i6o Methods of Observing Micro-organisms 



water and counterstained with a saturated aqueous methylene-blue 

 solution for half a minute, washed again with water, dried, and 

 mounted in Canada balsam. 

 Abbott's method of staining spores is as follows: 



1. Stain deeply with methylene-blue, heating repeatedly until the stain 



reaches the boiling point- — one minute. 



2. Wash in water. 



3. Wash in 95 per cent, alcohol containing 0.2 to 0.3 per cent, of hydrochloric 



acid. 



4. Wash in water. 



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. 



Moller* 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 r : 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, sul- 

 phuric 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. 



FioccaJ 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. A very 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 



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



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



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



