90 BACTERIOLOGY. 



bacilli. Under ordinary circumstances the stain will not penetrate 

 the sheath, but if it can be made to penetrate, it is not readily 

 removed. The cover-glass preparation must be heated to a tem- 

 perature of 210° 0., for half an hour, or passed as many as twelve 

 times through the flame of a Bunsen burner, or exposed to the 

 action of strong sulphuric acid for several seconds, and then a few 

 drops of a watery solution, of an aniline dye may be applied in the 

 usual way. 



To double stain spore-bearing bacilli the cover-glass preparations 

 may be floated, for from twenty minutes to an hour, on Ehrlich's 

 fuchsine-aniline-water, or on the Ziehl-JSTeelsen solution. The stain 

 must be heated — by preference in a capsule placed in a sand-bath — 

 until steam rises. The fuchsine is removed from the bacilli by 

 rinsing in water and washing in weak hydrochloric acid, and then 

 the preparations are washed again in water, and floated for a few 

 minutes on a watery solution of methylene blue. They are again 

 rinsed in water, dried, and mounted. Neisser's decolorising solution 

 consists of 25 parts of hydrochloric acid to 75 parts of alcohol. 



Staining of Flagella. 



Koch first stained flagella by floating the cover-glasses on a 

 watery solution of h8ematox}din. From this they were transferred 

 to a 5 per cent, solution of chromic acid, or to Muller's fluid, by 

 which the flagella obtain a brownish-black coloration. The author 

 succeeded in demonstrating and photographing flagella in prepara- 

 tions stained with a saturated solution of gentian violet in absolute 

 alcohol ; but these methods are now superseded owing to the much 

 more satisfactory method introduced by Lbffler. 



Lbffler's method depends upon the employment of a mordant. 

 Lbfiler tried tannate of iron, and after a number of experiments 

 the following method was introduced. An aqueous solution of 

 ferrous sulphate is added to an aqueous solution of tannin (20 per 

 cent.), until the mixture turns a violet-black colour, then 3 or 4 cc. 

 of a 1 in 8 aqueous solution of logwood are added. This constitutes 

 the mordant, and a few drops of carbolic acid may be added, and the 

 solution kept in well-stoppered bottles. The dye consists of 1 cc. of 

 a 1 per cent, solution of caustic-soda, added to 100 cc. of aniline 

 water, in which 4 or 5 grammes of either methyl violet, methylene blue, 

 or fu.chsine, are dissolved. A cover-glass preparation is made in 

 the ordinary way, the bacteria being diffused in water, and then 

 spread out in a very thin film. After drying and very carefully 

 fixing, the film is covered with the mordant, and the cover-glass 



