EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 47 



The intensity of the stain must then be increased by warm- 

 ing the preparation till it steams or boils, then allowing the 

 warm stain to act on the specimens for from three to five 

 minutes; the preparation may also be left in the cold stain 

 over night. Decolorization may be effected with a 25 per 

 cent, solution of sulphuric acid used till the red color disap- 

 pears, or a 30 per cent, solution of nitric acid, which operates 

 very rapidly. If the red color persists after washing in 

 water, dip in the acid again. After either acid the prepara- 

 tion is to be washed in alcohol until the' last trace of the 

 stain has been removed. An excellent decolorizing agent 

 is a 3 per cent, solution of hydrochloric acid in alcohol, 

 used for about a minute. With any of these acid solutions 

 the decolorization can be accomplished more perfectly than 

 with Gabbett's solution, where the operation of the de- 

 colorizing agent is masked. The contrast-stain may be 

 omitted entirely if it is desired. A suitable contrast-stain 

 after fuchsin staining is a solution of methylene-blue ; after 

 gentian-violet staining, Bismarck brown. 



Those who have had experience in staining tubercle ba- 

 cilli soon discover that the bacilli exhibit some differences 

 in their resisting power to strong acids. One encounters 

 occasionally bacilli that are perfectly stained side by side 

 with others that are more or less completely decolorized. 

 These facts show the necessity of practice with any method, 

 and of exercising caution and judgment in making a' diag- 

 nosis where the number of bacilli happens to be scanty. 

 If tubercle bacilli are not found in the first preparation, 

 other preparations should be made. Sometimes a large 

 number of cover-glasses must be examined. 



Various expedients have been devised to concentrate 

 tubercle bacilli when only a small number may be present 

 in a sample of sputum. In Biedert's method about 15 c.c. 

 of sputum are mixed with 5 c.c. of distilled water, 4 to 8 



