14 CARL GOTTFRIED HARTMAN 



ably having been lost in the body cavity. Ninety-two hours later 

 the animal was killed and embryos in the eleven somite stage 

 were found in the remaining uterus. Female No. 117 was oper- 

 ated February 13, and 20 degenerate eggs were taken from the 

 left uterus. On March 17, the remaining uterus furnished 3 

 eggs. The ovary was much enlarged and densely studded with 

 large corpora lutea. The eggs were mostly fertilized. In the 

 remaining cases, even where copulation was not observed, eggs 

 were nearly always found, both after the first and the second 

 oestrus periods, but were unfertilized and hence worthless. 



Nos. 81 to 88 were killed in the field, where aseptic operation 

 was impossible. 



4) Fixing and staining. Four fixing fluids were most often 

 used: Carnoy's, Bouin's, Fleming's and Hill's. Carnoy's fluid 

 gave only fairly good results. Eggs fixed in Bouin's solution 

 always proved usable: shrinkage does not occur, but the cell- 

 elements are not so well fixed as with Hill's mixture. Fleming's 

 solutions, both weak and strong, fix beautifully but cause shrink- 

 age of the egg. The larger blastocysts invariably collapse in 

 this fluid. I have found Hill's picro-nitric-aceto-osmic mixture 

 to be a perfect fixing fluid for the opossum egg; it penetrates the 

 tough shell of the egg rapidly and fixes the cell structures without 

 causing shrinkage. The osmic acid stains the fat granules black 

 in this as efficiently as does Fleming's solution. Hill's solution 

 is made as follows: Mayer's picro-nitric acid, 96 cc, 1 per cent 

 osmic acid 2 cc; glacial acetic acid, 2 cc. Some specimens of 

 every batch of eggs, except those of No. 144, were fixed in this 

 last mentioned mixture. 



It is important that the eggs be carried up into the higher 

 percentages of alcohol by degrees to prevent collapse of the egg 

 envelopes. Since the shell prevents penetration, this envelope 

 may advantageously be removed with fine pointed needles. In 

 the case of older blastocysts a hole may be punched in one side. 



Toto preparations were stained in Dehi field's haematoxylin and 

 sections in iron-alum haemotoxylin ; the latter were counter- 

 stained with orange G or eosin. A few sections of blastocysts 

 were stained in safranin. 



