FERTILIZATION OF NEREIS. 1 97 



the needle some five or ten times its original length. In these 

 later stages the method of removal by centrifuging is more 

 practical. 



The experiments were made during the summer of 1919 at the 

 Marine Biological Laboratory at Woods Hole, Mass. Sperm and 

 eggs were obtained from animals caught the evening before and 

 kept in the upper compartment of the refrigerator during the 

 night. Sperm dilutions of about 1/400 to 1/500 were used for 

 most of the experiments. This dilution is not such as will 

 insure that only one spermatozoon will come in contact with 

 each egg (Lillie, '15), but it is sufficiently dilute so that poly- 

 spermy rarely results. Polyspermy was noted in a few of the 

 eggs in experiments 6 and 7. The eggs were placed in a dilute 

 suspension of chinese ink, at once fertilized and transferred to 

 the coverslip which forms the roof of the moist chamber. The 

 coverslips had been previously carefully cleaned of all traces of 

 oil or grease in order to allow the drop of water to spread, thus 

 forming a thin film which compresses the eggs slightly as is neces- 

 sary in order to hold the egg firmly during the operation. The 

 apparatus was always in complete readiness for the operation 

 and it was usually possible to locate promptly an attached 

 spermatozoon and to carefully push or rub it from its point of 

 attachment to the egg. Spermatozoa remained immobile after 

 detachment. Two or three operations were usually carried out 

 on each coverslip. More were not attempted because the delay 

 would allow too great an evaporation of water in the necessarily 

 imperfect moist chamber, thus causing greater compression of the 

 egg and also greater concentration of the sea water. The posi- 

 tion of the operated eggs among others was plotted, the coverslip 

 removed and placed under a binocular microscope, the selected 

 eggs were isolated and removed to separate dishes for observation. 

 Frequently the remainder of the eggs on the cover slip was also 

 removed for cleavage counts (Control 2). These were the last 

 to be removed and in some cases had by that time undergone 

 considerable compression (in other cases the cover slip was slightly 

 flooded to prevent this). This may in part account for the low 

 percentage cleavage recorded frequently under Control 2. 



The eggs were then kept under observation to note the forma- 



