naked-eye appearances, the diphtheritic from the pseudo-diphtheritic inflammation, as well as the formerly existing confusion 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 injected into animals without producing diphtheritic paralysis. But 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 porcelain filter in order to remove all the living germs, the filtered liquid would produce exactly the same symptoms, and consequently the microbe only acted through its toxines.
The toxine is produced by the cultivation of the virulent diphtheritic 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 incubating 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 98º 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 filtered 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 centimetre, 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 methods for producing immunity to the infectious diseases affecting 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 70º C. (158º F.). Subsequently Behring commenced his experiments regarding 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-
