246 GREGOR T. POPA. 



various forms. This layer flows in and out of middle piece and 

 forms the sensillse. 1 



2. STAINING METHODS AFTER FIXATION. 



Spermatozoa of Arbacia and Nereis were also studied by the 

 more usual methods of fixing and staining. The fixing methods 

 used were: 



1. Corrosive sublimate, saturated solution in distilled water, 100 



parts, plus glacial acetic acid, 8 parts. 



2. Vapor of osmic acid, 2 per cent, aqueous solution. 



3. Flemming, strong solution. 



4. Formalin, 10 per cent. 



5. Heat, after rapid evaporation. 



The following stains were employed : 



1. Iron-hsematoxylin (Heidenhain) and eosin. 



2. Iron-haematoxylin (Heidenhain) and safranin. 



3. Fuchsin. 



These procedures applied to the study of the spermatozoa, 

 when united with the observations on living material, give us 

 the conviction that there is no one fixing fluid which preserves 

 perfectly the form and the structure of the spermatozoa.- The 

 fastest acting fixing agents (as vapor of osmic acid) and the 

 most penetrating fluids (as acetic acid plus ^-bichromate) change 

 the size, the configuration of the spermatozoa and the mutual 

 relations of the substances in it. The outlines of the head 

 slightly change, the heads swell, the sensillae disappear, the ring 

 changes its shape, and the lipochromatic substance migrates. 



1 I have strong reasons to believe that Ballowitz has observed also the migration 

 of the peripheral substance of the spermatozoa, "Untersuch. iiber die Struktur 

 der Spermatozoon der Fische. Amphibien, Reptilien," Arch. f. mikr. Anal., 36 Bd., 

 1890, S. 225. His explanation is not satisfactory. He believes that the change 

 of color in various parts of the head of the spermatozoon is a consequence of stain 

 diffusion in the medium (p. 239). But the changes can be better observed by the 

 method of Victoria blue, fuchsinand Janus green than by his method; and one can 

 also observe the accumulation of the substance in the connecting region even on 

 spermatozoa not stained at all. 



2 Students of spermatozoa note the same thing. This fact is demonstrated 

 particularly clearly by C. Pictet, "Recherches sur la spermatogenese chez quelques 

 inverte bres de la Mediterranee," Milleil a. d. Zool. St. Neapel., 10 Bd., 1891-93, 

 S. 75- 



