THE SERUM TREATMENT OF DIPHTHERIA. 515 



naked-eye appearances, tlie diplitlieritic from the pseudo-diph- 

 theritic inflammation, as well as the formerly existing confu- 

 sion regarding the identity of all such inflammations, at first 

 inspired doubt in the minds of many investigators that the Klebs- 

 Loeffler bacillus caused diphtheria, especially as it could be in- 

 jected into animals without producing diphtheritic paralysis. 

 Bat in 1888 Roux and Yersin found that such injections would 

 kill animals if the bacilli were introduced in sufficient number 

 and sufficient degree of virulence ; and, further, that the growth 

 of the bacilli in culture fluids produced poisonous substances, 

 called toxines, so that if the cultures were passed through a por- 

 celain filter in order to remove all the living germs, the filtered 

 liquid would produce exactly the same symptoms, and conse- 

 quently the microbe only acted through its toxines. 



The toxine is produced by the cultivation of the virulent diph- 

 theritic bacillus in broth, in contact with the air. Flat-bottomed 

 flasks that have lateral tubes, and that contain a thin layer of a 

 two-per-cent peptonized alkaline broth, are sterilized in an incu- 

 bating oven, and then a fresh culture of very virulent diphtheritic 

 bacillus is added to the broth. The flasks are kept in the oven at 

 a temperature of 98 F,, and by means of the lateral tubes moist 

 air that has been passed through a wash-bottle is constantly kept 

 passing over the broth ; within from three to four weeks a culture 

 that is rich in toxines is obtained, a thick layer of bacilli covering 

 the bottom of the flask. All diphtheritic bacilli do not furnish 

 the same quantity of toxine in cultures, nor is the power of the 

 toxine the same in cultures that are apparently made under the 

 same conditions. When the cultures are completed they are fil- 

 tered by a Chamberland filter, and the clear liquid is kept at the 

 ordinary temperature in well-filled, stoppered bottles, that are 

 protected from the light. A dose of one tenth of a cubic centi- 

 metre, about a drop and a half, of this toxine usually kills a 

 guinea pig weighing five hundred grammes, or one pound, within 

 from forty-eight to sixty hours. 



Pasteur's success in producing immunity to chicken cholera, 

 anthrax, and rabies by the inoculation of toxines produced by 

 those diseases has stimulated other investigators to seek out meth- 

 ods for producing immunity to the infectious diseases afl'ecting 

 man. Prof. Carl Fraenkel first immunized guinea pigs against 

 diphtheria by injecting them, with great care, with diphtheria 

 toxine modified by heating it at a temperature of 70 C. (158 

 F.). Subsequently Behring commenced his experiments regard- 

 ing the production of immunity, and injected animals with a 

 mixture of the toxines and iodine trichloride, though to-day he 

 injects very small doses of pure toxine at sufficient intervals for 

 the animals to rest comfortably. Brieger and Wassermann pro- 



