1 66 Methods of Observing Bacteria 



Glanders bacilli appear dark violet on a colorless back- 

 ground; typhoid bacilli intense dark red violet. 



Method of Staining Spores. It has already been pointed 

 out that the peculiar quality of the spore capsules pro- 

 tects them to a certain extent from the influence of stains 

 and disinfectants. On this account they are much more 

 difficult to color than the adult bacteria. Several methods 

 are recommended, the one generally employed being as 

 follows : Spread the thinnest possible layer of material upon 

 a cover-glass, dry, and fix. Have ready a watch-crystalful 

 of Ehrlich's solution, preferably made of fuchsin, and drop 

 the cover-glass, prepared side down, upon the surface, where 

 it should float. Heat the stain until it begins to steam, 

 and allow the specimen to remain in the hot stain for from 

 five to fifteen minutes. The cover is then transferred to a 

 3 per cent, solution of hydrochloric acid in absolute alcohol 

 for about one minute. Abbott recommends that the cover- 

 glass be submerged, prepared side up, in a dish of this solu- 

 tion and gently agitated for exactly one minute, removed, 

 washed in water, and counterstained with an aqueous solu- 

 tion of methyl or methylene-blue. 



In such a specimen the spores should appear red, and the 

 adult organisms blue. 



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. 



* "Manual of Bacteriology," London, 1897. 



