2. M E T H O D S
79
impairment of their oxygen supply, and they may get dislodged when
the chamber is handled. The gas-phase and the medium are under
complete control and can be varied at any time without disturbing
the cultures. Some workers have sought to do away with the metal
grid by using a floating "raft" of lens paper or rayon fabric and these
methods must now be described.
3. "Raft" Methods
Chen (1954) discovered that a certain brand of lens paper (G. T.
Gurr, London) is more or less non-wettable and will float if carefully
handled. He used the Fell apparatus and put up to four small cultures
on a 25 x 25 mm raft of lens paper floating on serum in the watch-glass
(Fig. 8g). Richter (1958) found that any sort of lens paper will float if
it is first treated with silicone and he successfully cultured many mature
organs (chiefly of the bat) on siliconed lens paper floating on 4 ml of
medium in a 125 ml conical flask. Shaffer (1956) used a rayon fabric
(curtain net) treated with silicone to make it float.
An interesting and useful adaptation of the lens-paper raft method is
that described by Lash, Holtzer and Holtzer (1957), in which a hole
is punched through the middle of the raft and covered with a millepore
filter. Tissues can then be cultured on either or both sides of the filter.
The methods and modifications of it have been particularly useful in
elucidating the influence of the proximity of one tissue on the behaviour
and development of another (see Chapter 13 and Vol. 2, Chapter 4).
One limitation of the raft method is that not more than about
four small cultures can be put in any one vessel or the raft will sink.
The rafts are also rather tricky to handle. When the medium is to be
changed, the raft must be lifted off the old medium and put on to a
dish of new medium with considerable care. Chen noted that the top
surface of the paper must never be allowed to get wet all over, otherwise it would sink and become useless. Shaffer found with his siliconed
rayon that once this became coated with protein from the medium it
would no longer float, so the experiments were rather short-lived.
As pointed out in the preceding section, the "wetness" of the cultures
is a most important matter. A balance has to be struck between the
supply of oxygen and the supply of water-soluble nutrients, chiefly
glucose. The proper wetness can only be judged by experience and it is
therefore important that it should at any rate be under control. The
chief defect of the raft methods is that this factor is not controllable,
and in point of fact most cultures on rafts are much too wet. The grid
method offers complete control in this respect and it is also much easier
to handle.
79
impairment of their oxygen supply, and they may get dislodged when
the chamber is handled. The gas-phase and the medium are under
complete control and can be varied at any time without disturbing
the cultures. Some workers have sought to do away with the metal
grid by using a floating "raft" of lens paper or rayon fabric and these
methods must now be described.
3. "Raft" Methods
Chen (1954) discovered that a certain brand of lens paper (G. T.
Gurr, London) is more or less non-wettable and will float if carefully
handled. He used the Fell apparatus and put up to four small cultures
on a 25 x 25 mm raft of lens paper floating on serum in the watch-glass
(Fig. 8g). Richter (1958) found that any sort of lens paper will float if
it is first treated with silicone and he successfully cultured many mature
organs (chiefly of the bat) on siliconed lens paper floating on 4 ml of
medium in a 125 ml conical flask. Shaffer (1956) used a rayon fabric
(curtain net) treated with silicone to make it float.
An interesting and useful adaptation of the lens-paper raft method is
that described by Lash, Holtzer and Holtzer (1957), in which a hole
is punched through the middle of the raft and covered with a millepore
filter. Tissues can then be cultured on either or both sides of the filter.
The methods and modifications of it have been particularly useful in
elucidating the influence of the proximity of one tissue on the behaviour
and development of another (see Chapter 13 and Vol. 2, Chapter 4).
One limitation of the raft method is that not more than about
four small cultures can be put in any one vessel or the raft will sink.
The rafts are also rather tricky to handle. When the medium is to be
changed, the raft must be lifted off the old medium and put on to a
dish of new medium with considerable care. Chen noted that the top
surface of the paper must never be allowed to get wet all over, otherwise it would sink and become useless. Shaffer found with his siliconed
rayon that once this became coated with protein from the medium it
would no longer float, so the experiments were rather short-lived.
As pointed out in the preceding section, the "wetness" of the cultures
is a most important matter. A balance has to be struck between the
supply of oxygen and the supply of water-soluble nutrients, chiefly
glucose. The proper wetness can only be judged by experience and it is
therefore important that it should at any rate be under control. The
chief defect of the raft methods is that this factor is not controllable,
and in point of fact most cultures on rafts are much too wet. The grid
method offers complete control in this respect and it is also much easier
to handle.