STUDIES OF FERTILIZATION 545 



To consider the latter method first: The matter was first 

 brought clearly to my attention in the experiment fisted in table 

 2. In the 34th washing the supernatant fluid tested to 1/20 

 dilution; it was poured ofi^, leaving 1.5 cc. in the tube, and 10.5 cc. 

 of sea-water was added. AVhen the eggs were stirred up they at 

 once disintegrated, liberating the red pigment so that the fluid 

 which in previous washings had been colorless became bright red. 

 When this fluid was tested with a fresh sperm suspension it was 

 absolutely without agglutinating effect. The dilution of the 20 

 power fluid of the 34th washing was 8 times, so that even if the 

 eggs had suddenly ceased the production of the agglutinating 

 substance the fluid would have contained 2.5 agglutinating units 

 and should have given at least a 10-second agglutination reaction; 

 but it was absolutely negative. The agglutinating substance 

 had therefore been neutralized by some substance escaping from 

 the disintegrating eggs. 



In the experiment listed in table 3, the repetition of this result 

 was of course looked for. On the morning of August 24, after 

 35 washings on previous days, I noticed that pigment was begin- 

 ning to diffuse from the eggs in tubes A and B, but it had not 

 extended above the 1.5 cc. mark. The eggs were beginning to 

 break down. Without disturbing them, I tested the colorless 

 supernatant fluid and got undiluted an 8-second reaction in A 

 and a 7-second reaction in B. Both tubes were then vigorously 

 shaken until the eggs were disintegrated and the fluid colored with 

 the escaping pigment. Tested in 4 minutes A was negative for 

 agglutination, B gave a 4-second reaction. Tube B was again 

 shaken and in 11 minutes more its agglutinating action had also 

 disappeared. 



There can, therefore, be no doubt that the egg contains in its 

 interior a substance that unites with the spermophile group of 

 the fertilizin. 



In these experiments it was interesting to note that after the 

 agglutinating action had disappeared, the solution was very 

 strongly chemotactic, so that the drop injected into the sperm 

 suspensions formed a ring of active, entirely unagglutinated 

 spermatozoa. The chemotactic substance is certainly distinct 



