1 88 Methods of Observing Micro-organisms 



and boil it for at least fifteen minutes, after which it is 

 decolorized, either with 3 per cent, hydrochloric or 2-5 per 

 cent, acetic acid, washed in water, and counterstained blue. 



Muir and Ritchie * recommend that cover-films be pre- 

 pared and stained as for tubercle bacilli (q. V.), decolor- 

 ized with a i per cent, sulphuric acid solution in water or 

 methyl alcohol, then washed in 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. 



Mollerf finds it advantageous to prepare the films, before 

 staining, by immersion in chloroform for two minutes, fol- 

 lowing this by immersion in 5 per cent, chromic acid solu- 

 tion for one-half to two minutes. 



The exact technic is as follows: 



1. Treat the spread with chlororoform 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. 



Anjeszky { 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 



* "Manual of Bacteriology," London, 1897. 



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



i Ibid., Feb. 27, 1898, xxm, No. 8, p. 329. 



