D. W. STAINER, J. M. C. CORKILL, AND M. J. SCHOLTE
Connaught Medical Research Laboratories, University of Toronto, Toronto, Ontario
Received June 1, 1968
A new medium has been developed which does not contain any meat digest products and is suitable for the deep-culture production of high-potency diphtheria toxins. The main ingredient is a commercially available, tryptic digest of casein known as N.Z. Amine type A. It has been found that when the iron, calcium, and phosphate levels in the medium are care- fully controlled by suitable additions of ferrous sulphate, calcium chloride, and inorganic phosphate solutions, high levels of diphtheria toxin are regularly produced (200 Lf/ml in a 48-hour growth period).
Canadian Journal of Microbiology, 14, 1155 (1968)
The production of diphtheria toxin in sub- merged culture has been described by many authors (2, 4, 5, 6, 10). Corynebacterium diphtheriae cultured in media containing enzymatic digests of beef muscle has yielded diphtheria toxin with titers greater than 200 Lf1/ml in 48 hours. Yoneda developed a totally synthetic medium consisting of only three amino acids and showed that if the cultural conditions were carefully controlled, toxins of high potency could be produced (15). Raynaud et al. (9) also used a synthetic medium for deep culture toxin production but in small volumes. For the commercial production of diphtheria toxin, however, both of the above-mentioned synthetic media are impractical and too expensive for routine use. There is a need for an easily prepared medium without meat digests which will yield good toxin production by C. diphtheriae in deep culture, since it has been shown by Stainer that diphtheria toxoids, prepared from toxins produced by growth of C. diphtheriae in beef broths, frequently contain antigenic material of bovine origin (12, 13). These extraneous proteins cause anaphylactic responses to beef serum in sensitized guinea pigs and could possibly give untoward reactions in human subjects who receive repeated doses of such diphtheria toxoids.
The original semisynthetic medium of Mueller (7) was composed of an acid digest of casein (Casamino acids) and for many years the production of diphtheria toxins for conversion to toxoids has been carried out in still culture using this medium. These toxoids do not possess any beef-sensitizing properties. Unfortunately, in deep culture, Mueller's medium did not give good toxin yields (30-40 Lf/ml), and much work has been done to develop a medium which would be free of sensitizing antigens and which would support diphtheria toxin production in submerged culture. Early work in this laboratory, using a tryptic rather than an acid digest of casein, gave quite promising results, although the Lf levels obtained were still much less than those produced in meat digest broths (1). This paper describes the further modification of the casein digest medium which is inexpensive, easy to prepare, and which yields high titers of diphtheria toxin.
Methods
Preparation of Papain Digest of Beef Broth (P.D.B. Broth)
This was described previously (12).
Seed Culture Preparation and Growth Conditions
A modified P.W. 8 strain of C. diphtheriae was used throughout. The cultures for seeding the flasks or fermenters were obtained by subculturing from Loeffler slopes into 100 ml P.D.B. broth in 500-ml Erlenmeyer flasks and incubating the cultures on a shaker for 24 hours at 34-35 °C. The volume of seed used was 4 ml per liter. Growth conditions were as described previously (12).
Preparation of Enzymatic Digest of Casein Medium
The basic ingredient of the medium was an enzymatic digest of casein known as N.Z. Amine type A as supplied by the Sheffield Chemical Corporation, Norwich, New York. For a 60-liter lot the medium was prepared in the following manner. [end of text]
1 Lf, limit flocculation.