Simultaneous measurement of metabolic activity and membrane integrity in marine bacterioplankton determined by confocal laser-scanning microscopy

Pirker, Heidemarie; Pausz, Clemens; Stoderegger, Karen E.; Herndl, Gerhard J.

doi:10.3354/ame039225

Cited by 11 publications

(15 citation statements)

References 31 publications

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“…In addition, the discrimination of viable cells did not result in a higher correlation with bacterial production (Table 1), as expected from previous reports (Pearce et al 2007, Falcioni et al 2008). Like Pirker et al (2005), our study showed that the PI stain is not the most reliable method of examining bacterial activity in the field. There is still a debate about the estimation of active and viable bacterioplankton, which is in part semantic (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Another easy and helpful technique is nucleic acid double staining (NADS), which is based on simultaneous staining with SYBR Green (staining all cells) and Propidium Iodide (PI, staining only membrane-damaged cells) (Barbesti et al 2000) that allows discrimination of live (PI-impermeable) vs. dead (PI-positive) cells in natural assemblages (Grégori et al 2001, Falcioni et al 2008. Although some authors (Pirker et al 2005) have found that PI-positive cells can also uptake organic substrates, the proportion of viable (PI-impermeable) cells could be considered an acceptable cutoff in the continuum from inactive to active cells.…”

Section: Introductionmentioning

confidence: 99%

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Exploring the relationship between active bacterioplankton and phytoplankton in the Southern Ocean

Ortega−Retuerta¹,

Reche²,

Pulido-Villena³

et al. 2008

Aquat. Microb. Ecol.

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Bacterioplankton are a heterogenous community composed of cells with different physiological states. The consideration of the active fraction of bacterioplankton as a potential factor affecting the strength of the relationship between bacteria and phytoplankton in the Southern Ocean was evaluated in waters around the Antarctic Peninsula. We estimated active bacterioplankton from uptake of 3 H-Leucine (bacterial production [BP]) and using vital stains to estimate their proportion within the total bacterioplankton community, based on their relative nucleic acid content (high [HNA] vs. low [LNA]), and by nucleic acid double staining (NADS), based on their membrane permeability. Then we performed a comparative analysis between total and active bacterioplankton and chlorophyll a (chl a) in this area. Staining with NADS suggested that 61% of all bacteria were viable, a higher proportion of the total bacterial community than previously reported for the Southern Ocean. HNA bacteria comprised 45% of all bacteria, indicating that 16% of bacteria may be viable but with LNA. BP was more strongly related to abundance of LNA cells than NADS-viable or HNA bacteria. The relationship between chl a and bacterial abundance (BA) did not increase when considering the abundance of HNA or NADS-viable cells alone, showing that viability/activity of stains did not enhance the linkage between BA and phytoplankton biomass in the Southern Ocean. In contrast, the relationship between chl a and BP was stronger than those reported in the literature, suggesting that, in this region, BP is closely dependent on phytoplankton.KEY WORDS: Active bacteria · NADS · HNA · LNA · Bacterial production · Chlorophyll a · Southern OceanResale or republication not permitted without written consent of the publisher

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Exploring the relationship between active bacterioplankton and phytoplankton in the Southern Ocean

Ortega−Retuerta¹,

Reche²,

Pulido-Villena³

et al. 2008

Aquat. Microb. Ecol.

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“…Particulate phosphate (PartP) was measured using the Strickland and Parsons procedure (1972) for standard DIP, following high-temperature persulfate wet-oxidation at 120 • C and 1 bar (Pujo-Pay and Raimbault, 1994). Sequential filtration was carried out on 1 to 1.2 l samples through different porosity polycarbonate filters (0.2, 0.6, and 2 µm; 47 mm) using Sartorius systems and very low vacuum (drop by drop).…”

Section: Methodsmentioning

confidence: 99%

“…Applying various methods (measurements of the natural abundance of nucleoid-containing cells by combined epifluorescence and phase-contrast microscopy; detection of the reduction of the fluorogenic dye, 5-cyano-2,3-ditolyl tetrazolium chloride; nucleic acid double staining (SYBR Green + propidium iodide); determination of membrane integrity by confocal laser-scanning microscopy), it has been shown that at any given time, a significant fraction of the bacterioplankton community has minimal or no metabolic activity (Zweifel and Hagström, 1995;Sherr et al, 1999;Gregori et al, 2001;Pirker et al, 2005). For this reason, µ estimates based on the HBP:HBB ratio could be underestimated.…”

Section: Growth Rates Estimatesmentioning

confidence: 99%

Growth and specific P-uptake rates of bacterial and phytoplanktonic communities in the Southeast Pacific (BIOSOPE cruise)

et al. 2007

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Abstract. Predicting heterotrophic bacteria and phytoplankton specific growth rates (µ) is of great scientific interest. Many methods have been developed in order to assess bacterial or phytoplankton µ. One widely used method is to estimate µ from data obtained on biomass or cell abundance and rates of biomass or cell production. According to Kirchman (2002), the most appropriate approach for estimating µ is simply to divide the production rate by the biomass or cell abundance estimate. Most methods using this approach to estimate µ are based on carbon (C) incorporation rates and C biomass measurements. Nevertheless it is also possible to estimate µ using phosphate (P) data. We showed that particulate phosphate (PartP) can be used to estimate biomass and that the P uptake rate to PartP ratio can be employed to assess µ. Contrary to other methods using C, this estimator does not need conversion factors and provides an evaluation of µ for both autotrophic and heterotrophic organisms. We report values of P-based µ in three size fractions (0.2-0.6; 0.6-2 and >2 µm) along a Southeast Pacific transect, over a wide range of P-replete trophic status. P-based µ values were higher in the 0.6-2 µm fraction than in the >2 µm fraction, suggesting that picoplankton-sized cells grew faster than the larger cells, whatever the trophic regime encountered. Picoplankton-sized cells grew significantly faster in the deep chlorophyll maximum layer than in the upper part of the photic zone in the oligotrophic gyre area, suggesting that picoplankton might outcompete >2 µm cells in this particular high-nutrient, low-light environment. P-based µ attributed to free-living bacteria (0.2-0.6 µm) and picoplankton (0.6-2 µm) size-fractions were relatively low (0.11±0.07 d −1 and Correspondence to: S. Duhamel (solange.duhamel@univmed.fr) 0.14±0.04 d −1 , respectively) in the Southeast Pacific gyre, suggesting that the microbial community turns over very slowly.

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“…Pirker et al (58) combined microautoradiography and PI to simultaneously assess the metabolic activity and viability of individual bacterioplankton cells in the coastal and open North Sea. In this case, the overwhelming majority (97%) of cells taking up glucose and leucine were also PI positive.…”

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confidence: 99%

Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death

Falcioni

Papa

Gasol

2008

Appl Environ Microbiol

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Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 g ml ؊1 of propidium iodide, simultaneous to a 10؋ concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.Heterotrophic prokaryotes (Bacteria and Archaea) play a relevant role in planktonic marine microbial food webs (e.g., reference 5). As a relevant difference from other planktonic organisms, prokaryotes are known to sometimes be inactive or dead, but in the way that they are commonly enumerated (e.g., with DAPI [4Ј,6Ј-diamidino-2-phenylindole]), these cells are accounted for in budgets for prokaryote biomass since they are not distinguished from live and active cells.From an ecological point of view, at least three cell categories within microbial communities can be distinguished: the viable and active cells, which play a functional role and participate in biomass production at the time of sampling; the live but inactive cells (often called dormant cells), which do not participate in production at the time of sampling but have the potential for doing so; and the dead and therefore inactive cells, which do not have a role in the cycling of chemical elements, even though they might be retaining nutrients. The discrimination of these cellular categories is not without dispute: for example, the "active" term is often confounded with the "live" one and the "inactive" with the "dead" one (26). Moreover, different intrinsic levels of metabol...

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Simultaneous measurement of metabolic activity and membrane integrity in marine bacterioplankton determined by confocal laser-scanning microscopy

Cited by 11 publications

References 31 publications

Exploring the relationship between active bacterioplankton and phytoplankton in the Southern Ocean

Exploring the relationship between active bacterioplankton and phytoplankton in the Southern Ocean

Growth and specific P-uptake rates of bacterial and phytoplanktonic communities in the Southeast Pacific (BIOSOPE cruise)

Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death

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