BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS 115 



the cover-glass and held over the flame until the stain boils; 

 fresh stain is added, the boiling continued for a minute. 

 Then the excess of stain is removed with edge of filter-paper. 

 Decolorize in 25 per cent, nitric or 2 per cent, hydrochloric 

 acid. The excess of acid is then washed out with 95 per 

 cent, alcohol until no further color is imparted to the alcohol, 

 and the smear is gray or light pink in color. The preparation 

 is then washed with water and counterstained with aqueous 

 methylene-blue for ten to thirty seconds. 



The Rapid Method (B. FrankeVs Method, Modified by Gab- 

 bet). The principle is to combine with the contrast stain the 

 decolorizing agent; but the preparations are not permanent; 

 the method, however, is very useful. 



Two solutions are required: one of Ziehl's carbolfuchsin; 

 the other, Gabbet's acid methylene-blue. (See Formula No. 

 x, on p. 51.) 



The cover-glass containing the dried sputum is passed 

 three times through the flame, as described in the general 

 directions. It is then placed in the carbolfuchsin solution 

 five minutes (cold), or two minutes in the hot, immediately 

 transferred to the second solution, the acid blue, where it 

 remains one minute, then washed in water. The preparation 

 is dried between filter-paper and mounted. Examined with 

 oil-immersion. 



Slow Method. The above method may also be used with- 

 out heating, though in this case a much longer time is required 

 before the bacilli take up the stain. The preparation is left 

 in a small dish or beaker full of carbolfuchsin for eight to 

 ten hours, and then decolorized and counterstained in the 

 way described above. The method is less liable to produce 

 artefacts than the quick method, but is not much used on 

 account of the time it takes. 



Examination in Urine. In urine, owing to the almost in- 

 evitable contamination with the smegma bacillus, special 

 methods are necessary to avoid error. The preparation may 

 be left in 97 per cent, alcohol for eight hours, when the 

 smegma bacillus will have become decolorized, or Pappenheim's 



