A Cytological Study of Artificial Parthenogenesis etc. 149 



tonic solution, the same result may be obtained by preventing the 

 oxidations in the eggs for about three hours, either by putting them 

 in sea water containing a little potassium cyanide, or simply by re- 

 moving the oxygen from the sea water by means of a stream of puri- 

 fied hydrogen. 



Technique. 



The eggs of Strongylocentrotus purpumtus were the only kind 

 employed, and in all cases were obtained by taking out the gonads 

 from a female and allowing them to remain in a dish of sterilized 

 sea water for a few hours. The ripe eggs gradually drop out of the 

 ovaries and fall to the bottom of the dish where they may be col- 

 lected by means of a pipette. 



Membrane formation was effected by allowing the eggs to re- 

 main in a mixture of 50 cc.s. sea water -f- 2.9 cc.s. of N/10 butyric 

 acid from 90 to 150 seconds, according to the temperature. The eggs 

 were then transferred back into normal sea water and if they had 

 been in the butyric acid solution for the correct length of time all 

 of them formed membranes. As it was desired to study the cyto- 

 logical effects of butyric acid alone, some of these eggs were allowed 

 to develop without further treatment but at 15 C. very few of them 

 completed even the first division. 



When the eggs from the butyric acid solution had remained in 

 normal sea water for about 20 minutes, they were then placed in a 

 mixture of 50 cc.s. sea water + 8 cc.s. of 2y 2 N-NaCl and exposed 

 to the action of this solution for times varying from 30 to 60 mi- 

 nutes. The length of exposure necessary depends mainly upon the 

 temperature, but also on the length of time the eggs have remained 

 in the sea water after removal from the butyric acid solution. Finally, 

 the eggs were again transferred into normal seawater and, if the 

 time of exposure to the hypertonic solution had been correctly chosen, 

 practically all them developed and gave rise to free swimming larvae. 



The various stages in the formation of spindles and segmentation 

 were followed under the low power of the microscope and embryos 

 were obtained at all stages of development. 



The material was fixed in FLEMMING'S strong solution, and im- 

 bedded in paraffin in the usual way. Sections were generally cut 

 3 u in thickness and stained on the slide by one of the three follow- 

 ing methods: 



