CULTIVATION OF BACILLUS LEPRAE. 79 



presence of another acid-fast bacillus occurring in the spleen of leprous 

 subjects. The fact that the acid-fast bacillus isolated from the two 

 different cases is of similar morphology and is evidently the same 

 organism in both instances would render it extremely improbable that it 

 was a contamination from either the spleen or from the air during the 

 inoculation of the plates. 



This paper represents a brief resume of my work on this subject up 

 to the present time and is preliminary to a more extensive research 

 already outlined. One of the first problems to be confronted is the cul- 

 tivation of the acid-fast organism in pure culture with some well-known 

 species of bacteria for the symbiotic organism and the amoeba. It is now 

 well known that amoebae can not be cultivated without the presence of 

 symbiotic bacteria. In the experiments described above the organisms 

 growing in symbiosis with the amoebae were unidentified. In as much 

 as we know nothing regarding the morphology or biological properties of 

 the leprosy bacillus on artificial media I shall first investigate the prop- 

 erties of the acid-fast organism growing in symbiosis with the amoebae and 

 a well-known species of bacteria such for example as the cholera 

 spirillum or the typhoid bacillus. In this manner if the leprosy 

 bacillus colonizes, I will probably be able to differentiate its colonies 

 from those of the cholera or typhoid organisms (constituting the sym- 

 biotic bacteria), whose characters are so well known. 



SUPPLEMENTARY NOTE. 



Since presenting this paper I have succeded in obtaining a growth of 

 an acid-fast organism similar to that obtained in the first two cases 

 described, from leprous nodules in the ear of three living subjects. I have 

 also modified the media by using plain agar alone without the beef extract 

 but the same amount of sodium chloride as was employ.ed in the first 

 experiments. The finished product should have a reaction of 1 per cent 

 alkaline to phenolphthalein. The technique of the method of cultivation 

 has not been changed. At the present time the acid-fast organisms 

 grown from the first two cases are developing well on the tenth sub- 

 culture and those cultivated from the ear are being grown on the fifth. 

 All efforts have failed to grow the bacillus on any media without the 

 association of amoebae. 



Kepeated inoculations of the leprous material on the media containing 

 amoebae seems to be necessary in most cases in order to produce a primary 

 growth of the acid fast bacillus. This may be due to the change in 

 environment of the amoebae caused l)y the addition of the blood and tissue 

 cells from the spleen. Eepeated inoculations of the plates probably 

 allow the amoebge to become gradually accustomed to this change in the 

 media and in this way perhaps a satisfactory relationship for growtli, 

 between both the amoebae and the acid-fast bacillus is obtained. 



(For the discussion of this paper before the Philippine Islands Medical 

 Association see p. 141.) 



