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Title: Optimization of a Method To Quantify Soil Bacterial Abundance by Flow Cytometry

Abstract

ABSTRACT Bacterial abundance is a fundamental metric for understanding the population dynamics of soil bacteria and their role in biogeochemical cycles. Despite its importance, methodological constraints hamper our ability to assess bacterial abundance in terrestrial environments. Here, we aimed to optimize the use of flow cytometry (FCM) to assay bacterial abundances in soil while providing a rigorous quantification of its limitations. Soil samples were spiked with Escherichia coli to evaluate the levels of recovery efficiency among three extraction approaches. The optimized method added a surfactant (a tetrasodium pyrophosphate [TSP] buffer) to 0.1 g of soil, applied an intermediate degree of agitation through shaking, and used a Nycodenz density gradient to separate the cells from background debris. This procedure resulted in a high (average, 89%) level of cell recovery. Recovery efficiencies did not differ significantly among sites across an elevation gradient but were positively correlated with percent carbon in the soil samples. Estimated abundances were also highly repeatable between technical replicates. The method was applied to samples from two field studies and, in both cases, was sensitive enough to detect treatment and site differences in bacterial abundances. We conclude that FCM offers a fast and sensitive method to assay soil bacterialmore » abundance from relatively small amounts of soil. Further work is needed to assay differential biases of the method across a wider range of soil types. IMPORTANCE The ability to quantify bacterial abundance is important for understanding the contributions of microbial communities in soils, but such assays remain difficult and time-consuming. Flow cytometry offers a fast and direct way to count bacterial cells, but several concerns remain in applying the technique to soils. This study aimed to improve the efficiency of the method for soil while quantifying its limitations. We demonstrated that an optimized procedure was sensitive enough to capture differences in bacterial abundances among treatments and ecosystems in two field studies.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Univ. of California, Irvine, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)
OSTI Identifier:
1595495
Alternate Identifier(s):
OSTI ID: 1612541
Grant/Contract Number:  
SC0016410; DEB-1457160; MRI-1126749
Resource Type:
Published Article
Journal Name:
mSphere
Additional Journal Information:
Journal Name: mSphere Journal Volume: 4 Journal Issue: 5; Journal ID: ISSN 2379-5042
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES; Microbiology; bacterial cell count; ecosystem types; extraction procedure; flow cytometry; soil

Citation Formats

Khalili, Banafshe, Weihe, Claudia, Kimball, Sarah, Schmidt, Katharina T., Martiny, Jennifer B. H., and Suen, ed., Garret. Optimization of a Method To Quantify Soil Bacterial Abundance by Flow Cytometry. United States: N. p., 2019. Web. https://doi.org/10.1128/mSphere.00435-19.
Khalili, Banafshe, Weihe, Claudia, Kimball, Sarah, Schmidt, Katharina T., Martiny, Jennifer B. H., & Suen, ed., Garret. Optimization of a Method To Quantify Soil Bacterial Abundance by Flow Cytometry. United States. https://doi.org/10.1128/mSphere.00435-19
Khalili, Banafshe, Weihe, Claudia, Kimball, Sarah, Schmidt, Katharina T., Martiny, Jennifer B. H., and Suen, ed., Garret. Wed . "Optimization of a Method To Quantify Soil Bacterial Abundance by Flow Cytometry". United States. https://doi.org/10.1128/mSphere.00435-19.
@article{osti_1595495,
title = {Optimization of a Method To Quantify Soil Bacterial Abundance by Flow Cytometry},
author = {Khalili, Banafshe and Weihe, Claudia and Kimball, Sarah and Schmidt, Katharina T. and Martiny, Jennifer B. H. and Suen, ed., Garret},
abstractNote = {ABSTRACT Bacterial abundance is a fundamental metric for understanding the population dynamics of soil bacteria and their role in biogeochemical cycles. Despite its importance, methodological constraints hamper our ability to assess bacterial abundance in terrestrial environments. Here, we aimed to optimize the use of flow cytometry (FCM) to assay bacterial abundances in soil while providing a rigorous quantification of its limitations. Soil samples were spiked with Escherichia coli to evaluate the levels of recovery efficiency among three extraction approaches. The optimized method added a surfactant (a tetrasodium pyrophosphate [TSP] buffer) to 0.1 g of soil, applied an intermediate degree of agitation through shaking, and used a Nycodenz density gradient to separate the cells from background debris. This procedure resulted in a high (average, 89%) level of cell recovery. Recovery efficiencies did not differ significantly among sites across an elevation gradient but were positively correlated with percent carbon in the soil samples. Estimated abundances were also highly repeatable between technical replicates. The method was applied to samples from two field studies and, in both cases, was sensitive enough to detect treatment and site differences in bacterial abundances. We conclude that FCM offers a fast and sensitive method to assay soil bacterial abundance from relatively small amounts of soil. Further work is needed to assay differential biases of the method across a wider range of soil types. IMPORTANCE The ability to quantify bacterial abundance is important for understanding the contributions of microbial communities in soils, but such assays remain difficult and time-consuming. Flow cytometry offers a fast and direct way to count bacterial cells, but several concerns remain in applying the technique to soils. This study aimed to improve the efficiency of the method for soil while quantifying its limitations. We demonstrated that an optimized procedure was sensitive enough to capture differences in bacterial abundances among treatments and ecosystems in two field studies.},
doi = {10.1128/mSphere.00435-19},
journal = {mSphere},
number = 5,
volume = 4,
place = {United States},
year = {2019},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1128/mSphere.00435-19

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Cited by: 2 works
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Works referenced in this record:

Recovery and quantification of bacterial cells associated with streambed sediments
journal, October 2008


Prokaryotes: The unseen majority
journal, June 1998

  • Whitman, W. B.; Coleman, D. C.; Wiebe, W. J.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 12
  • DOI: 10.1073/pnas.95.12.6578

Past, present and future applications of flow cytometry in aquatic microbiology
journal, August 2010


Microbial decomposers not constrained by climate history along a Mediterranean climate gradient in southern California
journal, June 2018

  • Baker, Nameer R.; Khalili, Banafshe; Martiny, Jennifer B. H.
  • Ecology, Vol. 99, Issue 6
  • DOI: 10.1002/ecy.2345

Estimates of viral abundance in soils are strongly influenced by extraction and enumeration methods
journal, February 2013

  • Williamson, Kurt E.; Corzo, Krysten A.; Drissi, Camelia L.
  • Biology and Fertility of Soils, Vol. 49, Issue 7
  • DOI: 10.1007/s00374-013-0780-z

New cell extraction procedure applied to deep subsurface sediments: Cell extraction of deep subsurface sediments
journal, June 2008

  • Kallmeyer, Jens; Smith, David C.; Spivack, Arthur J.
  • Limnology and Oceanography: Methods, Vol. 6, Issue 6
  • DOI: 10.4319/lom.2008.6.236

Cost-effective ecological restoration: Cost-effective ecological restoration
journal, August 2015

  • Kimball, Sarah; Lulow, Megan; Sorenson, Quinn
  • Restoration Ecology, Vol. 23, Issue 6
  • DOI: 10.1111/rec.12261

Flow cytometry for microbial sensing in environmental sustainability applications: current status and future prospects
journal, July 2004


Flow cytometric analysis of bacteria- and virus-like particles in lake sediments
journal, March 2006


Bias in bacterial diversity as a result of Nycodenz extraction from bulk soil
journal, October 2011


Flow cytometric analysis of benthic prokaryotes attached to sediment particles
journal, November 2009

  • Amalfitano, Stefano; Puddu, Stefano Fazi, Alberto
  • Journal of Microbiological Methods, Vol. 79, Issue 2
  • DOI: 10.1016/j.mimet.2009.09.005

Soil bacterial quantification approaches coupling with relative abundances reflecting the changes of taxa
journal, July 2017


A rapid flow cytometry method to assess bacterial abundance in agricultural soil
journal, April 2015


Flow-cytometric quantification of microbial cells on sand from water biofilters
journal, October 2018


Microbial control over carbon cycling in soil
journal, January 2012


Evaluation of quantitative and qualitative recovery of bacterial communities from different soil types by density gradient centrifugation
journal, April 2006

  • Maron, Pierre-Alain; Schimann, Heidy; Ranjard, Lionel
  • European Journal of Soil Biology, Vol. 42, Issue 2
  • DOI: 10.1016/j.ejsobi.2005.08.003

Dispersal alters bacterial diversity and composition in a natural community
journal, October 2017

  • Albright, Michaeline B. N.; Martiny, Jennifer B. H.
  • The ISME Journal, Vol. 12, Issue 1
  • DOI: 10.1038/ismej.2017.161

Evaluation of quantitative recovery of bacterial cells and DNA from different lake sediments by Nycodenz density gradient centrifugation
journal, March 2010


Flow Cytometric Assessment of Bacterial Abundance in Soils, Sediments and Sludge
journal, June 2016


Dominant plant species shape soil bacterial community in semiarid sandy land of northern China
journal, January 2018

  • Wang, Shaokun; Zuo, Xiaoan; Zhao, Xueyong
  • Ecology and Evolution, Vol. 8, Issue 3
  • DOI: 10.1002/ece3.3746

Microbial abundance and composition influence litter decomposition response to environmental change
journal, March 2013

  • Allison, Steven D.; Lu, Ying; Weihe, Claudia
  • Ecology, Vol. 94, Issue 3
  • DOI: 10.1890/12-1243.1

Fluorescence as an alternative to light-scatter gating strategies to identify frozen–thawed cells with flow cytometry
journal, August 2014


An efficient and rapid method for the enumeration of heterotrophic prokaryotes in coastal sediments by flow cytometry
journal, October 2014

  • Lavergne, Céline; Beaugeard, Laureen; Dupuy, Christine
  • Journal of Microbiological Methods, Vol. 105
  • DOI: 10.1016/j.mimet.2014.07.002

The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems
journal, March 2008


Enumeration of soil bacteria with the green fluorescent nucleic acid dye Sytox green in the presence of soil particles
journal, November 2004


Cell Size Distributions of Soil Bacterial and Archaeal Taxa
journal, September 2013

  • Portillo, Maria C.; Leff, Jonathan W.; Lauber, Christian L.
  • Applied and Environmental Microbiology, Vol. 79, Issue 24
  • DOI: 10.1128/AEM.02710-13