CULTIVATION OF THE BACILLUS 81 



B, phlei^ except that redistilled water was used in 

 place of the glycerine and saline for extraction. We 

 found that Johne's bacillus grew on the glycerine- 

 saline extract medium, and on that containing the 

 residue. It also grew on that containing the residue 

 after extraction with redistilled water, but it failed to 

 grow on the medium containing the distilled-water 

 extract. From this we concluded that the ** essential 

 substance " is only very slightly, if at all, extracted 

 by redistilled water, but that it is soluble in a 

 glycerine - saline solution, although in the above 

 case it is clear that some of it remained in the 

 residue. 



A further series of experiments was made, using 

 ethyl alcohol as the solvent. Two grammes of dry, 

 powdered B. phlei were placed in a Soxhlet 

 apparatus with loo c.c. of absolute alcohol and ex- 

 tracted for three hours. The residue was dried in 

 an incubator, and the alcoholic extract evaporated to 

 dryness, leaving a dark yellowish, sticky mass. The 

 extract and residue were then weighed separately, 

 and it was found that the bacilli were reduced in 

 weight from 2 grammes to i'25 grammes, the difference 

 being represented by the extract. (In later experiments 

 we have extracted very much larger quantities of 

 various acid-fast bacilli, sometimes using as much as 

 100 grammes of dry bacilli, and we have found that 

 the proportion of extract to residue varies consider- 

 ably, the proportion of extract usually being lower 

 than that given above.) Media were prepared with 

 the residue and with the extract thus obtained : some 

 contained only i or J per cent., and some i per cent., 

 of the extract or residue. Tubes of each were inocu- 

 lated with a young growth of Johne's bacillus and 

 incubated at 39° C. Good growths were obtained on 



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