Chapter XVI: Some Embryological Methods 119 



Amphibian eggs are so friable that they must be sectioned in 

 celloidin (see, however, Johnson's asphalt-rubber method, chap, v, 

 memorandum 9). 



Stain the eggs in borax -carmine (reagent 32, Appendix B), then pass 

 them through the alcohols (35, 50, 70, 95, 100), leaving them half an 

 hour in each (if necessary decolorize in acidulated 70 per cent, alcohol); 

 thence into ether-alcohol for an hour or more, followed by thin celloidin 

 (12 hours) and thick celloidin (6 hours). Imbed in the usual manner, 

 but clear in the block and section as directed in memorandum 10, 

 chap. vii. Older embryos may be sectioned in paraffin. 



6. For Early Stages of the Mammalian Embryo rabbits are commonly 

 employed because they breed readily, especially in the spring of the 

 year, and the observer can note the exact time when the female is 

 covered if she has been kept separate from the buck until she comes 

 into heat. The period of gestation is 4 weeks and impregnation takes 

 place again immediately after littering. The two uteri of the rabbit 

 diverge as two anterior horns from the single median vagina and each ter- 

 minates in front in a narrow, coiled tube, the oviduct or Fallopian tube. 

 To obtain the early stages the abdomen is slit open from pubis to ster- 

 num, the intestinal tract is cut away or pushed to one side, and each ute- 

 rus and oviduct carefully removed and stretched out along a glass plate. 

 The segmenting ova are found in the oviduct up to nearly 70 hours from 

 the time of copulation. After that period of time they must be looked 

 for in the uterus. Fecundation takes place about 9 hours after coition. 

 While in the oviduct, with the aid of a lens they may sometimes be seen 

 through its walls. A segmenting ovum once located, a transverse cut is 

 made to one side of it through the wall of the oviduct, and the ovum 

 which is very small is gently squeezed out by compressing the oviduct 

 behind it. With a spear-headed needle or the point of a scalpel the 

 ovum is conveyed to the fixing fluid. In case segmenting ova are not 

 visible from the exterior of the oviduct, the latter must be slit open 

 carefully with a pair of fine-pointed scissors, and the eggs sought for 

 by means of a lens. In case no red corpora lutea are visible on the 

 surface of the ovary, indicating a recent discharge of ova from the 

 Graafian follicles, further search is useless. 



Rabbit ova of 18 hours show 4 blastomeres; 36 to 48 hours, advanced 

 segmentation; 72 hours (about 0.6 mm. in diameter; in anterior end of 

 uterus) show the fully segmented ovum an outer layer of clear, cubical 

 cells, an inner mass of irregular granular cells; 72 to 90 hours show 

 enlarged blastodermic vesicle and establishment of embryonic area; 

 fifth and sixth days (0.8 to 4 mm.) show germinal layers; seventh day, 

 primitive streak; 8th day, medullary folds. 



The earlier stages (up to 70 hours) may be fixed for from 5 to 8 min 



