Till-: HACILLUS OF TUBERCULOSIS 315 



two preparations of each specimen, in the careful manner described 

 above, is usually sufficient to demonstrate the presence of the bacilli 

 when they are present in the sputa, and they are usually found to be 

 present to this extent in fairly well-developed cases of pulmonary 

 tuberculosis, and in many cases which are in the incipient stage. There 

 are, however, undoubted cases of incipient pulmonary tuberculosis 

 which require the examination of many preparations before the 

 tubercle bacillus can be found; and cases also occur in which the sputum 

 for a time does not contain the bacilli, which were, nevertheless, 

 present at an earlier period, and which again later appear. Therefore, 

 if cases occur which may be still regarded as possibly tuberculosis, 

 further examinations of the sputum should be made. It should also 

 be constantly borne in mind that the demonstration of the presence 

 of tubercle bacilli in the sputum proves about as conclusively as any- 

 thing can the existence of some degree of tuberculosis; but that the 

 absence of tubercle bacilli or the failure to find them microscopically 

 does not positively exclude the existence of the disease. Here tuberculin 

 can be made use of. 



Staining of Tubercle Bacilli in Tissues. Thin sections of tuberculous 

 tissues may be stained by the same methods recommended for cover- 

 glass preparations, except that it is best not to employ heat to any 

 extent. Fixation in bichloride of mercury is better than in alcohol. 

 Formalin is a bad fixative, as it makes the tissues hold the fuchsin with 

 as much tenacity as the bacilli. Both paraffin and celloidin may be 

 used for embedding, but the former is better. 



EHRLICH'S METHOD. Place the paraffin sections in aniline fuchsin 

 and leave at 37 C. for from six to twelve hours, or at about 80 C. for 

 three to five minutes, the sections are then washed in water; then 

 decolorize by placing them for about half a minute in dilute nitric acid 

 (10 per cent.), or in 3 per cent, hydrochloric acid in alcohol; wash in 

 60 per cent, alcohol until no more color is given off; counterstain for 

 two or three minutes in a saturated aqueous solution of methylene blue, 

 or, better, with haematoxylin; wash in water; dehydrate with absolute 

 alcohol; clear in oil of cedar or xylol, and mount in xylol balsam. 



METHOD OF ZIEHL-NEELSON. Stain the section in warmed carbol- 

 fuchsin solution for one hour; the temperature to be not over 45 to 

 50 C. Decolorize for a few seconds in 5 per cent, sulphuric acid, 

 then in 70 per cent, alcohol, and from this on as in the Ehrlich method. 



Inoculation of Animals. The inoculation of suspected material into 

 guinea-pigs sometimes produces tuberculosis when no bacilli could be 

 detected by microscopic examination. The material may be injected 

 into the subcutaneous tissues, into the peritoneal cavity, or into the 

 mammary gland of a pregnant guinea-pig. 



Cultivation. This is so difficult and requires so much time that it is 

 not used except in important investigations upon the nature of the 

 tubercle bacilli. 



