Staining 66 1 



method, that the latter, which colors all micro-organisms stained, 

 the same blue-black color, and hence is not differential, is never 

 used for diagnostic purposes. 



Ehrlich's Method, or the Koch-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 11 



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. 



This method, requiring twenty-four hours for its completion, is no longer used. 



Ziehl's Method. — Among clinicians, Ziehl's method of staining with carbol- 

 fuchsin has met with just favor. It is as follows: After having been spread, 

 dried, and fixed, the cover-glass is held in the bite of an appropriate forceps (cover- 

 glass forceps), or the slide spread at one end is held by the other end as a handle, 

 and the stain (fuchsin, i; alcohol, 10; 5 per cent, phenol in water, 100) dropped 

 upon it from a pipet. As soon as the entire smear is covered with stain, it is held 

 over the flame of a spirit lamp or Bunsen burner until the stain begins to vola- 

 tilize a httle. When vapor is observed the heating is sufficient, and the temper- 

 ature can be maintained by intermittent heating. 



If evaporation take place, a ring of encrusted stain at the edge prevents the 

 prompt action of the acid. To prevent this, more stain should now and then be 

 added. The staining is complete in from three to five minutes, after which the 

 specimen is washed off with water, and then with a 3 per cent, solution of hydro- 

 chloric acid in 70 per cent, alcohol, 25 per cent, aqueous sulphuric, or 33 per cent, 

 aqueous nitric acid solution dropped upon it for thirty seconds, or until the red 

 color is extinguished. The acid is carefully washed off with water, the specimen 

 dried and mounted in Canada balsam. Nothing will be colored except the tuber- 

 cle baciUi, which appear red. 



■ Gobbet's Method. — Gabbet modified the method by adding a little methylene 

 blue to the acid solution, which he makes according to this formula : 



Methylene blue 2 



Sulphuric acid 25 



Water 75 



In Gabbet's method, after staining with carbol-fuchsin, the specimen is washed 

 with water, acted upon by the methylene-blue solution for thirty seconds, washed 

 again with water until only a very faint blue remains, dried, and finally mounted 

 in Canada balsam. The tubercle bacilli are colored red; the pus-corpuscles, 

 epithelial cells, and unimportant bacteria, blue. 



Pappenheim,* having found bacilli stained red by Ziehls' method in the sputum 

 of a case which subsequent postmortem examination showed to be one of gan- 

 grene of the lung without tuberculosis, condemns that method as not being 

 sufficiently differential, and recommends the following as superior to methods in 

 which the mineral acids are employed: 



1. Spread the film as usual. 



2. Stain with carbol-fuchsin, heating to the point of steaming for a few minutes. 



■ 3. Pour off the carbol-fuchsin and without washing — 



* "Berl. klin. Wochenschrift," 1898, No. 37, p. 809. 



