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. 
