CULTIVATION OF TUBERCLE BACILLUS 265 



lesions, and yet the material from such was virulent on 

 inoculation. This was supposed to be due either to the 

 paucity of the bacilli or possibly to the presence of spores. 

 Recently observations have been brought forward by Much 

 which may throw important light on this subject. Briefly put, 

 his conclusions are that the tubercle virus exists in three forms 

 (a) the ordinary bacillary form stainable by the Ziehl method ; 

 (6) a fine bacillary form which is not acid-fast, often showing 

 granules in its interior ; and (c) free granules which also fail to 

 stain with the Ziehl method. The two last forms can be stained 

 by Gram's method when the stain is applied for a long time. 

 Much gives three modifications of Gram's method, the following 

 being one which has been found by others to be specially 

 suitable : 



Methyl violet B. N. 10 c. c. of a saturated alcoholic solution in 100 c.c. of 

 a 2 per cent, watery solution of carbolic acid ; stain by boiling over the 

 name for a few minutes or at 37 C. for 24-48 hours, then treat with Lugoe's 

 iodine for 1-5 minutes, 5 per cent, hydrochloric acid for one minute, 3 

 per cent, hydrochloric acid 10 seconds, and complete the decolorisation 

 with ;i mixture of acetone and alcohol in equal parts. 



Much claims that by such a method bacilli and granules can 

 be found in tubercular lesions when the Ziehl method gives 

 a negative result. He also found that, w^ien bacilli from a 

 culture were added to sterilised milk and incubated, the acid-fast 

 forms disappeared whilst those stainable with Gram's method 

 remained; and that when this had occurred the milk when 

 injected into an animal produced tuberculosis in which acid-fast 

 bacilli were demonstrable. His statements have received con- 

 firmation by other observers, e.g. Wirths and Treuholtz, but as 

 yet it is not possible to give a definite pronouncement on the 

 whole subject. If the bacillus can pass into a form not demon- 

 strable by the method usually employed but still virulent, we 

 have manifestly to deal with a fact of the highest importance. 

 There seems to be no doubt that in certain conditions more 

 tubercle bacilli can be demonstrated in the tissues by Much's 

 method than by the ordinary carbol-f uchsin method. 



Cultivation. The medium first used by Koch was inspissated 

 blood serum (vide p. 40). If inoculations are made on this 

 medium with tubercular material free from other organisms, 

 there appear in from ten to fourteen days minute points of growth 

 of dull whitish colour, rather irregular, and slightly raised above 

 the surface (it is advisable to plant on the medium an actual 

 piece of the tubercular tissue and to fix it in a wound of the 



