Department of Biology

Aarhus University Seal / Aarhus Universitets segl

Mark Lever

Improving the accuracy of flow cytometric quantification of microbial populations in sediments: Importance of cell staining procedures

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Improving the accuracy of flow cytometric quantification of microbial populations in sediments : Importance of cell staining procedures. / Deng, Longhui; Fiskal, Annika; Han, Xingguo; Dubois, Nathalie; Bernasconi, Stefano Michele; Lever, Mark Alexander.

In: Frontiers in Microbiology, Vol. 10, No. APR, 720, 2019.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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Deng, Longhui ; Fiskal, Annika ; Han, Xingguo ; Dubois, Nathalie ; Bernasconi, Stefano Michele ; Lever, Mark Alexander. / Improving the accuracy of flow cytometric quantification of microbial populations in sediments : Importance of cell staining procedures. In: Frontiers in Microbiology. 2019 ; Vol. 10, No. APR.

Bibtex

@article{4a8ef1c97a3b4ee39bcbd44ff19c88bb,
title = "Improving the accuracy of flow cytometric quantification of microbial populations in sediments: Importance of cell staining procedures",
abstract = "The accuracy of flow cytometric (FCM) quantifications of microbial populations in sediments varies with FCM settings, cell extraction and staining protocols, as well as sample types. In the present study, we improve the accuracy of FCM for enumerating microorganisms inhabiting diverse lake and marine sediment types based on extensive tests with FCM settings, extraction buffer chemical compositions, cell separation methods, and staining procedures. Tests on the FCM settings, (e.g., acquisition time, rates of events) and salinity of extraction solutions show minor impacts on FCM enumerations and yields of cell extraction, respectively. Existing methods involving hydrofluoric acid (HF) treatment to release sediment-attached cells into solution prove effective on both marine and freshwater samples. Yet, different staining techniques (direct staining of cell extracts, staining of membrane-filtered cell extracts) produce clear differences in cell number estimates. We demonstrate that, while labor-intensive membrane-staining generates high cell staining efficiency and accurate cell counts that are consistent across FCM and epifluorescence microscopy-based (EFM) quantification methods, accurate cell counts determined by more time- and labor-efficient direct staining require consideration of dye concentration, sample dilution, and lithology. Yet, good agreement between the two staining methods can be achieved through sample-specific adjustments of dye concentrations and sample dilutions during direct staining. We thus present a complete protocol for FCM-based cell quantification, that includes all steps from the initial sample fixation to the final enumeration, with recommendations for buffer compositions, direct and membrane-based staining procedures, and the final FCM assay. This protocol is versatile, accurate, and reliable, as is evident from good agreement with cell quantifications by EFM and quantitative polymerase chain reaction (qPCR) of 16S rRNA genes across a wide range of sedimentary sample types.",
keywords = "Cell counts, Epifluorescence microscopy, Flow cytometry, Lacustrine, Marine, Microbial populations, Staining technique",
author = "Longhui Deng and Annika Fiskal and Xingguo Han and Nathalie Dubois and Bernasconi, {Stefano Michele} and Lever, {Mark Alexander}",
note = "Funding Information: This study is funded by the Swiss National Science Foundation (project 205321_163371 “Role of bioturbation in controlling microbial community composition and biogeochemical cycles in marine and lacustrine sediments” awarded to ML). Publisher Copyright: {\textcopyright} 2007 - 2019 Frontiers Media S.A. All Rights Reserved. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.",
year = "2019",
doi = "10.3389/fmicb.2019.00720",
language = "English",
volume = "10",
journal = "Frontiers in Microbiology",
issn = "1664-302X",
publisher = "Frontiers Media S.A",
number = "APR",

}

RIS

TY - JOUR

T1 - Improving the accuracy of flow cytometric quantification of microbial populations in sediments

T2 - Importance of cell staining procedures

AU - Deng, Longhui

AU - Fiskal, Annika

AU - Han, Xingguo

AU - Dubois, Nathalie

AU - Bernasconi, Stefano Michele

AU - Lever, Mark Alexander

N1 - Funding Information: This study is funded by the Swiss National Science Foundation (project 205321_163371 “Role of bioturbation in controlling microbial community composition and biogeochemical cycles in marine and lacustrine sediments” awarded to ML). Publisher Copyright: © 2007 - 2019 Frontiers Media S.A. All Rights Reserved. Copyright: Copyright 2019 Elsevier B.V., All rights reserved.

PY - 2019

Y1 - 2019

N2 - The accuracy of flow cytometric (FCM) quantifications of microbial populations in sediments varies with FCM settings, cell extraction and staining protocols, as well as sample types. In the present study, we improve the accuracy of FCM for enumerating microorganisms inhabiting diverse lake and marine sediment types based on extensive tests with FCM settings, extraction buffer chemical compositions, cell separation methods, and staining procedures. Tests on the FCM settings, (e.g., acquisition time, rates of events) and salinity of extraction solutions show minor impacts on FCM enumerations and yields of cell extraction, respectively. Existing methods involving hydrofluoric acid (HF) treatment to release sediment-attached cells into solution prove effective on both marine and freshwater samples. Yet, different staining techniques (direct staining of cell extracts, staining of membrane-filtered cell extracts) produce clear differences in cell number estimates. We demonstrate that, while labor-intensive membrane-staining generates high cell staining efficiency and accurate cell counts that are consistent across FCM and epifluorescence microscopy-based (EFM) quantification methods, accurate cell counts determined by more time- and labor-efficient direct staining require consideration of dye concentration, sample dilution, and lithology. Yet, good agreement between the two staining methods can be achieved through sample-specific adjustments of dye concentrations and sample dilutions during direct staining. We thus present a complete protocol for FCM-based cell quantification, that includes all steps from the initial sample fixation to the final enumeration, with recommendations for buffer compositions, direct and membrane-based staining procedures, and the final FCM assay. This protocol is versatile, accurate, and reliable, as is evident from good agreement with cell quantifications by EFM and quantitative polymerase chain reaction (qPCR) of 16S rRNA genes across a wide range of sedimentary sample types.

AB - The accuracy of flow cytometric (FCM) quantifications of microbial populations in sediments varies with FCM settings, cell extraction and staining protocols, as well as sample types. In the present study, we improve the accuracy of FCM for enumerating microorganisms inhabiting diverse lake and marine sediment types based on extensive tests with FCM settings, extraction buffer chemical compositions, cell separation methods, and staining procedures. Tests on the FCM settings, (e.g., acquisition time, rates of events) and salinity of extraction solutions show minor impacts on FCM enumerations and yields of cell extraction, respectively. Existing methods involving hydrofluoric acid (HF) treatment to release sediment-attached cells into solution prove effective on both marine and freshwater samples. Yet, different staining techniques (direct staining of cell extracts, staining of membrane-filtered cell extracts) produce clear differences in cell number estimates. We demonstrate that, while labor-intensive membrane-staining generates high cell staining efficiency and accurate cell counts that are consistent across FCM and epifluorescence microscopy-based (EFM) quantification methods, accurate cell counts determined by more time- and labor-efficient direct staining require consideration of dye concentration, sample dilution, and lithology. Yet, good agreement between the two staining methods can be achieved through sample-specific adjustments of dye concentrations and sample dilutions during direct staining. We thus present a complete protocol for FCM-based cell quantification, that includes all steps from the initial sample fixation to the final enumeration, with recommendations for buffer compositions, direct and membrane-based staining procedures, and the final FCM assay. This protocol is versatile, accurate, and reliable, as is evident from good agreement with cell quantifications by EFM and quantitative polymerase chain reaction (qPCR) of 16S rRNA genes across a wide range of sedimentary sample types.

KW - Cell counts

KW - Epifluorescence microscopy

KW - Flow cytometry

KW - Lacustrine

KW - Marine

KW - Microbial populations

KW - Staining technique

UR - http://www.scopus.com/inward/record.url?scp=85068141328&partnerID=8YFLogxK

U2 - 10.3389/fmicb.2019.00720

DO - 10.3389/fmicb.2019.00720

M3 - Journal article

AN - SCOPUS:85068141328

VL - 10

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

SN - 1664-302X

IS - APR

M1 - 720

ER -