1889.] MICROSCOPICAL JOURNAL. 91 



BACTERIOLOGY.* 



Staining Tubercle-Bacilli. — The Koch-Ehrlich'sf method is 

 probably the most trustworthy of any in use. According to this method, 

 specimens to be examined for tubercle-bacilli are prepared and stained 

 in the following manner : 



The cover-glasses must be thoroughly cleansed in some cleaning mix- 

 ture to free them from any adherent particles of grease or dirt that might 

 pi'event the substance to be examined from sticking to the glass. The 

 suspected material must then be spread on a cover-glass in the thinnest 

 possible layer. If a hard tubercular nodule is to be examined it must 

 be crushed and completely broken up before spreading it on the cover- 

 glass. In case of sputum the more solid, yellowish masses should 

 be taken, excluding as much as possible the accompanying mucus. The 

 film thus prepared must now be allowed to dry in the air of the room 

 without extra heat. As soon as it is thoroughly dry the cover-glass 

 with the film uppermost is passed three times moderately quickly through 

 the flame of a spirit-lamp or Bunsen burner, in order to fix the film so 

 that it may not be washed away during the staining process. 



After heating, the cover-glass must be floated film downwards on the 

 surface of the staining fluid, care being taken that no air-bubbles lie be- 

 neath it, which would protect the specimen from exposurre to the stain- 

 ing fluid at this point. The cover-glass should remain in the stain for 

 from 12 to 24 hours at the temperature of the room. 



The stain consists of a solution composed of 100 c.cm. of aniline 

 water, 11 c.cm. of a saturated alcoholic solution of methyl violet (or 

 fuchsin), and 10 c.cm. of absolute alcohol. The aniline water is made 

 by adding about 5 c.cm. of aniline oil to 100 c.cm. of distilled water, 

 and shaking the two thoroughly together. From 3 to 4 per cent, of 

 aniline is taken up by the water ; the remainder adheres to the bottom 

 of the vessel in the form of thick drops. This saturated solution of an- 

 iline is obtained in about one-half hour, when it is filtered. The filtrate 

 should be as transparent and colorless as water'. It is better to prepare 

 afresh a few c.cm. of the stain each time that it is required, for owing 

 to the aniline water it will not keep for any length of time. The satu- 

 rated alcoholic solution of methyl violet (or fuchsin) is obtained' by 

 pouring about 100 c.cm. of absolute alcohol upon about 20 grms. of dry 

 methyl violet (or fuchsin) in a well-stoppered glass vessel and shaking 

 frequently. 



After the necessary time has elapsed the cover-glass is removed from the 

 stain by means of fine forceps. The preparation is now stained very dark, 

 almost black. It is immediately placed in nitric acid diluted with three 

 to four parts of water ; in this it is freely moved about for some seconds 

 until it becomes of a greenish-blue color, when it is transferred to a ves- 

 sel containing 60 per cent, alcohol. It is left in the alcohol for several 

 minutes until no more of the staining fluid is washed out, when it is 

 ready for the next staining process. 



In preparations treated with nitric acid and alcohol the tissue elements 

 are quite colorless, or of a very light blue tint, while the tubercle-bacilli 



* Conducted by V. A. Moore, assistant in the laboratory of the Bureau of Animal Industry, 

 t Volume xcv of the New Sydenham Society. Microparasites in Disease. London, 1886. 



