168 BACTERIOLOGY 



Nitric acid in from 20 to 30 per cent, watery solution. 



Sulphuric acid in from 5 to 10 per cent, solution in water. 



Hydrochloric acid in from 1 to 3 per cent, solution in 

 alcohol. 



NOTE. For details as to the technique of hardening and 

 cutting sections and staining bacteria in tissues, the student 

 is referred to Mallory and Wright's Pathological technique. 



Method of Staining the Tubercle Bacillus. Select from the 

 sputum of a tuberculous subject one of the small, white, 

 cheesy masses which it is seen to contain. Spread this 

 upon a cover-slip, dry and fix it in the usual way. The slip 

 is now to be taken by its edge with forceps and the film 

 covered with a few drops of either the solution of Koch- 

 Ehrlich or that of Ziehl. It is then held over a gas-flame, 

 at first some distance away, gradually being brought nearer 

 until the fluid begins to boil. After it has bubbled once or 

 twice it is removed from the flame, the excess of stain washed 

 away in a stream of water, then immersed in a 30 per cent, 

 solution of nitric acid in water, and allowed to remain until 

 all color has disappeared. This takes longer in some cases 

 than in others. One can always determine if decolorization 

 is complete by washing off the acid in a stream of water. 

 If the preparation is still distinctly colored, it should be 

 immersed again in the acid; if of only a very faint color, 

 it may be dipped in alcohol, again washed in water, and 

 stained with some contrast-color. If, for example, the 

 tubercle bacilli have been stained with fuchsin, methylene- 

 blue forms a good contrast-stain. In making the contrast- 

 stain the steps in the process are exactly those followed in 

 the ordinary staining of cover-slip preparations in general: 

 the slip containing the stained tubercle bacilli is carefully 

 rinsed in water, and a few drops of the methylene-blue 



