328 A MANUAL OF BACTERIOLOGY 



material, as the bacilli may be very scanty, or practically impossible 

 to find, e.g. in lupus. Sections should be prepared either by the 

 freezing or the paraffin method, stained with haematoxylin, and 

 counter-stained with eosin, or orange-rubin, or with the Ehrlich- 

 Biondi mixture. 



In order to demonstrate the tubercle bacillus in fresh tissue 

 smears may be made and stained like sputum, or sections prepared 

 and stained in warm carbol-fuchsin for about ten minutes. For 

 frozen sections the stain may be contained in a watch-glass or small 

 glass capsule, and is warmed until it steams, but not boiled, on a 

 piece of asbestos cardboard or a sand-bath. Paraffin sections 

 should be fixed to the slides with glycerin albumin, and may be 

 stained by flooding with the carbol-fuchsin and warming on asbestos 

 cardboard, or a heated penny, for ten minutes. After staining, the 

 sections are washed in water and are then decolorised in 25 per 

 cent, sulphuric acid. This is a longer process than with sputum, 

 and the sections after being in the acid for a few seconds are washed 

 in water and then returned to the acid, and this alternate rinsing in 

 acid and in water is repeated until they are nearly colourless when 

 placed in water. It is not necessary to remove the colour absolutely ; 

 a faint pink remaining does not matter. After rinsing in fresh water 

 to remove all the acid, the sections are counter-stained in Loffler's 

 methylene blue for two minutes, rinsed in methylated spirit, passed 

 through absolute alcohol somewhat rapidly to avoid removing too 

 much of the blue, cleared in cedar oil or xylol, and mounted in 

 balsam. The sections may also be counter-stained with haema- 

 toxylin or Bismarck brown. 



Instead of using the strong acid solution for decolorising, an 

 acid alcohol solution may be used with advantage, or 2 per cent, 

 aqueous hydrochloride of anilin may be employed. 



Gram's method may also be used, but is, of course, not distinctive 

 for the tubercle bacillus. 



Sections may also be first stained with Ehrlich's or other haema- 

 toxylin solution, then stained with warm carbol-fuchsin, washed, 

 treated with 2 per cent, aqueous anilin hydrochloride for a few 

 seconds, decolorised with 75 per cent, alcohol until the red colour 

 is no longer apparent (15-30 minutes), and counter-stained with an 

 aqueous solution of orange. 



Where a positive diagnosis is important, a small piece of the 

 tissue may be inserted under the skin of the thigh or abdomen of 

 a guinea-pig. If tuberculous, the animal will show signs of tuber- 

 culosis in two or three weeks (see below, " Urine "). 



Films of pure cultivations of the tubercle bacillus may be stained 



