DIPHTHERIA. 69 



itation with alcohol and a current of carbonic acid gas, secured a 

 white substance, and the injection of from 0.1 to 0.2 gram of this 

 substance subcutaneously in guinea-pigs, caused marked pain, fol- 

 lowed by a fibrinous swelling with hemorrhages into the muscles and 

 oedema, terminating in necrosis. From these studies Loeffler con- 

 cluded that the poison belongs to the enzymes. 



Eoux and Yersin found that bouillon cultures from which the 

 bacillus had been removed by filtration through a Chamberland filter 

 are poisonous, especially cultures that are four or five weeks old. 

 The results obtained varied with the amount of the fluid, the species 

 of animal, and the method of administration. The effects observed 

 were a serous exudate into the pleural cavity, marked acute inflam- 

 mation of the kidney, fatty degeneration of the liver, especially 

 after injection into a blood vessel, and cedematous swelling in the 

 surrounding tissue after subcutaneous inoculation. In some in- 

 stances, paralysis, generally in the posterior extremities, followed. 

 The action of the poison was slow, and death, as a rule, occurred 

 days, and in some cases, weeks after treatment, and was preceded by 

 marked emaciation. The cultures first employed were seven days 

 old ; older cultures (six weeks) contain more of the toxin, and the 

 symptoms appear within a few hours. In cultures especially rich in 

 the poison, a small amount (from 0.2 to 2 c.c.) injected under the skin 

 in guinea-pigs, suffices to induce the symptoms. Heating to 100" 

 for twenty minutes renders the poison inert, and a temperature of 58" 

 maintained for two hours markedly lessens its virulence. The toxin 

 is precipitated by absolute alcohol, and is carried down mechanically 

 by the addition of calcium chlorid to the filtered cultures. The 

 great toxicity of this substance is indicated by the statement of Eoux 

 and Yersin, that 0.4 mg. suffices to kill eight guinea-pigs or two 

 rabbits ; and that 0.02 g. of the calcium chlorid precipitate, containing 

 about 0.2 mg. of the pure poison will kill a guinea-pig within four 

 days. 



Brieger and Frankel employed cultures of bouillon and pepton 

 containing from five to six per cent, of glycerin, and others contain- 

 ing two per cent, of blood serum ; the latter were found to be most 

 suitable. In all cases they confirmed the statement of Eoux and 

 Yersin, that the cultures, at first alkaline, become strongly acid, and 

 finally again alkaline, with the exception that the glycerin cultures 

 remain acid. For the removal of the bacteria two methods were 

 employed. First, the bacilli were destroyed by heat ; when a tem- 

 perature of 100° was employed the cultures became inert, but it was 

 found that exposure for from three to four hours to 50° was sufficient 

 to destroy the germ, while the virulence of the toxin was not affected. 

 The second method of removing the bacteria consisted in filtration 

 through a Chamberland filter. The germ-free filtrate could be heated 

 to 50° without loss of toxicity, while a temperature of 60° rendered 



