BACILLUS TETANI 461 



6r blood clot produced, the spores may develop and tetanus ensue. 

 These experiments may explain, cases of so-called cryptogenic tetanus. 



Tetanus Toxin. The pathogenicity of the tetanus bacillus depends 

 entirely upon the soluble toxin which it produces. This toxin is produced 

 in suitable media by all strains of virulent tetanus bacilli, individual 

 strains showing less variation in this respect than do the separate strains 

 of diphtheria bacilli. While partial aerobiosis does not completely elimi- 

 nate toxin formation, anaerobic conditions are by far more favorable for 

 its development. 



The medium most frequently employed for the production of tetanus 

 toxin is neutral or slightly alkaline beef -infusion bouillon containing five- 

 tenths per cent NaCl and one per cent pepton. Glucose, sodium formate, 

 or tincture of litmus may be added, but while these substances increase 

 the speed of growth of the bacilli they do not seem to enhance the de- 

 gree of toxicity of the cultures. Glucose is said even to be unfavorable for 

 strong toxin development. It is important, too, that the bouillon shall 

 be freshly prepared. 1 There does not seem to be any direct relationship 

 between the amount of growth and the degree of toxicity of the cultures. 

 Under anaerobic conditions in suitable bouillon and grown at 37.5 C., 

 the maximum toxin content of the cultures is reached in from ten days 

 to two weeks. After this time the toxin deteriorates rapidly. 



Tetanus toxin has been produced without resort to anaerobic 

 methods by several observers, notably by Debrand, 2 by cultivating the 

 bacilli in bouillon in symbiosis with Bacillus subtilis. By this method, 

 Debrand claims to have produced toxin which was fully as potent as 

 that produced by anaerobic cultivation. 



The tetanus toxin, in solution in the bouillon cultures, may be sepa- 

 rated from the bacteria by filtration through Berkefeld or Chamberland 

 filters. Since the poison in such filtrates deteriorates very rapidly, 

 much more rapidly even than diphtheria toxin, various methods have 

 been devised to obtain the toxin in the solid state. The most useful of 

 these is precipitation of the poison out of solution by oversaturation 

 with ammonium sulphate. 3 Very little of the toxin is lost by this method 

 and, thoroughly dried and stocked in vacuum tubes, together with an- 

 hydrous phosphoric acid, it may be preserved indefinitely without dete- 

 rioration. The precipitate thus formed is easily soluble in water or 



1 Vaillard et Vincent, Ann. de 1'inst. Pasteur, 1891. 



2 Debrand, Ann. de 1'inst. Pasteur, 1890, 1902. 



3 Brieger und Cohn, Zeit. f . Hyg., xv. 



