THE TUBERCLE BACILLUS 483 



one most frequently employed is that of Pappenheim. 1 The preparations 

 are stained in hot carbol-fuchsin as before; the carbol-fuchsin is then 

 poured off without washing and the preparation immersed in a solution 

 made by saturating a 1 per cent alcoholic solution of rosolic acid with 

 methylene-blue and adding 20 per cent of glycerin. In such prepara- 

 tions 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 

 settle 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 is described by Rosenau 2 as consisting of equal parts of liquor sodse 

 chlorinatse and a 15 per cent solution of caustic soda. The formula for 

 liquor sodse chlorinatse he gives as: 



Sodium carbonate 600 



Chlorinated lime 400 



Distilled water 4,000 



If sputum is poured into a 10 to 15 per cent solution of antiformin 

 and allowed to stand for several hours, most of the other elements of 

 the sputum, cells, and bacteria, will dissolve out, and acid-fast bacilli 

 be left in the residue. Strangely enough they are not killed by this- 

 process and if sufficiently washed may be cultivated or can produce 

 lesions in guinea-pigs. 



Isolation and Cultivation. Tubercle bacilli are not easily cultivated. 

 Their slowness of growth precludes isolation by plating. The first isola- 

 tions by Koch 3 were made upon coagulated blood serum from tuber- 

 culous tissue. 



Isolation from tuberculous material may be aided by inoculation 

 into guinea-pigs. These animals will withstand the acute infection 



1 Pappenheim, Berl. klin. Woch., 1898. 



2 Rosenau, "Preventive Medicine and Hygiene," D. Appleton, N. Y., 1913; 

 Uhlenhuth, Berl. klin. Woch., No. 29, 1908. 



3 Koch, loc. cit. 



