354 TISSUE CULTURE 



type of explant do not grow so well as those made from the trans- 

 lucent tissue. They tend to become necrotic in the centre of the 

 explant ; a phenomenon which can be avoided entirely in small 

 explants if due care is taken at the outset. 



Place 1 drop of plasma, taken up in a fine sterile pipette, in the 

 centre of each coverslip, and transfer an explant to each with 

 iridectomy knives. Add 1 drop of embryonic extract to each, and 

 with the utmost speed, mix the two media, spreading it evenly 

 over the surface, but not as far as the edge of the coverslip. This 

 can be done quite easily with iridectomy knives, and usually there 

 is time to mix twelve cultures before the plasma clots. In summer 

 the plasma clots rather more quickly. The evenness with which 

 the medium is spread before clotting has an important effect on 

 the distribution of the growth at the circumference of the explant. 

 Invert a waxed cavity slide over the coverslip and, with pressure, 

 it will adhere to the wax. The whole preparation is then inverted 

 and rapidly sealed with the wax mixture. 



Incubate cultures of fowl tissues at 38-5° to 39° C. and mam- 

 malian tissues at 37-5° C. 



Sub-culturing. Every forty-eight hours the explants in hanging- 

 drop cultures should be washed in Tyrode solution and fresh 

 medium added. The wax sealing the culture is carefully removed 

 with a hot scalpel and the coverslip taken up with sterile forceps 

 and placed, culture upwards, in a Petri dish, remembering that 

 only the region of the coverslip adjacent to the medium is sterile. 

 Four tangential cuts across the periphery of the zone of new 

 growth are made with iridectomy knives, and the culture lifted 

 out with a minimum of the old medium adhering to it. The cells 

 which have migrated nearest to the extreme edge of the medium 

 are usually degenerate and are in this way also discarded. Wash 

 the explants in Tyrode solution for at least ten minutes, and cut 

 them into smaller fragments if necessary. The cultures are then 

 treated as original explants prepared with fresh coverslips and 

 media. 



Maximow {Contrib. Embryol. Carneg. Instit., xvi, 1925, p. 47) 

 has devised a useful variation of the hanging-drop technique where 

 it is desirable not to disturb the explant and new growth during 

 sub-culturing. A small sterile coverslip, holding the culture, is 

 fixed with a drop of sterile saline to the surface of a larger cover- 

 slip. Both are inverted over a large cavity slide. The inner 

 coverslip remains sterile and may be detached for immersion, with 

 its explant, in Tyrode solution prior to renewing the medium and 

 re-sealing. Coverslip cultures may also be placed in small numbers 

 in a Petri dish containing moist filter paper, with a large watch- 

 glass inverted over them in addition to the cover of the Petri dish 

 itself (see Barta, Archiv. f. exper. Zellforsch., iv, 1927, p. 600). 

 Coverslips of clear sheet mica are useful for the intermediate 



