162 VICTOR E. EMMEL 



once inverted, placed over the chamber of a hanging drop slide and 

 immediately sealed with vaseline. The entire operation, occupy- 

 ing but a few minutes, was always performed inside an operating 

 chamber maintained at a temperature of from 38 to 40°. In some 

 cases, as an additional precaution, the air in this chamber was 

 always kept moist by means of evaporation from open vessels 

 filled with warm water. To counteract still further the possible 

 loss of moisture from the cultures, for a part of the experiments, 

 small drops of various fluids (cf. p. 152) were placed in the bot- 

 tom of the culture chambers. The important fact of the absence 

 of coagulation in these cultures has been previously noted (p. 152). 



As already stated the temperature throughout the experiments 

 was maintained at 38 to 40°, both during the preparation and 

 the incubation of the cultures as well as during the microscopical 

 examination of the blood cells. The cytoplasmic constrictions, 

 consequently, cannot be due to excessive heat such as was early 

 described by Schultze ('65) who found that a temperature of 52° 

 caused the non-nucleated erythrocytes to extrude globular proc- 

 esses and beaded filaments which became separated from the 

 original corpuscles, whereas the erythrocytes at a temperature 

 of 38 to 40° remained unchanged. 



In the course of a critical analysis of factors which might 

 have artificially produced the constriction phenomena, it suggested 

 itself that since the erythroblasts tend to settle toward the lower 

 surface of the hanging drop, possibly their contact with the sur- 

 face film of the plasma might result in changes in surface tension 

 sufficient to cause the cytoplasmic constrictions. To test this 

 matter cultures were made in which the contact of the corpuscles 

 with this surface film should be eliminated. After attempting 

 several methods, the most satisfactory results were finally at- 

 tained with the preparations in which the under surface of the 

 culture drop had been brought into contact with a small circular 

 cover-glass, the rim of which had been slightly thickened so as to 

 prevent any compression of the cells (cf. footnote 4, p. 159). 

 The erythroblasts which were thus resting on a glass surface 

 instead of the culture drop film were observed, however, to un- 

 dergo cytoplasmic constrictions similar to those occurring in 

 the hanging drop cultures. 



