72 BINUCLEATE CELLS IN TISSUE CULTUHES. 



satioii of tlu" Napor about ihv walls of the moist chamber on the depressed slide. 

 By using a shde with a deep depression, containing a little distilled water, this evapo- 

 ration was lessened. 



Cultures planted in a hanging drop of this media, ui)()n a sterik', clean cover-slip, 

 inverted over a depressed slide, which was sealed with \aseline, grew xvvy well at a 

 temperature of 39° to 40°. However, it was found that if a small (juantity of 

 extract of chick embryo (Carrel, 1913) were added to the media better growths were 

 obtained, /. c, cells migrated out from the original ])iece earlier, growth was more 

 rapid and ^•igorous, mitoses were more freciuent, and a larger p(>rcentage of growths 

 was obtained. Hence this addition to the media was generally made. Ordinary 

 bouillon had a similar activating effect. 



The embryonic extract was ))repare(l as follows: After the embryo had been 

 removed from the egg, under sterile conditions, and with as little contamination with 

 yolk as possible, it was placed in a Petri dish containing 10 c.c. of sterile Locke's 

 media and washed. The tissue to be planted having been dissected out and removed 

 to another dish of media, the remainder of the embryo was cut up and placed in a 

 small, sterile test tube with a little media, and carefully ground up with a glass rod. 

 This mixture was next centrifugalized, and the supernattuit fluid added to the 

 culture, generally in the proportion of equal parts of this fluid and Locke. Too 

 high a proportion of embryonic extract was undesirable on account of its richness 

 in food material, in that it produced a cell overloaded with fat globules, which 

 interfered with observation. 



The advantages of glycosahne over plasma have been noted by Lewis and Lewis 

 (1912a, p. 10). It is more transparent and practically all of the growth is upon the 

 lower surface of the cover-slip — not scattered throughout the hanging drop, as in 

 the case of the plasma clot. The cells, unim]X'ded by the fibrin network, migrate 

 freely along the cover-slip, ujjon which they spread themselves flat and thin, thus 

 facilitating observation. The ([uantit}^ of fat being much less than in plasma- 

 grown cells, the cj'toplasmic constituents, such as the centrosphere and mitochondria, 

 are much more easily observed and studied. There are, too, the additional ^■erv 

 considerable advantages that the media is more convenient to handle, fixed prepa- 

 rations are more easily made and are not marred by stained fibrin and coagulated 

 albumen, and experimental operations, such as staining with vital dyes, are more 

 satisfactorily carried out. 



Immediately after planting, the cultures were transferred to a warm box, kej^t 

 at a constant temperature of 39 to 40 degrees bj' means of an electric thermo- 

 regulator. With the microscope inside this box it was not necessary to remove the 

 cultures from their warm environment for purposes of observation, and it was owing 

 to this manifest advantage that the earlier method of observation of the living 

 cultures upon a warm stage outside the incubator was discontinued. In addition, 

 the warm-stage method of heating, from one side only, was found to be inferior to 

 that of the warm box, in which the culture was completelj^ surrounded by an 

 environment of uniform temperature. 



Illumination was fiu-nished by daA'light, Tungsten globe, or Welsbach burner. 

 A raj' filter, consisting of a glass vessel filled with a solution of copper sulphate or 



