420
T. G. HSU
B. THE CELL CYCLE
From the time a cell is born, i.e. when a mother cell divides into two
daughter elements, to the time the daughter cells divide again, the
cell completes its reproduction cycle, or cell cycle. Actually, cell cycle
does not necessarily mean the time span between two successive
mitoses; it denotes the time from any point in the cycle to the same
point in the next cycle. Using mitosis as an end point is merely a
contingency because there are morphological criteria to aid determinations.
In mammalian cells in culture, the time required for a cell cycle can
be directly computed by cinematographic analysis. However, this
method, though accurate, is tedious. Since it has been shown (Hsu,
1960) that great variation exists in the time required for completing
the cell cycle between two sister cells, cine data, after statistical analysis,
only represent mean values, or population parameters. An easier way
to estimate generation time is cell counting. When a population doubles
its size, the time interval is considered as the generation time. In dealing
with cell populations, it is naturally important to ascertain the phase
of growth, e.g. whether the population is in the logarithmic growth
phase or has already reached the plateau of the growth curve. Usually
in autoradiography. Fixed material, both sectioned and squashed, can
be used for the autoradiograph. Generally the material is not stained,
and either stripping film (Doniach and Pelc, 1950), or the more recent
liquid film (Messier and Leblond, 1957) is applied. Some investigators
prefer the liquid film NTB because it is easy to use and it does not give
heavy background grains caused by friction or other causes. The
device of Mazia and Bucher (1960) to immerse the stripping film in
alcohol before pealing greatly reduces background grains. In our
laboratories we have been using the standard stripping film A R 10
from Kodak. Staining is usually applied after exposure and development.
For chromosome work the popular method of staining is still the Feulgen
reaction. However, our procedure involves a rapid succession of fixation
with acetic acid, staining with acetorcein, squashing as soon as the
cells are suspended in the stain, chilling with dried ice, prying off the
coverslip, and washing the preparations with alcohol. The slides are
then air-dried and are ready for application of the film. When the
autoradiograph is eventually viewed with dark-phase-contrast microscope, the chromosomes appear dark while the grains are highly
refractile. No superimposition of two negatives is necessary in photomicrography (Fig. 5).
T. G. HSU
B. THE CELL CYCLE
From the time a cell is born, i.e. when a mother cell divides into two
daughter elements, to the time the daughter cells divide again, the
cell completes its reproduction cycle, or cell cycle. Actually, cell cycle
does not necessarily mean the time span between two successive
mitoses; it denotes the time from any point in the cycle to the same
point in the next cycle. Using mitosis as an end point is merely a
contingency because there are morphological criteria to aid determinations.
In mammalian cells in culture, the time required for a cell cycle can
be directly computed by cinematographic analysis. However, this
method, though accurate, is tedious. Since it has been shown (Hsu,
1960) that great variation exists in the time required for completing
the cell cycle between two sister cells, cine data, after statistical analysis,
only represent mean values, or population parameters. An easier way
to estimate generation time is cell counting. When a population doubles
its size, the time interval is considered as the generation time. In dealing
with cell populations, it is naturally important to ascertain the phase
of growth, e.g. whether the population is in the logarithmic growth
phase or has already reached the plateau of the growth curve. Usually
in autoradiography. Fixed material, both sectioned and squashed, can
be used for the autoradiograph. Generally the material is not stained,
and either stripping film (Doniach and Pelc, 1950), or the more recent
liquid film (Messier and Leblond, 1957) is applied. Some investigators
prefer the liquid film NTB because it is easy to use and it does not give
heavy background grains caused by friction or other causes. The
device of Mazia and Bucher (1960) to immerse the stripping film in
alcohol before pealing greatly reduces background grains. In our
laboratories we have been using the standard stripping film A R 10
from Kodak. Staining is usually applied after exposure and development.
For chromosome work the popular method of staining is still the Feulgen
reaction. However, our procedure involves a rapid succession of fixation
with acetic acid, staining with acetorcein, squashing as soon as the
cells are suspended in the stain, chilling with dried ice, prying off the
coverslip, and washing the preparations with alcohol. The slides are
then air-dried and are ready for application of the film. When the
autoradiograph is eventually viewed with dark-phase-contrast microscope, the chromosomes appear dark while the grains are highly
refractile. No superimposition of two negatives is necessary in photomicrography (Fig. 5).