238 FIELD. [Vol. XI. 



Technique, 



Owing to the extreme delicacy of the cells involved, and the 

 extreme distortion caused by the great majority of the fixing 

 fluids in common use for other tissues, I have endeavored to 

 record with great care the methods which I have found best 

 adapted. Pictet has already given figures showing the effect 

 upon spermatozoa of the reagents most commonly employed 

 (18). All the points described have been studied {A) on the 

 fresh material, teased in sea-water on the slide. The fresh 

 material teased on the slide was then treated with the various 

 reagents run under the cover glass, and the reaction watched 

 through the microscope. (B) The carefully killed and hardened 

 material has been studied by dissociation methods, and {C) by 

 means of sections cut in paraffin. 



A. Fresh Material on the Slide ; for Structure and Development 



of the Spermatozoon. 



I. Neutral dahlia and methyl green. — To a watchglassful of 

 sea-water add a small quantity of concentrated aqueous solution 

 of dahlia. Filter very carefully, several times. Place the living 

 spermatozoa in a drop of this liquid on the slide. After three 

 to five minutes add a drop of a dilute solution of methyl green 

 prepared in the same way. Examine under cover-glass. The 

 nucleus is stained a delicate green ; the mitosome and centro- 

 some violet. This method gives a minimum distortion ; (Figs. 

 14 and 27; compare with Figs. $B and 4 D) ; but unfor- 

 tunately the results are very transitory, for after an hour or so 

 the colors fade and finally the nucleus swells {Fig. 28) and 

 bursts, leaving only the tail and mitosome (Figs. 53, 54) 

 (frequently also the centrosome) visible. 



If the dahlia in solution in distilled water is added to the 

 salt water under the cover-glass, a precipitate of granules of 

 dahlia is formed, which destroys the value of the preparation. 



The value of this method lies in its delicacy, and for that 

 reason very clean and very dilute stains are necessary. The 

 solutions of dahlia and methyl-green in sea-water must be 

 freshly prepared. 



