212 CARNEGIE INSTITUTION OF WASHINGTON. 



By another set of experiments it was shown that both kinds of spermatozoa 

 withstand a relatively great increase in alkalinity of the medium in which they 

 are placed. In these experiments was used a 0.62 m NaCl solution, neutral to 

 phenolphtalein; the alkalinity of the solution was increased by the addition 

 of carefully measured quantities of a standardized N/2 solution of NaOH In 

 a solution of N/GOO NaOH in 0.62 vi NaCl, the movements of the eupyrene 

 spermatozoa were practically normal, while activity among the a])yrene was 

 increased. When the NaOH content was increased, making a N/400 solu- 

 tion, there occurred a complete paralysis of both kinds of spermatozoa. 



In the course of a set of experiments, the object of which was to determine 

 the normal reactions of the spermatozoa in pure sea-water, two general facts 

 of importance were discovered. In the first place, the reactions of the sper- 

 matozoa, under the conditions of the experiment, take place very much more 

 slowl}^ than do those of the spermatozoa of Nereis or Arbacia, and very often 

 with less definitive results. In the second place, variations in the rapidity 

 of the reactions and in their duration and intensity were caused bj^ at least 

 four factors: (1) the condition of the animal; (2) the portion of the sperm-duct 

 from which the sperm-fluid was drawn; (3) the concentration of the mixture 

 of sj^ermatozoa and sea-water; and (4) the condition of the sea-water itself. 

 To the first of these factors was due a considerable amount of trouble and lack 

 of uniformity in results during the first half of the season, when the attention 

 of the writer was confined entirely to S. gigas (costatusf). For some reason, 

 which is not yet fully understood, there has been for the last two seasons a 

 steady decrease in the number of the mature males and females of this species, 

 the so-called conch. Considerable difficulty was experienced in collecting a 

 sufficient number of males and those which were obtained were subnormal; in 

 these cases the reactions of their spermatozoa showed many puzzling dispari- 

 ties. Before the end of the season, however, S. hituberculatus was found in 

 great numbers, and not only were the mature males more health}^, but they 

 were more economical on account of their smaller size, in that a fresh specimen 

 could be used for each experiment. Again, the use of S. hituberculatus elimi- 

 nated the second factor, since the sperm-ducts of that species will not supply 

 ordinarily more sperm-fluid than enough for one experiment. In the case of 

 S. gigas, however, the sperm-ducts are so large that they can be tapped suc- 

 cessively at the distal, median, and proximal portions, and it is unquestionably 

 true that the spermatozoa from these three regions showed differences in the 

 rate and intensity of their reactions, those from the distal end being the more 

 active. 



The concentration of the mixture of sperm-fluid and sea-water has an impor- 

 tant bearing upon the agglutination phenomenon. When the sperm sus- 

 pension is too thin, agglutination (in Lillie's sense) of the eupyrene sperma- 

 tozoa invariably fails to take place. In order to assure a maximum degree of 

 activity and, at the same time, to keep the culture from being too dense for 

 microscopical examination, a certain standard concentration was determined 

 empirically. The suspension which was found best for these requirements 

 was a mixture of a column of sperm-fluid, 7 mm. high, in the end of a pipette 

 which was 1 mm. in diameter, with 20 c.c. of pure sea-water, or any other 

 aqueous solution which was used. The variations which arose by the use of 

 this method were not great enough to be of any consequence. This standard 

 suspension was used in all subsequent experiments. 



A considerable amount of variation in the results obtained in these experi- 

 ments was unquestionably due to slight changes in the alkalinity and the 

 carbon-dioxide content of the sea-water used. To obviate this difficulty only 

 the purest obtainable filtered sea-water was used, and this was constantly 



