482 PATHOGENIC MICROORGANISMS 



minutes, at the end of which time all elements in the preparation except 

 the acid-fast bacilli will be decolorized and counterstained. 



Tubercle bacilli in very young culture are often not acid-fast and it 

 is not always possible to demonstrate acid-fast bacilli in pus from cold 

 abscesses in sputum, in serous exudates, and in granulomatous lesions 

 of the lymph nodes which can be shown by animal inoculation to be 

 tuberculous. Much 1 demonstrated in such material Gram-positive 

 granules which lay singly in short chains or in irregular clumps, and 

 which he believed to be non-acid-fast tubercle bacilli. He found similar 

 granules in cultures of tubercle bacilli which showed on further incuba- 

 tion numerous acid-fast bacillary forms. His work has been repeatedly 

 confirmed, and there seems little doubt but that these granules are really 

 tubercle bacilli. Their demonstration is not, however, of great diag- 

 nostic value, as other bacilli form granules of the same appearance. 

 Small rods and splinters are also found which stain by Gram's method, 

 but not by carbol-fuchsin. 2 



To find "Much's granules," smears or sections are steamed in a 

 solution of methyl violet B.N. (10 c.c. of saturated alcoholic solution 

 of the dye in 100 c.c. of distilled water containing 2 per cent phenol). 

 They are then treated with Gram's iodine solution 1-5 minutes; 5 per 

 cent nitric acid 1 minute; 3 per cent hydrochloric acid 10 seconds; ab- 

 solute alcohol and acetone equal parts, until decolorized. The granules 

 may be stained by other modifications of Gram's method. Weiss 3 

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

 is mixed with three parts of Ziehl's carbol-fuchsin and filtered; slides 

 are stained for 24 to 48 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 t6 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 important to apply to 

 suspected specimens one or the other of the stains devised for the 

 differentiation of the smegma bacillus from Bacillus tuberculosis. The 



l Much, Berl. klin. Woch., 1908, xlv, 700. 

 2 Liebermeister, Deutsche med. Woch., 1909, xxxv, 1324, 

 3 Weiss, Berl. klin, Woch., 1909, xlvi, 1797, 



