150 Methods of Observing Bacteria 



I have not found that spores usually color so easily, 

 and for many species the best method seems to be to place 

 the prepared cover-glass in a test-tube half full of carbol- 

 fuchsin : 



Fuchsin 1 



Alcohol 10 



Five per cent aqueous solution of phenol crys- 

 tals 100 



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. 



Holier t 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. 



Anjeszky J 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 to 

 ten minutes. 



*" Manual of Bacteriology," London, 1897. 

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

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



