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A system was developed for the identification of mycobacteria such as Mycobacterium tuberculosis and M. avium, by thin layer chromatography of 55Fe-labelled mycobactin. Approximately 2 × 103 mycobacteria were detected within 24 h and little operator time or skill was required. M. avium, M. intracellulare and M. scrofulaceum were found to have lower requirements for iron than other mycobacteria and this may influence their growth in host organisms.