FERTILIZATION REACTION IN ECHINARACHNIUS PARMA. 13 



must in no wise be confused with agglutination produced by egg 

 secretions. What is significant is that the agglutinin produced 

 by eggs behaves in a characteristic fashion, may be quantitatively 

 studied, and is a necessary part of the fertilization reaction. 



A. Sperm Agglutinin Production in Echinarachnius. 



Eggs of Echinarachnius charge sea-water with a substance that 

 agglutinates Echinarachnius sperm. If a drop of this charged 

 sea-water be added to a sperm suspension the sperm rapidly 

 form clumps. The eggs give off this agglutinating substance in 

 the absence of the jelly hulls enclosing them. Stale eggs give 

 off relatively little and immature eggs none, of the agglutinin. 

 i. Quantitative Method for Determining Agglutinin Production by 

 Ova. 



By the method of successive dilutions of the sea-water taken 

 from above Echinarachnius eggs after they have settled one may 

 obtain quantitative estimates of the agglutinating power of the 

 egg-secretion. This method, used by Lillie on Arbacia, briefly is 

 as follows : A series of successive dilutions of the sea-water above 

 Echinarachnius eggs is made, each dilution being one half the 

 preceding. Usually I started with a i/ioo dilution of the agglu- 

 tinin charged sea- water; a half dilution gives 1/200. Thus the 

 series i/ioo, 1/200, 1/400, 1/800, etc., is made. As noted below, 

 series of 1/2, 1/4, 1/8, 1/16, etc., were likewise employed, so too, 

 i/io, 1/20, 1/40, etc. The agglutinating power of these dilutions 

 is tested on sperm suspensions until the reaction comes negative. 

 The last dilution to give a positive reaction rates the power of 

 the agglutinin. 



To observe the agglutination, drops of sperm suspension are 

 mounted on a slide the cover slip of which is supported by glass 

 rods, the slide being placed on the stage of the microscope in 

 focus under low power. Into this sperm suspension, by means of 

 a capillary pipette attached to a rubber tube, is blown a drop of 

 the sea-water from the eggs. This microscopic method, as will 

 be shown beyond, is indispensable for a comparative study of the 

 agglutination reaction, or indeed for accurate quantitative de- 

 terminations. 



Agglutination mayalso be observed byadding two or threedrops 



