Staining 159 



1. Fix and harden in Muller-formol solution. 

 Paraffin imbedding. 



f Orcein D o.i 



2. Staining overnight in \ Officinal sulphuric acid 2. 



\ 70 per cent, alcohol 100. 



3. Washing in 70 per cent, alcohol for a short time to remove the excess o£ 



orcein. 



4. Washing in water. 



5. Staining in polychrome methylene-blue ten minutes to two hours. 



6. Washing in distilled water. 



7. Thorough differentiation in glycerin-ether i : 2-5 water until the tissues 



become pale blue. 



8. Washing in distilled water. 



9. Seventy per cent, alcohol. 

 10. Absolute alcohol. 



. II. 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 diflacult 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-crystalf ul of Ehrlich's 

 solution, preferably made of fuchsin, and drop the cover-glass, 

 prepared side doAvn, 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-fuchsin: 



Fuchsin , r 



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 bacilh {q.v.), decolorized with a i per cent, 

 sulphuric acid solution in water or methyl alcohol, then washed in 



* "Manual of Bacteriology," London, 1897. 



