1. Animals immunized with the formalinized filtrates of young toxic cultures of B. botulinus produce an antitoxic serum poor in precipitins.
2. Animals immunized with the formalinized filtrates of old and partly autolyzed toxic cultures produce an antitoxic serum containing precipitins.
3. Animals immunized with toxin-free autolyzed bacteria produce a serum free from antitoxin but rich in specific precipitins.
4. Animals immunized with the filtrates of an atoxic variant produce a serum free from antitoxin but rich in precipitins for the homologous toxin.
5. Animals immunized with the washed bacteria of the atoxic variant produce a serum that contains no antitoxin, but is rich in precipitins for the homologous toxin.
6. Removal of the precipitins by flocculation with a non-toxic antigen does not materially reduce the antitoxic value of a serum.
7. Removal of the proteins of the antigen by add coagulation removes the specific precipitable substance.
8. All the sera that contain precipitins produce the specific flocculus when combined with homologous toxins, anatoxins, or with the filtrates of the atoxic variant. The flocculation is restricted within the type. The amount of the precipitate and the width of the zone vary approximately with the estimated amount of bacterial protein in the antigen that is used for the immunization of animals.
We conclude, therefore, that the toxin-antitoxin flocculation is a specific bacterial precipitation phenomenon.
