ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 261 



vessel and a little sterilised water added. The mucus was allowed to 

 dry slowly at from 18-25°. After a few days the dried mucus and 

 water were sown on plates of washed agar. About 1 plate in 10 gave a 

 successful culture. Each of these served as a starting point for obtaining 

 the pure mixed culture by progressively eliminating the bacteria. 



Each time the amoeba was sown at the bottom of the tube, and the 

 symbiotic bacterium at the top. The plate was kept at 25°. After a 

 few days the amceba reached the upper part, and from here the amoebas 

 were taken for the next culture. 



Cultivating the Bacillus of Leprosy.* — E. R. Rost cultivates 

 Bacillus lepra and also other acid-fast bacilli on media from which 

 chlorine has been removed. The medium is made by distilling beef 

 extract, or by passing a current of superheated steam from the autoclave 

 over boiling beef extract, or by passing superheated steam over the beef 

 extract soaked in pumice stone in bottles inside the autoclave. 



By the last procedure a growth of B. tuberculosis is obtained in from 

 1-3 days, of B. lepras in from 3-5 days. The characteristic appearance 

 is a curly white, stringy, heavy deposit at the bottom of the tubes, which 

 is hard to shake up, but, when shaken up, appears as a curly white stringy 

 shred in the tubes. 



A satisfactory solid medium is obtained by dialysing nutrient agar 

 in frequently changed warm distilled agar ; by this means the sodium 

 chloride is disposed of, and on the surface of the medium the acid-fast 

 group of bacteria grow with greatest ease. The bacillus of leprosy grows 

 at first as a white and later as a yellow, or brick-red, curly thick growth, 

 very much like the bacillus of tubercle on the glycerinised nutrient agar. 



The author then calls attention to the staining reactions of B. lepras, 

 and states that it may be differentially diagnosed from other acid-fast 

 bacteria as follows : (1) It retains the stain of acid dyes much more than 

 any of the other bacteria of this class. It retains the stain of carbol- 

 fuchsin even after decolorisation in 25 p.c. nitric acid. (2) It is more 

 irregular than the tubercle bacillus, and not curved, and is somewhat 

 smaller. (3) It contains small oval spores within itself, which are highly 

 refractile, and the end of the bacillus may be open where some have 

 presumably escaped. (4) It has a beady appearance, due to the presence 

 of these oval spores. (5) Like the B. tuberculosis, it may grow out into 

 cultures into long, branching filaments, but there are often oval spores 

 separate in the cultures, and these may be alone visible at times. (6) 

 In the body it is found in great numbers inside epithelial cells, generally 

 in the middle of the cells, whereas the B. tuberculosis is found in small 

 numbers inside giant cells at the polar ends. 



In order to obtain pure cultures from a given case, a tube of the 

 medium is inoculated with a piece of leprous tissue, and incubated at 

 100° F. In from 3-5 days the thick deposit is examined. It is usually 

 found to contain the bacilli of leprosy and other organisms. The tube 

 is then placed in a warm Petri dish of the dialysed medium. In from 

 3-5 days colonies of B. lepras may be picked out in the usual way. 



Then follows the method of making the toxin, or leprolin. 



* Brit. Med. Journ., 1905, i. pp. 294-6. 



