156 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY. 



entire the yolk and embryo. The time of exposure to any killing fluid 

 should vary with the age of the embryo. 



I have tried a variety of stains. Of the many carmine combinations 

 used, I have found Grenadier's aqueous alum carmine the most service- 

 able. The best results are gained from material killed with hot water. 

 Of the hsematoxylins, I have found Ehrlich's acetic acid-alum mixture 

 as good as any, and the most reliable. It stains material fixed by any of 

 the methods mentioned, the best results being obtained after fixing with 

 Perenyi's fluid or with Flemming's weaker mixture. I have found, how- 

 ever, that the staining is much better if the hsematoxylin is followed by 

 an aniline dye. Of several tried I have found orange G of Griibler 

 much the best ; consequently I have used this excellent combination in 

 most of my work. The results are precise and clear, and the manipula- 

 tion of staining is very simple. Sections 6|, 10, or 13^ micra in thickness 

 are stained on the slide in Ehrlich's mixture from 20 to 30 minutes, then 

 washed in acidulated alcohol until they are very pale, next neutralized 

 in a two per cent solution of bicarbonate of soda, then thoroughly washed 

 in distilled water and put into a saturated aqueous solution of orange G. 

 This is allowed to act from 10 to 20 minutes, the time being determined 

 by watching the action of the stain. The slide, after draining, is trans- 

 ferred directly to absolute alcohol, which sets the stain and at the same 

 time washes out the excess. After clearing in xylol, the sections are 

 mounted in xylol balsam, the stain being thoroughly permanent. 



In studying the larval organ I have depended entirely upon serial 

 sections, for the gland is too small to permit satisfactory dissections, and 

 the shell of the appendage does not clear readily enough to permit study 

 of the entire organ in situ. The first larva was obtainable in abimdance, 

 so that various methods could be tried, but the older larvae are more dif- 

 ficult to get, and as a matter of fact all of the older larvae which I had 

 were already preserved when they came into my hands. 



The most successful results with the first larva were obtained with 

 the weaker Flemming's mixture, Perenyi's fluid, corrosive sublimate, or 

 Kleinenberg's picro-sulphuric mixture. I give these reagents preference 

 in the order named ; the last, however, is not available in the older larval 

 or the adolescent stages, when the shell comes to contain considerable 

 quantities of lime salts. 



Vox this stage several stains have been used. I finally settled upon 

 Ehrlich's acetic acid-alum hajmatoxylin, however, as the most reliable 

 stain for the gland. For a double stain I have used this followed by acid 

 fuchsin or orange G, the latter being, in my opinion, preferable. 



