Flow cytometric analysis of bacteria- and virus-like particles in lake sediments
Introduction
Flow cytometry provides a method for high-speed multi-parametric data acquisition and analysis. During the last two decades, it has been successfully used to analyze and count pelagic microbial communities of organisms such as protists, small algae, bacteria and viruses (Chisholm et al., 1988, Button and Robertson, 1989, Courties et al., 1994, Marie et al., 1999a, Marie et al., 1999b, Lindström et al., 2002, Rose et al., 2004), to identify and quantify population DNA content and/or to investigate the cell cycle (Boucher et al., 1991, Marie et al., 1996, Marie et al., 1997, Gasol et al., 1999), to identify populations of interest using molecular probes (Simon et al., 1995, Wallner et al., 1996, Lange et al., 1996), to assess cellular physiology (Jochem, 2000, Lebaron et al., 2001), etc. Reviews have been published of the accuracy of this technique applied to the field of aquatic sciences and ecology in particular, and to the modification/optimization of the apparatus and procedures (Olson et al., 1991, Yentsch et al., 1983, Davey and Kell, 1996, Porter et al., 1996, Dubelaar et al., 1999, Veldhuis and Kraay, 2000, Vives-Rigo et al., 2000, Collier and Campbell, 2000, Gruden et al., 2004).
Since Marie et al., 1999a, Marie et al., 1999b, viruses in the water column have been counted using benchtop flow cytometers on several occasions (Marie et al., 1999a, Marie et al., 1999b, Brussaard et al., 2000, Chen et al., 2001, Jacquet et al., 2002a, Jacquet et al., 2002b). Previously, viruses in aquatic environments were investigated using either transmission electron microscopy (TEM) or epifluorescence microscopy (EFM). Estimates of viral levels were first obtained using TEM after ultrafiltration or ultracentrifugation procedures (Bergh et al., 1989, Borsheim et al., 1990, Sime-Ngando et al., 1996). Since 1959, TEM has also been used to visualize phages, and characterize their morphology (Field, 1982). The use of EFM combined with the development of a variety of highly fluorescent nucleic acid specific dyes soon became the accepted method, because it involved a faster and less expensive technology. Nowadays, viruses (especially bacteriophages) are still usually counted by EFM using fluorochromes, such as SYBR Green I, SYBR Green II, SYBR Gold or Yo-Pro I (Xenopoulos and Bird, 1997, Marie et al., 1999a, Marie et al., 1999b, Shopov et al., 2000, Hewson et al., 2001a, Hewson et al., 2001b, Hewson et al., 2001c, Chen et al., 2001, Middelboe et al., 2003, Wen et al., 2004).
There have only been a few studies comparing the efficiency of the different techniques (such as EFM, FCM and TEM) for direct total counts of viruses in aquatic pelagic ecosystems. However, these comparisons make it possible to conclude that all these methods are fairly suitable for counting viruses (Hara et al., 1991, Hennes and Suttle, 1995, Weinbauer and Suttle, 1997, Marie et al., 1999a, Marie et al., 1999b, Bettarel et al., 2000, Chen et al., 2001), even though it seems that FCM has been reported to be as efficient as EFM or between 1 and 2 times more efficient (Marie et al., 1999a, Marie et al., 1999b, Brussaard et al., 2000, Chen et al., 2001, Dorigo et al., in revision), which in turn is reported to be up to seven times more efficient than TEM (Hara et al., 1991, Hennes and Suttle, 1995, Weinbauer and Suttle, 1997, Noble and Fuhrman, 1998, Bettarel et al., 2000). To the best of our knowledge, only Noble (2001) found TEM and EFM to have exactly the same efficiency levels for counting bacteriophages. The advantages and disadvantages of the three methods mentioned above, and a comparison of their efficiency can also be found in Weinbauer's excellent review on prokaryotic viruses (Weinbaueur, 2004).
Viruses are now considered to constitute an important component of aquatic microbial communities. They have been shown to be the most abundant biological compartment, and to play a crucial role in bacterial mortality, diversity and diversification in the pelagos (Wommack and Colwell, 2000, Weinbauer, 2004). Typically, viral infections are responsible for 20–50% of daily prokaryotic mortality, and they are a major source of dissolved organic matter. There has been little investigation of their importance in the sediment domain, where even basic information, such as their temporal dynamics and spatial distribution, is almost non-existent. However, sediments play a key role in the aquatic carbon cycle, and a high proportion of carbon degradation may be mediated by benthic processes (Glud and Middelboe, 2004). Since the work of Paul et al. (1993) and Maranger and Bird (1996), it has been known that high concentrations of viruses can occur in lake and marine surface sediments, typically reaching concentrations 10 to 1000 fold higher than those in the water column above, these densities being generally related to the trophic status of the ecosystem. In theory, aquatic sediments could provide an optimal environment (and hence constitute a reservoir) for virus development, since potential hosts (typically bacteria) are found in higher numbers than in the water column above, concentrations of organic matter are relatively high, and the distances between cells are very small (Wiggins and Alexander, 1985). Although, Danovaro and Serresi (2000) found large quantities of viruses and bacteria in a variety of sediments in the Eastern Mediterranean Sea, the low virus-to-bacterium ratios and their inverse relationship with trophic status suggest that the role played by viruses in controlling deep-sea benthic bacterial assemblages and biogeochemical cycles may be less relevant than in the pelagic systems. In another study, Danovaro et al. (2002) found that the lowest viral counts were obtained at stations where the largest cell sizes and the lowest bacterial growth and turnover rates were reported. These authors have suggested that the bacterial doubling time may play an important role in limiting virus development in sediments, and may influence the life strategies of benthic viruses. Recently, Middelboe and colleagues (Middelboe et al., 2003, Glud and Middelboe, 2004) clearly showed that the benthic viral community can be very dynamic, morphologically diverse and coupled with benthic bacterial activity. In freshwater ecosystems, high viral abundance but low virus-to-bacterium ratios have also been reported in lake sediments, suggesting that these particles are again only loosely related (Maranger and Bird, 1996, Lemke et al., 1997; Gessner et al. personal communication). At least, it seems that viral activity has only limited impact on benthic biogeochemical cycling (Middelboe, 2005). Clearly, only scant information about benthic viral ecology is available, and there are still no obvious conclusions about the importance and role of the viriobenthos.
To date, and to the best of our knowledge, viral abundance in aquatic sediments has always been determined using EFM or TEM. In this paper, we set out i) to propose an alternative way of counting viruses using flow cytometry combined with optimization of the extraction/fixation/dilution/staining procedure and ii) to report for the first time some typical concentrations of viruses and bacteria in the lake sediments of the two largest natural French lakes: Lakes Bourget and Geneva.
Section snippets
Study sites
Lake Geneva (46°27′N, 06°32′W, 372.05 m altitude) is the largest natural western European lake, and is located between the eastern part of France and Switzerland. It is a mesotrophic lake. It is elongated in shape (72.3 and 13.8 km in length and width respectively) and west–east orientated, with an area of 580.1 km2, a total volume of 89 × 109 m3, maximum and average depths of 309.7 and 152.7 m respectively, and a water residence time of approximately 11.4 years. It has a catchment area of about
Extraction of viruses and bacteria from the sediments
The first series of tests dealt with the extraction of the bacterial and viral particles contained in the lake sediment. One of the first extraction steps reported in the literature consists of sonicating the sediment sample after adding sodium pyrophosphate. The different studies all agree that sonicating for 3 min efficiently dislodged viruses and bacteria from the sediment, but none made it clear whether adding ice to the water bath or not made for better efficiency. Our results clearly show
Discussion
It is only a few years since Marie et al., 1999a, Marie et al., 1999b reported the successful use of a benchtop flow cytometer with a low-power argon-ion laser to detect and count viruses in seawater. We can now propose another application for this device, i.e., the detection and counting of viruses in aquatic (lake) sediments. There are many advantages of being able to use flow cytometry rather than epifluorescence microscopy or transmission electron microscopy. The most important advantage is
Acknowledgements
SD was supported by an INRA contract. This study was funded by the DYLACHEM project. The flow cytometer has been funded by both INRA and University contracts. We are grateful to Monika Ghosh for improving the English. Mathias Middelboe is acknowledged for useful advice.
References (70)
- et al.
Flow cytometric detection of viruses
J. Virol. Methods
(2000) Diagnostic virology using electron microscopy
Adv. Viral. Res.
(1982)- et al.
Flow cytometry for microbial sensing in environmental sustainability applications: current status and future prospects
FEMS Microbiol. Ecol.
(2004) - et al.
Enumeration of small ciliates in culture by flow cytometry and nucleic acid staining
J. Microbiol. Methods
(2002) Enumeration of viruses
- et al.
Combination of rRNA-targeted hybridization and immuno-probes for the identification of bacteria by flow cytometry
Syst. Appl. Microbiol.
(1996) Ecology of prokaryotic viruses
FEMS Microbiol. Rev.
(2004)- et al.
High abundances of viruses found in aquatic environments
Nature
(1989) - et al.
A comparison of methods for counting viruses in aquatic systems
Appl. Environ. Microbiol.
(2000) - et al.
Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy
Appl. Environ. Microbiol.
(1990)
Flow cytometric determination of phytoplankton DNA in cultures and oceanic populations
Mar. Ecol., Prog. Ser.
Optimization of procedures for counting viruses by flow cytometry
Appl. Environ. Microbiol.
Kinetics of bacterial processes in natural aquatic systems bases on biomass as determined by high-resolution flow cytometry
Cytometry
Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR Gold
Appl. Environ. Microbiol.
A novel free living prochlorophyte abundant in the oceanic euphotic zone
Nature
Flow cytometry in molecular aquatic ecology
Hydrobiol.
Smallest eukaryotic organism
Nature
Viral density and virus-to-bacterium ratio in deep-sea sediments of the eastern Mediterranean
Appl. Environ. Microb.
Determination of virus abundance in marine sediments
Appl. Environ. Microbiol.
Higher abundance of bacteria than of viruses in deep Mediterranean sediments
Appl. Environ. Microbiol.
Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analysis
Microbiol. Rev.
Vertical profiles of virus-like particles and bacteria in the water column and sediments of Chesapeake Bay, USA
Aquat. Microb. Ecol.
Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters
Cytometry
Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria
Appl. Environ. Microb.
Virus and bacteria dynamics of a coastal sediment: implication for benthic carbon cycling
Limnol. Oceanogr.
Factors influencing the loss of bacteria in preserved seawater samples
Mar. Ecol., Prog. Ser.
Abundance of viruses in marine waters: assessment by epifluorescence and transmission electron microscopy
Appl. Environ. Microbiol.
Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy
Limnol. Oceanogr.
Virus-like particles associated with Lyngbya majuscula (Cyanophyta; Oscillatoria) bloom decline in Moreton Bay, Australia
Aquat. Microb. Ecol.
Effects of concentrated viral communities on photosynthesis and community composition of co-occurring benthic microalgae and phytoplankton
Aquat. Microb. Ecol.
Virus-like particle distribution and abundance in sediments and overlying waters along eutrophication gradients in two subtropical estuaries
Limnol. Oceanogr.
Bacterial diversity in shallow oligotrophic marine benthos and overlying waters: effects of virus infection, containment, and nutrient enrichment
Microb. Ecol.
Effect of inorganic and organic nutrient addition on a coastal microbial community (Isefjord, Denmark)
Mar. Ecol., Progr. Ser.
Flow cytometric analysis of an Emiliana huxleyi bloom terminated by viral infection
Aquat. Microb. Ecol.
Cited by (109)
Pseudomonas ability to utilize different carbon substrates and adaptation influenced by protozoan grazing
2023, Environmental ResearchEffects of intermittent flow on biofilms are driven by stream characteristics rather than history of intermittency
2022, Science of the Total EnvironmentCitation Excerpt :The resulting extract was analyzed as a glucose equivalent by a phenol and sulphuric acid assay (DuBois et al., 1956). Bacterial abundances were measured according to the protocol by Duhamel and Jacquet (2006). Bacterial (prokaryotic) cells were detached, filtered, and diluted (100-200×).
Vulnerability of seagrass blue carbon to microbial attack following exposure to warming and oxygen
2019, Science of the Total EnvironmentA flow cytometry method for bacterial quantification and biomass estimates in activated sludge
2019, Journal of Microbiological MethodsCitation Excerpt :Each treatment was analysed in triplicate, with a paired control (sub-samples without mechanical mixing or sonication) per replicate. The nucleic acid dyes SYBR Green I, SYBR Green II, SYBR Gold and SYTO 9, which preferentially bind to double stranded DNA (dsDNA); single stranded DNA (ssDNA), RNA and dsDNA; and ssDNA and RNA respectively (SYBR Gold and SYTO 9), were tested separately, at varying dilutions (1:30, 1:100 and 1:200 v/v of each dye's stock solution and dimethyl sulfoxide (DMSO), chosen in accordance with Duhamel and Jacquet (2006) and Foladori et al. (2010)). Those that target both DNA and RNA, and/or have better extinction coefficients and quantum yields at 488 nm, may be expected to result in a greater signal to noise ratio and therefore achieve higher counts (Table S1).
Physisorption and chemisorption of T4 bacteriophages on amino functionalized silica particles
2018, Journal of Colloid and Interface Science