HUNGERFORD: ANATOMY OP SIMULIUM VITTATUM. 373 



Cutting and Mounting. When the flies were infiltrated they 

 were imbedded and cut in serial sections of from 4 to 15 

 microns in thickness. 



The dry paraffin ribbons were placed on an absolutely clean 

 slide and floated with prepared egg albumen. The albumen 

 was then drained ofi" and the slide held just enough below the 

 surface of tepid distilled water to float out the folds and allow 

 the ribbons to be arranged. The slides were then air-dried 

 for one week, after which they were warmed enough to melt 

 down the paraffin and placed in xylol for ten minutes. From 

 the xylol they were passed down through the alcohols to the 

 grade used in the stain. 



Staining. Five stains were used in the studies made with 

 the Simitlium fly. 



I. For thin sections to be studied for parasitic forms 

 Gemsa's Lasung was used. This stain was diluted 1 drop to 

 1 cc. of water made faintly alkaline with weak potassium 

 carbonate (1%). The sections were stained with this for 

 fifteen minutes. (With this stain chromatin is stained blue 

 and protozoa and bacteria pinkish.) Dift'erentiation was made 

 with tap water. After dehydrating in the alcohols they were 

 transferred to xylol and from the xylol to the balsam mount. 



II. For ordinary work the sections of any thickness were 

 run down to 50'v alcohol and stained for twelve hours in 

 borax carmine, dift'erentiated in acid alcohol, dehydrated and 

 mounted as above. 



III. Iron hsematoxylin was used in the usual way. 



IV. Good results were obtained by overstaining with Dela- 

 field's hsematoxylin and difi'erentiating with acid alcohol as 

 suggested by Hewett. 



V. The best results were obtained by running the sections 

 dovvm to 25% alcohol and staining in ferric alum for 4 hours, 

 then Hjedenhain's hsematoxylin (5% aq. solution) for 12 

 hours. Following this they were bleached in ferric alum, 

 placed in orange g. and dipped back and forth in the two 

 stains till proper results were obtained as determined by fol- 

 lowing the action of the stains under the low-power compound. 

 Then they were rehydrated, passed up through xylol, and 

 mounted. 



