326 A MANUAL OF BACTERIOLOGY 



red colour may be present in addition to the tubercle bacilli. Hair 

 and keratinised material generally, such as horny epithelium, and 

 red blood-corpuscles, retain the red colour after the foregoing 

 treatment, and the spores of bacteria are also liable to retain the 

 red somewhat persistently. These exceptions are not, however, 

 likely to prove a source of error, for the tubercle bacilli should be 

 recognised not only by their red colour, but also by their charac- 

 teristic size, shape, and general appearance. It is conceivable that 

 acid-fast bacilli not tubercle might be present in sputum, but such 

 an event is a very unlikely one. For the microscopical examination, 

 a ^-inch with good illumination is sufficient when the tubercle bacilli 

 are present in any number. When they are scanty it is necessary 

 to use a yV'inch oil-immersion, and this is the better lens in any 

 case. (See" Plate IX, b, and Plate X, a.) 



If tubercle bacilli are not found, other specimens should be pre- 

 pared and examined. It is only by repeated examinations on different 

 occasions that the negative evidence, the absence of tubercle bacilli, 

 becomes of any value. 



The tubercle bacillus is occasionally not acid-fast ; 1 probably 

 the bacilli in such cases are degenerate, and, like all degenerate 

 bacteria, fail to stain well. Spengler claims that the following 

 method will stain these and "splitter" forms: (1) Stain with 

 warm carbol-fuchsin by the ordinary method, avoiding overheating ; 

 (2) pour off the stain without washing and treat with picric acid 

 alcohol (equal parts of saturated aqueous picric acid and absolute 

 alcohol) ; (3) after 3 seconds rinse with 60 per cent, alcohol ; 



(4) treat with 15 per cent, nitric acid until yellow (about 30 seconds) ; 



(5) rinse again with 60 per cent, alcohol ; (6) counter-stain with 

 the picric acid alcohol until yellow ; (7) wash with distilled water. 

 This is an excellent method, and thick films may be used. In 

 material which has been preserved a long time, e.g. sputum with 

 carbolic, or tissue in spirit, the bacilli may be much less acid-fast 

 than in fresh material. 



Various methods have been recommended for the solution of 

 the sputum and the examination of the sediment of the bacilli. 

 In one method 5 c.c. of sputum are mixed with 50 c.c. of normal 

 KOH solution ; the mixture is warmed in a water-bath to 60-65 C. 

 until the sputum is dissolved (about 3 hours) ; 50 c.c. of cold water 

 are next added, the whole is well shaken, and again warmed for 

 ^ hour. Petroleum ether, 2 c.c., is next added, the whole is well 

 shaken, and is then kept at 60 C. until the ether has separated. 

 The bacilli will be concentrated in the fluffy layer at the junction 

 1 See Lancet, 1908, vol. i, p. 1222. 



