588 PATHOGENIC MICROORGANISMS 



either with 5 per cent nitric acid, 5 to 20 per cent sulphuric acid, or 1 

 per cent hydrochloric acid, until most of the red color has disappeared 

 (a few seconds), and the preparation appears pale pink. This results in 

 decolorization of all microorganisms with the exception of members of 

 the acid-fast group. Thorough washing in 80 to 95 per cent alcohol 

 is now employed to complete the decolorization. The preparation is 

 then rinsed in water and counterstained with 1 per cent aqueous methy- 

 lene-blue. 



Tubercle-bacillus staining has been further simplified by Gabbett, 10 

 who combines decolorization and counterstaining. In this method 

 preparations are stained with Ziehl's carbol-fuchsin as in the preceding; 

 they are then rinsed in water and covered with a solution containing 

 methylene-blue 1 gram, concentrated sulphuric acid 25 grams, and 

 distilled water 100 c.c. This is allowed to act for from two to four min- 

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

 the acid-fast bacilli will be decolorized and counterstained. 



Another excellent stain for tubercle bacilli, which has the advantage 

 of greater clearness of contrast over the carbol-fuchsin stain is that of 

 Hermann in which Crystal Violet is used. It is described in the section 

 on staining. 



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 n 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. 12 



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.o. of distilled water containing 2 per cent phenol). 



10 Gabbett, Lancet, 1887. 



11 Much, Ben. klin. Woch., 1908, xlv, 700. 



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



