THE preparation of diphtheria toxin on a large scale is, in spite of the numerous investigations which have been carried out on this subject, still a very uncertain operation. Without doubt the cause of this must be sought to a considerable extent in the fact that the usual culture media are of a very complicated nature and always contain besides the simple compounds such as sugars, inorganic salts etc., meat extract and one or other of the various kinds of peptone.Most of the experiments on the dissimilative metabolism of C. diphtheriae mentioned in the literature have been carried out on media of much simpler composition.It is a striking fact, however, that in all these experiments little or no attention is paid to the formation of toxin as such. Only a few investigators make any mention of this, yet as a rule the quantity of toxin formed is so small as to be of no practical value.In both the older and the more recent literature there is a very large number of communications in which different " routine " culture media are described and attention is drawn to some of the important factors affecting toxin formation. The older literature is conveniently summarized in the monograph " Diphtheria " published by the Medical Research Council [1923]. After an extensive discussion of all the data then available the authors come to the conclusion: " At the moment it is only by adhering rigidly to certain formulae of technique that one may hope to arrive at a satisfactory product and, even with the most careful work and attention to detail, some batches of toxin do not come up to the standard of potency demanded by the maker of antitoxin. Certain factors are still beyond control, and until these are elucidated, the problem of making toxin on an entirely satisfactory basis will not be solved."In spite of the very great amount of work which has been carried out on this subject since 1923, the verdict pronounced above still holds in its entirety.A very extensive study on the formation of toxin was carried out by Pope [1932]. He started from a so-called "semi-synthetic" medium, in which Difcoproteose-peptone was employed as the source of nitrogen in conjunction with a few inorganic salts. Various sources of carbon such as sugars, organic acids etc., were added to a relatively simple "standard solution" and the effects of these additions on the formation of toxin investigated. Further attention must be drawn to a publication by Strom [1935]. This investigator examined the influence of various substances, when added to a simple stock solution, on the course of pH and the formation of toxin after inoculation with a diphtheria culture. The problem of the biochemistry of toxin production will be elucidated only when the following conditions are fulfilled.1. The " stock solution " used must be as simple as possible; for this purpose the "semi-synthetic" medium described by Pope is eminently suitable.
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