Tetanus. 337 



resistance of the spore to heat that enabled Kitasato to kill off the 

 contaminating organisms and obtain pure cultures from the sur- 

 viving spores. The spores will survive desiccation for years, 

 retaining their virulence, and may live 2>^ months in putrefying 

 material. The addition to the culture medium of i>^ to 2 per 

 cent, of glucose makes the growth much more rapid and abundant, 

 and causes opacity in the medium. The upper portion clears up 

 in 6 or 7 days by the precipitation of the bacilli as a grayish mass. 

 In a glucose culture medium growth is not prevented by the pres- 

 ence of oxygen at the surface. The colonies formed in gelatine 

 plate cultures show an opaque centre with fine divergent rays, and 

 a similar radiating growth is shown in deep stick-cultures. At 

 the end of the second week the gelatine begins to liquefy and form 

 a little gas, and finally the whole mass becomes soft and sticky. 

 The bacillus does not liquefy blood serum. Cultures have a dis- 

 agreeable aromatic odor. 



The bacilli stain readily in aniline colors and by Gram's 

 method. The spores may be stained by Ziehl's method. To 10 

 parts of a 10 per cent, alcoholic solution of basic fuchsin, add 100 

 parts of a watery solution of carbolic acid. Float the cover glass 

 upon this, heating gently for three to five minutes until steam 

 begins to rise, wash well in water, and decolorize in nitric or sul- 

 phuric acid, 25 per cent, solution, then in 60 per cent, alcohol to 

 remove color from albuminous background. Wash in water and 

 mount. By placing the specimen for two minutes in a watery 

 solution of methylene blue a contrast is obtained, the bacillus blue 

 and the spore red. 



Outside the animal body the bacillus has a saprophytic life in 

 rich garden mould, street dust, stables, yards and drains, and the 

 cracks of floors. Nicolaier failed to obtain it in soil from forests 

 and from the deeper layers of garden earth. Marchesi found it 

 to a depth of two metres but no more. Again it is much more 

 abundant in tropical countries than in temperate and cold ones, 

 and appears to be, to a great extent limited to particular localities. 

 It has been found in the intestinal contents of man and horse 

 (Babes, Sormani), and in horse manure, and this mingling with 

 the surface soil and generating an abundance of ammonia deter- 

 mines the anaerobic conditions which favor the growth of the 

 microbe. This serves to explain the remarkable prevalence of 

 22 



