2 . M E T H O D S
77
the medium had to be done by sucking it out of the sponge with a fine
pipette, which was difficult and generally unsatisfactory. Furthermore
sponges present an enormous surface area on to which constituents of
the medium were frequently adsorbed.
2. "Grid" Methods
Trowell (1954) introduced the method of using a metal grid to support the cultures at the surface of a fluid medium (Fig. 8e). The grid
is square, with the edges bent over to form legs 4 mm high, the top
surface being 25 x 25 mm. The grid stands in a shallow dish (internal
diameter 43 mm) and sufficient medium is added to reach and just wet
the top of the grid. A piece of lens paper is then placed on the grid and
the cultures are planted on the wet paper. The grids must be made of
some non-corrodible and non-toxic metal. Originally they were made
from the tantalum wire gauze which is used in surgery (Trowell,
1954), but later it was found better to use "expanded metal" instead
of wire gauze because it is much more rigid. This is a continuous
sheet of metal 0-003 in thick with 1-5 mm holes. The "expanded"
metal used can be stainless steel (Trowell, 1959) or, better, titanium
(Trowell, 1961b). The grid described carries up to 25 cultures in 5-6
ml of medium. If only two or three cultures are needed, a flat disk of
expanded metal (17 mm in diameter) can be placed in a 4 cm watch
glass and filled up below with about 0-5 ml of medium, as described
by Merchant, Kahn and Murphy (1960) and shown in Fig. 8f.
In TrowelPs method, the culture dish is placed in a sealed culture
chamber which is then filled with a controlled gas-phase. For embryonic
organs and for the retina 5°/o C 0 2 in air is used, while for all other
mature organs 5% C 0 2 in oxygen is used. The simplest type of culture
chamber is made of Perspex (Lucite) (Trowell 1954, 1961b, and Fig.
8e). This is suitable for short-term experiments; it has a gas volume of
30 ml and the gas has to be replenished twice every 24 h owing to
accumulation of C 0 2 . The Trowell Type II chamber (Trowell, 1959)
is made of aluminium and has a built-in gas reservoir (170 ml) so that
the gas-phase needs to be replenished only at 3-day intervals. This is
convenient for long-term experiments, particularly over week-ends.
Prop (1961) has described a modified Trowell Type II chamber which
can be made from a screw-capped jar. For culture of embryonic organs
or small fragments of certain mature organs which do not have a high
oxygen requirement (e.g. prostate) it is sufficient to keep the culture
dish in air in a Petri dish (Franks, 1959).
There is always some "outgrowth" due to cell migration along the
cellulose fibres of the lens paper, but relative to the size of the culture
it is very small. If, after the cultures have been removed at the end of
77
the medium had to be done by sucking it out of the sponge with a fine
pipette, which was difficult and generally unsatisfactory. Furthermore
sponges present an enormous surface area on to which constituents of
the medium were frequently adsorbed.
2. "Grid" Methods
Trowell (1954) introduced the method of using a metal grid to support the cultures at the surface of a fluid medium (Fig. 8e). The grid
is square, with the edges bent over to form legs 4 mm high, the top
surface being 25 x 25 mm. The grid stands in a shallow dish (internal
diameter 43 mm) and sufficient medium is added to reach and just wet
the top of the grid. A piece of lens paper is then placed on the grid and
the cultures are planted on the wet paper. The grids must be made of
some non-corrodible and non-toxic metal. Originally they were made
from the tantalum wire gauze which is used in surgery (Trowell,
1954), but later it was found better to use "expanded metal" instead
of wire gauze because it is much more rigid. This is a continuous
sheet of metal 0-003 in thick with 1-5 mm holes. The "expanded"
metal used can be stainless steel (Trowell, 1959) or, better, titanium
(Trowell, 1961b). The grid described carries up to 25 cultures in 5-6
ml of medium. If only two or three cultures are needed, a flat disk of
expanded metal (17 mm in diameter) can be placed in a 4 cm watch
glass and filled up below with about 0-5 ml of medium, as described
by Merchant, Kahn and Murphy (1960) and shown in Fig. 8f.
In TrowelPs method, the culture dish is placed in a sealed culture
chamber which is then filled with a controlled gas-phase. For embryonic
organs and for the retina 5°/o C 0 2 in air is used, while for all other
mature organs 5% C 0 2 in oxygen is used. The simplest type of culture
chamber is made of Perspex (Lucite) (Trowell 1954, 1961b, and Fig.
8e). This is suitable for short-term experiments; it has a gas volume of
30 ml and the gas has to be replenished twice every 24 h owing to
accumulation of C 0 2 . The Trowell Type II chamber (Trowell, 1959)
is made of aluminium and has a built-in gas reservoir (170 ml) so that
the gas-phase needs to be replenished only at 3-day intervals. This is
convenient for long-term experiments, particularly over week-ends.
Prop (1961) has described a modified Trowell Type II chamber which
can be made from a screw-capped jar. For culture of embryonic organs
or small fragments of certain mature organs which do not have a high
oxygen requirement (e.g. prostate) it is sufficient to keep the culture
dish in air in a Petri dish (Franks, 1959).
There is always some "outgrowth" due to cell migration along the
cellulose fibres of the lens paper, but relative to the size of the culture
it is very small. If, after the cultures have been removed at the end of