Staining 703 



The best result will be secured if the examination be made on 

 the same day, for if the Tbacilli are few they occur most plentifully 

 in small flakes of caseous matter, which are easily found at first, 

 but which break up and become part of a granular sediment that forms 

 in decomposed sputum. 



The sputum should be poured into a watch-glass and held over a 

 black surface. A number of grayish-yellow, irregular, translucent 

 fragments somewhat smaller than the head of a pin can usually 

 be found. These consist principally of caseous material from the 

 tuberculous tissue, and are the most valuable part of the sputum 

 for examination. One of the fragments is picked up with a pointed 

 match-stick and spread over the surface of a perfectly clean cover- 

 glass or slide. If no such fragment can be found, the purulent part 

 is next best for examination. 



The material spread upon the glass should not be too small in 

 amount. Of course, a massive, thick layer will become opaque 

 in staining, but should the layer be spread, as is often advised, 

 "as thin as possible," there may be so few bacilli upon the glass 

 that they are found with difficulty. 



The film is allowed to dry thoroughly, is passed three times 

 through the flame for fixation, and is then stained and examined. 



Where examination by these means fails to reveal the presence 

 of bacilli because of the small number in which they occur, recourse 

 may be had to the use of caustic potash or, what is better, anti- 

 formin (q.v.) for digesting the sputum. A considerable quantity 

 of sputum is collected, receives the addition of an equal volume 

 of the antiformin, is permitted to stand until the formed elements 

 and pus-corpuscles have been dissolved, is then shaken and poured 

 into centrifuge tubes and whirled for fifteen to thirty minutes. 

 The sediment at the bottom of the tubes is then spread upon the 

 glasses and stained and will often reveal the bacilli which, having 

 been freed from the viscid materials in the sputum, are thrown 

 down in masses by the centrifuge. 



The purpose of the staining being the discovery of the tubercle 

 bacillus, success is only possible when the method employed en- 

 ables that particular micro-organism to be recognized, as such, 

 so soon as it is seen. This can be accompHshed by taking advantage 

 of the " acid-proof ' quality of the micro-organism, which permits 

 it to take up the penetrating stains employed, but does not permit 

 it to let them go again in the bleaching agents, and assume the 

 counter stain. It is owing to this peculiarity that the tubercle 

 bacillus alone is colored blue by the Koch-Ehrlich method, and the 

 tubercle bacillus alone red by the Ziehl method, and it is because 

 no advantage is taken of the acid-proof peculiarity in using Gram's 

 method, that the latter, which colors all micro-organisms stained, 

 the same blue-black color, and hence is not differential, is never 

 used for diagnostic purposes. 



