THE TUBERCLE BACILLUS 589 



They are then treated with Gram's iodine solution one to five minutes; 

 5 per cent nitric acid one minute; 3 per cent hydrochloric acid ten sec- 

 onds; absolute alcohol and acetone equal parts, until decolorized. The 

 granules may be stained by other modifications of Gram's method. 

 Weiss 13 has devised a combination stain. One part of Much's methyl 

 violet is mixed with three parts of ZiehFs carbol-fuchsin and filtered; 

 slides are stained for twenty-four to forty-eight hours in the mixture. 

 They are then decolorized as in Much's method and counterstained with 

 Bismarck brown or safranin 1 per cent. Both acid-fast and Gram- 

 positive forms are stained by this method and in the red may be seen 

 blue-black granules. 



While the acid-fast group of bacteria is composed of a number of 

 organisms to be mentioned later, a few only of these offer difficulties of 

 differentiation from the tubercle bacillus. Those to be considered 

 practically are the bacillus of leprosy and that of smegma. The latter 

 bacillus, because of its distribution, is not infrequently found to con- 

 taminate feces, urine, or even sputum, and it is sometimes desirable to 

 apply to suspected specimens one or the other of the stains devised 

 for the differentiation of the smegma bacillus from Bacillus tuber- 

 culosis. The one most frequently employed is that of Pappenheim. 14 

 The preparations are stained in hot carbol-fuchsin as before; the 

 carbol-fuchsin is then poured off without washing and the preparation 

 immersed in solution made by saturating a 1 per cent alcoholic solu- 

 tion of rosolic acid with methylene-blue and adding 20 per cent of 

 glycerin. In such preparations tubercle bacilli remain red, smegma 

 bacilli appear blue. 



Stained by Gram, tubercle bacilli retain the gentian- violet. 



When tubercle bacilli are very sparsely present in sputum and other 

 material it may be impossible to find them by direct examination, 

 and often the only method of finding them will be animal inoculation. 

 However, a number of methods have been devised by which the bacilli 

 may be concentrated in such a way that they may be found even when 

 a few only are present. One of these is to add peroxide of hydrogen to 

 the sputum. By this the mucus is dissolved out and the solid particles 

 sstth or may be centrifugalized. A method very commonly employed 

 to-day is that which depends on the use of "antiformin." This is a 

 preparation used extensively for the cleansing of vats in breweries. It 



13 Weiss, Bcrl. klin. Woch., 1909, xlvi, 1797. 



14 Pappenheim, Berl. klin. Woch., 1898. 



