ZIEHL-NEELSEN METHOD 325 



be chosen, and sufficient material should be used so as to form a 

 distinct but not too thick film ; a little experience will soon decide 

 the right amount ; too thin a film should be avoided. Preparations 

 may also be made by smearing the sputum on a cover-glass or 

 between two cover-glasses instead of using slides. Whichever plan 

 is adopted, the film is dried and fixed in the usual manner (generally 

 by heat), and then stained by one of the following methods : 



(a) Ziehl-N eelsen mzthod. Film specimens on slides are most 

 conveniently stained by flooding with filtered, undiluted carbol- 

 fuchsin and warming for 2 to 5 minutes on a piece of asbestos 

 cardboard supported on a tripod, or on a heated penny (p. 110), or 

 slides or cover-glasses flooded with the stain may be held in the 

 forceps and carefully warmed over a flame, or the preparations 

 may be immersed in a watch-glass or dish of the stain, covered, and 

 placed in the warm incubator for half an hour. In no case must 

 the stain bs allowed to boil, or the bacilli may lose their staining 

 power ; it should only bs warmed sufficiently to steam (50-60 C.), 

 and with slides or cover-glasses as evaporation takes place more 

 stain (always filtered), or better, 5 per cent, carbolic, should be added. 

 After staining, the preparations are rinsed in water and are then 

 decolorised by treating with 25 per cent, sulphuric or 30 per cent, 

 nitric acid. The preparation may be flooded with the acid, but a 

 better method is to immerse the preparation in a pot (Fig. 20, 

 p. 110) containing the acid. In the acid the colour changes after 

 a few seconds to a yellowish brown, the preparation is then rinsed 

 in water, and some of the pink colour returns. The treatment with 

 acid and with water alternately is repeated until the preparation 

 is nearly colourless when rinsed in water. With sputum this is 

 usually the case after three or four rinses in the acid, but it varies 

 with the thickness of the film and with the number of tubercle 

 bacilli present ; when these are absent the film often decolorises 

 more readily than when there are many. The presence of blood 

 renders the decolorisation difficult. After decolorising and washing, 

 the preparations are stained for one minute in Loffler's methylene 

 blue, washed in water, and mounted in water, or, better, dried and 

 mounted in Canada -balsam or cedar oil. When the preparation is 

 made on the slide, after washing and drying, it can be examined 

 directly without a cover-glass with the oil-immersion after applying 

 a drop of cedar oil, unless a permanent specimen is desired, in which 

 case it should be mounted in Canada-balsam. 



The tubercle bacilli appear as delicate red rods, often beaded or 

 segmented, on a blue background composed of cells, mucus, and 

 putrefactive or other bacteria. Occasionally here and there a little 



