1911.] Cultivating the Mycobacterium enteritidis, etc. 537 



No growth was observed in the absence of oxygen or with considerable 

 excess of oxygen. If sub-cultures which have been kept anaerobically for 

 three months at 39° C. are placed under aerobic conditions, the cultures 

 grow, showing that although the bacillus does not grow anaerobically it is 

 not killed by the absence of oxygen. 



Growth occurs between 28° C. and 43° C, or perhaps a little beyond 

 these limits, the optimum being about 39° C, but it is always slow. The 

 reaction of the medium should be distinctly alkaline ; the degree of 

 alkalinity possessed by new laid eggs is very suitable, and if this is in any 

 way lessened in the medium there is a marked diminution in the rapidity 

 and amount of the growth. As has been stated, no growth occurs on any of 

 the artificial media in general bacteriological use, such as peptone bouillon, 

 agar, gelatine, serum, potato, or egg, even when such substances as glycerine, 

 sugars, amino-acids, fresh blood, etc., are added. It is absolutely essential 

 that certain previously detailed bacteria or extracts from them be added to 

 one or other of the media, before any growth of Johne's bacillus takes place, 

 and this is equally true for strains of Johne's bacillus which have been 

 freshly isolated from the animal body and for strains which have been 

 cultivated on artificial media for 15 months or more. 



Of the bacilli tested, undoubtedly the most suitable for adding to the 

 medium is the timothy-grass bacillus; certain strains of human tubercle 

 bacilli are also very good. 



On the egg timothy-grass bacillus medium previously described, Johne's 

 bacillus, when taken from the animal body, will grow as tiny discrete 

 colonies which usually become visible in three to five weeks. At first the 

 colonies are round, smooth, and dull stone white, they are slightly heaped up, 

 •and as growth increases this becomes more marked, while the colour turns 

 to a dull light yellow. Later, the colonies may coalesce and the growth 

 show some wrinkling, while the colour may change to a light yellowish 

 brown. The degree of pigmentation seems to be considerably influenced by 

 the amount of pigment in the timothy grass bacillus incorporated in the 

 medium, and possibly also, though to a less extent, by the colour of the egg 

 itself. 



If the first culture taken from the diseased tissue is sub-cultured on to 

 a fresh tube of medium, visible growth occurs on this in a few days, and may 

 reach its maximum in about two months instead of three. The growth now 

 appears as a heaped-up continuous line along the needle track, with only an 

 occasional discrete colony at the margins. Wrinkling of the surface may 

 also be more marked, especially if the culture is growing very well on a tube 

 which is a little dry. When the timothy-grass bacillus in the medium is 



