THE ANAEROBIC BACILLI 731 



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 eliminate 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. Glu- 

 cose, 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 degree of toxicity of the cultures. Glu- 

 cose is said even to be unfavorable for strong toxin development. 

 It is important, too, that the bouillon shall be freshly prepared. 11 

 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 mechanical 

 anaerobic methods by several observers, notably by Debrand, 12 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 

 separated from the bacteria by filtration through Berkefeld or Cham- 

 berland 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 solu- 

 tion by saturation with ammonium sulphate. 13 Very little of the 

 toxin is lost by this method and, thoroughly dried and stocked in 

 vacuum tubes, together with anhydrous phosphoric acid, it may be 

 preserved indefinitely without deterioration. The precipitate thus 

 formed is easily soluble in water or salt solution, and therefore 



11 Vaillard et Vincent, Ann. de Pinst. Pasteur, 1891. 



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



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



