Cultivation 769 



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 c.c. 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 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 moist and orange- 

 yellow in color, while in the water of condensation, though 

 growth apparently has not occurred, the detached bacilli col- 

 lect 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 multipli- 

 cation 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 pep- 

 tone 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 mul- 

 tiplication of the lepra bacillus. 



" Bacillus leprse 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 

 49 



