EFFECTS OF INJURY UPON SPERM. 215 



which were retained so long were not used. The experimental 

 work was usually started as early in the morning as possible so 

 as to use the material in a fresh condition. A few experiments 

 were carried out shortly after removing the animals from the 

 Eel Pond. Since no better results were secured by working at 

 night, the material was usually kept until morning when condi- 

 tions for work were better. 



C. Removal of Germ Cells. 



The abundant eggs and sperm were removed by snipping the 

 sides of the body w r all at intervals, with scissors. If no water 

 is used the sperms push out as a white mass nearly free from 

 liquid and the eggs may be secured as a thick greenish mass. As 

 a rule, however, the operation was conducted under water. In 

 all cases the usual precautions were taken to avoid the introduc- 

 tion of stray sperm cells. The worms were thoroughly washed 

 in a jet of salt water and, in the earlier experiments, in boiled 

 salt water as well, a precaution which I afterwards found to be 

 unnecessary since the sea water from the laboratory supply was 

 repeatedly tested and found to contain no active Nereis sperm. 

 The bodies of the animals used were quickly removed from the 

 dishes of sperm and eggs so as to avoid, as far as possible, any 

 contamination from body fluids, and every precaution was taken 

 to avoid abnormalities due to outside influences such as strong 

 sunlight, bacterial action, mechanical agitation, etc. 



D. Culture Methods. 



An effort was made to use a minimal quantity of sperm for 

 the process of insemination, in order to avoid, as far as possible, 

 errors due to polyspermy. In the case of the injured sperm, a 

 large quantity was often necessary to secure a small percentage 

 of fertilized eggs. Usually a small number of eggs was insemi- 

 nated first in a small quantity of water to which a liberal supply 

 of water was soon added. In this manner I was able to secure a 

 sufficient number of fertilized eggs to make preservation possible. 

 Up to the time when the larvae begin to swim, the water was 

 changed several times. After this period it is very difficult to 

 renew the water without losing many of the larva?. An effort 



