74
A . M O S C O N A , O . A . T R O W E L L A N D E . N . W I L L M E R
C. O R G A N S ON A P L A S M A C L O T
Organs or organ pieces, other than skin and adipose tissue, have to
be supported at the surface of the medium, and the easiest way, of
course, is to use a solid medium. Plasma, coagulated by embryo
extract, was used for this purpose by Strangeways and Fell (1926).
They were the first to culture organs on the surface of the medium and
to use the method to study the in vitro morphogenesis of chick-embryo
rudiments. They used about 1-5 ml of medium in a small centrifuge
tube, but this was an ill-designed arrangement because of the small
surface available and the difficulty of access for observation and transplantation. Obviously it would be much better to have the medium in
a shallow dish which would give free access to a large surface, and the
method was soon modified in this way. Champy and Morita (1928)
used a small crystalizing dish in a moist culture chamber, and Fell
and Robison (1929) used a watch-glass in a moist Petri dish. The latter
apparatus proved so simple and successful that it soon became the
standard method for culture of embryonic organ rudiments, and it has
been used in the Strangeways Laboratory and elsewhere for many
classical studies of morphogenesis in vitro. This "watch-glass" method,
as it is generally called, therefore merits a detailed description. The
apparatus is shown schematically in Fig. 8c, and the following
particulars are taken from Fell (1951). The floor of an 8 cm Petri dish
is covered with a thick layer of cotton wool which has a 1 in diameter
hole in the middle. The wool is wetted with 10 ml of distilled water so
as to provide a moist chamber. A 4 cm diameter watch-glass is placed in
the hole in the cotton wool carpet. Cock plasma is placed in the watchglass, followed by an equal volume of embryo extract. After mixing, the
plasma clots and up to four cultures are placed upon it. The total volume
of medium originally used was 0-5 ml, but in later work with larger
cultures (limb bones) this was increased to 0-7-1*2 ml (Fell and
Mellanby, 1952). The cultures are transplanted to a watch-glass of
fresh medium every 2-3 days. The instruments used for dissecting out
the organ rudiments are a mounted sewing needle and a cataract
knife which has been ground down to a sharp and very flexible point.
The gas-phase is uncontrolled (air) and, as the Petri dish lid is not
sealed, C O a escapes from the plasma and the p H of the medium is
initially quite alkaline. This may be a good thing because a relatively
small volume of medium is used and lactic acid accumulates rapidly
owing to the high rate of glycolysis in embryonic tissues. Another
advantage of the unsealed lid is that C 0 2 does not accumulate in the
gas-phase. A, point of criticism is that water must distil from the wet
cotton-wool to the medium, because the vapour pressures are different.
A . M O S C O N A , O . A . T R O W E L L A N D E . N . W I L L M E R
C. O R G A N S ON A P L A S M A C L O T
Organs or organ pieces, other than skin and adipose tissue, have to
be supported at the surface of the medium, and the easiest way, of
course, is to use a solid medium. Plasma, coagulated by embryo
extract, was used for this purpose by Strangeways and Fell (1926).
They were the first to culture organs on the surface of the medium and
to use the method to study the in vitro morphogenesis of chick-embryo
rudiments. They used about 1-5 ml of medium in a small centrifuge
tube, but this was an ill-designed arrangement because of the small
surface available and the difficulty of access for observation and transplantation. Obviously it would be much better to have the medium in
a shallow dish which would give free access to a large surface, and the
method was soon modified in this way. Champy and Morita (1928)
used a small crystalizing dish in a moist culture chamber, and Fell
and Robison (1929) used a watch-glass in a moist Petri dish. The latter
apparatus proved so simple and successful that it soon became the
standard method for culture of embryonic organ rudiments, and it has
been used in the Strangeways Laboratory and elsewhere for many
classical studies of morphogenesis in vitro. This "watch-glass" method,
as it is generally called, therefore merits a detailed description. The
apparatus is shown schematically in Fig. 8c, and the following
particulars are taken from Fell (1951). The floor of an 8 cm Petri dish
is covered with a thick layer of cotton wool which has a 1 in diameter
hole in the middle. The wool is wetted with 10 ml of distilled water so
as to provide a moist chamber. A 4 cm diameter watch-glass is placed in
the hole in the cotton wool carpet. Cock plasma is placed in the watchglass, followed by an equal volume of embryo extract. After mixing, the
plasma clots and up to four cultures are placed upon it. The total volume
of medium originally used was 0-5 ml, but in later work with larger
cultures (limb bones) this was increased to 0-7-1*2 ml (Fell and
Mellanby, 1952). The cultures are transplanted to a watch-glass of
fresh medium every 2-3 days. The instruments used for dissecting out
the organ rudiments are a mounted sewing needle and a cataract
knife which has been ground down to a sharp and very flexible point.
The gas-phase is uncontrolled (air) and, as the Petri dish lid is not
sealed, C O a escapes from the plasma and the p H of the medium is
initially quite alkaline. This may be a good thing because a relatively
small volume of medium is used and lactic acid accumulates rapidly
owing to the high rate of glycolysis in embryonic tissues. Another
advantage of the unsealed lid is that C 0 2 does not accumulate in the
gas-phase. A, point of criticism is that water must distil from the wet
cotton-wool to the medium, because the vapour pressures are different.