DEPARTMENT OF MARINE BIOLOGY. 213 



replaced and not allowed to stand. Eventually an artificial sea-water was 

 used which is based upon F. W. Clarke's analysis of the sea-water at Tortu- 

 gas, and is composed of the following elements: 100 c.c. of 0.6 m NaCl -|- 17 

 c.c. of 0.4 7n MgCl2 + 3.5 c.c. of 0.4 tn CaCls + 2.1 c.c. of 0.62 m KCL.^ The 

 behavior of the spermatozoa of Stromhus in this medium corresponds very 

 closely to that in pure filtered sea-water. It is proposed in the future to sub- 

 stitute this medium for the other, since it can be made up in a large quantity 

 at one time, neutral to phenolpthalein, thus doing away with any changes in 

 the composition of the medium. As mentioned above, in certain experiments, 

 the object of which was to determine the effect of increased alkalinity on the 

 behavior of the spermatozoa, a 0.62 m NaCl solution was used as a medium. 

 The effect of neutral sodium chloride is to cause a primary stimulation of both 

 kinds of spermatozoa, which is followed by a depression of their activity, 

 shortening the duration of the reaction. Another important effect of sodium 

 chloride will be mentioned below. 



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

 of both kinds of spermatozoa in pure normal sea-water under a raised cover, 

 the following technique was finally adopted: The specimen was opened and 

 the sperm-ducts were exposed; this region was washed Avith pure sea-water 

 and then the ducts were ruptured, permitting a flow of sperm-fluid. The 

 required amount of the latter necessary for a standard suspension, as given 

 above, was drawn up into a chemically clean pipette and was discharged into 

 20 c.c. of the purest obtainable filtered sea-water contained in a chemically 

 clean watch-glass. The thorough mixing of the suspension was completed 

 by drawing up the contents of the watch-glass into the pipette and then dis- 

 charging it, repeating the operation at least three times. A portion of the 

 suspension was then transferred to a slide upon which were permanently 

 sealed, by means of a thin layer of paraffin, two parallel glass supports for the 

 cover, both of them slightly longer than the width of the cover itself and about 

 1 mm. thick. A cover-glass was then placed upon the culture and a suflricient 

 amount of the suspension was added or withdrawn to bring it flush with the 

 free edges of the cover. Observations, together with data upon the time and 

 the room temperature, were carefully recorded. Loss by evaporation was 

 made up by the addition, as required, of small quantities of distilled water 

 tested free from chlorides and sulphates. This treatment had no appreciable 

 effect upon the results of the experiment, owang to the behavior of both kinds 

 of spermatozoa in hypotonic and hypertonic solutions and to the effect of 

 surface tension in the culture, a phenomenon which will be described later. 

 After the normal behavior of the spermatozoa had been satisfactorily deter- 

 mined, cultures such as just described were always used as controls in experi- 

 ments in which the conditions were varied, as, for instance, when using a 

 0.62 m NaCl solution or CO2 charged with sea-water instead of pure sea-water. 

 The same technique was adhered to when artificial sea-water was used as the 

 medium. 



The following description of the behavior of both kinds of spermatozoa of 

 Stromhus in pure sea-v/ater, in cultures of this kind, is based upon the obser- 

 vation of many experiments, the results of which were quite uniform. It 

 must be remembered that this description only applies when the following 

 conditions have been fulfilled : First, the animal must be fresh, the sperm-ducts 

 full, and the sperm-fluid must be drawn only from the distal portion of the 

 ducts; second, the concentration of the spermatozoa in sea- water must be 



^See Mayer, A. G. Papers from the Tortugas Laboratory of the Carnegie Institution of 

 Washington, vol. 6, 1914. 



