Staining 157 



5. Stain for eight to ten seconds in anilin-fuchsin solution. 



6. Wash in water. 



7. Dry. 



8. Mount in balsam. 



The spores are blue; the bacteria, red. 



MoUer* finds it advantageous to prepare the films, before staining, 

 by immersion in chloroform for two minutes, following this by 

 immersion in 5 per cent, chromic acid solution for one-half to two 

 minutes. 



The exact technic is as follows: 



1. Treat the spread with chloroform for two minutes. 



2. Wash with water. 



3. Treat with 5 per cent, solution of chromic acid for one-half to two minutes. 



4. Wash in water. 



5. Stain with carbol-fuchsin, slowly heating until the fluid boils. 



6. Decolorize in 5 per cent, aqueous sulphuric acid. 



7. Wash well with water. 



8. Stain in a i : 100 aqueous solution of methylene-blue for thirty seconds. 

 The spores should be red and the bacilli blue. 



Anjeszkyt recommends the following method of staining spores, 

 which is said always to give good results even with anthrax bacilli: 

 A cover-glass is thinly spread with the spore-containing fluid and 

 dried. While it is drying, some 0.5 per cent, hydrochloric acid is 

 warmed in a porcelain dish over a Bunsen flame until it steams well 

 and bubbles begin to form. When the solution is hot and the smear 

 dry, the cover-glass is dropped upon the fluid, which is allowed to act 

 upon the unfixed smear for three or four minutes. The cover is 

 removed, washed with water, dried, and fixed for the first time, then 

 stained with Ziehl's carbol-fuchsin solution, which is warmed twice 

 until fumes arise. The preparation is allowed to cool, decolorized 

 with a 4-5 per cent, sulphuric acid solution, and counterstained for 

 a minute or two with malachite green or methylene-blue. The whole 

 procedure should not take longer than eight or ten minutes. 



Fioccat suggests the following rapid method: "About 20 cc. of 

 a 10 per cent, aqueous solution of ammonium are poured into a 

 watch-glass, and 10 to 20 drops of a saturated solution of gentian 

 violet, fuchsin, methyl blue, or safranin added. The solution is 

 warmed until vapor begins to rise, then is ready for use. Avery 

 thinly spread cover-glass, carefully dried and fixed, is immersed for 

 three to five minutes (sometimes ten to twenty minutes), washed in 

 water, washed momentarily in a 20 per cent, solution of nitric or 

 sulphuric acid, washed again in water, then counterstained with an 

 aqueous solution of vesuvin, chrysoidin, methyl blue, malachite 

 green, or safranin, according to the color of the preceding stain. 

 This whole process is said to take only from eight to ten minutes, and 

 to give remarkably clear and beautiful pictures." 



*"Centralbl. f. Bakt. u. Parasitenk.," Bd. x, p. 273. 



t Ibid., Feb. 27, 1898, xxiii, No. 8, p. 329. 



t "Centralbl. f. Bakt. u. Parasitenk.," July i, 1893, xiv, No. i. 



