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The microbiota of the water and biofilms in DUWLs can be very complex [2, 4]. If the biofilms are not regularly removed they can build up complex microbial communities composed of heterotrophic environmental Gram-negative bacteria in the water. Opportunistic pathogens such as Legionella and Pseudomonas species have gained the most attention, but other heterotrophic species such as mycobacteria, staphylococci (S. aureus, S. epidermidis), and a number of Gram-negative rods and fungi may also be present [2, 4, 7]. Potential pathogens grow rapidly (within 2 days) on ordinary solid bacteriological media in 36 °C. Samples could be incubated at room temperature (22 °C) but grow slower [8]. A longer incubation time (1 week) will increase the number of CFU due to the slow-growing nature of most heterotrophic bacteria for which the water lines are their natural habitat [4]. High numbers of slow-growing bacteria therefore indicate poor water quality, the presence of a biofilm, and a rich inner life of the DUWL.

A special concern relating to biofilms and the microbiota in water lines such as in DUWLs is the presence of protozoa/amoebae [13]. There are various examples, with the most recognized being Acanthamoeba species. Protozoans are of special interest in Legionella pathogenicity (see below).