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

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DOI

  • Longhui Deng, Swiss Federal Institute of Technology Zurich
  • ,
  • Annika Fiskal, Swiss Federal Institute of Technology Zurich
  • ,
  • Xingguo Han, Swiss Federal Institute of Technology Zurich
  • ,
  • Nathalie Dubois, Swiss Federal Institute of Aquatic Science and Technology, Swiss Federal Institute of Technology Zurich
  • ,
  • Stefano Michele Bernasconi, Swiss Federal Institute of Technology Zurich
  • ,
  • Mark Alexander Lever

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.

Original languageEnglish
Article number720
JournalFrontiers in Microbiology
Volume10
IssueAPR
ISSN1664-302X
DOIs
Publication statusPublished - 2019
Externally publishedYes

Bibliographical 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:
© 2007 - 2019 Frontiers Media S.A. All Rights Reserved.

Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.

    Research areas

  • Cell counts, Epifluorescence microscopy, Flow cytometry, Lacustrine, Marine, Microbial populations, Staining technique

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