Extracellular Production of Toxin by Clostridium botulinum.

Abstract

It is well known that bacterial free filtrates containing the toxin of Clostridium botulinum increase in toxicity when mixed with certain substances such as normal blood serum and non-specific antitoxins. The observed increases have, as a rule, been relatively small (2 to 10 fold) and the observations have been variously interpreted by the several investigators. The possibility that the toxin might be produced extracellularly by the action of the enzymes of the organism on a suitable substrate has not been seriously entertained by the more prominent workers in this field; on the contrary, the belief is generally held that the toxin is produced only intracellularly.

From our own work with Clostridium botulinum (Type A) we have been led to the conclusion that the toxin is produced entirely intracellularly, or nearly so, in some media, while it may be produced both intracellularly and extracellularly in certain other media.

If a bacterial free filtrate of the botulinum organism is mixed with sterile skimmed milk and the mixture incubated at 37° C. until proteolysis ensues, a material increase in toxicity takes place. This is illustrated by the example given in Table I. In this experiment 1 part of the culture filtrate was mixed with 4 parts of sterile skimmed milk and the mixture incubated at 37° C. for 4 days. As a control, some of the same filtrate was incubated alone at the same temperature for the same period of time. After incubation, series of guinea pigs were inoculated with, (1) the incubated filtrate mixed with 4 parts of sterile physiological salt solution, (2) the incubated filtrate mixed with 4 parts of sterile skimmed milk within an hour previous to inoculation, and (3) the incubated filtrate-milk mixture referred to previously.