The paper briefly describes the development of a third marker in the single tube technique used in the confirmation of E. coli in water. It has been shown that the third marker, is totally compatible with the markers of gas production from mannitol and indole from tryptophane. The principle of the third marker is to utilise the E. coli specific enzyme, beta-d-glucuronidase, which produces the photoluminescent methylumbelliferone.