io8 Journal of Agricultural Research voi. iv, N0.2 



caterpillars, if small, were either punctured or split open dorsally before being put 

 into the solution or, if large, were cut crosswise into three or four pieces and allowed 

 to fix for 48 hours. After using the fixing fluid, the caterpillars were immersed in 

 95 per cent alcohol, then in 100 per cent alcohol, and next in cedar oil, in which the 

 material was cleared for 48 hours. The specimens were then embedded in paraffin, 

 and sections were cut of the thickness of 2, 3, 4, or 5/i. 



The process of staining and differentiation used is based upon Wolbach's modifica- 

 tion of Giemsa's method.' The various processes to which the cut sections were sub- 

 jected according to this method can be followed best in a numerical series. 



(i) Xylol; (2) absolute alcohol; (3) 95 per cent alcohol; (4) iodin alcohol (100 c. c. 

 of 70 per cent alcohol plus 3 or 4 c. c. of a sattu-ated alcoholic solution of iodin); (5) 

 95 per cent alcohol; (6) distilled water; (7) hyposulphite of soda (0.5 per cent) in dis- 

 tilled water; (8) washing sections in running tap water for five minutes; (9) rinsing in 

 distilled water; (10) staining with Giemsa's solution, changing stain twice at one- 

 half hour intervals, leaving sections in third solution overnight (about 12 to 15 hours); 

 (11) acetone-colophonium mixture for about one minute (this differentiates and 

 consists of 30 gm. of colophonium to 200 c. c. of acetone) ; (12) acetone-xylol mixture, 

 which stops the destaining and consists of 70 c. c. of xylol and 30 c. c. of acetone; 

 (13) xylol; (14) mounting in thick cedar oil. 



The Giemsa's solution was made from the stains of Griibler's manufacture. At 

 first the mixture was bought all ready "made up " by local chemists, but the results 

 were unsatisfactory and too variable to be depended upon. It w-as not until the 

 writer made his own mixture that Giemsa's stain was a complete success. 



The stock solution is made up as follows: 



Azur II eosin 3 gm. 



Azur II o. 8 gm. 



Methyl alcohol (c. p.) 375 gm. 



Glycerin (c. p.; Merck's, sp. gr., 1.250) 125 gm. 



The staining solution is made when needed from the stock solution, as follows: 



Distilled water 100 c. c. 



Methyl alcohol 4 c. c. 



Stain, 40 drops from an eye dropper; 0.5 per cent sodium carbonate, 2 drops from 

 an eye dropper. 



Another stain following the corrosive-sublimate fixation and giving very good 

 results is Unna's polychrome blue, which consists of a i per cent aqueous solution of 

 methylene blue to which has been added i gm. of sodium carbonate. This is allowed 

 "to ripen" for one week. Following are the steps in the staining process to which 

 the sections are subjected : 



(i) 5 per cent aqueous w. g. eosin for 20 minutes; (2) Unna's polychrome blue 

 10 c. c. and 100 c. c. of water till the sections areadeep blue; (3) tap w^ater; (4) differ- 

 entiation in 95 per cent alcohol containing 10 per cent of resin; (5) absolute alcohol, 

 xylol, Canada balsam. 



Some good slides were obtained by fixing with Kahle's fluid and by staining with 

 iron haematoxylin and orange G : but this method was not as satisfactory as another 

 one suggested by Prof. Gary N. Calkins, in which the material is fixed for an hour in a 

 fluid consisting of 20 per cent of glacial acetic acid and 80 per cent of saturated aqueous 

 corrosive sublimate. The sections are mordanted for 12 hours in a 4 per cent solution of 

 ferric alum, after which they are stained in a 0.5 per cent solution of aqueous iron 

 haematoxylin for 12 to 24 hours. No counterstain is used, for by differentiating with 

 the ferric alum the staining can be stopped at a point where both nucleus and 

 cytoplasm are nicely colored. 



1 Wolbach, S. B. The filterable viruses. A summary. In Jour. Med. Research, v. 27 (n. s. v. 22), 

 no. I, p. I-2S, I fig. 



