Staining 149 



Zieler* recommends for the staining of the typhoid, 

 glanders and other difficultly stainable bacteria, the follow- 

 ing method of demonstration in the tissues :- 



1. Fix and harden in Miiller-formol solution. 

 Paraffin imbedding. 



2. Staining overnight in Orcein D. (Griibler), 0.1 



Officinal " schwef elsaiire " (sulphuric acid), 2.0 



70 per cent, alcohol, 100.0 



3. Washing in 70 per cent, alcohol for a short time to remove 



the excess of 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 1 : 2-5 water until 



the tissues become pale blue. 



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 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. 



* "Centralbl. f allg Path, u path. Anat .," Bd. xiv, Xo. 14, p 561 



