Abstract
Flow cytomtery (FCM) has become a standard approach to enumerate viruses in water research. However, the nature of the fluorescent signal in flow cytometric analysis of water samples and the mechanism of its formation, have not been addressed for bacteriophages expected in wastewaters. Here we assess the behaviour of fluorescent DNA-staining dyes in aqueous solutions, as well as sensitivity and accuracy of FCM for enumeration of DNA-stained model bacteriophages λ, P1, and T4. We demonstrate that in aqueous systems fluorescent dyes form a self-stabilized (pseudolyophilic) emulsion of auto-fluorescing colloid particles. Sample shaking and addition of surfactants enhance auto-fluorescence due to increased dispersion and, in the presence of surfactants, stabilization of the dye emulsion. Bacteriophages with genome sizes <100 kbp (i.e. λ & P1) did not generate a distinct population signal to be detected by one of the most sensitive FCM instruments available (BD LSR Fortessa™ X-20), whereas the larger T4 bacteriophage was resolved as a distinct population of events. These results indicate that the use of fluorescent dyes for bacteriophage enumeration by flow cytometry can produce false positive signals and lead to wrong estimation of total virus counts by misreporting colloid particles as virions, depending on instrument sensitivity.
Highlights
Viruses are the most numerous microbial group and have a fundamental impact on aquatic ecosystem dynamics (Steward et al, 2013; Kauffman et al, 2018)
Critical to the presence of possible artifacts analysed by Flow cytometry (FCM), this dye produces small crystals or amorphous mass, which may lead to uneven distribution of the SYBR fluorophore among the aliquots used for sample staining (Fig. 2)
Numerous fluorescing SYBR® Green I particles were observed by microscopy (Fig. 4), and FCM signal was more intense when compared to controls with no surfactant added
Summary
Viruses are the most numerous microbial group and have a fundamental impact on aquatic ecosystem dynamics (Steward et al, 2013; Kauffman et al, 2018). With the development of sensitive fluorescent dyes, TEM was replaced by epifluorescent microscopy (EFM) (Noble and Fuhrman, 1998; Patel et al, 2007), which has demonstrated even higher counts, compared with TEM (Hermes and Suttle, 1995; Weinbauer and Suttle, 1997). Though sensitive, these direct methods are labor intensive and time consuming. Flow cytometric virus enumeration has become a standard approach in water research (Marie et al, 1999)
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