744 Leprosy 



trypsin medium. After tlie tliird or fourth generation the bacilli may Jje grown 

 without difficulty upon glycerinated serum agar prepared in the following 

 manner : 



"Twenty grams of agar, 3 gm. of sodium chlorid, 30 cc. of glycerin, and 500 

 cc. of distilled water are thoroughly mixed, clarified, and sterilized in the usual 

 way. To tubes containing 10 c c. of this material is added in proper proportion a 

 solution of unheated turtle muscle infusion. Five hundred grams of turtle 

 muscle are cut into fine pieces and placed in a flask with 500 cc. of distilled water. 

 This is kept in the ice-chest for forty-eight hours and then filtered through gauze 

 to remove the tissue. The filtrate is then passed through a Berkefeld filter for 

 purposes of sterilization. By means of a sterile pipet, 5 cc. of the muscle filtrate 

 is added to the agar mixture which has been melted and cooled to 42°C. _ The 

 tubes are now thoroughly agitated and allowed to solidify in the slanted position. 



"This medium is perfectly clear or of a light amber color, and admirably suited 

 to the cultivation of the BaciUus lepra, once the initial culture has been started. 

 Growth is luxuriant and reaches its maximum in forty-eight to sixty hours. On 

 the surface of this medium the growth is moist and orange-yellow in color, while 

 in the water of condensation, though growth apparently has not occurred, the 

 detached bacilli collect in the dependent parts in the form of feathery masses 

 without clouding the fluid. 



"Ordinary nutrient agar may be used with trypsin as a plating medium instead 

 of the inspissated serum where bits of tissue are employed. With the addition of 

 I per cent, of tryptophan it answers every purpose, whether the bacilli are planted 

 with tissue or alone. It also serves to start multiplication of lepra bacilli that 

 are contaminated at the time of plating. In the latter case the medium is 

 'surface seeded' with an emulsion of the tissue juices in the same manner as in 

 preparing 'streak' plates. The leprosy colonies in the thinner parts of the loop 

 track are well separated and easily distinguished from those of other species by 

 their color and by their appearance only after two to five days. 



"In using an agar medium it is well to leave out the peptone and to titrate the 

 reaction to 1.5 per cent, alkaline in order to prevent too profuse growth of the 

 associated bacteria; besides, an alkaline medium seems best adapted for the 

 multiplication of the lepra badillus. 



"Bacillus leprae will also grow on the various blood-agar media once they are 

 accustomed to artificial conditions. The Novy-McNeal agar for the cultivation 

 of trs^panosomes gives a luxuriant growth of the organism if 2 per cent, glycerin 

 has been added; without the glycerin, growth is very scant. Fluid media are 

 not suited for the artificial cultivation of leprosy bacilli unless they are kept upon 

 the surface. Like the tubercle bacilli they require abundant oxygen. . . 



"Ordinarily the growth of Bacillus lepras is very moist, and in this respect 

 unlike that of Bacillus tubertulosis, except possibly the avian strain. Sometimes 

 when the medium is devoid of water of condensation, the growth is dry and occa- 

 sionally wrinkled, though it is easily removed from the surface of the medium. 



"The chromogenic property of lepra cultures is a constant and characteristic; 

 feature of the rapidly growing strains. The color varies in the degree of intensity 

 depending upon the medium employed. If glycerinated agar (without peptone) 

 is used, the colonies are faint lemon, while on inspissated blood-serum they are 

 deep orange. It is noteworthy that the growth in the tissues and in the first 

 dozen or so generations on artificial media is entirely without pigment." 



Although each of the workers upon leprosy has begun by asserting 

 that he had certainly cultivated the specific organism, a time comes 

 when a more extended acquaintance with the bacteriology of the 

 disease seems to cause him to doubt the results of his own work. 

 This is particularly true of this work of Duval, which was prosecuted 

 with enthusiasm, carried conviction with it, and then was partially 

 repudiated by its author, for in the discussion before the 17th Inter- 

 national Medical Congress in London in 1913, Duval is reported as 

 saying that "he knew less of the bacteriology of leprosy now than he 

 did some four years ago. He had made several mistakes, had 



