METHODS OF INVESTIGATING BACTERIA. 783 



are stained blue. The clubs of actinomyces can be 

 stained reddish-blue by treatment first with Wedl's 

 orseille (Yirchow's Archiv, vol. 74), and then with 

 gentian violet. 



Tubercle bacilli are best stained by the following To stain 

 method (see p. 261): aniline oil and water are well 

 shaken up together, and then filtered through moistened 

 thick filter paper ; concentrated alcoholic fuchsine or 

 gentian violet solutions are then cautiously dropped into 

 the filtered aniline water till a slight cloudiness is noted, 

 which later again disappears (about 10 to 20 drops of 

 the stain to 10 ccm. of aniline water). The cover glasses, 

 prepared with sputum in the manner above-mentioned, 

 are then floated on this colouring material for from twelve 

 to twenty- four hours, or, if the solution is kept at a tem- 

 perature of 30 to 35 C., for one to two hours ; in the case 

 of sections, it is best to stain them for twenty-four hours, 

 and then to warm the solution for one to two hours. The 

 cover glasses and sections are then washed in the first 

 instance in acidulated alcohol (100 ccm. alcohol, 20 ccm. 

 water, and 20 drops of concentrated hydrochloric acid) ; 

 after a short time (a half to two minutes) the red or violet 

 colour is removed from all the thinner parts of the pre- 

 parations, which are now carefully washed in water, the 

 cover glasses dried, and then stained in dilute watery solu- 

 tion of methylene blue if fuchsine has been employed in 

 the first instance, or with a solution of vesuvine if gentian 

 violet has been used. After five to fifteen minutes 

 they are washed in water, dried, and mounted in Canada 

 balsam. Sections are placed for half an hour in the 

 methylene blue or vesuvine solution, and then treated in 

 the usual manner; if the preparations are to be preserved 

 we must employ oil of bergamot instead of oil of cloves. 

 In this way we obtain preparations in which the tubercle 

 bacilli are stained red or violet, and the cells and cell 

 nuclei blue or brown. The only other organisms which 

 show the same staining reactions as tubercle bacilli are, 

 so far as we know at present, the leprosy bacilli. Some 

 other materials also retain the stain in this method, such 

 as the epidemic structures, the spores of the mould fungi, 



