470 



METHODS AND FORMULAS 



DS 23.20 



tion of tubercle bacilli than do some of the 

 more recent methods. These, though they 

 give good preparation, do tend to cause 

 certain errors of diagnosis through the 

 ability of other bacteria to withstand the 

 lower concentration of acids nowadays 

 employed. 



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 looked over carefully to see 

 if any small yellowish particles exist in it; 

 if they do, one of these particles should be 

 carefully extracted with a sterile bacterio- 

 logical wire loop and utilized in making 

 the preparation. If no such particles are 

 visible to the naked eye, it is, of course, 

 possible that tubercle bacilli will be pres- 

 ent, but due consideration should be given 

 to some method of concentrating these ba- 

 cilli before making the smear. The stand- 

 ard method of concentration is to hy- 

 drolize the sputum to the extent necessary 

 with the aid of a weak solution of potas- 

 sium hypochlorite, which is known to be 

 without action on tubercle bacilli. For a 

 long time, a proprietary compound known 

 as antiformin, which is a strongly alkaline 

 solution of potassium hypochlorite, was 

 also used for the same purpose. About an 

 equal quantity of the selected solution 

 and the sputum are placed in a centrifuge 

 tube, the tube incubated in a serological 

 water bath (37°C.) for about ten minutes, 

 and then centrifuged rapidly. The smear 

 is made from the denser portions which 

 remain at the bottom of the tube. 



Whichever method is employed, the 

 quantity removed by the sterile loop 

 should be about the size of a large pin- 

 head. That is, a great deal more should 

 be employed than is used for a simple 

 bacterial smear from a known culture. 

 This pinhead of material must be spread 

 over the largest possible area of the shde. 

 This is best done by pressing another shde 

 on it and then drawing the two slides 

 apart with a lateral motion. Both slides 

 are then dried in air and flamed as has 

 been described in the discussion of previ- 

 ous bacteriological preparations. 



The solutions required for the original 

 Neelsen technique are the phenol-magenta 



solution of Ziehl (Chapter 20, DS 11.43), a 

 25% solution of nitric acid, and the poly- 

 chrome methylene blue of Loffler (Chap- 

 ter 20, DS 11.44). The shde is first flooded 

 with the magenta solution and then placed 

 on a metal sheet, where it should be 

 warmed by a bunsen flame to the point at 

 which it is steaming, but at which no 

 bubbles have appeared. If it shows signs 

 of drying, fresh quantities of the magenta 

 solution should be added to it. It may 

 either be left at this temperature for three 

 to five minutes (Neelsen's original recom- 

 mendation) or, as is customary in modern 

 practice, it may be raised to steaming, per- 

 mitted to cool, again raised to steaming, 

 permitted to cool, and so on, until four 

 such cycles have been completed. The 

 slide is then washed in tap water until no 

 further magenta comes away and placed 

 in 25% nitric acid until it is almost com- 

 pletely decolorized. It cannot be decolor- 

 ized too far, but there will usually be, even 

 after prolonged exposure to the acid, a 

 faint pink coloration of the background. 

 The slide is now washed in running water 

 until all the acid is removed, and then 

 treated with a blue stain for about two 

 minutes, to provide a contrasting colora- 

 tion of any other bacteria present. It 

 should then be washed thoroughly, dried, 

 and examined in the customary manner. 



It must be emphasized that this tech- 

 nique as described is specifically designed 

 to show tubercle bacilU, and is so violent 

 that many bacteria which are acid-fast to 

 less strong acids will be decolorized. 



Demonstration of the flagella of 



Proteus vulgaris by the method 



of Tribondeau, Fichet, and 



Dubreuil 1916 



Examination of the abbreviated form 

 of this technique (DS 23.215 Tribondeau, 

 Fichet, and Dubreuil 1916) below would 

 give one the impression that it was a sim- 

 ple matter to stain the flagella of bacteria. 

 In point of fact it is one of the most diffi- 

 cult preparations known to the science of 

 microtomy. 



In the first place it is necessary to secure 

 a culture in which the organisms are ac- 

 tively motile and growing; it is useless to 



