The University of Southampton
University of Southampton Institutional Repository

Optimized routine flow cytometric enumeration of heterotrophic flagellates using SYBR Green I

Optimized routine flow cytometric enumeration of heterotrophic flagellates using SYBR Green I
Optimized routine flow cytometric enumeration of heterotrophic flagellates using SYBR Green I
Heterotrophic flagellates (HF) are the major consumers of bacteria in aquatic ecosystems and dominate heterotrophic
nanoplankton in numbers and in biomass. A DNA-staining based flow cytometry (FC) protocol to enumerate HF was described by Zubkov et al. (2007), but has not yet been widely adopted. We tested extensively the method and its limitations using a wide range of sample types and trying several fixation and conservation alternatives. We evaluated simplification of some steps of the method, seeking the best compromise between precision and the quality of distinction of HF from bacteria and phytoplankton in the cytograms. We found that a flow rate of 120-220 ?L min–1 without using a syringe-pump enhanced machine modification, and running times of 8-10 min allowed enumeration of HF even at values below 102 cells mL–1. SYBR Green I, at final concentrations of 1:10000 and a minimum staining time of 10 min at room temperature in the dark, was adequate for staining and detecting HF. No significant differences were found between cell numbers obtained from freshly analyzed samples and those previously frozen in liquid-N. FC and epifluorescence microscopy (EpiM) were in
good agreement and FC yielded lower variability between replicate samples than EpiM. One limitation we encountered was that, in the presence of large bacteria and/or bacterial aggregates, enumeration was difficult.
However, in absence of bacterial aggregates samples with Bact/HF ratios > 1000, HF could be well-enumerated.
1541-5856
329-339
Christaki, Urania
4587f9b6-aaba-4951-9548-37e723c05748
Courties, Claude
56041e95-57b7-462e-a9f8-ef705d6b850a
Massana, Ramon
482d6ecf-5206-4bb6-8bc0-66561f52b1a8
Catala, Philippe
67d6e35b-f895-4e94-9730-aa21bd1d06d2
Lebaron, Philippe
0bbb16e0-a835-4d30-858b-c43f2faf5e34
Gasol, Josep M.
15fcba14-0b79-4871-85ad-c098bdd7d582
Zubkov, Mikhail V.
b1dfb3a0-bcff-430c-9031-358a22b50743
Christaki, Urania
4587f9b6-aaba-4951-9548-37e723c05748
Courties, Claude
56041e95-57b7-462e-a9f8-ef705d6b850a
Massana, Ramon
482d6ecf-5206-4bb6-8bc0-66561f52b1a8
Catala, Philippe
67d6e35b-f895-4e94-9730-aa21bd1d06d2
Lebaron, Philippe
0bbb16e0-a835-4d30-858b-c43f2faf5e34
Gasol, Josep M.
15fcba14-0b79-4871-85ad-c098bdd7d582
Zubkov, Mikhail V.
b1dfb3a0-bcff-430c-9031-358a22b50743

Christaki, Urania, Courties, Claude, Massana, Ramon, Catala, Philippe, Lebaron, Philippe, Gasol, Josep M. and Zubkov, Mikhail V. (2011) Optimized routine flow cytometric enumeration of heterotrophic flagellates using SYBR Green I. Limnology and Oceanography: Methods, 9, 329-339. (doi:10.4319/lom.2011.9.329).

Record type: Article

Abstract

Heterotrophic flagellates (HF) are the major consumers of bacteria in aquatic ecosystems and dominate heterotrophic
nanoplankton in numbers and in biomass. A DNA-staining based flow cytometry (FC) protocol to enumerate HF was described by Zubkov et al. (2007), but has not yet been widely adopted. We tested extensively the method and its limitations using a wide range of sample types and trying several fixation and conservation alternatives. We evaluated simplification of some steps of the method, seeking the best compromise between precision and the quality of distinction of HF from bacteria and phytoplankton in the cytograms. We found that a flow rate of 120-220 ?L min–1 without using a syringe-pump enhanced machine modification, and running times of 8-10 min allowed enumeration of HF even at values below 102 cells mL–1. SYBR Green I, at final concentrations of 1:10000 and a minimum staining time of 10 min at room temperature in the dark, was adequate for staining and detecting HF. No significant differences were found between cell numbers obtained from freshly analyzed samples and those previously frozen in liquid-N. FC and epifluorescence microscopy (EpiM) were in
good agreement and FC yielded lower variability between replicate samples than EpiM. One limitation we encountered was that, in the presence of large bacteria and/or bacterial aggregates, enumeration was difficult.
However, in absence of bacterial aggregates samples with Bact/HF ratios > 1000, HF could be well-enumerated.

Full text not available from this repository.

More information

Published date: 2011
Organisations: Marine Biogeochemistry

Identifiers

Local EPrints ID: 336368
URI: https://eprints.soton.ac.uk/id/eprint/336368
ISSN: 1541-5856
PURE UUID: fca863df-fa64-4e0b-97d6-9ef8da8a7b89

Catalogue record

Date deposited: 22 Mar 2012 10:40
Last modified: 16 Jul 2019 22:09

Export record

Altmetrics

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×