STl.DIES OF FERTILIZATION 533 



zoa consists in mounting some drops of a sperm suvspension be- 

 neath a long cover slip supported by glass rods, and injecting a 

 drop of the fluid to be tested into the suspension. It then forms 

 a clear drop within the milky suspension, and reaction at once 

 begins at its borders. This method gives incomparably more 

 delicate results than Pfeffer's method of using capillary glass tubes. 

 The drop is confined above by the cover and below by the sUde 

 and diffusion takes place only at its margins; in this way a gradi- 

 ent is established. In the case of the capillary glass tubes diffu- 

 sion is so slight from the open ends that no delicate reaction can 

 be expected. So after a number of trials the capillary tube 

 method was abandoned and the injected drop method was used 

 exclusively. 



A. NEREIS 



1. Aggregation with reference to CO 2 



As introduction, the reaction of a fresh suspension of the sper- 

 matozoa of Nereis to a 1 per cent dilution of sea-water saturated 

 with CO2 will first be described from a specific experiment: 



June 26, 1912. A ripe male Nereis was placed in a dry watch 

 crystal and snipped with scissors ; two drops of the dry sperm were 

 mixed in 10 cc. sea-water at 9.11^ a.m. and made a milky suspen- 

 sion which aggx-egated freely in thirty seconds. Some drops of 

 this were then mounted beneath a cover slip supported by glass 

 rods about 1 mm. in diameter. A drop of the 1 per cent CO2 

 sea- water was injected at 9.13. In withdrawing the pipette a 

 trail of the CO2 sea-water is left extending to the margin. In a 

 few seconds the following configuration developed (fig. 4-/). It 

 consists essentially of a dense aggregation of very active sperma- 

 tozoa in the form of a ring within the margin of the original drop, 

 and a line extending from the drop to the edge where the pipette 

 was introduced and withdrawn. In this case the ring is open 

 below and a linear aggregation extends from the opening towards 

 the margin of the suspension. The ring and the linear aggrega- 

 tion are separated from the general sperm suspension by a clear 

 area devoid of spermatozoa 1.5 to 2 mm., in width. , This area 



