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Determining exact viral titers in a given sample is essential for many environmental and clinical applications, e.g., for studying viral ecology or application of bacteriophages for food safety. However, virus quantification is not a simple task, especially for complex environmental samples. While clonal viral isolates can be quantified with relative high accuracy using virus-specific methods, i.e., plaque assay or quantitative real-time PCR, these methods are not valid for complex and diverse environmental samples. Moreover, it is not yet known how precisely laser-based methods, i.e., epifluorescence microscopy, flow cytometry, and nanoparticle tracking analysis, quantify environmental viruses. In the present study, we compared five state-of-the-art viral quantification methods by enumerating four model viral isolates of different genome and size characteristics as well as four different environmental water samples. Although Nanoparticle tracking analysis combined with gentle staining at 30 °C could be confirmed by this study to be a reliable quantification technique for tested environmental samples, environmental samples still lack an universally applicable and accurate quantification method. Special attention has to be put on optimal sample concentrations as well as optimized sample preparations, which are specific for each method. As our results show the inefficiency when enumerating small, or single-stranded DNA or RNA viruses, the global population of viruses is presumably higher than expected.

Linked to diverse biological processes, groundwater ecosystems deliver essential services to mankind, the most important of which is the provision of drinking water. In contrast to surface waters, ecological aspects of groundwater systems are ignored by the current European Union and national legislation. Groundwater management and protection measures refer exclusively to its good physicochemical and quantitative status. Current initiatives in developing ecologically sound integrative assessment schemes by taking groundwater fauna into account depend on the initial classification of subsurface bioregions. In a large scale survey, the regional and biogeographical distribution patterns of groundwater dwelling invertebrates were examined for many parts of Germany. Following an exploratory approach, our results underline that the distribution patterns of invertebrates in groundwater are not in accordance with any existing bioregional classification system established for surface habitats. In consequence, we propose to develope a new classification scheme for groundwater ecosystems based on stygoregions.