202 PATHOGENIC BACTERIA. 



a considerable distance around it becomes greenish- 

 brown. (See Frontispiece.) No other known organism 

 produces the same appearance upon potato. 



The organism loses its virulence if cultivated for many 

 generations upon artificial media. 



That this bacillus is the cause of glanders there is no 

 room to doubt. L,6ffler and Schiitz have succeeded by 

 the inoculation of horses and asses in producing the 

 well-known disease. 



The organisms when in cultures can be stained with 

 the watery anilin-dye solutions, but are difficult to stain 

 in tissues. They do not stain by Gram's method. 



The chief difficulty in staining the bacillus in tissues 

 is the readiness with which it gives up the stain in the 

 presence of decolorizing agents. Loffler at first accom- 

 plished the staining by allowing the sections to lie for 

 some time (five minutes) in the alkaline methylene-blue 

 solution, then transferring them to a solution of sulphuric 

 and oxalic acids 



Concentrated sulphuric acid, 2 drops ; 



5 per cent, oxalic-acid solution, i drop ; 



Distilled water, 10 c.cm. 



for five seconds, then transferring to absolute alcohol, 

 xylol, etc. The bacilli appear dark blue upon a paler 

 ground. This method gives very good results, but has 

 been largely superseded by the use of Kuhne's carbol- 

 methylene blue : 



Methylene blue, 1.5 



Alcohol, 10. 



5 per cent, aqueous phenol solution, 100. 



Kiihne's method of staining is to place the section in the 

 stain for about half an hour, wash in water, decolorize 

 carefully in hydrochloric acid (10 drops to 500 c.cm. of 

 water), immerse at once in a solution of lithium carbonate 

 (8 drops of a saturated solution of lithium carbonate in 10 

 c.cm. of water), place in a bath of distilled water for a few 



