Detection and Identification of Free-Living Protozoa Present in Drinking Water

This chapter focuses on optimizing and applying cultivation-independent methods to obtain information about the presence and identity of free-living protozoa in river water (RW), treated water (TW), and tap water (Tap) in The Netherlands. This is the first description of protozoal diversity in freshwater by using molecular techniques. The PCR products were purified and cloned into Escherichia coli JM109 by using the Promega pGEM-T easy vectors system. Free-living protozoa represented a major part of the total eukaryotic community. The clone libraries show that the eukaryotic community in surface water, treated water, and tap water consisted, respectively, of 44, 62 and 20% of free-living protozoa. Blast analysis of the clones confirmed the presence of a highly diverse group of free-living protozoa in all three water types. Further optimization of terminal restriction fragment length polymorphism (T-RFLP) assays is needed to achieve better resolution, distributing the T-RFs of dominant free-living protozoa over the T-RFLP pattern from 50 to 500 bp. Clone libraries are a powerful tool to determine the diversity and identity of eukaryotic communities, but this technique is time-consuming. The application of real-time PCR assays for selected protozoa genera and cultivation methods will further extend one's knowledge of free-living protozoa serving as hosts for L. pneumophila in tap water installations.