Example 8 



Demonstration of Tubercle Bacilli in Sputum by 

 the Technique of Neelsen 



When Neelsen published his original technique for the demonstration of 

 tubercle bacilli, the standard magenta solution used for the staining of bacteria 

 was that proposed in the previous year by Ziehl. As a result, Neelsen's tech- 

 nique was referred to as a modification of Ziehl and to this day, the hyphen- 

 ated term "Ziehl-Neelsen" is applied to almost any method for the demon- 

 stration of tubercle bacilli in sputum regardless of the author of the technique. 



It is proposed here to describe the original technique of Neelsen, leaving it 

 to the technician to determine which of the many other methods given in 

 Gray's Microtechnique can be more readily applicable to his problem. It may be 

 said in favor of the technique of Neelsen that it gives a far better differenti- 

 ation of tubercle bacilli than do some of the more recent methods which, 

 although they give good preparations, tend to cause certain errors of diagnosis 

 through the ability of other bacteria to withstand the lower concentration of 

 acids employed today. 



It is to be presumed that the sputum collected from the patient will have 

 been placed at the disposal of the technician in the glass vessel in which it was 

 secured. It should be examined to see whether or not any small yellowish 

 particles exist in it, and if they do, one of these particles should be extracted 

 carefully with a sterile bacteriological wire loop and utilized for the preparation. 

 Even if no such particles are visible to the naked eye, it is possible that tubercle 

 bacilli will be present, but due consideration should be given to some method 

 of concentrating these bacilli before making the smear. The standard method 

 of concentration is to hydrolyze the sputum to the extent necessary with the 

 aid of a weak solution of potassium hypochlorite, which is known to be with- 

 out action on tubercle bacilli. For a long time a proprietary compound known 

 as antiformin, which is a strongly alkaline solution of sodium hypochlorite, 

 has also been employed for the same purpose. About an equal quantity of the 

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

 incubated in a serological water bath for about 10 minutes and then centri- 

 fuged rapidly, the smear being made from the denser portions which 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 



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