506 PATHOGENIC MICROORGANISMS 



for an unusual time. A differentiation from tubercle bacilli by virtue of 

 greater ease of decolorization is of value only in the hands of those 

 having much experience with these bacilli, and follows no regular laws 

 of acid-strengths or time of apphcation which can be generally apphed 

 by the inexperienced. In tissues, the bacilli are easily stained by the 

 methods used for staining tubercle bacilli. The sections are left in the 

 Ziehl carbol-fuchsin solution either from two to twelve hours at incu- 

 bator temperature or for twenty-four hours at room temperature. 

 Subsequent treatment is that employed in the case of tuberculous tissue 

 sections (see p. 112). 



Cultivation. — Cultivation of the leprosy bacillus has not met with 

 success. Hansen and others who have approached the problem with 

 a thorough knowledge of the microorganism, combined with a com- 

 petent bacteriological training, have failed in all their attempts. 

 Numerous positive results reported by observers have always lacked 

 adequate confirmation. Recently, Ilost,i of the British Army Medical 

 Corps, has claimed success in cultivation of leprosy bacilH upon salt-free 

 bouillon, his point of departure being the previous observation that 

 salt-free media favored the growth of tubercle bacilli. His results have 

 not been confirmed. 



In 1909 Clegg^ succeeded in growing an acid-fast bacillus from 

 leprous tissue, obtaining his results by inoculating leprous material 

 upon agar plates upon which ameba coli had been grown in symbiosis 

 with other bacteria. On such plates the acid-fast bacilli multiphed, 

 and, subsequently, pure cultures were obtained by heating the cultures 

 to 60° C, which destroyed the ameba coli and other bacteria. These 

 results were confirmed by other workers and, soon after that, Duval ^ 

 not only succeeded in repeating Clegg's experiments, but obtained cul- 

 tures of an acid-fast bacillus directly from leprous lesions without the 

 aid of ameba. He first observed that the leprosy organism would multi- 

 ply around a transplanted piece of leprous tissue upon ordinary blood 

 agar tubes upon which influenza bacilli and meningococci were grown. 

 He concluded that such growth depended upon chemical changes in 

 the media and believed the formation of amino-acids essential for the 

 initial growth. The method he subsequently described depended upon 

 supplying these substances either by adding tryptophan to nutrient 

 agar or by pouring egg albumen and human blood serum in Petri dishes, 



1 Rost, Brit. Med. Jour., 1, 1905. 



^ Clegg, Philippine Jour, of Sc, iv, 1909. 



' Diwal, Jour. Exp. Med., xii, 1910, and ibid., 15, 1912. 



