Submerged Culture Production of Diphtheria Toxin

Abstract

IT has been shown^1–3 that high-titre diphtheria toxin can be produced by growing the Park Williams No. 8 strain of C. diphtheriae in shaking flasks, and one of us⁴ has reported the production of 100 Lf. u./ml. toxins in stirred and aerated cultures. Recently, using the latter technique, we have been able to prepare high-titre toxins (Lf. 150–200 u./ml.) by growing the organism for 48 hr. in 14-litre amounts of tryptic digest medium1 in 20-litre ‘Pyrex’ flasks stirred by either a glass or a ‘Birmabright’ (a magnesium–aluminium alloy) stirrer with air bubbling through the cultures from an ‘Aerox’ filter (porosity P 28) at the rate of one-sixth litre of air per litre of medium per minute. Such culture filtrates contain 1,500–1,700 Lf./mgm. protein nitrogen and are, therefore, very suitable starting material for the further purification of the diphtheria toxin. Moreover, the antigen composition of the culture filtrates, concentrated by ultrafiltration, when examined by a gel diffusion test against antitoxin⁵, was found to be as complex as in surface-culture filtrates; but the amounts of the contaminating antigens were less, thus explaining the higher purities obtained from submerged cultures.