Forcipulata 549 



mentation. The usual steps are then taken to aerate the egg culture 

 properly by changes of water. It is to be noted that fertilized eggs settle 

 more rapidly than unfertilized ones. 



Most Asterias eggs are not harmed by such physical disturbances as 

 those involved in pouring them from dish to dish or transferring them 

 by a wide-mouthed pipette. Some, however, are exceedingly sensitive to 

 such manipulations between the time they are shed and the breaking 

 down of the germinal vesicle; thereafter they are not affected. 



Several cubic centimeters of eggs may be obtained from a fully ripe 

 female. Eggs are about 110/x in diameter, yellow in color, and opaque. 

 Cytological details may be seen only vaguely in living eggs. 



The optimum percentage of normal cleavage occurs in eggs fertilized 

 during the first %-hour period following the formation of the first polar 

 body, which is about the time when eggs are fertilized under natural 

 conditions. 



To obtain sperm, remove a plume of a male, rinse it in seawater to 

 wash away body fluid, and place it in a dry vessel. The seminal fluid 

 soon flows out. 



The concentration of sperm is an important factor when fertilizing 

 Asterias eggs, since they are unusually sensitive to polyspermy. In 

 theory, the writer mixes one drop of "dry" sperm in 25 cc. of seawater, 

 and uses two drops of this sperm suspension for every 250 cc. of water 

 containing eggs. But since the consistency of dry sperm and the size 

 of the drop differ from one experiment to another, in practice the proper 

 suspension is determined by the eye, according to the degree of cloudi- 

 ness. 



Early in an experiment there are several criteria which indicate that 

 an egg-set will cleave normally: The majority of the eggs are shed in 

 the germinal vesicle stage, and oogonia are relatively few in number; 

 the irregular shape of freshly shed eggs soon gives place to a rounded 

 form; and the germinal vesicle breaks down in practically all cases. If 

 these conditions are not satisfied the set should be discarded. When 

 eggs in good condition are fertilized, most of them form distinct, well 

 lifted membranes. 



The temperature of the seawater at Woods Hole during late May and 

 June varies from about 13 to 18 C; hence much of the work is done 

 at about 15 , unless temperature is controlled. At 15 about 4 hours 

 are required from shedding to first cleavage. Eggs will not develop at 

 25 , and the lower limit is about io°. 



F. R. Lillie (1919) found that the addition of NaOH to seawater 

 during the summer, when eggs frequently do not fertilize, aids the fertili- 

 zation reaction. During May and June, however, when eggs are in 

 optimum condition, the use of NaOH is unnecessary. 



