288 KANSAS UNIVERSITY SCIENCE BULLETIN. 



very thin and delicate and is easily penetrated ; but it increases 

 in thickness as the cell grows, even adding in final stages new 

 and separate layers, until days are sometimes required for the 

 most penetrating fluid to reach the interior. Occasionally I 

 succeeded in making an opening in the membrane by piercing it 

 with a fine needle, but usually such attempts resulted in com- 

 pletely crushing the egg. One soon learns in working with 

 such delicate material that the less it is handled the better. 

 Even the force required to draw the eggs into a pipette will 

 often break the membrane and allow the contents to flow out. 

 To avoid these difficulties I left them in one vessel through the 

 different processes, carefully drawing off the fluids from them 

 and adding others. Even embedding was managed in this 

 way ; instead of lifting them from the clearing agent and plac- 

 ing them into melted paraffin, thus running the risk of crush- 

 ing them at the very end of the process, I removed the fluid as 

 completely as possible and then poured the paraffin upon them. 



Of all the fixatives I used, Gilson's aceto-nitric sublimate mix- 

 ture and Merkel's fluid proved most satisfactory'-. These came 

 near working in all stages of the eggs' growth. Gilson's fluid 

 was excellent for the oocytes and even the mature egg, com- 

 pletely filled with yolk. The vitelline body shows at its best 

 with this fixative, there being no shrinkage, and the material 

 staining perfectly. 



Merkel was used largely for the early stages, before the eggs 

 had left the ovarian sac. The oogonia and young odcytes are 

 well preserved and stain readily with iron-haematoxylin. For 

 this stage the Merkel fluid is better than Gilson, but those 

 eggs in the follicle are not always successfully fixed. Passing 

 this stage, however, Merkel works well once more ; the mature 

 egg is very well fixed by it, and also the segmenting egg. 



Flemming has been used in preserving the ovary in situ, but 

 not for a study of the finer structures. 



Kleinberg's picro-sulphuric fixed the mature egg very well, 

 but great care must be exercised in using it. If left in too long, 

 the egg becomes greatly distorted and shrunken. 



6. Stains. 



Eggs fixed in Gilson and stained in Ehrlich's h^ematoxylin 

 and acid fuchsin show the vitelline body very distinctly, but 

 no radiations can be seen. 



