296 KING. [Vol. XVII. 



through the various ascending grades of alcohol (remaining in 

 each about ten hours) and preserved in 80 per cent alcohol. 



A third method of fixation which gives good results with the 

 ovarian Qgg, but which I have not tried with the fertilization 

 stages, is that used successfully by Jordan on the Qgg of the 

 newt, namely, immersion for a few seconds only (from thirty 

 to forty) in distilled water at a temperature of 80° C. The 

 eggs are then transferred to 35 per cent alcohol and passed 

 sloivly through the various ascending grades of alcohol to 80 

 per cent alcohol, in which they are preserved. 



If Flemming's solution (strong) be diluted one-half with distilled 

 water, it gives fairly good preparations ; but owing to the diffi- 

 culty of staining after its use, this method was seldom employed. 



The preparation for sectioning is the same for all methods 

 of fixation. From 80 per cent alcohol the eggs are put into 

 90 per cent alcohol for half an hour ; 95 per cent alcohol for 

 one hour ; absolute alcohol for two hours ; turpentine from two 

 to three hours ; fifteen minutes each in paraffin with a melting 

 point of 48° C, of 52° C, and of 56° C. (a longer time makes 

 the eggs brittle) ; and imbedded in paraffin with a melting 

 point of from 56° to 58° C. 



Owing to the large amount of pigment in the upper hemi- 

 sphere, the two poles of the ^g^ are readily distinguished and 

 orientation in any desired plane is easy. The eggs were cut 

 either parallel to an axis passing through the light and dark 

 poles or at right angles to this axis. 



Heidenhain's iron-haematoxylin, which has given such bril- 

 liant results with many kinds of eggs, cannot be used to advan- 

 tage on the Q'g^ of Bufo, as the yolk colors as deeply and 

 retains the stain almost as tenaciously as does the chromatin. 

 Staining the entire egg with borax-carmine, the method recom- 

 mended by Schultze, Pick, and Jordan, gives good preparations ; 

 but far more satisfactory results can be obtained by staining 

 the sections fastened to the slide by Meyer's albumen fixative, 

 with the following solution : 



Saturated solution of Bleu de Lyon in 95 per cent alcohol . . . i part 



Borax-carmine (Griibler's) 2 parts 



90 per cent alcohol i part 



