Tubercle Bacilli in Sputum 205 



also been employed for the same purpose. About equal quantities of the 

 selected solution and the sputum are placed in a centrifuge tube. The tube 

 is incubated in a serological water bath for about ten min and then centri- 

 fuged rapidly, the smear being made from the denser portions that remain 

 at the bottom of the tube. 



Whichever method is employed, the quantity of material removed by 

 the sterile loop should be about the size of a large pinhead. That is, a 

 great deal more material should be employed than in the preparation of a 

 simple bacterial smear from a known culture for the reason that large 

 areas have to 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 readily 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 bac- 

 teriological preparations. 



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

 magenta solution of Ziehl, 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 min or, as is cus- 

 tomary in modern practice, it may be raised to steaming, permitted to 

 cool, again raised to steaming, 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 completely decolorized. It cannot 

 be decolorized too far, but usually there will be found, even after pro- 

 longed exposure to the acid, a faint pink coloration of the background. 

 The slide is next washed in running water until all the acid is removed. 

 Finally, it is treated with a blue stain for about 2 min to provide a con- 

 trasting coloration of any other bacteria present and washed thoroughly, 

 dried, and examined in the customary manner. 



It must be emphasized that this technique as described is designed 

 specifically to show tubercle bacilli and is so violent that many bacteria 

 that are acid-fast to less strong acids will be decolorized. 



SUMMARY 



1. Smear yellow flecks or centrifuged concentrate from sputum between 

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



