308 THE TUBERCLE BACILLUS 



2. Pour off the stain and treat for a few seconds with a 33 per cent, solution 

 of nitric acid (distilled water 2 volumes, pure nitric acid 1 volume) or a 25 per 

 cent, solution of sulphuric acid (distilled water, 3 volumes, pure sulphuric 

 acid, 1 volume), [or 25 per cent, hydrochloric acid (Eastwood)]. The pre- 

 paration now assumes a yellowish tint. 



[This method of decolourization appears to be perfectly satisfactory in the case 

 of bacilli from cultures and was moreover the method adopted by Eastwood in his 

 work for the English Commission. But it is undoubtedly true that tubercle bacilli 

 direct from human and animal tissues in sputum, for example will sometimes 

 lose the stain in these strong acids. In searching for the tubercle bacillus therefore 

 in fresh material in which its presence is suspected it is recommended that a 2 '5 per 

 cent, solution of sulphuric or hydrochloric acid be used and that the film be not 

 exposed to the acid for a longer time than is absolutely necessary. ] 



3. Wash freely in water : the preparation should now be pale pink, and if 

 the pale pink colour does not appear, the decolourization has been insufficient 

 and the film must be treated with acid again. 



4. Pour a few drops of absolute alcohol on the film : when decolourization 

 is complete the film should be a very faint pink colour. 



By using alcohol decolourization can be pushed much further than would be possible 

 with acid since the latter would ultimately decolourize the tubercle bacillus. A 

 further advantage in using alcohol is that it decolourizes the smegma bacillus and 

 thus a possible source of error is eliminated. 



5. Wash well in water. Stain for a few moments with an aqueous solution 

 of methylene blue. 



6. Wash in water. Dry. Mount in balsam. 



Note. When it is merely a question of searching for the tubercle bacillus it is a 

 great advantage not to counterstain the background after decolourizing with alcohol ; 

 the tubercle bacilli are much more easily seen when they appear stained deep red 

 on an unstained or faintly pink background. 



For this purpose the above procedure is stopped at the end of Stage 4 and, after 

 washing, the preparation is examined in water. If after examination it be thought 

 desirable to keep the film it may be counterstained with blue and treated as described 

 above. 



This simpler method is particularly applicable when the bacilli are likely to be 

 present in small numbers ; in any case it renders the detection of the bacilli more 

 rapid, and beginners will find it of great use. 



2. Ehrlich's method. 



1. Stain the film for 5 minutes in the warm with aniline- violet. 



2. Decolourize in 33 per cent, nitric acid for a few seconds. 



3. Wash in water : continue the decolourization with absolute alcohol. 



4. Stain for a few seconds in the cold in a saturated aqueous solution of 

 vesuvin. 



5. Wash. Dry. Mount. 



The tubercle bacilli are stained violet, other structures brown. 



3. Gabbe's method. 



This method is merely a modification of Ziehl's but is less reliable and more 

 difficult. 



1. Stain with carbol-fuchsin as above. 



2. Decolourize and counterstain at the same time by dipping the film for a minute 

 in the following solution. 



Methylene blue, - - 2 grams. 



25 per cent, sulphuric acid, - - 100 c.c. 



3. Wash. Dry. Mount. 



The methods described by Stocquart, by Pithion and Roux (of Lyon) are modifica- 

 tions of the above but are of no interest. 







