Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death
ABSTRACT 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 desi...
Published in: | Applied and Environmental Microbiology |
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Language: | English |
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American Society for Microbiology
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Online Access: | http://dx.doi.org/10.1128/aem.01668-07 https://journals.asm.org/doi/pdf/10.1128/AEM.01668-07 |
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crasmicro:10.1128/aem.01668-07 2024-09-15T18:26:23+00:00 Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death Falcioni, Tania Papa, Stefano Gasol, Josep M. 2008 http://dx.doi.org/10.1128/aem.01668-07 https://journals.asm.org/doi/pdf/10.1128/AEM.01668-07 en eng American Society for Microbiology https://journals.asm.org/non-commercial-tdm-license Applied and Environmental Microbiology volume 74, issue 6, page 1767-1779 ISSN 0099-2240 1098-5336 journal-article 2008 crasmicro https://doi.org/10.1128/aem.01668-07 2024-08-26T04:06:49Z ABSTRACT 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 μg 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. Article in Journal/Newspaper Northwest Atlantic ASM Journals (American Society for Microbiology) Applied and Environmental Microbiology 74 6 1767 1779 |
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Open Polar |
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ASM Journals (American Society for Microbiology) |
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crasmicro |
language |
English |
description |
ABSTRACT 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 μg 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. |
format |
Article in Journal/Newspaper |
author |
Falcioni, Tania Papa, Stefano Gasol, Josep M. |
spellingShingle |
Falcioni, Tania Papa, Stefano Gasol, Josep M. Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death |
author_facet |
Falcioni, Tania Papa, Stefano Gasol, Josep M. |
author_sort |
Falcioni, Tania |
title |
Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death |
title_short |
Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death |
title_full |
Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death |
title_fullStr |
Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death |
title_full_unstemmed |
Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death |
title_sort |
evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death |
publisher |
American Society for Microbiology |
publishDate |
2008 |
url |
http://dx.doi.org/10.1128/aem.01668-07 https://journals.asm.org/doi/pdf/10.1128/AEM.01668-07 |
genre |
Northwest Atlantic |
genre_facet |
Northwest Atlantic |
op_source |
Applied and Environmental Microbiology volume 74, issue 6, page 1767-1779 ISSN 0099-2240 1098-5336 |
op_rights |
https://journals.asm.org/non-commercial-tdm-license |
op_doi |
https://doi.org/10.1128/aem.01668-07 |
container_title |
Applied and Environmental Microbiology |
container_volume |
74 |
container_issue |
6 |
container_start_page |
1767 |
op_container_end_page |
1779 |
_version_ |
1810466872913559552 |