
as those of the Carrel-flask method, but the tissues are explanted into
a plasma coagulum placed along the side of a tube which may be shaped
43
from the cells which remain in the central explant. Activity per cell is
perhaps the most useful manner of expressing the data, and in a steadystate population this is probably not very different from activity per
unit DNA, so long as the culture is reasonably uniform, not multiplying
too rapidly, and when there are no dead cells. Cultures using solid
pieces of tissue, however, are seldom uniform.
For the maintenance of an adequate oxygen supply, for the regulation
of pH, especially in a bicarbonate-buffered medium, or for other purposes, Carrel flasks and various modifications of them have a great
advantage over hanging-drop cultures in that they can readily be
filled with known gas mixtures (e.g. 5°/o C 0 2 + 9 5 % 0 2 , or 5% C 0 2
in air) instead of normal air, and the mixture can be renewed as and
when necessary. Sterile hypodermic needles passed through the rubber
cap which is usually used to seal the flask provide ready means of ingress
and egress.
Many tissues cannot readily be grown when fragments of them are
explanted in a fluid medium directly on to the bottom of Carrel flasks,
since they are either too easily displaced or are damaged by the
relatively large volume of fluid. However, if such tissues are covered
with a layer of well-washed and leached Cellophane then a copious
growth may occur between the Cellophane and the glass and particularly on the Cellophane (Evans and Earle, 1947; Shannon and
Earle, 1951). Opinions differ as to whether the Cellophane should or
should not be perforated so as to form a lattice, as it was in the earlier
experiments along these lines. The use of Cellophane in this way
eliminates the necessity for the capricious and indeterminate plasma
clot and must be regarded as having been a major advance, even though
it has now, in its turn, been largely replaced by the newer methods for
obtaining mass cultures of cells (see p. 60).
If the bottom of a Carrel flask is made optically flat, or has a large hole
drilled in it which is then sealed with a coverslip, the outgrowing cells
can be satisfactorily observed and photographed, especially with the
help of that highly useful instrument for Tissue Culture purposes, the
inverted microscope.
D. R O L L E R TUBES
In order to offset any effects caused by the stagnation of the supernatant fluid and to assist in establishing greater uniformity in the activity
of the tissues than is possible in Carrel flasks, the roller tube was
developed (Gey, 1933). This technique is based on the same principles
as those of the Carrel-flask method, but the tissues are explanted into
a plasma coagulum placed along the side of a tube which may be shaped