STAINING OF TUBERCLE BACILLI IN TISSUES. 175 



been in the stain a sufficient length of time it, with the 

 slide to which it is cemented, is washed in water until 

 the surplus stain is removed; it is then subjected to the 

 action of a combined decolorizer and contrast-stain made 

 as follows: 



Methyl-blue . 2.25 grammes. 



Absolute alcohol 30 c.c. 



Sulphuric acid . . . 12 " 



Water (distilled) . 100 " 



Dissolve the methyl-blue in the alcohol, add the acid 

 to the water, mix the two solutions, and let stand, with 

 occasional shaking, for twelve hours; then filter. 



This solution is allowed to act upon the tissue for a 

 few seconds, and as soon as the blue color predominates 

 over the red, as seen by transmitted light, the section is 

 immediately washed in water. Generally the red color 

 reappears, and the section must be again subjected to 

 the action of the blue solution and again washed in 

 water. This must be repeated until the blue almost, 

 if not quite completely and permanently, replaces the 

 red stain. This is the most important part of the pro- 

 cess, and entirely satisfactory results are only obtained 

 after some practice. The tendency is usually not to 

 replace sufficiently the fuchsin with the methyl-blue, 

 and in consequence the red color of the bacilli is masked 

 by the red of the surrounding tissues. Unless all acid 

 is thoroughly removed by the final washing in water 

 the stain is not permanent. The section is then com- 

 pletely dehydrated with absolute alcohol, after taking 

 up the excess of water on the slide with blotting-paper. 

 The alcohol is followed by turpentine, and the process 

 is completed by mounting in xylol-balsam. 



In case it is desired to stain sections cut by the freez- 



