STUDIES OF FERTILIZATION 527 



of unit concentration. Thus any given solution could be rated 

 as of 10, 100, or 6400, etc., agglutinating strength depending on 

 the dilution test. 



The most delicate method of determining the reaction is the 

 raised cover-slip method described in a previous paper ('13). 

 The sperm-suspension used as indicator should be about 1 per 

 cent (i.e., one drop dry sperm to about 99 drops sea-water) and 

 should be not more than 10 minutes old. A few drops of this 

 are mounted on a glass slide beneath a long cover-slip supported 

 by glass rods about 1 to 1.5 mm. in diameter. The shde is placed 

 on the stage of a microscope in focus under a magnification of 

 about 40 diameters and a drop of the solution to be tested is blown 

 into the sperm suspension with a capillary pipette attached to 

 a flexible rubber tube. The agglutination reaction begins within 

 less than a second if the solution be of considerable agglutinating 

 power, or within 2 or 3 seconds if the solution is diluted nearly to 

 the unit reaction. In the latter case it lasts only 4 to 6 seconds. 

 The masses formed are relatively small in the latter case, and 

 larger and denser proportionately with the higher concentrations. 

 The reaction is accompanied by a strong stimulation of the 

 spermatozoa, and the reversal of the reaction by slackening of 

 movement, which is more or less proportional to the strength 

 of the solution, so that after agglutination with a strong solution 

 the spermatozoa are practically paralyzed for a time. For this 

 reason the agglutination masses formed by a strong solution do 

 not break up readily and it is difficult to fix the exact cessation of 

 the reaction; but with concentrations up to about 20 units the 

 beginning and end of the reaction are both clearly marked. For 

 a more detailed account of the reaction with higher concentrations, 

 see Study V (Lillie '13, p. 550). 



As an important detail of technique, I would say that for the 

 determination of the more delicate reactions it is desirable to 

 have the sperm suspensions free from blood corpuscles or other 

 foreign particles. I therefore proceed as follows: the sea-urchin 

 is dried slightly with a towel and is opened by a circular cut on 

 the oral side of the equator. If it be a ripe male the thick sperm 

 will begin to exude from the genital pores. The animal is then 



