Staining 357 



Diagnosis of Tuberculosis. In all cases where the 

 detection of tubercle bacilli in pus or secretions is a matter 

 of clinical importance, it must be remembered that the 

 quantity of material examined by the staining method is 

 extremely small, so that a few bacilli in a relatively large 

 quantity of matter can easily escape discovery. It is, 

 therefore, important to supplement the microscopic ex- 

 amination by the inoculation of a guinea-pig with a 

 considerable quantity (1-5 c.c.) of the suspected material, 

 and at the end of about three weeks kill it and look for 

 evidences of the disease in its tissues. 



Staining the Bacillus in Sections of Tissue. Ehrlich's 

 Method for Sections. Ehrlich's method must be recom- 

 mended as the most certain and best. The sections of 

 tissue, embedded in celloidin or paraffin, should be cemented 

 to the slide and then freed from the embedding substance. 

 They are then placed in the stain for from twelve to twenty- 

 four hours and kept at a temperature of 37 C. Upon 

 removal they are allowed to lie in water for about ten 

 minutes to wash away the excess of stain and to soften the 

 tissue (which, if not cemented to the glass, often shrinks 

 and becomes brittle). The washing in nitric acid (20 per 

 cent.) which follows may have to be continued for as long 

 as two minutes. Thorough washing in 60 per cent, alcohol 

 follows, after which the sections can be counterstained, 

 washed, dehydrated in 95 per cent, and absolute alcohol 

 cleared in xylol, and mounted in Canada balsam. 



Unna's Method for Sections. Unna's method is as follows : 

 The sections are placed in a dish of twenty-four-hour-old, 

 newly filtered Khrlich's solution, and allowed to remain 

 twelve to twenty-four hours at the room temperature 

 or one to two hours in the incubator. From the stain 

 they are placed in water, where they remain for about 

 ten minutes to wash. They are then immersed in acid 

 (20 per cent, nitric acid) for about two minutes, and 

 become greenish-black. From the acid they are placed 

 in absolute alcohol and gently moved to and fro until 

 the pale blue color returns. They are then washed in 

 three or four changes of clean water until they become 

 almost colorless, and then removed to the slide by means 

 of a section-lifter. The water is absorbed with filter paper, 

 and then the slide is heated over a Bunsen burner until the 

 section becomes shining, when it receives a drop of xylol 

 balsam and a cover-glass. 



