Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures
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 marin...
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doaj-807331ab99c94c2e84183395418ffb7c2020-11-25T00:15:58ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-04-011010.3389/fmicb.2019.00720441088Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining ProceduresLonghui Deng0Annika Fiskal1Xingguo Han2Nathalie Dubois3Nathalie Dubois4Stefano Michele Bernasconi5Mark Alexander Lever6Institute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, Zurich, SwitzerlandInstitute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, Zurich, SwitzerlandInstitute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, Zurich, SwitzerlandSurface Waters Research-Management, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, SwitzerlandDepartment of Earth Sciences, ETH Zürich, Zurich, SwitzerlandDepartment of Earth Sciences, ETH Zürich, Zurich, SwitzerlandInstitute of Biogeochemistry and Pollutant Dynamics, ETH Zürich, Zurich, SwitzerlandThe 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.https://www.frontiersin.org/article/10.3389/fmicb.2019.00720/fullmicrobial populationslacustrinemarinecell countsstaining techniqueflow cytometry |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Longhui Deng Annika Fiskal Xingguo Han Nathalie Dubois Nathalie Dubois Stefano Michele Bernasconi Mark Alexander Lever |
spellingShingle |
Longhui Deng Annika Fiskal Xingguo Han Nathalie Dubois Nathalie Dubois Stefano Michele Bernasconi Mark Alexander Lever Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures Frontiers in Microbiology microbial populations lacustrine marine cell counts staining technique flow cytometry |
author_facet |
Longhui Deng Annika Fiskal Xingguo Han Nathalie Dubois Nathalie Dubois Stefano Michele Bernasconi Mark Alexander Lever |
author_sort |
Longhui Deng |
title |
Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures |
title_short |
Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures |
title_full |
Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures |
title_fullStr |
Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures |
title_full_unstemmed |
Improving the Accuracy of Flow Cytometric Quantification of Microbial Populations in Sediments: Importance of Cell Staining Procedures |
title_sort |
improving the accuracy of flow cytometric quantification of microbial populations in sediments: importance of cell staining procedures |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2019-04-01 |
description |
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. |
topic |
microbial populations lacustrine marine cell counts staining technique flow cytometry |
url |
https://www.frontiersin.org/article/10.3389/fmicb.2019.00720/full |
work_keys_str_mv |
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