Enumerating Viruses by Using Fluorescence and the Nature of the Nonviral Background Fraction

Pollard, Peter

doi:10.1128/aem.01268-12

Cited by 5 publications

(7 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hence, when high gain levels are used to enumerate small-particle virions by FCM, hydrophobic fluorophores may generate various levels of false positive ‘virus’ signals. The same phenomenon was observed earlier by Pollard (2012), who compared the excitation and emission spectra of organic matter in water, in parallel with intact virus particles, and confirmed that about 70% of the fluorescent signal was associated with the matrix itself independently of the presence or absence of virus. Although Pollard did not use flow cytometry, his findings contribute to our observations that fluorescent colloid dye particles, present in dye-stained virus suspensions, can comprise a significant portion of the FCM signal.…”

Section: Discussionsupporting

confidence: 87%

Colloid chemistry pitfall for flow cytometric enumeration of viruses in water

Dlusskaya

Atrazhev²,

Ashbolt

2019

Water Research X

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 87%

Colloid chemistry pitfall for flow cytometric enumeration of viruses in water

Dlusskaya

Atrazhev²,

Ashbolt

2019

Water Research X

View full text Add to dashboard Cite

show abstract

“…Although the isolation methods employed in this study unveiled a wide range of VLP morphotypes, no quantitative assessments were undertaken. To further our understanding of viral dynamics within the sponge holobiont, quantitative studies that count the number of VLPs per known tissue area, perform quantitative transmission electron microscopy (qTEM) ( Brum, Schenck & Sullivan, 2013 ), flow cytometry (Brussaard, 2004; Pollock et al, 2014 ) or fluorescent staining ( Leruste, Bouvier & Bettarel, 2012 ; Pollard, 2012 ) should also be performed.…”

Section: Discussionmentioning

confidence: 99%

Morphological characterization of virus-like particles in coral reef sponges

Pascelli

Laffy

Kupresanin

et al. 2018

PeerJ

View full text Add to dashboard Cite

Marine sponges host complex microbial consortia that vary in their abundance, diversity and stability amongst host species. While our understanding of sponge-microbe interactions has dramatically increased over the past decade, little is known about how sponges and their microbial symbionts interact with viruses, the most abundant entities in the ocean. In this study, we employed three transmission electron microscopy (TEM) preparation methods to provide the first comprehensive morphological assessment of sponge-associated viruses. The combined approaches revealed 50 different morphologies of viral-like particles (VLPs) represented across the different sponge species. VLPs were visualized within sponge cells, within the sponge extracellular mesohyl matrix, on the sponge ectoderm and within sponge-associated microbes. Non-enveloped, non-tailed icosahedral VLPs were the most commonly observed morphotypes, although tailed bacteriophage, brick-shaped, geminate and filamentous VLPs were also detected. Visualization of sponge-associated viruses using TEM has confirmed that sponges harbor not only diverse communities of microorganisms but also diverse communities of viruses.

show abstract

“…Phages can be quantified via fluorescence-based microscopy or cell counting when stained with a fluorophore ( Figure 4 ; Wen et al, 2004 ; Pollard, 2012 ). Common fluorescent stains are SYBR Green I and II ad SYBR Gold ( Shibata et al, 2006 ; Patel et al, 2007 ).…”

Section: Methods To Study Phages In Bwt Systemsmentioning

confidence: 99%

Bacteriophages in Biological Wastewater Treatment Systems: Occurrence, Characterization, and Function

Runa

Wenk

Bengtsson³

et al. 2021

Front. Microbiol.

View full text Add to dashboard Cite

Phage bacteria interactions can affect structure, dynamics, and function of microbial communities. In the context of biological wastewater treatment (BWT), the presence of phages can alter the efficiency of the treatment process and influence the quality of the treated effluent. The active role of phages in BWT has been demonstrated, but many questions remain unanswered regarding the diversity of phages in these engineered environments, the dynamics of infection, the determination of bacterial hosts, and the impact of their activity in full-scale processes. A deeper understanding of the phage ecology in BWT can lead the improvement of process monitoring and control, promote higher influent quality, and potentiate the use of phages as biocontrol agents. In this review, we highlight suitable methods for studying phages in wastewater adapted from other research fields, provide a critical overview on the current state of knowledge on the effect of phages on structure and function of BWT bacterial communities, and highlight gaps, opportunities, and priority questions to be addressed in future research.

show abstract

Enumerating Viruses by Using Fluorescence and the Nature of the Nonviral Background Fraction

Cited by 5 publications

References 21 publications

Colloid chemistry pitfall for flow cytometric enumeration of viruses in water

Colloid chemistry pitfall for flow cytometric enumeration of viruses in water

Morphological characterization of virus-like particles in coral reef sponges

Bacteriophages in Biological Wastewater Treatment Systems: Occurrence, Characterization, and Function

Contact Info

Product

Resources

About