2 . M E T H O D S
75
Measurement shows that the amount of distillation is small, and indeed
it may be a good thing as it compensates for the slight drying of the
medium which occurs during the setting-up process.
Another popular type of culture vessel consists of a standard "embryological watch-glass", 3*5 cm square, with a glass lid sealed on with
paraffin wax (Fig. 8d). This was introduced by Rudnick (1938) and
later adopted by Gaillard. In the method developed by Gaillard
(1951) the clot is made by mixing 2 parts human plasma, 1 part human
placental serum, 1 part human baby brain-extract and 6 parts saline.
The clot is therefore much weaker than that used by Fell. About four
organ fragments, 0-25-0-5 mm in diameter, are cultured on 0-75 ml
of medium and transplanted to fresh medium every 3 days. Since the
chamber is sealed, the p H does not become as alkaline as in Fell's
method, and C 0 2 accumulates during the culture period, but not to
any harmful extent. Gaillard (1957) also devised a very shallow chamber
of similar type, his ' 'piano-parallel" chamber, in which cultures could
be examined and photographed under the low power of the microscope.
As it stands the plasma clot method has certain limitations, which are
that chemically defined media cannot be used, the medium cannot
be changed without transplanting the cultures, samples of medium
cannot be removed for analysis, and chemicals cannot be added to the
medium during the course of the experiment. A more serious disadvantage, which is particularly evident when mature organs are used,
is that the cultures digest the clot and slowly sink into a pool of liquefied
plasma which eventually impairs their oxygen supply. Attempts were
made to overcome this difficulty by laying a sheet of tunica albuginea
on the plasma clot (Martinovitch, 1951) or by laying 4-5 thin glass
rods (100/x in diameter) on the clot and placing the culture on the rods
(Martinovitch, 1953), but neither of these devices has been generally
adopted.
D. O R G A N S O N A G A R
The idea of using an agar medium was taken from the bacteriologists
and it at once avoided the trouble of clot-liquefaction and allowed the
use of chemically defined media. The method was introduced by Spratt
(1947) who used a medium composed of Ringer-albumen-agar (final
agar concentration 0-3°/o) in the apparatus of Fell (Fig. 8c). Later,
a chemically defined medium consisting of bicarbonate-buffered saline
with glucose, eleven amino acids, ten vitamins and agar at 0-4°/o was
used (Spratt, 1948). Wolff and Haffen (1952a,b) used Gaillard's
apparatus (Fig. 8d) with a medium of Tyrode, chick-embryo extract
and agar at 0*5%. In later work, the embryo extract was replaced by
75
Measurement shows that the amount of distillation is small, and indeed
it may be a good thing as it compensates for the slight drying of the
medium which occurs during the setting-up process.
Another popular type of culture vessel consists of a standard "embryological watch-glass", 3*5 cm square, with a glass lid sealed on with
paraffin wax (Fig. 8d). This was introduced by Rudnick (1938) and
later adopted by Gaillard. In the method developed by Gaillard
(1951) the clot is made by mixing 2 parts human plasma, 1 part human
placental serum, 1 part human baby brain-extract and 6 parts saline.
The clot is therefore much weaker than that used by Fell. About four
organ fragments, 0-25-0-5 mm in diameter, are cultured on 0-75 ml
of medium and transplanted to fresh medium every 3 days. Since the
chamber is sealed, the p H does not become as alkaline as in Fell's
method, and C 0 2 accumulates during the culture period, but not to
any harmful extent. Gaillard (1957) also devised a very shallow chamber
of similar type, his ' 'piano-parallel" chamber, in which cultures could
be examined and photographed under the low power of the microscope.
As it stands the plasma clot method has certain limitations, which are
that chemically defined media cannot be used, the medium cannot
be changed without transplanting the cultures, samples of medium
cannot be removed for analysis, and chemicals cannot be added to the
medium during the course of the experiment. A more serious disadvantage, which is particularly evident when mature organs are used,
is that the cultures digest the clot and slowly sink into a pool of liquefied
plasma which eventually impairs their oxygen supply. Attempts were
made to overcome this difficulty by laying a sheet of tunica albuginea
on the plasma clot (Martinovitch, 1951) or by laying 4-5 thin glass
rods (100/x in diameter) on the clot and placing the culture on the rods
(Martinovitch, 1953), but neither of these devices has been generally
adopted.
D. O R G A N S O N A G A R
The idea of using an agar medium was taken from the bacteriologists
and it at once avoided the trouble of clot-liquefaction and allowed the
use of chemically defined media. The method was introduced by Spratt
(1947) who used a medium composed of Ringer-albumen-agar (final
agar concentration 0-3°/o) in the apparatus of Fell (Fig. 8c). Later,
a chemically defined medium consisting of bicarbonate-buffered saline
with glucose, eleven amino acids, ten vitamins and agar at 0-4°/o was
used (Spratt, 1948). Wolff and Haffen (1952a,b) used Gaillard's
apparatus (Fig. 8d) with a medium of Tyrode, chick-embryo extract
and agar at 0*5%. In later work, the embryo extract was replaced by