1890.] MICKOSCOPICAL JOURNAL. 63 



Triple Staining of Sections Containing Tubercle Bacilli.* — 



The sections are first stained in Delafield'shEematoxylin solution, (i) after 

 which they are allowed to remain in a considerable quantity of water for 

 some hours to remove all traces of the alum. They are then dehydrated 

 in alcohol and stained for ten minutes in carbol-fuchsin, (2) after rins- 

 ing in water they are placed in fluorescine-alcohol to extract the fuchsin 

 from the tissues, then in pure alcohol, ethereal oil, and xylol. From 

 this thev are placed in auramin aniline oil (3) for a few minutes until 

 a yellowish tint has been obtained, when they are rinsed in pure aniline 

 oil and passed successively through a bath of some ethereal oil, xylol, 

 and finally mounted in balsam. 



The nuclei appear violet, the cell protoplasm yellow, and the bacilli 

 red. This method shows the cell boundaries very sharply defined, 

 and gives generally excellent tissue pictures. The dark nuclear stain- 

 ing, however, does not admit of the greatest possible number of bacilli 

 being brought into view, as some may be covered up by the dark nuclei. 

 This method is, therefore, especially to be used for the staining of ma- 

 terial in which the bacilli are in considerable numbers. 



Kuhne's Modification of Gram's Method. f — The sections are 

 stained in an alcholic solution of methyl violet, diluted one-sixth with 

 I per cent, watery solution of ammonium carbonate, or in Victoria blue 

 for five minutes (the latter solution is not to be diluted). After stain- 

 ing, the sections are thoroughly rinsed in water and thus transferred to 

 Gram's solution, in which they should remain for 2 or 3 minutes, when 

 they are again rinsed in water and placed in fluorescine-alcohol to ex- 

 tract the stain from the tissues. They are now rinsed in clear alcohol, 

 cleared in oil of cloves or aniline oil, and finally passed through a bath 

 of thin ethereal oil and xylol. Mount in Canada balsam. 



The bacteria which take this stain appear sharply stained in the tis- 

 sue, which is free from deposit and quite decolorized. The prepara- 

 tions are durable if the ethereal oil has been absolutely removed from 

 the sections by xylol and then mounting in xylol-damar balsam, or in 

 Canada balsam free from oil. 



A very beautiful double stain may be obtained by first staining the 

 sections fifteen minutes in carmine solution (Caccati). After stain- 

 ing rinse in water and then stain as described above. 



In a second modification of Gram's method, the dehydrated, un- 

 stained specimens, or those previously stained with carmine are placed 

 for ten minutes in concentrated watery violet solution to which HCl. 

 (one drop in fifty) has been added. The sections are then well rinsed 

 in water, treated as usual with Gram's solution, again rinsed in water, 

 dipped for a few seconds into absolute alcohol in order to remove the 

 adherent water, and finally put into pure aniline oil, which will pro- 

 vide for the extraction and difierentiation, and at the same time will 

 absorb the last trace of water. After the decolorization has been ac- 

 complished they are transferred into some ethereal oil, then to xylol, 

 and finally mounted in balsam. 



(/) DelafielcCs Solution of Hcetnatoxylin. — To 206 c.c. of a con- 

 centrated solution of ammonia alum is added 12,5 c.c. of absolute al- 

 cohol in which 2 grams of h^ematoxylin has been dissolved. This so- 



*Ibid., p. 28. f Ibid., p. 33. 



