412 APPENDIX. 



as the sections become a pale blue colour they are transferred to a solution 

 of lithium water made of the strength of about I part of lithium carbonate 

 to 20 of water ; they are then thoroughly washed in pure water, de- 

 hydrated in absolute alcohol in which a little methyl blue may be 

 dissolved, placed in aniline oil, which may or may not contain a small 

 portion of methyl blue in solution, and rinsed in pure aniline oil. They are 

 after this treatment transferred to terebene, where they are left for about a 

 couple of minutes. They are then washed in two lots of xylol and mounted 

 in Canada balsam. Almost any organisms may be stained by this method, 

 even the glanders bacillus coming out fairly distinctly. 



Another great stand-by of bacteriologists is fuchsin. With this reagent 

 almost every bacillus may be brought into prominence. It is especially 

 useful for the bacilli of tubercle, leprosy, and mouse septicaemia. 



Ziehl-Neelsen Carbol- Fuchsin Method for Tubercle Bacilli. 

 The Ziehl-Neelsen method of staining the tubercle bacillus is a modification 

 of the Weigert-Ehrlich method. The sections or cover glasses are stained in 

 Neelsen's solution, made as follows : Fuchsin i part, is dissolved in 10 parts 

 of absolute alcohol, to this solution are added I oo parts of a 5 per cent, watery 

 solution of carbolic acid, and the mixture is heated until steam rises pretty 

 freely. Cover glass preparations are stained in three or four minutes, or 

 even less ; sections are usually sufficiently deeply stained in seven or eight 

 minutes. In the cold they may be left for twelve or even twenty-four hours. 

 The superfluous fluid is drained off and the preparations are placed for a second 

 or two in alcohol (90 per cent.), then in a 25 per cent, solution of sulphuric 

 acid, when the pink tinge should immediately be replaced by a yellowish 

 brown. The preparations are then washed in alcohol, and if they are 

 sufficiently decolorized they are transferred to a solution of lithium 

 carbonate. They may afterwards be stained with a watery solution of 

 methylene blue, cleared up with clove oil or with aniline oil, terebene, and 

 xylol, and mounted in Canada balsam. Exceedingly good results are obtained 

 by this method, which is preferable in many respects to the aniline oil 

 method. In place of sulphuric acid nitric or hydrochloric acid may be 

 used. 



The Kilhne-Gram Staining Method. 



Kiihne's modification of Gram's method is, perhaps, superior to the 

 original. Instead of using Weigert's saturated alcoholic solution of methyl 

 violet (or gentian violet) in 100 parts of aniline water and 10 parts of abso- 

 lute alcohol, he stains with a 2 per cent, solution of gentian voilet in dilute 

 alcohol, to which has been added one sixth of its bulk of a I percent, watery 

 solution of ammonium carbonate, or with a similar solution of Victoria blue 

 without the ammonium carbonate, for about five or ten minutes. The pre- 

 parations are then rinsed in water, and are placed in Gram's solution made up 

 of iodine I gramme, iodide of potassium 2 grammes, distilled water 300 cc. 

 for two or three minutes ; they are again washed in water, dehydrated with 

 fluoresceine alcohol, which is prepared by dissolving I gramme of yellow 

 fluoresceine in 50 cc. of absolute alcohol, the part undissolved being allowed 

 to settle at the bottom of the bottle. The section is washed in pure alcohol, 

 then with aniline oJl, and mounted in xylol balsam. 



The Kiihne-Unna Method. 

 Kiihne describes a modified method of making dry preparations adopted 



