hungerford: anatomy of simulium vittatum. 371 



used only to verify the other studies by giving a different 

 point of view. 



SECTIONAL STUDY. 



Killing and Fixing. In order to determine the best method 

 of fixing and staining the adult flies it was necessary to try 

 a number of fixations and stains. 



The flies were divided into lots and killed and fixed in the 

 following ways : 



Six lots were treated with picro-aceto-sublimate for 12, 18, 

 and 24 hours respectively. When they had been in the fluid 

 the specified length of time, the liquid was carefully drawn oflF 

 with a pipette, and the vials filled with 70 per cent grain 

 alcohol. At the end of 24 hours the alcohol was changed by 

 the pipette method as above, and this process of changing the 

 alcohol repeated for 3 days. At the end of that time the 70 

 per cent was replaced with 85 per cent alcohol and the lots 

 stored. 



Six lots were captured and placed in picro-formal for 12 

 hours, 18 hours, and until they sank, respectively. They were 

 then washed out with 70 per cent alcohol as for the picro- 

 aceto-sublimate and stored in 85 per cent alcohol. 



Two lots were fixed in Fleming's stronger mixture. The 

 flies were allowed to remain in the fixing fluid until they sank. 

 Then they were changed to water and washed for three days. 

 From the water they were transferred to 95 per cent alcohol. 



Six lots of live sand flies were fixed in Gilson's chloro-aceto- 

 sublimate for 10 minutes, 20 minutes, and until they sank. 

 They were then washed in 70 per cent alcohol for several days 

 and stored in 85 per cent alcohol. 



Six lots were fixed in Zenker's fluid for 6 hours, 12 hours, 

 and until they sank, respectively. They were then washed out 

 in water and transferred to alcohol containing tincture of 

 iodine. 



In the above series the chloro-aceto-sublimate gave the best 

 fixation. 



C. G. Hewett, '07, in his work with the house fly used Hen- 

 ning's solution made up as follows: Nitric acid 16 parts, 

 chromic acid (.5 per cent) 16 parts, picric acid saturated in 

 water 12 parts, and absolute alcohol 42 parts. After fixing 

 the material he washed out in iodine water. This method he 

 finds not only fixes but to a certain extent softens the chitin, 



