ORIGIN OF PRIMORDIAL GERM CELLS 489 



MATERIAL AND METHODS 



In the selection of a fixative for the germ-cells, the yolk ma- 

 terial, mitochondria, and attraction-sphere, in addition to the 

 nucleus and general cytoplasm, have to be taken into considera- 

 tion. The same fixing fluid cannot be relied upon in all stages, 

 but in order to meet the changing cell content, a variation is 

 necessary. For instance, the yolk-laden germ-cell of the very 

 young embryos requires very different treatment from the almost 

 yolk-free cell of the later stages, if equally good results in both 

 cases are desired. So that in the case of embryos over 20 somites 

 in development, in which the germ-cells contain only a moderate 

 amount of the primary vitellus, a fixing fluid containing osmic 

 acid may be used to advantage. Osmic acid resembles potassium 

 bi-chromate, with which it is frequently combined, in being an ex- 

 cellent cytoplasmic preservative. It has its drawbacks, however, 

 in being a poor penetrant, amd in blackening the yolk of the germ- 

 cells. This last property of osmic acid is a decided disadvantage 

 in the early stages, in which the cytoplasm of the germ-cells is 

 stuffed with yolk spheres, and an advantage in the case of the 

 older embryos, in which the yolk is reduced in amount, since the 

 blackening of the vitellus facilitates identification. 



Fixatives such as Benda's^ Meves' modification of Flemming's 

 fluid, and Bensley's acetic-osmic-bichromate mixture, all of which 

 contain osmic acid, were found to be best adapted for use in the 

 case of older embryos, in which there is little yolk material re- 

 maining. They preserve such cytoplasmic structures as mito- 

 chondria and the attraction-sphere excellently, and, thanks to 

 their osmic acid content, bring out the yolk spheres plainly. 

 However, in the younger embryos, from the primitive streak 

 stage to 20 somites, they were found to be not nearly so efficient. 

 In these stages the yolk content of the germ-cells is so great that 

 the value of any fixative containing osmic acid is seriously di- 

 minished. In these young embiyos a mixture of equal parts of 

 5 per cent trichloracetic acid and 5 per cent sublimate was found 

 to be excellent. The mitochondria are not presented by this 



