136 MICROBIOLOGY 



the most important of this group. The Ziehl-Neelson method 1 

 is the one most used. Thin smears of the suspected material 

 are made on cover-glasses or slides, fixed by heat and stained 

 in carbol-fuchsin solution. The slides or cover-glasses may 

 be flooded with the stain, and gently heated over a flame until 

 steam is given off, or else the cover- glass may be inverted upon 

 the surface of the staining fluid, in a porcelain dish or watch- 

 glass, and this heated until it steams. The heat is continued 

 for three to five minutes. Decolorize with either five per cent 

 nitric acid, 10% sulphuric acid, or 3% hydrochloric acid for 

 three to five seconds. A 3^ hydrochloric acid in 95% alcohol 

 is an excellent decolorizer. It has been recommended as 

 being of value in differentiating tubercle bacteria from other 

 acid fast organisms. It should not be used therefore in 

 staining saprophytic acid fast organisms. The treatment 

 with the acid is continued until subsequent washing with 

 water will give only a faint pink color to the preparation. 



If the stain is for tubercle bacteria, after treating with 

 the acid wash with 95% alcohol until no further color can be 

 removed. If, after prolonged washing with alcohol, a red 

 color still remains in very thick places only upon the smear, 

 it may be disregarded. If a counter stain is desired wash the 

 preparation in water and stain with Loeffler 's methylene blue 

 from 10 to 30 seconds. Again rinse in water, dry and mount. 

 By this method the tubercle bacteria are colored red, other 

 "bacteria and cellular elements which may be present are 

 stained blue. 



Gabbett 2 has devised a rapid method in which the de- 

 colorization and counterstaining are accomplished by one 

 solution. The specimen is prepared and stained with carbol 

 fuchsin as in the preceding method. It is then immersed for 

 one minute directly in the following solution : 



Methylene blue 2 gram* 



Sulphuric acid 25 per cent (sp. grav. 1018) 100 cc. 



1 Ziehl. Deut. med.'woch., 1882, p. 451. 

 Neelson. Deut. med. Woch., 1882. 



2 Gabbett. Lancet, 1887- i, p. 757. 



