166 SPECIAL BACTERIOLOGY 



the light and kept at ordinary temperature. One-tenth c.c. of a toxin 

 so prepared is usually fatal in forty-eight hours to a guinea-pig weighing 

 500 grammes. It loses its activity after a time, though very slowly if 

 kept in the manner above mentioned. 



There is considerable variance in the degrees of virulence of different 

 cultures of diphtheria bacilli. This varying virulence tends to explain 

 the protean nature of the disease and the differing character of the 

 various epidemics. It also helps to explain the occurrence of diphtheria 

 bacilli in ' rhinitis fibrinosa ' and other benign affections. 



Experiment animals are easily immunized. Behring, and later Roux, 

 immunized horses with diphtheria toxin which had its poisonous proper- 

 ties weakened by the addition of a solution of trichloride of iodine, or 

 iodide of potassium. 



A serum possessing very high immunizing properties is obtained from 

 the horse by introducing into that animal large quantities of diphtheria 

 toxin as follows : 



ROUX'S METHOD OF IMMUNIZING A HORSE. 



Subject. Seven-year old horse, weighing about 400 kilogrammes. 

 The toxin used was very active ; ^ c.c. killed in forty-eight hours a 

 guinea-pig weighing 500 grammes. The point of injection was under 

 the skin of the neck or behind the shoulders. 



Days of Injection. Injection of ^^ tt j^ dk Reaction. 



1 % c.c. 1 10 No reaction. 



2 \ ,, 110 do. 

 4, 6, 8 \ 110 do. 

 13, 14 1 110 do. 



17 i Pure toxin. Slight oedema, no fever. 



22 1 do. do. 



23 2 do. do. 

 25 3 do. do. 

 28 5 do. do. 

 30, 32, 36 5 do. do. 

 39, 41 10 do. do. 



43 46 48 50 30 do Well-marked oedema, disappearing in 



43, 46, 48, 5C twenty-four hours. 



53 60 do. do. 



57, 63, 65, 67 60 do. do. 



72 90 ,, do. do. 



80 250 do. do. 



In eight to ten days after the last injection 5 to 6 litres of blood 



are taken from the jugular vein with a sterilized trocar, and placed in 

 the ice-chest, when a clear serum forms. 



Behring preserves the serum thus obtained by adding 0*5 per cent, 

 of carbolic acid. Schering's serum is preserved with 0*4 per cent, of 



