106 MICKOSCOPIC METHODS 



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 methylene -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. 



Fraenhel'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 , , 



Mtric 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 

 leprosy bacilli ought to be bright red, and the. tissue blue or 

 brown, according to the contrast stain used. Other bacteria 

 which may be present are also coloured with the contrast stain. 



The Staining of Spores. — If bacilli containing spores' are 

 stained with a watery solution of a basic aniline dye the spores 

 remain unstained. The spores either take, up the stain less 

 readily than the protoplasm of the bacilli, or they have a resisting 

 envelope which prevents the stain from penetrating to the proto- 

 plasm. Like the tubercle bacilli, when once stained they retain 

 the colour with considerable tenacity. The following is the 

 simplest method for staining spores : — 



