SPERM FILTRATES AND DIALYZATES. 305 



Preparation of Filters. Berkefeld and Mandler diatomaceous 

 filters were used: in preliminary experiments the coarse and 

 medium grade of Berkefeld filters; and in all other work Mandler 

 filters, tested to six to twelve pounds air pressure in the size 

 two and one half by five eighths inches. The latter are used 

 generally in bacteriological work. These were boiled in five 

 per cent, aqueous solutions of sodium bicarbonate, washed and 

 boiled repeatedly in redistilled water until the wash water was 

 neutral in reaction. Finally they were thoroughly washed in 

 filtered sea-water. After this a stream of sea-water was passed 

 through the filters until samples of such water produced no 

 injurious effect on unfertilized ova and no disturbance of fertiliza- 

 tion or of development. Such filters were considered "clean." 

 Every filter was subjected to this treatment each time that it 

 was used. The necessity for these precautions was indicated by 

 a variety of experiences. The first sea-water passed through a 

 boiled and washed filter may be sufficiently hypotonic to cause 

 cytolysis of ova within twenty-four hours. Unless all alkali is 

 removed the filtered sea-water may produce activation of ova. 

 The use of acid in the cleaning of filters is prohibitive because the 

 filtrates would then contain traces of heavy metal dissolved from 

 the filter bands. The impurities in question are often too slight 

 for detection by chemical tests, but are only too clearly revealed 

 by their effects upon unfertilized or fertilized ova. 



Preparation of Sperm Filtrates. Five and ten per cent, sus- 

 pensions of spermatozoa were prepared by adding to a definite 

 volume of dry sperm a measured quantity of sea-water. These 

 were allowed to stand at room temperature (fifteen degrees 

 centigrade at Pacific Grove and twenty-two degrees centigrade 

 at Woods Hole) for from five minutes to four hours. To ensure 

 the greatest activity of the spermatozoa the carbon dioxide 

 generated by them was prevented from accumulating by a 

 thorough aeration of the suspensions. The latter were then 

 either clarified first by centrifuging or by filtering through filter 

 paper, or were transferred directly to a diatomaceous filter. 

 Contamination of the filtrates by back suction was prevented by 

 collecting the filtrates into Pyrex test tubes set in the suction 

 flasks. 



