Triple-Sugar Iron Agar Medium for the Identification of the Intestinal Group of Bacteria

Abstract

containing an excess of iron (Mueller and Miller, J. Immunol., in press). For purposes of attempted purification it has been grown in a heart infusion medium containing glucose, tryptic digest of casein, and an excess of reduced iron. The crude toxins obtained from this medium contain 200,000 to 800,000 mouse M.L.D. per ml (ca. 5 X 104 mg N2 per M.L.D.). Several lots of the crude toxin have been purified by precipitation with cadmium chloride according to Eaton and Gronau, by precipitation with 0.5 saturated ammonium sulfate followed by dialysis at 0 to 5 C against 0.9 per cent NaCl, or by precipitation with cadmium chloride and reprecipitation with ammonium sulfate. The best preparation (table 1, toxin no. 4) contained 0.023 X 10-mg N2 per M.L.D. The relatively constant ratio, Lf/M.L.D., indicated that losses during purification did not result from inactivation of toxin; rather, losses resulted largely from incomplete elution from the cadmium precipitate. Although the N2/M.L.D. ratio represents a toxin of a hundredfold greater purity than any previously obtained, we have as yet no evidence that a limit of purification has been reached. Further, these data do not yet exclude the possibility that the toxic entity is nonprotein and that it is carried through the purification procedures by adsorption on inactive protein. Indirect evidence, however, argues against this assumption: 80 per cent of the total nitrogen of the purified preparations is precipitated by 5 per cent trichloroacetic acid and 70 per cent by 1 per cent metaphosphoric acid; and the total nitrogen, calculated as protein, constitutes .100±2 per cent of the dry weight in the purified preparations.