No. 2.] THE EGG OF BUFO LENTIGINOSUS. 297 



Eggs fixed with hot water or corrosive-acetic stain in from 

 one to three hours ; those fixed with chromic-acetic stain much 

 more slowly and must remain in the solution from eighteen to 

 twenty-four hours ; if the material is not fresh, two or three 

 days may be required to give satisfactory results. When re- 

 moved from the solution the sections appear to have taken 

 only the borax-carmine, but a good differentiation can be 

 obtained by a careful use of 90 per cent alcohol which has been 

 strongly acidulated. When rightly differentiated, the sections 

 should appear a reddish-purple to the naked eye. 



At various stages in their development the eggs react some- 

 what differently toward this stain. In general, the chromatin, 

 cytoplasm, and nuclear sap of the ovarian ^^g take the car- 

 mine, the yolk granules, nucleoli, and egg-membrane staining 

 blue. In the fertilization stages the chromatin as well as the 

 yolk granules and egg-membrane take the Bleu de Lyon, the 

 cytoplasm and amphiaster showing a stronger affinity for 

 the borax-carmine. 



III. Maturation of the Ovum. 

 I. The Unripe Ovarian Egg. 



Normally segmenting eggs of the same female have been 

 found to vary in size from 0.6 mm. to 1.5 mm. in diameter, and 

 comparative measurements of a large number of eggs seem to 

 show that the average diameter of the fertilized o.^^ and of the 

 ovarian Q.gg just before the beginning of the hibernation period 

 is I.I mm. From these facts it seems probable that the eggs 

 attain their maximum growth before the animal goes into its 

 winter sleep, and that little, if any, significance can be attached 

 to the fact that eggs taken from a female late in the fall usu- 

 ally show a considerable variation in size. 



At the time of hibernation the ovarian egg is nearly spheri- 

 cal and is surrounded by three distinct membranes (PI. XXVIII, 

 Fig. 2). The two outer ones (Fig. 2, A, C) are thin and homo- 

 geneous ; follicle cells and blood corpuscles are frequently found 

 between and under them. These membranes do not properly 



