Staining 715 



easily found at first, but which break up and become part 

 of a granular sediment that forms in decomposed sputum. 



The sputum should be poured into a watch-glass and 

 held over a black surface. A number of grayish-yellow, 

 irregular, translucent fragments somewhat smaller than the 

 head of a pin can usually be found. These consist prin- 

 cipally of caseous material from the tuberculous tissue, 

 and are the most valuable part of the sputum for exam- 

 ination. One of the fragments is picked up with a pointed 

 match-stick and spread over the surface of a perfectly 

 clean cover-glass or slide. If no such fragment can be found, 

 the purulent part is next best for examination. 



The material spread upon the glass should not be too 

 small in amount. Of course, a massive, thick layer will be- 

 come opaque in staining, but should the layer spread be, 

 as is often advised, " as thin as possible," there may be so 

 few bacilli upon the glass that they are found with diffi- 

 culty. 



The film is allowed to dry thoroughly and is then passed 

 three times through the flame for fixation. 



Ehrlich's Method, or the Kock-Ehrlich Method. Cover- 

 glasses thus prepared are floated, smeared side down, or 

 immersed, smeared side up, in a small dish of Ehrlich's 

 anilin-water gentian violet solution: 



Anilin 4 



Saturated alcoholic solution of gentian violet 1 1 



Water 100 



and kept in an incubator or paraffin oven for about twenty- 

 four hours at about the temperature of the body. Slides 

 upon which smears have been made can be placed in Coplin 

 jars containing the stain and stood away in the same manner. 

 When removed from the stain, they are washed momentarily 

 in water, and then alternately in 25 to 33 per cent, nitric acid 

 and 60 per cent, alcohol, until the blue color of the gentian 

 violet is entirely lost. A total immersion of thirty seconds is 

 enough in most cases. After final thorough washing in 60 

 per cent, alcohol, the specimen is counterstained in a dilute 

 aqueous solution of Bismarck brown or vesuvin, the excess of 

 stain washed off in water, and the specimen dried and mounted 

 in balsam. The tubercle bacilli are colored a fine dark blue, 

 while the pus-corpuscles, epithelial cells, and other bacteria, 

 having been decolorized by the acid, will appear brown. 



