250 GREGOR T. POPA. 



to the action of osmic acid vapor, one sees under the microscope 

 very fine round droplets, black and refractile, evidently origi- 

 nating in the intercellular substance. The preparations by osmic 

 acid give us the impression that there is on the surface of and 

 between the spermatozoa a substance which contains fat and which 

 surrounds the cells as a mantle. 



5. SUDAN III. 



Sudan III. shows in the same way as osmic acid, the presence 

 of fatty globules accumulated between the spermatozoa after 

 treating them with the reagents. 



6. THE MACROCHEMICAL TEST WITH VICTORIA BLUE. 



If i per cent. Victoria blue in distilled water is mixed with 

 ether, one obtains, after shaking, an emulsion which, after 10-30 

 minutes, separates in two distinct layers: the wow-stained ether 

 above, and the strong colored Victoria blue below. If to the 

 same mixture one adds dry sperm, after shaking, the ether 

 separates again, but now it carries with it the fatty substances 

 from the spermatozoa. These substances are already stained by 

 Victoria blue, and, therefore, the ether in the upper part of the 

 test tube is blue. If, instead of spermatozoa, we use only the 

 fluid separated after centrifuging a sperm suspension, we may 

 have a complete transfer of color into the ether, the lower layer, 

 representing the water of the Victoria blue solution, being now 

 uncolored. This reaction corroborated by the tests of Schu- 

 macher and the reactions with Sudan III. and osmic acid demon- 

 strates that around and between spermatozoa there is a substance 

 rich in unsaturated and neutral fats. 



7. OBSERVATIONS ON LIVING SPERMATOZOA IN SEA-WATER. 



Observations are made with the oil immersion lens. By 

 varying the focus, the strength of the light and the eye pieces, 

 one can see quite clearly many of the facts obtained by staining 

 methods. Without doubt, the value of this kind of observation 

 is very great, especially when combined with the above mentioned 

 methods; more and more one reaches the conviction that there 

 are in the spermatozoa two different zones: a darker one at the 



