AGGLUTINATION OF SPERMATOZOA. 269 



and in 5\ franciscanus. This, as Lillie (1921) later determined, 

 is identical with iso-agglutination. 



2. MATERIAL AND METHODS. 



Material. The animals used in this work represent two phyla, 

 Echinodermata and Mollusca. Belonging to the first are Stron- 

 gly ocentrotus purpuratus, S. franciscanus, Arbacla punctidata, 

 Asterias ochracea, and Lcpasterias csqualis; and to the second 

 Katharina tunicata, Ishnochiton magdalenensis, Mopalia muscosa, 

 Cryptochiton, and Abalone. 



Mctlwds. Every precaution is taken to prevent contamination 

 of the gametes with body fluids. With the three species of sea- 

 urchins this consists of washing animals and dissecting instru- 

 ments with tap water, cutting around the oral disc and removing 

 all body contents except the gonads, and then washing the cavity 

 thoroughly with sea-water. The animals are then placed on their 

 aboral surfaces in Syracuse watch glasses and allowed to shed 

 their gametes through the germinal pores. A second method, sug- 

 gested by Dr. O. C. Glaser, consists of washing the animals in 

 tap water, rubbing off the spines, and drying with a towel. They 

 are then allowed to shed as in the first method. Practically dry 

 gametes can be obtained by this method. In the case of the star- 

 fish and molluscs the same method of sterilization is employed. 

 The rays of the starfish are removed and the gonads, stripped 

 from each ray, are transferred to a finger bowl of filtered sea- 

 water and thoroughly washed. In the molluscs the gonads lie just 

 beneath a dorsal shell and can be reached in one of two ways. 

 The first consists of removing the ventral muscle and viscera with 

 the exception of the gonads, and the second of removing the shell. 

 The exposed gonads can then be thoroughly washed with sea- 

 water, removed with blunt forceps to Syracuse watch glasses or 

 finger bowls, and ruptured. In this manner dry gametes can be 

 procured. 



One ( i ) per cent, suspensions of spermatozoa are made by add- 

 ing to 99 drops of filtered sea-water I drop of dry sperm. 

 Pipettes known to deliver the same number of drops per c.c. are 

 used. All suspensions are used within ten minutes or discarded. 

 Standard egg-water is prepared by allowing one volume of dry 



