Various laboratory media were examined for use in trimethoprim-sensitivity testing. The most suitable were Wellcotest Sensitivity Test Agar and Wellcome Nutrient Agar, because these were the least antagonistic to the action of trimethoprim. Thymine as well as thymidine were found to antagonise trimethoprim activity against wild-type bacteria, and both these compounds are absent from the above media.

A procedure was devised for the identification of thymineless mutants, which are inherently resistant to trimethoprim. The two Wellcome media were found to contain an inhibitor that prevented the uptake of thymine but not of thymidine by thymineless mutants. The action of this inhibitor could be partially reversed by the addition of deoxyadenosine to the medium.

Because thymine and thymidine both antagonise the action of trimethoprim, a two-stage testing scheme was devised to distinguish between three types: trimethoprim-sensitive thymine-independent, trimethoprim-resistant thymine-independent, and trimethoprim-resistant thymine-requiring organisms.


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