I04 PROBLEMS OF FERTILIZATION 



filled with the solution to be tested into sperm suspen- 

 sions and observing the reactions of the spermatozoa 

 to the open end of the tube. Sea-water was charged 

 to saturation with CO2, and various dilutions of such 

 charged sea-water were employed. The capillary-tube 

 method, however, though giving positive results, proved 

 inadequate, on account of the very slight diffusion from 

 the open end of the tube. It was then found that the 

 injection of a drop of the solution into a sperm suspen- 

 sion mounted beneath a raised cover slip gave results 

 at least ten times more delicate. Such a drop is con- 

 fined above and below by the glass surfaces, and by 

 diffusion a CO2 gradient is established around its 

 margin.^ 



If a drop of a I per cent CO2 solution in sea-water^ 

 be introduced into a fresh milky sperm suspension of 

 Nereis the following configuration develops in a few 

 seconds (Fig. 14a). A ring of densely aggregated, very 

 active spermatozoa forms near the margin of the origi- 

 nal drop, and a similar linear aggregation extends from 



^ The insistence of certain investigators on the Pfeffer capillary- 

 tube method (e.g., Loeb, 1916, p. 93) for studying chemotaxis of sper- 

 matozoa is difficult to understand. The effectiveness of the Pfeffer 

 method depends upon diffusion from the open end of the tube, and the 

 gradient is therefore estabhshed for the greater part without the tube. 

 The amount of diffusion depends on the diameter of the tube, which 

 is rarely stated by investigators; the walls of the tube, moreover, 

 provide a source of thigmotactic stimulation, thus interfering with the 

 pure chemotactic reaction. My method of confining a drop in the me- 

 dium between glass surfaces i to 2 mm. apart provides a gradient more 

 surely than the tube method, and one which lasts sufficiently long for 

 all practical purposes. It is, moreover, much simpler and more easily 

 controlled, and is not complicated by the thigmotactic factor. 



' CO2 saturated sea-water diluted a hundred times with normal 

 sea-water. 



