CULTIVATION OF TUBERCLE BACILLUS. 241 



one of the most powerful solutions ought to be employed, e.g. 

 gentian-violet or fuchsin, along with aniline-oil water or solution 

 of carbolic acid. Further, such staining solutions require to be 

 applied for a long time, or the staining must be accelerated by 

 heat, the solution being warmed till steam arises and the 

 specimen allowed to remain in the hot stain for two or three 

 minutes. One of the best and most convenient methods is the 

 Ziehl-Neelsen method (see p. 104). The bacilli present this 

 further peculiarity, however, that after staining has taken place 

 they resist decolorising by solutions which readily remove the 

 colour from the tissues and from other organisms which may 

 be present. Such decolorising agents are sulphuric or nitric 

 acid in 20 per cent solution, or 2 per cent solution of hydro- 

 chloric acid in 80 per cent alcohol. Preparations can thus be 

 obtained in which the tubercle bacilli alone are coloured by the 

 stain first used, and the tissues can then be coloured by a con- 

 trast stain. Within recent years certain other bacilli have been 

 discovered which present the same staining reactions as tubercle 

 bacilli; they are therefore called " acid-fast " (vide infra). Tu- 

 bercle bacilli, also stain by Gram's method, but the results are 

 inferior to those obtained with carbolic fuchsin. 



Bulloch and Macleod, by treating tubercle bacilli with hot alcohol and 

 ether, extracted a wax which gave the characteristic staining reactions of the 

 bacilli themselves. The remains of the bacilli, further, when extracted with 

 caustic potash, yielded a body which was probably a chitin, and which was 

 acid-fast when stained for twenty-four hours with carbol-fuchsin. 



Cultivation. The medium first used by Koch was inspissated 

 blood serum (vide p. 43). If inoculations are made on this 

 medium with tubercular material free from other organisms, 

 there appear from the tenth to fourteenth day minute points of 

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

 above the surface. Koch compared the appearance of these to 

 that of small dry scales. In such cultures they usually reach 

 only a comparatively small size and remain separate, becoming 

 confluent only when many occur close together. In sub-cultures, 

 however, growth is more luxuriant and may come to form a dull 

 wrinkled film of whitish colour, which may cover the greater 

 part of the surface of the serum and at the bottom of the tube 

 may grow over the surface of the condensation water on to the 



