FERTILIZATION REACTION IN ECHINARACHNIUS PARMA. 3! 



July 10, 11:05 A.M. 7 c.c. of Arbacia eggs from four females in 50 c.c. of sea- 

 water. 12:15, 40 c.c. pipetted off and filtered (40 c.c. of fresh sea-water added to 

 the eggs). 4:00 P.M. Arbacia egg-water (first washing) gave an 8-second reac- 

 tion with Arbacia sperm at 1/12,800 dilution. This same suspension gave a 3-4- 

 second reaction on Echinarachnius sperm at 1/800 dilution. 



July 16, n:oo A.M. 7 c.c. of Arbacia eggs and ovarian tissue put in 50 c.c. of 

 sea-water; strained giving 55 c.c. 1:40 P.M. 37 c.c. of supernatant sea-water 

 pipetted off and filtered, 80 c.c. of sea-water being added to eggs. 2:30 P.M. 

 Supernatant sea-water withdrawn gave at 1/6,400 dilution a 6-second reaction 

 with Arbacia sperm; faintly positive for Echinarachnius at i/ioo dilution. 



Hetero-agglutinin Production of Washed Arbacia Eggs. 

 In the second place, if Arbacia eggs be repeatedly washed the 

 hetero-agglutinating substance drops out before the iso-agglu- 

 tinin. A single washing may suffice to remove the hetero- 

 agglutinin. Certainly washing during twelve hours is sufficient 

 entirely to remove the hetero-agglutinin. 



July 12, 6:30 P.M. Arbacia eggs in 250 c.c. of sea-water; eggs settle at the 7-c.c. 

 mark. 140 c.c. poured off and 140 c.c. sea-water added. 



7:30 P.M. 220 c.c. sea-water poured off, 220 c.c. added. 



8:30 P.M. 190 c.c. sea-water poured off, 190 c.c. added. 



10:00 P.M. 140 c.c. sea-water poured off, 140 c.c. added. 



July 13- 



8:15 A.M. 210 c.c. sea-water poured off, 210 c.c. added. 



9U5 A.M. 150 c.c. sea-water poured off, 150 c.c. added. 



10:10 A.M. 200 c.c. sea-water poured off, 200 c.c. added. 



11:45 A.M. 240 c.c. sea- water poured off, 240 c.c. added. 



At 11:45 A.M. 2 c.c. of eggs left. At 12:30 P.M. this supernatant sea-water 

 gave a 6-second reaction at i/ioo dilution with Arbacia sperm. With Echinarach- 

 nius sperm the last positive reaction made with undiluted supernatant sea-water 

 was at 9:50 P.M., July 12. 8:05 A.M., July 13, test was negative with Echinarach- 

 nius sperm. 



The eggs used July 16 in the experiment cited above were farther washed as 

 follows: 2:35, 3:06 and 5:15 P.M. by removing 90, 94 and 93 c.c. of sea-water, re- 

 placing in each case an amount equal to that withdrawn. The last positive effect 

 with undiluted egg-water on Echinarachnius sperm was at 3:06 P.M. At 5:15 

 P.M. the last washing gave a positive reaction with Arbacia at 1/1,600 dilution. 



In these washing experiments because eggs are lost, it might 

 be argued that the hetero-agglutinin disappears because of the 

 insufficient number of eggs left to charge the sea-water. But 

 since the activity of the hetero-agglutinin in any case is inconstant, 

 its loss in no wise paralleling that of the iso-agglutinin, this 

 argument is scarcely tenable. Nothing is surer than the varia- 

 tion in hetero-agglutinin production by equal bulks of eggs from 

 zero upward. 



