214 CARNEGIE INSTITUTION OF WASHINGTON. 



a close approximation to that which was described above as being the stan- 

 dard; third, the sea-water must be pure and freshly drawn and must be free 

 from any excess of carbon dioxide or hydroxyl ions above that which is nor- 

 mally present, or, better still, an artificial sea-water, made up according to the 

 formula given above, should be used; and fourth, the technique described 

 should be followed in making the culture. Under these conditions, if the cul- 

 ture is observed immediately after being started, it wall be seen that the 

 eupyrene spermatozoa, which lie in tufts or bunches, are very slowly active, 

 while the apyrene are totally inactive. Within 1 minute a great increase is 

 noticeable in the activity of the eupyrene spermatozoa, and within 3 to 5 

 minutes they will have reached their maximum activity; if the tufts are still 

 intact they will be seen swirling around and lashing the apyrene spermatozoa 

 about. It very often happens, however, that even before maximum activity 

 is reached the tufts are broken up and the spermatozoa begin to swarm about 

 freely. When this occurs it will be noticed that the apyrene spermatozoa begin 

 to become active, generally about 5 minutes after the culture was started. 

 This activity does not imply much actual motility; it is usually confined to a 

 beating of the undulating membranes. At this time, too, the effect of surface 

 tension becomes visible to the naked eye, in that two dense streaks of the 

 eupyrene spermatozoa are formed parallel to the free edges of the culture and 

 about 2 mm. distant from them. That the formation of these streaks or 

 bars is due to the effect of surface tension is shown by the fact that the free 

 eupyrene spermatozoa can be seen under the microscope to be actually drawn 

 away from the free edges of the culture, no matter in which direction they may 

 be swimming. 



It will be noticed that there has been no aggregation of the spermatozoa 

 in the sense in which LilHe uses the word. In a 0.62 m NaCl solution, however, 

 aggregations visible to the naked eye can be seen 2 minutes after the culture 

 is started. The aggregations become very dense and definitive, but within 

 8 or 9 minutes they begin to break up, and free spermatozoa are given off in 

 increasing numbers. This phenomenon is quite different from that of the 

 agglutination of the eupyrene spermatozoa; it is never a permanent condition 

 and apparently takes place only while the tufts are still intact. Another 

 effect of sodium chloride is that within one-half of a minute activity is usually 

 apparent among the apyrene spermatozoa, and these continue active after the 

 eupyrene spermatozoa have ceased to move. In these sodium chloride cul- 

 tures all activity ceases within 2 hours, sometimes within a shorter period than 

 that. 



Within the next 30 to 40 minutes the effect of surface tension becomes more 

 and more evident in that the bars mentioned above recede farther and farther 

 from the free edges of the culture until they meet and form a dense mass 

 across the middle of the culture. If, during this time, the culture is examined 

 under the microscope, it Avill be seen that the apyrene spermatozoa, and these 

 lie at the lowest focus, have gradually become less active, except at the free 

 edges. In the middle region of the culture, above the apyrene spermatozoa, 

 lies the dense mass of active eupyrene spermatozoa. When this configuration 

 has been reached a number of agglutinations are formed in the mass of eup}^- 

 rene spermatozoa, and these continue to increase in size. Soon after the 

 agglutinations are formed it will be seen that the apyrene spermatozoa lying 

 immediately beneath them become active, and this time the activity is con- 

 tinued. 



About an hour after the culture is started, in its middle region and at high 

 focus, there is a dense mass of still very active eupyrene spermatozoa; beneath 

 these are still denser agglutinated masses, between which may be seen active 



