352 THE LEPROSY BACILLUS 



On glycerin-potato (primary culture). After incubation for 10 days at 37 C. 

 very small, yellowish, hardly visible colonies appeared. 



On glucose-glycerin-agar. Small, colourless, irregularly circular colonies. 



On coagulated serum. Small, greyish-yellow, irregularly circular colonies. 



On fish broth. A viscous precipitate adhering to the sides of the vessel. 



Bacilli from these cultures were agglutinated by the blood of lepers in dilutions 

 of 1 in 70 to 1 in 1000 and only in dilutions of 1-30 or 1-40 by the blood of other 

 persons. 



[Rost employed a medium of the following composition : 



Distilled volatile alkaloid of rotten fish, - 250 c.c. 



Weak Lemco broth (without peptone or salt), - - - 250 c.c. 

 Milk, 50 c.c. 



which he sowed with material from cases of nodular leprosy and obtained in 3 days 

 a slight stringy growth at the bottom of the tube which on microscopical examination 

 proved to be masses of acid-fast bacteria. Sub-cultures were sown on agar and 

 in broth (no salt or peptone) and a growth was obtained in 48 hours. 



[Clegg grew the parasite of leprosy symbiotically with amoebae and their symbiotic 

 bacteria on an agar medium. After destroying the amcebse and bacteria by heat 

 at 60 C. for half an hour pure cultures of the organism were obtained by sub -cul- 

 tivating on ordinary media agar, potato, milk, etc. 



[Duval used egg-albumin or human blood serum poured into sterile Petri 

 dishes and inspissated for 3 hours at 70 C. 



[The excised leprous nodule is cut into thin slices ('5-1 mm.) and distributed over 

 the surface of the albumin. After sowing, the medium is bathed in a 1 per cent. 

 solution of trypsin added with a pipette but the tissue must not be submerged. 

 Incubate at 20 C. 1 for a week to 10 days trypsin being added from time to time 

 as evaporation necessitates. 



[Sub-cultures may be sown on the albumin- trypsin medium but after sub- 

 cultivating three or four times growth can be obtained on a glycerin- agar. 



Agar. 20 grams. 



Salt, - - 3 



Glycerin, 30 c.c. 



Distilled water, - 500 c.c. 



Mix, clear and sterilize in the usual manner. 



To 10 c.c. of the agar at 42 C. add 5 c.c. of unheated turtle muscle infusion : 

 Turtle muscle cut into fine pieces, - 500 grams. 



Water, - ... - 500 c.c. 



Keep in the ice chest for 48 hours : filter through gauze and then through a Berkefeld 

 filter. 



[Duval claims that the organism he cultivated was the true leprosy parasite 

 because with its aid he was able to produce the lesions of leprosy in a monkey. 



[Twort has introduced a method of cultivation based upon the addition of 

 sterilized tubercle bacilli to an egg medium. Growth is not visible to the 

 naked eye for about 6 weeks. 



[The material used was the nasal discharge and scrapings from a typical leper. 



[The nasal discharge was first placed in a 2 per cent, solution of ericolin a gluco- 

 side at 38 C. for 1 hour to destroy contaminating organisms and the sediment 

 was then used for sowing the culture medium. 



[The culture medium. Cultivations of the tubercle bacillus on Dorset's egg medium 

 were steamed and the growth scraped off (care being taken to avoid the medium 

 containing the waste products). The tubercle bacilli were ground up into an emul- 

 sion with glycerin and normal saline solution, steamed for 30 minutes and added 

 to the yolk and white of new-laid eggs in the following proportions : 



Eggs. 75 parts. 



8 per cent, saline solution, 95 parts. 



Mix well and add tubercle bacilli 1 per cent, and glycerin 5 per cent, or less. 



[The medium was tubed, heated to 60 C. for 1 hour and on the following morning 



[ l In his original experiments Duval incubated at 37 C. but now finds room tempera- 

 ture more suitable.] 



