A Comparison of Methods for Counting Viruses in Aquatic Systems

Bettarel, Yvan; Sime-Ngando, Télesphore; Amblard, Christian; Laveran, H.

doi:10.1128/aem.66.6.2283-2289.2000

Cited by 97 publications

(84 citation statements)

References 36 publications

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“…Instead, FISH should be regarded as a more reliable method for Bacteria and Archaea. There is knowledge about the unspecific staining properties of DAPI (Bettarel et al 2000, Zampino et al 2004) and the determination of bacterial abundance is still affected by the subjectivity of each researcher. Our observation that most of the small particles (small cocci) were not hybridized reflects a long lasting (> 20 yr) discussion.…”

Section: Methodological Remarksmentioning

confidence: 99%

“…Most likely, this 'underestimation' is not due to methodological problems of the FISH method itself (Wagner et al 2003, Aman & Fuchs 2008. In contrast, DAPI -as the typical reference dye for FISH -may also stain viruses (Bettarel et al 2000) or even detrital and sub-micrometer particles, thus potentially leading to imprecise estimates of bacterial numbers, i.e. an overestimation of total bacterial abundance.…”

Section: Introductionmentioning

confidence: 99%

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New image analysis tool to study biomass and morphotypes of three major bacterioplankton groups in an alpine lake

Posch

Franzoi²,

Prader³

et al. 2009

Aquat. Microb. Ecol.

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We present an image analysis routine to determine the contribution of distinct morphotypes to the total abundance and biomass (carbon) of freshwater bacterioplankton and to the fraction of cells detected by fluorescence in situ hybridization via catalyzed reporter deposition (CARD-FISH). The method was tested on bacterial assemblages from an alpine lake (Piburger See, Austria) at characteristic time points during the limnological year. Although on average 51% of 4', 6'-diamidino-2-phenylindole (DAPI)-stained objects were hybridized with the oligonucleotide probe EUB I-III, we detected on average 80% of total biomass determined from DAPI staining. The assemblage was numerically dominated by cocci and rods < 0.6 µm (mean cell volume = 0.024 µm 3 ). Only a minor part of these morphotypes could be hybridized (18 and 50%, respectively). In contrast, larger rods (0.087 µm ) showed much higher probabilities to be detected by CARD-FISH. These morphotypes per se formed the highest contribution to total biomass, which explained the high detection efficiency of biomass with CARD-FISH. In addition, we determined the seasonal dynamics of morphotype distributions within 3 distinct phylogenetic lineages. Actinobacteria were predominately small rods and cocci, whereas bacteria from the Cytophaga -Flavobacterium-Bacteroides group formed mainly large rods, cocci and filaments. Betaproteobacteria showed the highest morphological variability. Within all lineages, distinct spatio-temporal dynamics of dominant morphotypes were observed. Thus, the approach presented here will allow for more detailed studies of the amount of carbon bound by different bacterial taxa. This is of relevance as distinct lineages can contribute much more to total bacterial biomass than to total bacterioplankton abundance. KEY WORDS: Bacterial biomass · Bacterial morphotype · CARD-FISH · DAPI · Image analysisResale or republication not permitted without written consent of the publisher

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Section: Methodological Remarksmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

New image analysis tool to study biomass and morphotypes of three major bacterioplankton groups in an alpine lake

Posch

Franzoi²,

Prader³

et al. 2009

Aquat. Microb. Ecol.

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“…Effect of centrifugation on virus counts in silty freshwater sediments. Mean ± SD of replicate samples (n = 3 for 1000 and 200 × g, 6 for 400 and 800 × g, 9 for 500 × g) Bettarel et al (2000) recorded a significant reduction in viral numbers in freshwater samples of 19% after DNase treatment; (2) in a culture containing T2-phages and marine plankton samples, 33 to 48% of the encapsulated viral DNA were sensitive to DNase (Jiang & Paul 1995); (3) some 10% of the < 0.2 µm fraction of DNA in marine water samples, which was assumed tocomprise viruses (i.e. 'coated DNA'), was digested by DNase (Maruyama et al 1993).…”

Section: Dnase Testmentioning

confidence: 99%

Optimization of extraction and estimation of viruses in silty freshwater sediments

Fischer¹,

Kirschner²,

Velimirov

2005

Aquat. Microb. Ecol.

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The present study focused on the optimization of procedures for the extraction of viruses from silty freshwater sediments for subsequent enumeration. Viral abundance in 2 different shallow backwater systems of the River Danube (Austria) ranged from 1.45 × 10 9 to 9.58 × 10 9 particles ml -1 wet sediment. The highest virus yields from the bulk of the sediments were obtained by 1 min sonication (3 × 20 s intervals, with 10 s interruptions). An increase in sonication time of up to 5 min decreased viral counts by an average of 15%. Since dissolved DNA within sediment samples could bind to the nucleic acid stain and thereby inflate viral estimates, sediment samples are often treated with DNase before the staining procedure. Moreover, they are usually centrifuged and diluted to a high extent in order to avoid interference of particulate material with virus counting. Centrifugation led to a reduction of viral numbers by 2 to 36% compared to untreated samples and did not reduce the background fluorescence; thus counting of viruses was not facilitated. Diluting 2000 × with Milli-Q water always provided an average of 19% lower viral numbers than diluting 4000 ×. Treatment with DNase had no significant effect on virus counting, with viral numbers in untreated samples being on average 96% of those in DNase-containing samples. Additionally, 2 different nucleic acid stains were compared -viruses stained with SYBR Gold fluoresced brighter than those stained with SYBR Green I and fluorescence lasted longer, while background fluorescence was reduced sufficiently, thus facilitating virus counting. Viral numbers using SYBR Gold were on average twice of those obtained with SYBR Green I. The mean efficiency of virus extraction was 88.8% using the protocol outlined in this paper, and was thus slightly higher than that obtained in previous sediment investigations. KEY WORDS: Virus extraction · Virus counting · Epifluorescence microscopy · Silty freshwater sedimentResale or republication not permitted without written consent of the publisher Aquat Microb Ecol 40: 207-216, 2005 Moreover, EFM yields higher and more precise viral counts than TEM (Hennes & Suttle 1995, Weinbauer & Suttle 1997, Noble & Fuhrman 1998. Enumeration of viruses by EFM is based on using highly fluorescent nucleic acid dyes such as DAPI (Hara et al. 1991, Weinbauer & Suttle 1997, Yo-Pro I (Hennes & Suttle 1995, Xenopoulos & Bird 1997, SYBR Green I (Noble & Fuhrman 1998) or SYBR Gold (Chen et al. 2001). Viral abundance in sediment systems is 10-to 1000-fold higher than in the overlying water column (e.g. Maranger & Bird 1996, Steward et al. 1996, Drake et al. 1998, Hewson et al. 2001, Lawrence et al. 2002, Fischer et al. 2003. Studies using EFM to estimate viruses in sediments applied a variety of protocols, without testing whether the procedures carried out before enumeration (e.g. virus dislodgement from particles, centrifugation, dilution) affect viral counts (Danovaro & Serresi 2000, RicciardiRigault et al. 2000, Danovaro et al. 2001, Fischer et a...

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“…More recently, flow cytometry (FCM) emerged as a new method, because it is a faster technology for direct counts. There have only been a few studies comparing the efficiency of these different techniques (4,11), even though FCM has been reported to be 1 and 2 times more efficient (11,26). It has been successfully used to analyze and count microbial communities such as protist, small algae, bacteria and viruses (3,14,25,27,35).…”

Section: Introductionmentioning

confidence: 99%

Virioplankton Abundance in Trophic Gradients of an Upwelling Field

Pereira¹,

Granato²,

Figueiredo³

et al. 2009

Braz. J. Microbiol.

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This work correlates time series of biological and physical variables to the marine viruses across trophic gradients within Arraial do Cabo upwelling system, Southeast of Brazil. The objective is to investigate the major controlling factors of virioplankton dynamics among different water masses. It was used an in situ and ex situ flow cytometry for accessing the plankton community. Viruses were highly correlated to bacteria and phytoplankton, but although the lack of direct correlation with physicals, upwelling turned out to be the main contributing factor to the highest values of viral abundance and virus:bacterial ratio.Our data suggest that the lowest temperature of upwelled South Atlantic Central Waters would help to maintain a high viral abundance and higher temperatures of Coastal and Tropical Waters might be another ecological niche allowing the co-existence.

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A Comparison of Methods for Counting Viruses in Aquatic Systems

Cited by 97 publications

References 36 publications

New image analysis tool to study biomass and morphotypes of three major bacterioplankton groups in an alpine lake

New image analysis tool to study biomass and morphotypes of three major bacterioplankton groups in an alpine lake

Optimization of extraction and estimation of viruses in silty freshwater sediments

Virioplankton Abundance in Trophic Gradients of an Upwelling Field

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