TUBERCLE BACILLI IN SPUTUM 105 



be searched in the interests of diagnostic accuracy. This pinhead of material 

 must be spread over the largest possible area of the slide, which is most read- 

 ily accomplished by pressing another slide on the first and then drawing the 

 two slides apart with a lateral motion. Both slides are dried in air and flamed 

 as has been described in previous bacteriological preparations. 



The solutions required for the original Neelsen technique are the carbol- 

 magenta solution of Ziehl (see p. 22), a 25 per cent solution of nitric acid, and 

 a 1 per cent methylene blue solution. The slide is first flooded with a large 

 quantity of the magenta solution and then placed on a metal sheet, where it 

 should be warmed underneath by a bunsen flame to the point where the stain 

 is steaming briskly but no bubbles have appeared. If it shows signs of drying, 

 fresh quantities of the magenta solution should be added to it. It either may 

 be left at this temperature for 3 to 5 minutes or, as is customary in modern 

 practice, it may be raised to steaming, permitted to cool, again raised to steam- 

 ing, permitted to recool, and so on, until four such cycles have been completed. 

 The slide is washed in a large volume of tap water until no further magenta 

 comes away, and then placed in 25 per cent nitric acid until it is almost com- 

 pletely decolorized. It cannot be decolorized too far, but usually there will be 

 found, even after prolonged exposure to the acid, a faint pink coloration of 

 the background. Now the slide is washed in running water until all the acid 

 is removed. Finally, it is treated with a blue stain for about two minutes to 

 provide a contrasting coloration of any other bacteria present and washed thor- 

 oughly, dried, and examined in the customary manner. 



It must be emphasized that this technique as described is designed specifi- 

 cally to show tubercle bacilli and is so violent that many bacteria which are 

 acid-fast to less strong acids will be decolorized. 



Summary 



1. Smear yellow flecks or centrifiiged concentrate from sputum between two 

 slides. Separate, dry, flame, and stain both slides. 



2. Flood smear with Ziehl's magenta, warm to steaming, and cool. Repeat 

 warming and cooling three times. 



3. Wash under tap and decolorize in 25 per cent nitric acid. 



4. Wash off acid and counterstain, if desired, in 1 per cent methylene blue for 

 two minutes. 



5. Wash in water and dry. 



