The maturation and fertilization of the egg of Cerebratulus. 427 



the early pilidium -stage, which is in about three or four days. From 

 this time on their development is irregular, and oftentimes, indeed, 

 no further development whatever takes place, although the embryos 

 may be kept alive for a period of two weeks or more. 



Several series of eggs fertilized as above described were placed 

 in various killing fluids at intervals of a few minutes from the time 

 of fertilization until the first cleavage. Of these fluids both corrosive- 

 acetic (2 "/o and 5 ^1^ acetic acid) and Boveri's picro-acetic gave 

 excellent results. After remaining for about five hours in the killing 

 fluid, the eggs were washed for several days in repeated changes of 

 70 "/o alcohol, and finally placed in 80 % alcohol. When ready for 

 imbedding they were made up into little packets, each wrapped in a 

 piece of the cast off "skin" of a frog or of a salamander, according 

 to the excellent method suggested by Prof. Boveri ^), 



To obtain great clearness of detail it was found desirable to cut 

 as thin sections as possible (3—6 in) even though each egg was 

 thereby cut into about twenty-five slices. Most of the important 

 structures of the egg, however, are found in a very much smaller 

 number of sections. The majority of these sections were stained with 

 iron-haematoxylin after the usual manner. Preparations obtained by 

 a preliminary staining for 12 — 24 hours in a strong aqueous solution 

 of Bordeaux red, and a subsequent staining with iron-haematoxylin 

 were the most beautiful of any. By this method the centrosomes, 

 chromosomes, and granules of the cytoreticulum are colored dark blue 

 or black, while the yolk-granules and centrospheres are colored red. 

 Bleu de Lyon brings out the yolk-granules nicely, 



1) This is a simple method of obtaining any desired number of 

 loose eggs, or other small objects, in a neat little package for con- 

 venience in transferring from one fluid to another. A bit of the cast 

 off "skin" (consisting usually of a single layer of epidermal cells) is 

 spread out on a concave slide and saturated with alcohol of the same 

 strength as that in which the eggs are preserved. A large number of 

 eggs are then drawn up into a pipette. After a moment the eggs will 

 settle at the lower end of the tube, and any desired number can be 

 forced out on the little sheet of frog's epidermis. Two opposite edges 

 of this sheet are folded over the eggs, and then the other two are 

 brought together, twisted about each other, and run through with a 

 very small pin to which a label is attached. This little bag with the 

 eggs closely packed together at the bottom may now be lifted by the 

 pin, transferred from one fluid to another, and imbedded with the 

 greatest ease. The single layer of epidermal cells forms no hindrance 

 in cutting the eggs. 



