108 MICROSCOPIC METHODS 



other mordant will stain the bacilli named, but the following 

 methods are most commonly used : 



Ziehl-Neelsen Carbol-Fuchsin Stain. 



Basic fuchsin . . . 1 part. 



Absolute alcohol . . . . 10 parts. 

 Solution of carbolic acid (1 : 20) . 100 



1. Place the specimen in this fluid, and having heated it till steam 

 rises, allow it to remain there for five minutes, or allow it to remain in 

 the cold stain for from twelve to twenty-four hours. (Films and paraffin 

 sections are usually stained with hot stain, loose sections with cold ; in 

 hot stain the latter shrink.) 



2. Decolorise with 20 per cent, solution of strong sulphuric acid, nitric 

 acid, or hydrochloric acid, in water. In this the tissues become yellow. 



3. Wash well with water. The tissues will regain a faint pink tint. 

 If the colour is distinctly red, the decolorisation is insufficient, and the 

 specimen must be returned to the acid. As a matter of practice, it is 

 best to remove the preparation from the acid every few seconds and 

 wash in water, replacing the specimen in the acid and re-washing till 

 the proper pale pink tint is obtained. Then wash in alcohol for half a 

 minute, and replace in water. 



4. Contrast stain with a saturated watery solution of methyleue-blue 

 for half a minute, or with saturated watery Bismarck-brown for from 

 two to three minutes. 



5. Wash well with water. In the case of films, dry and mount. In 

 the case of sections, dehydrate, clear, and mount. 



Fraenkel's Modification of the Ziehl-Neelsen Stain. 



Here the process is shortened by using a mixture containing 

 both the decolorising agent and the contrast stain. 



The sections or films are stained with the carbol-fuchsin as above 

 described, and then placed in the following solution : 



Distilled water ...... 50 parts. 



Absolute alcohol 30 ,, 



Nitric acid . . . . . 20 ,, 



Methylene-blue in crystals to saturation. 



They are treated with this till the red colour has quite disappeared and 

 been replaced by blue. The subsequent stages are the same as in No. 5, 

 supra. 



Leprosy bacilli are stained in the same way, but are rather 

 more easily decolorised than tubercle bacilli, and it is better 

 to use only 5 per cent, sulphuric acid in decolorising. 



In the case of specimens stained either by the original Ziehl- 

 Neelsen method, or by Fraenkel's modification, the tubercle or 



