This toxin had already been isolated by Kitasato. It is a toxalbumin, and is very sensitive to the action of heat. A temperature of 65° C., maintained for 30 minutes, renders it quite inactive; and it becomes oxidized and is destroyed by the action of the air in the presence of light. Brieger and Boer,[120] by precipitating with zinc chloride the filtered culture bouillon, obtained a pure, amorphous tetanus toxin, which they also considered as a toxalbumin, and which possesses exceedingly toxic properties. [120] _Deutsche Med. Wochenschr._, No. 49, Dec. 3, 1896. If a precipitate be caused to form in these toxic solutions, as, for instance, a precipitate of calcium phosphate, this carries down with it all the toxin present in the liquid. 0.0005 Gm. of this precipitate is surely fatal to a guinea-pig. Dozon and Cournemont have observed that even in doses of 300 to 400 Gm. of the filtered culture liquid, this toxin is not immediately toxic to a horse, but kills the animal only after a period of incubation of at least twenty-four hours. The blood of such an animal, however, is immediately and directly fatal to animals into which it is injected.[121] [121] _Compt. rend. Soc. Biol._, 1893, p. 294; _Ibid._, 1894, p. 878. Experiment has shown that animals that have been cured of tetanus possess no immunity whatever against tetanus; nevertheless Behring and Kitasato[122] first, and Wassermann and Kitasato later on, succeeded in preparing a _tetanus antitoxin_. To obtain this, the immunization of the animal, horse or cow, is effected by injecting increasing quantities of the toxin, more or less attenuated by mixing it with Gramm's iodine solution; the immunization is easily and rapidly accomplished by the process devised by Roux and Vaillard.[123] [122] _Deutsch. Med. Wochenschr._, 1890. [123] _Annal. Instit. Pasteur_, VII, p. 64. The immunized animals yield a serum which, mixed with tetanus cultures, renders these innocuous, and which enjoys an antitoxic power that borders on the marvelous.[124] A quintillionth of a cubic centimeter of the serum per gramme weight of a live mouse suffices to protect the animal from an otherwise fatal quantity of tetanus toxin.[125] [124] NOCARD: _Bull. de l'Acad. de Médecine_, Oct. 22, 1895. [125] NAILLARD: _Compt. rend. de l'Acad. de Sciences_, CXX, p. 1181. This serum is nevertheless powerless to preserve man in cases of acute tetanus; it confers an immediate, but only transitory, immunity. As to its mode of action, it appears to cause a permanent condition of excitation or of nutritive reaction of the cells, which makes these resistant to the poison. As in the case of the other toxins, the quantity of antitoxin necessary to protect an organism is so much greater the later the treatment is applied.