282 ARTIFICIAL PARTHENOGENESIS AND FERTILIZATION 



hours, and this process is accompanied by a characteristic 

 alteration in the color of the egg. It becomes first dark, then 

 rapidly black, and instead of the homogeneous appearance of 

 the protoplasm, there appear large drops or globules within it. 

 If we examine with a microscope such a culture of unfertilized 

 eggs after about twenty-four hours, we find two classes of eggs: 

 first the ripe but uniformly dark, dead eggs and, second, the 

 immature but living, normally colored eggs. We stated in a 

 previous chapter that the eggs which are taken from the ovary 

 of a starfish do not all ripen at once; many mature very slowly, 

 others practically never. It can now readily be observed that 

 the unripe eggs remain alive several days longer, until they even- 

 tually fall a prey to bacteria, while the ripe eggs mostly become 

 dark and die in from four to eight hours after maturation. 



Is the death of the ripe egg, which has not been made to 

 develop, due to intrinsic processes or to the bacteria contained 

 in the sea-water? A reliable method of determining this is 

 to make pure sterile cultures of the eggs in sea-water. This is 

 comparatively simple with starfish eggs. Eight flasks were 

 sterilized, filled with sterilized sea-water, and then heated again 

 for twenty minutes to 100 on three consecutive days. A 

 female starfish was thoroughly washed externally, one arm 

 opened, and an ovary removed with sterilized forceps and 

 brought into sterilized sea-water. From the thick stream of 

 eggs which immediately flowed out of the ovary a couple of 

 drops were quickly placed in each of the sterilized flasks with a 

 sterile pipette. A second series of eight flasks contained normal, 

 unsterilized sea-water and in each of these flasks also a couple 

 of drops of the same eggs were placed. A third series of flasks 

 was filled with sea-water, to which 2 c.c. of a thoroughly putrid 

 culture of old starfish eggs had been added in order to cause 

 from the first a vigorous development of bacteria. These 

 flasks too contained eggs from the same female as the sterilized 

 flasks. 



