NO. 3 EMBRYOLOGY OF FLEAS KESSEL 5 



does not rupture easily. The relatively thin chorion of the cat flea 

 egg was an added factor in making dechorionation in this species a 

 simple matter. This membrane collapses as soon as it is punctured and 

 is thereafter quite easily removed under the binocular by dissecting 

 it away with a fine pointed needle. 



Because the eggs of Nosopsylhis fasciatus and Hystrichopsylla 

 dippiei have thicker shells and therefore do not ordinarily collapse 

 when punctured, a different procedure was necessary for the removal 

 of their chorions. After heating, the surfaces of these eggs were 

 dried thoroughly with blotting paper and they were immersed in 

 liquid paraffin on a glass slide. When the paraffin had hardened, a 

 series of needle punctures was made completely around the egg 

 through both the paraffin and the chorion. Next, the adjacent punc- 

 tures in the paraffin were connected and the paraffin cap, thus formed, 

 was removed. Finally, the punctures through the exposed chorion 

 were connected and the upper surface of this shell was removed in one 

 piece. The shrunken vitellus with its attached embryo was lifted 

 through the resulting opening by means of a minute spatula. De- 

 chorionated eggs were fixed in Bouin's picro-formol-acetic fluid, 

 stained with alcoholic borax-carmine, destained in 70 percent acid 

 ethyl alcohol, cleared in cedar oil, and mounted in xylol-balsam. 



Eggs used for sectioning were neither heated nor dechorionated. 

 However, owing to the impermeability of the chorion, this membrane 

 was punctured before fixation. Puncturing was done in the same 

 Bouin's fluid which was to serve as the fixing reagent. Several punc- 

 tures were made in each egg. Tertiary butyl alcohol, as recommended 

 by Johansen (1935), was used for washing and dehydration. Im- 

 bedding was accomplished by a combination technique involving 

 modifications of two previously used methods. These were Boycott's 

 well-known paraffin-celloidin technique and Walls' (1932) adaptation 

 of the hot-celloidin process to animal tissues. From absolute tertiary 

 butyl alcohol, the eggs were placed in a 2 percent solution of celloidin 

 dissolved in ether-alcohol. Infiltration was done in small glass tubes, 

 3 mm. in diameter. These were cut into short lengths and closed at 

 one end. Such infiltration bottles, containing the eggs in 2 percent 

 celloidin, were placed, uncorked, in a metal cylinder capable of with- 

 standing high pressure. After being sealed, this cylinder was placed 

 in an electric oven with the temperature set at 6o° C. where the heat, 

 acting on the ether-alcohol solvent, provided the necessary pressure 

 to force the celloidin into the tissue. After 24 hours the cylinder was 

 removed from the oven, cooled, and opened. At this point the step 

 from Boycott's method was incorporated into the procedure. About 



