Staining Spirocfleta pallida in Tissue. 83 



1. Warthin and Starry's Cover-glass Method. 



1. Fix tissues in 4 per cent, formol. 



2. Wash thoroughly in distilled water. 



3. Imbed in paraffin (alcohol, xylol, paraffin). 



4. Cut; mount sections on cover-glasses with albumin fixative. 



5. Remove paraffin from section (xylol, alcohol, water). 



6. Place cover-glass in a saturated solution of ferric alum, or a 

 4 per cent, solution of ferrous ammonium sulphate, in incubator 

 for 1 to 2 hours. 



7. Wash in distilled water. 



8. Rinse cover-glass with section in a 2 per cent, silver nitrate 

 solution. Cover section with another perfectly clean cover-glass 

 which has also been rinsed in the silver solution, so that the cover- 

 glasses are held together by capillary attraction. Then place 

 them carefully on the bottom of a wide-mouthed dark bottle 

 covered with black paper, and cover them with the silver nitrate 

 solution. Cork tightly, and put into incubator for 3 to 24 hours. 



9. After impregnation pour off the silver nitrate solution 

 and rinse in distilled water without removing cover-glasses from 

 bottle, by pouring the water into the bottle, shaking gently, and 

 then pouring off. 



10. Pour reducing fluid (pyrogallic acid, 4 grams; 40 per cent, 

 formol, 5 cc; distilled water, 100 cc.) into the bottle. See that 

 fluid passes between cover-glasses by pressing upon them with a 

 glass rod, or by shaking. Reduction is almost instantaneous; 

 it should occur evenly over the section or brown lines will result. 

 After 2 to 3 minutes remove cover-glasses, separate, wipe off with 

 a cloth any precipitate on the albumin fixative about the section, 

 taking care not to touch the latter. 



1 1 . Wash in distilled water. 



12. Dehydrate in absolute alcohol, clear in xylol, then balsam. 

 The reduced section should have a faint, dull brownish-yellow 



color. If the color is bright yellow the organisms will be poorly 

 stained. The spirochetes should have a deep reddish brown color 

 contrasting sufficiently well with the background, if the pro- 

 cedure has been successful. 



