310 THE TUBERCLE BACILLUS 



7. Lustgarten's method (modified). 



Sabouraud, by slightly modifying the method devised by Lustgarten for staining 

 his so-called bacillus of syphilis, has perfected a method of staining the tubercle 

 bacillus which he affirms to be very delicate and precise. The method is as follows : 



1- Stain the film in Ziehl's solution in the cold for 1 or 2 hours or at 50 C. for 15 

 minutes. 



2. Treat the film for 1-3 seconds with a 1 *5 per cent, solution of potassium per- 

 manganate. 



3. Dip at once into a freshly prepared, saturated, aqueous solution of sulphurous 

 acid for a few seconds until the film is decolourized. 



The sulphurous acid solution can be conveniently prepared by bubbling the gas 

 from a cylinder of liquefied sulphurous acid through distilled water. 



4. Wash in water and counterstain with an aqueous solution of methylene blue 

 for 1-3 minutes. 



5. Wash in water. Dry. Mount in balsam. 



8. Koch's method. 



This, the earliest method used for the detection of the tubercle bacillus, is chiefly of 

 historical interest. 



1. Place the films for 1 day at room temperature or for a few hours at 45-50 C. 

 in the following bath : 



Saturated alcoholic solution of methylene blue, - 1 c.c. 



10 per cent, aqueous solution of potash, - 2 



DistiUed water, - 200 



2. Transfer the films to a saturated aqueous solution of vesuvin ; in about a 

 quarter of an hour a brown tint takes the place of the original blue colour save 

 in the tubercle bacilli which still retain the blue stain. 



B. The staining of sections. 



The methods just described are applicable with slight modification to the 

 staining of sections : but in this case it is essential that the staining should 

 always be done in the cold. 



1. Ziehl-Neelsen's method. 

 Method recommended. 



1. Stain the section in the cold for 15-30 minutes in Ziehl's fuchsin. 



2. Decolourize in the acid solution for a few seconds. Wash in water. 



3. Continue the decolourization with absolute alcohol until the section is 

 a pale pink colour. Wash in water. 



4. Stain the groundwork with an aqueous solution of methylene blue. 



5. Wash. Pass rapidly through absolute alcohol, clove oil, and xylol. 

 Mount in balsam. 



2. Kiihne's method. 

 Method recommended. 



The following unpublished method of Kuhne has been quoted by Borrel. 

 It is particularly useful for staining sections of lung. The action of the 

 hydrochloride of aniline, which is the decolourizing agent used, is not so rough 

 as that of mineral acids and does not alter the arrangement and shape of 

 the cells. 



1. Stain the section for 2 minutes in Bcehmer's hsematoxylin or hsematein 

 (p. 218) to stain the nuclei of the cells. Wash in distilled water. 



2. Stain in the cold with Ziehl's fuchsin for a quarter of an hour. 



3. Decolourize for 30-60 seconds in a 2 per cent, aqueous solution of aniline 

 hydrochloride. 



4. Continue the decolourization with absolute alcohol. 



The cells of the groundwork are now unstained with the exception of the nuclei. 





