Maximum production of the alpha toxin by Cl. welchii is dependent on the inclusion in the medium of several substances in addition to those required for growth. These factors include:
1. Some substance present in enzymatic digests of certain proteins such as casein and gelatine.
2. Glycerylphosphorylcholine and other substances that are present in extracts of pancreas.
3. The use of starch or dextrin as a carbohydrate source in the absence of other fermentable carbohydrates.
The omission of any one of these factors from the medium results in a very low yield of alpha toxin.