CULTURAL REACTIONS 411 



with a precipitating serum, though it is incapable of caUing forth the production of anti- 

 bodies (Laidlavv and Dudley 1925, Mueller 1926). The purest protein fractions appear 

 to be practically non-antigenic ; the fractions with larger molecular weight give rise on 

 injection to antibodies (for reviews, see Wells and Long 1932, Anderson 1932, Sabin 1932, 

 Calmette 1936, Seibert 1941, 1944). 



Cultmal Reactions. 



The acid-fast bacilli vary in the ease with which they grow under artificial 

 conditions. At one end of the scale arc the saprophytic acid-fast bacilli and the 

 cold-blooded tubercle bacilli, which grow well in 2 or 3 days on ordinary media ; 

 at the other end are the mammalian tubercle bacilli, which grow only on special 

 media, and which may take 2 weeks or more to form a layer of growth visible to 

 the naked eye ; in between come the avian bacilli, which grow poorly or not at 

 all on ordinary media, but which give a good growth in a few days on glycerinated 

 media. The most satisfactory media for cultivation of the tubercle bacilli are 

 inspissated serum, coagulated egg, and potato. The addition of 5 per cent, glycerine 

 to these media is of great value, enhancing the growth of all acid-fast organisms, 

 with the exception of the murine and partial exception of the bovine tubercle 

 bacillus. Its incorporation in nutrient agar or in broth renders these media 

 suitable for the growth of human and bovine bacilli, and, except for the murine 

 type, greatly improves the growth of other members of the group. 



One of the striking characteristics of the acid-fast group is the friable tenacious 

 consistency of the growth, and its adhesiveness to the medium. This is evident not 

 only in the process of subculturing the organisms, when a stout platinum loop 

 has to be used, but particularly in endeavouring to form a suspension of the 

 organisms in saline or other fluid. Instead of giving rise to a uniform turbidity, 

 they settle in granules to the bottom and leave the supernatant fluid clear ; to 

 produce a homogeneous suspension they must be ground up thoroughly in an 

 agate mortar — a process that may take anything from half an hour to several 

 days to complete. Similarly, it is difficult to make a uniform film of a culture 

 for microscopic examination ; even with a stout platinum loop it is impossible 

 to break up the growth completely, and the film remains granular. As a rule 

 the most difficult growths to emulsify are those of the human and bovine types ; 

 growths of the other types usually present less difficulty. Sometimes a creamy, 

 almost butyrous, growth is formed by members of the avian and cold-blooded 

 types, which can be emulsified rapidly ; but even with these suspensions the 

 disintegration of the bacillary clumps is rarely complete ; against an illuminated 

 dark-ground a fine granularity is visible to the naked eye. 



Another feature that is common to all the tubercle bacilli, though not to the 

 saprophytic acid-fast bacilli, is the rather pleasant, sweet, fruity odour of their 

 cultures. This odour, so far as we know, is peculiar to the tubercle bacilli ; it is 

 developed on all the usual media in which growth occurs, both solid and liquid ; 

 and is most readily detected in tubes that have been corked during incubation. 



In studying the cultural reactions of the acid-fast bacilli it is important to 

 realize that, on first isolation, the reactions of a given organism may seem to be 

 anomalous. It is often not till two or three generations, and sometimes more, 

 have been spent in artificial media that the characteristic reactions of the type 

 develop. Thus a cold-blooded bacillus, when first isolated, may grow very poorly 

 — almost like a human strain — but in 3 or 6 months in the laboratory it will become 

 accustomed to its new surroundings, and instead of giving a poor discrete growth 



