MORPHOLOGY 307 



Numerous methods of staining the tubercle bacillus have been suggested, 

 but they all depend upon the principle enunciated above. 



The various methods in most frequent use will now be described, but the necessity 

 for beginners to limit themselves to one method which they thoroughly understand 

 and upon the results of which they can rely cannot be too strongly emphasized. 

 The Ziehl-Neelsen method is by far the best. 



The tubercle bacillus stains by Gram's method but with difficulty and the 

 stain, carbol- or anilin-gentian-violet (Nicolle), must be allowed to act for, at 

 least, 10 minutes. The bacilli are always granular by this method. 



Much has shown that in cattle inoculated with tuberculosis, tuberculous nodules 

 are often seen, post mortem, in the lungs in which no bacilli can be demonstrated by 

 Ziehl's method, though the presence of bacilli in the lesions is proved by the result 

 of inoculations (the same is true of " cold abscesses " in man). If, however, Gram's 

 method of staining be adopted, leaving the preparations in the violet for 48 hours 

 and in the iodine solution for 24 hours, large numbers of bacilli can be seen in the 

 lesions. From this observation Much concludes that in addition to the acid-fast 

 tubercle bacillus there is a virulent form which is non-acid-fast. 



A. The staining of films. 

 1. Ziehl-Neelsen method. 



Method recommended. 



The principle of the method. If a film stained with carbol-fuchsin be 

 treated with a diluted mineral acid, the background and all the organisms, 

 with the exception of the tubercle bacillus (and also those of leprosy, verruga 

 and the " acid-fast " bacilli, pp. 350 and 345), will be decolourized. The 

 tubercle bacillus retains its red colour. If, now, the preparation be stained with 

 an aqueous solution of methylene blue, the background and the decolourized 

 organisms take up the blue while the tubercle bacillus remains red. 



Technique. 1. Spread, dry and fix a film on a cover-glass in the ordinary 

 way. Hold it in a pair of Cornet's forceps and flood it with a large drop of 



FIG. 195. Tubercle bacilli in sputum. Carbol-fuchsin and methylene blue. 



(Oc. 2, obj. jUh, Zeiss.) 



Ziehl's carbol-fuchsin (p. 138). Hold the cover-glass over a small flame (the 

 pilot light of a Bunsen, for example) and heat very gently until steam just 

 begins to rise ; continue the heating for two minutes, being careful not to 

 boil the stain and to see that the staining solution does not dry up. 



