324 BACTEKIOLOGY. 



Recognition of the Tubercle Bacillus. 



Ziehl-Neelsen method. — A large loopful of the sputum is 

 transferred to a wide cover-glass and thoroughly spread 

 over the surface. It is then allowed to dry in the air or 

 by moving it to and fro, over the flame, after which it is 

 fixed in the usual way. The cover-glass is held in the 

 forceps, specimen side up, and covered with carbolic fuchsin 

 solution. It is warmed over a low flame for 1 to 2 minutes, 

 avoiding actual ebullition. The dye should not be allowed 

 to dry down on the cover-glass. A drop or two of the stain 

 should be added, from time to time, to prevent this from 

 happening. The excess of dye is then washed off with 

 water and the specimen is placed in dilute nitric acid for 

 10 to 15 seconds. This dilute acid solution is prepared by 

 adding 3 or 4 drops of the concentrated acid to a watch- 

 glass full of water. When the color disappears it is at once 

 transferred to dilute alcohol (60 to 70 per cent.), where it is 

 moved about till it is almost decolored. After this it is 

 washed with water and stained for a few seconds with 

 methylene blue. The latter is washed off with water and 

 the specimen is then examined with the Ath inch oil immer- 

 sion objective. It should show the bright red tubercle 

 bacillus on a light blue back-ground. The ordinary bac- 

 teria that may be present are stained likewise blue. It is 

 evident, therefore, that the ordinary bacteria are readily 

 decolored by treatment with acid and alcohol and in this 

 respect differ from the tubercle bacillus. This is due to a 

 difference in the chemical composition or to a concentra- 

 tion of the protoplasm of the tubercle cell, rather than to 

 the presence of a denser cell-wall. 



Heavily stained preparations can be obtained by float- 

 ing the prepared cover-glass on slightly warmed carbolic 

 fuchsin solution for 15 to 30 minutes, then decoloring as 

 above. 



