608 CAROLINE M. HOLT 



and the thoughtful criticism which he has given so generously 

 throughout the progress of the investigation; to Miss Eleanor 

 Carothers to whom I am indebted for a part of the slides ; and 

 to my co-laborers, Dr. Phineas Whiting, and Mr. Robert 

 Hance, whose reports on other phases of the study have ap- 

 peared separately. 



TECHNIQUE 



Larvae and pupae of Culex pipiens were taken during the fall 

 and winter from tubs of water in the University greenhouses 

 and from the alligator-tank in the Vivarium. These were kept 

 in jars in the laboratory and a certain number of adults allowed 

 to emerge, to be identified as a check in the identification of the 

 pupae and larvae. Nothing but Culex pipiens was ever found in 

 this material. 



The observations for this portion of the work were all made 

 upon sections of pupae. The abdomens were cut off just pos- 

 terior to the thorax and immediately placed in fixing fluid. 

 Some twenty methods of preparation were tried out, including 

 fixation in six different modifications of Bouin and Flemming; 

 with heat, cold, and room temperature; and for varying lengths 

 of time. The method which gave the best results and which was 

 finally adopted was as follows : 



Fixation. One to six days at about 100° F. in "P. F. A. 3." 



Picric acid 75 parts 

 Formalin 15 parts 

 Acetic acid 10 parts 



Length of fixation after the first twenty-four hours seemed to make little 

 difference in the results. 



Dehydration. Very gradual, by drop method. 



Clearing in xylol. 



Embedding in paraffin. 



Staining. Iron haematoxylin, twelve to twenty-four hours. Cleared in 

 xylol and mounted in balsam. 



All of the slides upon which this study is based are in the col- 

 lection of the Zoology Laboratory of the University of Pennsyl- 

 vania, except one prepared by Dr. Stevens and now in the Bryn 



