PROTECTIVE INOCULATIONS. 313 



for from thirty-six to forty-eight hours, and proved that the method 

 could be successfully employed in immunizing sheep ; and the fact 

 was ascertained that blood serum from an immune animal could be 

 used with success in arresting diphtheritic infection in susceptible 

 animals. To preserve the serum, which they obtained from immun- 

 ized sheep, rabbits, and guinea-pigs, they added to it 0.5 per cent of 

 pure carbolic acid. For producing immunity they found that a 

 smaller amount of serum was required than w r as necessary for the 

 cure of an animal already infected. If the injection was made imme- 

 diately after infection, from one and a half to two times the amount 

 was required; eight hours after infection the amount was three times 

 as great, and twenty-four to thirty-six hours after infection the dose 

 required was eight times the immunizing dose. 



The immunizing value of blood serum from different animals was 

 estimated by finding the smallest dose which would protect an animal 

 from fatal infection by the minimum lethal dose of a culture the toxic 

 potency of which had been carefully determined. The value is ex- 

 pressed in figures which give the proportion required compared with 

 the body weight of the animal. Thus an immunizing value of one 

 hundred would mean that one gramme of the serum is sufficient to 

 protect an animal weighing one hundred grammes from the fatal 

 effect which would be produced in a control animal of the same weight 

 by infection with a virulent culture of the diphtheria bacillus in the 

 minimum doses required to produce this result. The cultures em- 

 ployed are made in bouillon containing one per cent of peptone ; they 

 are inoculated from agar cultures and are kept in the incubating oven 

 for two days. Cultures prepared in this way were found to be quite 

 uniform in their pathogenic virulence as tested upon guinea-pigs. But 

 when cultures are kept for some time there is an increase in virulence. 

 Thus a culture obtained from a fatal case of diphtheria which in 1890 

 killed guinea-pigs in three to four days, when injected subcutaneously 

 in the dose of 0.1 cubic centimetre (two-days-old bouillon culture), 

 at the end of a year was fatal to these animals in the dose of 0.025 

 cubic centimetre. This increase in virulence is ascribed to the fact 

 that the cultures were renewed at long intervals. 



More recently (1894) Behring has fixed a standard for what he 

 calls a normal therapeutic serum. This is a serum which when in- 

 jected into guinea-pigs in the proportion of 1 : 5,000 of bod}' weight 

 saves the animal from the fatal effects of ten times the minimum dose 

 of a culture in bouillon, two days old, which would kill a control ani- 

 mal not treated. 



In a subsequent communication (November, 1894) Behring states 



