A rigorous assessment and comparison of enumeration methods for environmental viruses

Abstract

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.

Comparison of interferometric light microscopy with nanoparticle tracking analysis for the study of extracellular vesicles and bacteriophages

“…As a first step towards evaluation of the Videodrop, we precisely determined the concentration of phages using three classical methods. We established that qPCR and EPI provide very similar concentrations, in contrast to a previous study in which the authors found significantly lower concentrations by EPI than qPCR (Kaletta et al 2020). The better agreement between the values obtained by the two methods in our study may have resulted from the use of a more concentrated staining solution, the addition of a flash freezing step to improve the staining, and/or the use of a different microscopic setup.…”

Adaptation of the polony technique to quantify Gokushovirinae, a diverse group of single‐stranded DNA phage

“…Quantifying gokushoviruses remains difficult due to methodological biases. Quantification of total viral communities by epifluorescence microscopy and flow cytometry after nucleic acid staining biases against viruses with small genomes and genomes that are single stranded (Tomaru and Nagasaki, 2007; Holmfeldt et al ., 2012; Kaletta et al ., 2020). RCA, which is the main method used to identify ssDNA phage genomes, preferentially amplifies these genomes over those of dsDNA phage, prohibiting inference of the relative abundance of different phage types from sequence data.…”

Container material dictates stability of bacteriophage suspensions: Light scattering and infectivity measurements reveal mechanisms of infectious titre decay

“…NTA and plaque counting form complementary, tandem measurements, which together reveal richer insights into the evolution of the phage suspension titre and aggregation state, compared to either technique alone. NTA yields individual particle information across statistically significant sample sizes (Malvern, 2017) and has been demonstrated for the study of bacteriophage (Hsiao et al, 2016; Kaletta et al, 2020) and viral suspensions (Du et al, 2010; Szakács et al, 2018). In contrast, dynamic light scattering (DLS) yields ensemble averages of diffusion coefficients (and extrapolated distributions of hydrodynamic radius).…”