Flocculation with Staphylococcal Toxin and Antitoxin

Abstract

Bronfenbrenner and Reichert 1 , 2 found that antitoxin obtained by immunizing animals against toxic filtrates of 24-day cultures of B. botulinus precipitated the filtrate while antitoxin prepared against toxic filtrates of 4-day cultures did not precipitate. They concluded that the precipitation in the Ramon test 3 may be influenced by the antibacterial antibody in the antitoxic serum. A similar observation was reported by the author 4 using the tetanal toxin-antitoxin system. It seemed interesting to see whether these findings might also influence the practical application of the Ramon test in the standardization of staphylococcal toxin and antitoxin.

In a preliminary experiment it was found that an appreciable amount of toxin was present in the 48-hour filtrate of a toxin-producing strain of staphylococcus cultivated in the broth described by Parker, Hopkins, and Gunther. 5 The cultures were incubated at 37°C in an atmosphere of 10% CO₂. A potent toxin was produced by this strain after 20 days' incubation at 37°C. White mice were killed almost immediately by the intravenous injection of 0.3 cc of undiluted filtrate. Rabbits were immunized with the formolized filtrates of 48-hour and 20-day cultures, respectively, as well as with filtrates of 48-hour and 20-day cultures of a cream-colored atoxic variant of the same strain of staphylococcus. Isolation of the atoxic variant was accomplished by repeated sub-culturing in lithium-chloride broth. As expected, the antisera prepared against the nontoxic filtrates were found to be entirely devoid of antitoxin as determined by the protection-test on mice, since even 0.5 cc of undiluted serum failed to neutralize the lethal toxin contained in 0.3 cc of a 6-day culture-filtrate.

Rane and Wyman, 6 instead of using weak toxins and relatively large amounts of antitoxin, obtained true flocculation with a strong toxin (hemolytic streptococcus) and relatively few units of antitoxin.