126 FRANK R. LILLIE. 



should enable us to classify it very closely. The present paper 

 is a contribution to this program. 



II. METHODS. 



The eggs of the sea-urchin, used in these experiments, should 

 be washed in two or three changes of sea-water to rid them of 

 blood or tissue secretions, and they should not be used for more 

 than about three hours after washing. The quantities used 

 should be small and constant for all experiments of a set; as a 

 practical matter not more than two drops of eggs from the layer 

 on the bottom of the container to 10 c.c. of sea-water. In more 

 delicate experiments egg concentration should be expressed as 

 number of eggs per c.c. of sea-water. 



Sperm concentration may be expressed in terms of dilution of 

 the dry sperm as I have done previously. For instance, one drop 

 (o.i c.c.) of dry sperm may be added to 25 c.c. of sea-water, 

 and one drop (0.04 c.c.) of this suspension used to fertilize one 

 drop of eggs in 7.5 c.c. sea-water. This may be expressed as one 

 drop of 1:25:7.5 c.c. Such an insemination is abundant but 

 not "heavy." It is equal to about 1.5 "units sperm concen- 

 tration" of study VIII. Sperm concentration is given for all 

 the experiments. A 1 : 25 sperm suspension should be prepared 

 immediately before using and should not be used for more than 

 about five minutes, as its fertilizing power diminishes rapidly. 

 The main point is to make sure that the inseminations of a 

 series are identical or comparable. 



A solution of I per cent, by weight of c.p. copper chloride in 

 distilled water was made; o.i per cent, in distilled water was 

 made from this, and the various concentrations actually employed 

 in the experiments were made immediately before each experi- 

 ment by adding a definite number of drops of the o.i percent, 

 solution to 100 c.c. of sea-water, using a pipette graduated in hun- 

 dredths of a c.c. on which the actual amount added was read off. 

 The strength of copper chloride most commonly employed was 5 

 drops of o.i per cent, in 100 c.c. of sea-water. The readings of the 

 5 drops ranged around 0.19 c.c.; in round numbers this was 

 calculated in as 0.2 c.c. and the amount of CuCU in such a 



