LARVAE OF ARBACIA PUNCTULATA 329 



second summer extended from the twelfth of June until the 

 first of August. 



Upon being brought into the laboratory, the sea-urchins were 

 placed in clean glass aquaria with running water, and used as 

 soon as possible, generally within twenty-four hours after being 

 received. Usually the experiments were begun at once. Each 

 sea-urchin was washed, first in fresh water, then in sterilized 

 sea-water, cut horizontally through the test, and inverted over 

 a Syracuse watch-glass. If the eggs were ripe, they were ex- 

 truded at once through the genital pores, and only those indi- 

 viduals which responded immediately were used. Eggs and 

 sperms were examined under the microscope, and the spermatozoa 

 employed only when they were very active, and the eggs only 

 when nearly one hundred per cent were mature. 



The solutions to be used in the experiment were previously 

 prepared and placed in separate beakers, while a similar series 

 of solutions were kept in readiness in a set of watch-glasses. 

 Into each of the former a portion of the eggs was dropped, 

 and into the latter sufficient of the sperm to give a slightly milky 

 suspension. About two drops of each sperm-suspension was 

 then added to its corresponding portion of eggs, and the mix- 

 ture quickly stirred and allowed to settle. By having every- 

 thing in readiness and by using a fresh pipette for each solu- 

 tion, the entire operation could be concluded within about five 

 minutes; consequently the eggs and sperm were subjected to 

 the action of the changed medium only a few seconds before 

 being brought together. This was highly desirable, in order to 

 eliminate as far as possible any specific effect of the altered 

 medium upon the unfertilized sex-products. 



As soon as the eggs had settled, the supernatant liquid was 

 siphoned off, fresh solution added and the process repeated 

 once every fifteen minutes during the first hour. The eggs, 

 which by this time had usually reached the two-cell stage, 

 were poured into finger-bowls and allowed to stand until they 

 swam at the surface of the water, when they were transferred 

 with as little of the liquid as possible to finger-bowls contain- 

 ing fresh solutions. In the cultures in which the blastulae did 



