156 Methods of Observing Micro-organisms 



8. Washing in distilled water. 



9. Seventy per cent, alcohol. 



10. Absolute alcohol. 



11. Xylol. 



12. Balsam. 



Glanders bacilli appear dark violet on a colorless background; 

 typhoid bacilli intense dark red violet. 



Method of Staining Spores. It has already been pointed out that 

 the peculiar quality of the spore capsules protects them to a certain 

 extent from the influence of stains and disinfectants. On this ac- 

 count 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 solution and 

 gently agitated for exactly one minute, removed, washed in water, 

 and counterstained with an aqueous solution of methyl or methylene- 

 blue. 



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

 organisms blue. 



A good simple method is to place the prepared cover-glass in a 

 test-tube half full of carbol-f uchsin : 



Fuchsin i 



Alcohol 10 



Five per cent, aqueous solution of phenol crystals 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 prepared and 

 stained as for tubercle bacilli (q.v.), decolorized 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. 



* "Manual of Bacteriology," London, 1897. 



