Distinguishing between living and nonliving bacteria: Evaluation of the vital stain propidium iodide and its combined use with molecular probes in aquatic samples

Williams, Scott A.; Hong, Yiguo; Danavall, D.C.A.; Howard-Jones, M.H; Gibson, Deidre; Frischer, Marc E.; Verity, Peter G.

doi:10.1016/s0167-7012(98)00014-1

Cited by 176 publications

(147 citation statements)

References 55 publications

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“…This observation agrees well with studies on selective and preferential grazing of protozoans on metabolically active bacteria (Del Giorgio et al 1996). Several studies had shown that bacterial cells with intact visible nucleoids were metabolically active and numbers of NucVB were tightly correlated with other parameters of bacterial metabolic activity (Williams et al 1998;Berman et al 2001). Results of experiments in Mazurian lakes previously described by Koton-Czarnecka and Chróst (2003) clearly demonstrated that PGB rates on live bacteria were almost two times higher than on dead bacteria.…”

Section: Discussionsupporting

confidence: 89%

Microbial production, utilization, and enzymatic degradation of organic matter in the upper trophogenic layer in the pelagial zone of lakes along a eutrophication gradient

Chróst

Siuda

2006

Limnology & Oceanography

100

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The major aim of this study was to evaluate the relationships between the rates of microbial activities (phytoplankton primary production, bacterial secondary production, bacterial utilization of organic matter, enzymatic activities, protozoan grazing on bacteria), bacterial numbers, and dissolved organic carbon concentrations and the trophic state index (TSI) of lakes in the upper trophogenic water layer in the pelagial zone along the trophic gradient (from oligo/mesotrophy to hypereutrophy) in 19 lakes of the Mazurian Lake District (northeastern Poland). Multiple regression analysis (analysis of variance-ANOVA) on all collected data and the TSI along eutrophication gradient showed that all studied microbial processes and parameters were very tightly coupled to the trophic conditions of the studied lakes. All studied microbial processes involved in utilization and enzymatic degradation of organic matter were strongly positively dependent on the intensity and rates of photosynthetic organic matter production and exudation that markedly increased along the eutrophication gradient of lakes. V max of alkaline phosphatase, aminopeptidase, and nonspecific esterase showed significant correlation with the TSI of the studied lakes. Protozoans removed a significant portion of bacterial production, i.e., from ϳ20% to 75-85% of newly produced bacterial biomass was simultaneously consumed by protozoans along the eutrophication gradient. These observations suggest that the importance of protozoan grazing on bacteria on regulation of bacterial production depends on lake productivity. The general working hypothesis that the intensity of microbial processes of organic matter can be tightly coupled to increasing eutrophication was proven in these studies.

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Section: Discussionsupporting

confidence: 89%

Microbial production, utilization, and enzymatic degradation of organic matter in the upper trophogenic layer in the pelagial zone of lakes along a eutrophication gradient

Chróst

Siuda

2006

Limnology & Oceanography

100

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“…In some cases, remaining fine sediment particles were removed by centrifugation (200 ϫ g) for 1 min. Total cell abundance was determined by epifluorescence microscopy after staining cells with DAPI (4Ј,6Ј-diamidino-2-phenylindole) (40).…”

Section: Methodsmentioning

confidence: 99%

Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

Frischer

Danforth

Healy

et al. 2000

Appl Environ Microbiol

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rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% ؎ 3.7% [mean ؎ standard deviation]) relative to direct DAPI (4,6-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r 2 ‫؍‬ 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.Over the past decade, 16S rRNA-targeted specific oligonucleotide probes have become a powerful tool for describing the structure of microbial communities in a variety of natural environments (1, 35). In planktonic environments, a number of straightforward hybridization techniques are available for utilizing oligonucleotide probes, including whole-cell fluorescence in situ hybridization (1,6,16,28,29) and direct wholecell blot hybridization (2, 5). These techniques simplify the use of 16S rRNA-targeted probes and therefore allow processing of larger numbers of samples required for conducting ecologically relevant studies. However, sediment and detritus often interfere with the enumeration of bacteria, hybridization, hybridization detection, and the extraction and purification of RNA (34). Thus, probe hybridization studies of sediment samples are more difficult and time-consuming than analogous studies in planktonic environments.Despite the difficulty involved, many studies have demonstrated the utility of applying 16S rRNA probe hybridization strategies in sediment environments (3,4,7,9,10,12,17,23,24,25,30). In general, these studies have relied on hybridization of probes to RNA extracted, purified, and immobilized onto charged nylon membranes or fluorescence in situ hybridization (41). To our knowledge, no studies have utilized more straight...

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“…Researchers are increasingly using combinations of several probes to further categorize bacteria into ecologically relevant fractions. For example, Williams et al (1998) have suggested a procedure for differentiating cells that are active from those that were recently active and those that are dead. The method has not yet been used with flow cytometry, but it certainly could be as it involves stains that have all been used in the past (Table 3).…”

Section: Single-cell Activitymentioning

confidence: 99%

Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

Gasol¹,

Giorgio²

2000

Sci. Mar.

777

713

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SUMMARY: Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.

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Distinguishing between living and nonliving bacteria: Evaluation of the vital stain propidium iodide and its combined use with molecular probes in aquatic samples

Cited by 176 publications

References 55 publications

Microbial production, utilization, and enzymatic degradation of organic matter in the upper trophogenic layer in the pelagial zone of lakes along a eutrophication gradient

Microbial production, utilization, and enzymatic degradation of organic matter in the upper trophogenic layer in the pelagial zone of lakes along a eutrophication gradient

Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

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