After some experimentation, a method was developed by which the embryo may be excised alive from the bursa of the parent and cultured in vitro; this proved very useful. A preliminary account of this method has already been given (Fell, 1940b), but owing to its important bearing on the problem of the nutrition of the embryo, it is repeated here in greater detail.

To extract the embryo is a delicate operation, for the disc of the parent measures only 3–5 mm. across. The pregnant adult is anaesthetised by Subjection to menthol, as above described. With a fine tenotomy scalpel and forceps the disc is separated from the arms and mouth skeleton, and turned so that its lower (oral) side is uppermost. The bursae will have come away with the disc, and in them the older embryos are usually to be seen moving about. The latter can be disentangled from the membranous walls of the bursa by directing a gentle stream of water from a hypodermic syringe into the bursae. When the disc is removed, the genital plates, gonads, and the younger attached embryos usually remain attached to the bases of the arms; these early embryos can be removed by cutting the attachment with fine scissors. The embryos are now pipetted through several washings of sterilised sea water. Each embryo is then placed in a small watch-glass (5 cm. in diameter) and covered by 2–3 mls. of “Erdschreiber” medium. The watch-glass is set in a larger Petri dish, together with a sterile swab of wet cotton wool to keep the contained air humid and thus minimise changes in the density of the culture medium through evaporation. The whole “set-up” is surrounded by a bath of flowing tap water to keep the temperature moderately constant. In practice the medium was renewed every fourth day, but a longer interval can be allowed. Aseptic technique must be used throughout, as the embryos are very susceptible to bacterial toxins. Embryos treated in this way have been successfully cultured for periods of three weeks; they continued to differentiate as if still within the bursa, but with the advantage that the development can be observed.

As described in my previous account, the embryos if cultured in sterile sea water instead of in “Erdschreiber” medium, even though the pH value be kept at its normal value for unsterilised sea water, underwent a retrograde development, unco-ordinated cell-division took place, and finally they died. The significance of this result is discussed in the section of this paper dealing with the nutrition of the embryo.

The composition of “Erdschreiber” medium, as given by Gross (1937), is as follows:—

The soil extract is prepared by autoclaving at a pressure of two atmospheres one kilogramme of garden soil in 1,000 mls. water for one hour. It is then decanted, filtered, and repeatedly sterilised till it becomes a clear, reddish-brown fluid. Further details are given