122 THE AMERICAN ARBACIA 



is sufficient to break the eggs into halves, and a much smaller force, 

 3,000 X g for two minutes will stratify them (Plate VII; also Fig. 12). 



In working with other eggs at other places, corrections must be 

 made for differences in density of the sea water and the eggs. 



The sugar-sea water medium described, in which the eggs are centri- 

 fuged,is quite harmless. Eggs may be kept in the solution for five hours, 

 and, when returned to sea water, cleave and develop as well as the con- 

 trols, and at the same rate. The eggs, however, cannot be fertilized in the 

 sugar solution, though the sperm are active and surround the eggs as 

 normally. This may be due to lack of sufficient calcium; it has been 

 found that eggs cannot be fertilized in Ca-free sea water, though the 

 sperm are active. 



In his pioneer experiments, Lyon (1907) .used gum arable for sus- 

 pending the eggs. He used a centrifugal force of 6,400 X g for one 

 or two minutes, the eggs remaining spherical, though he apparently 

 had a few eggs break into halves which he said could not be fertilized. 



b. Crushed Eggs. Homogenates 



When Arbacia eggs are crushed in a mortar after they have been frozen, 

 and then centrifuged, McClendon (1909 a) found that the material 

 separated into two layers, a fluid centripetal layer and a jelly-like 

 centrifugal layer. The chemical composition of the layers is given in 

 Table 7. References to more recent methods of obtaining egg homo- 

 genates by freezing and thawing are given by Runnstrom (1935 a). 

 Kopac (1943) has recommended the following treatment: 



1. Remove jelly layer by washing in two or more changes of 0.52 M NaCl. 



2. Transfer to i.o M solution of urea. 



3. Within I or 2 minutes wash eggs free of urea with 0.53 M KCl. 



4. Transfer eggs to a measured volume of citrated KCl solution containing 9 volumes 

 0.53 M KCl and i volume of 0.35 M Na-citrate. 



5. Immediately disintegrate eggs by flushing in the above solution through a fine 

 bore pipette. 



"The resulting suspension which includes all granules and nongranular residue 

 of the cells is then centrifuged gently to remove unbroken eggs and foreign particulate 

 debris. The supernatent is again centrifuged, at high speed, to separate the granules 

 and other formed elements. The sediment now contains pigment vacuoles, yolk 

 granules, and some mitochondria. The oil globules collect at the meniscus of the 

 centrifuge tube. In the absence of Ca ions, the granules are stable and may be pre- 

 served intact for considerable periods. The more or less nongranular supernatent 

 fluid may be separated from the sedimented granules by pipette transfer. This con- 

 tains most of the residue of the cytoplasmic matrix." 



