yoo Leprosy 



hence explains the advantage of adding tryptophan. The medium 

 most successfully employed by Duval was as follows: 



"Egg-albumen or human blood-serum is poured into sterile Petri dishes and 

 inspissated for three hours at 7oC. The excised leprous nodule is then cut into 

 thin slices, 2 to 4 mm. in breadth and 0.5 to i mm. in thickness, which are dis- 

 tributed over the surface of the coagulated albumin. By means of a pipette the 

 medium thus seeded with bits of tissue is bathed in a i per cent, sterile solution of 

 trypsin, care being taken not to submerge the pieces of leprous tissue. Sufficient 

 fluid is added to moisten thoroughly the surface of the medium. The Petri dishes 

 are now placed in a moist chamber at 37C., and allowed to incubate for a week or 

 ten days. They are removed from the plates from time to time, as evaporation 

 necessitates, for the addition of more trypsin. It will be noted that after a week 

 or ten days the tissue bits are partially sunken below the surface of the medium 

 and are softened to a thick, creamy consistence, fragments of which are readily 

 removed with a platinum needle. On microscopic examination of this material it 

 is noted that the leprosy bacilli have increased to enormous numbers and scarcely 

 a trace of the tissue remains. Separate lepra bacillus colonies are also discernible 

 on and around the softened tissue masses. . . . The colonies are at first gray- 

 ish white, but after several days they assume a distinct orange-yellow tint. . . . 

 Subcultures may be obtained by transferring portions of the growth to a second 

 series of plates or to slanted culture-tubes that contain the special albumin-trypsin 

 medium. After the third or fourth generation the bacilli may be 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 cc. 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 42C. 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 Bacillus 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 moit 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 bacillus. 



"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 trypanosomes 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 leprae is very moist, and in this respect 

 unlike that of Bacillus tuberculosis, except possibly the avian stain. 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 



