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

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

Full text not available from this repository. (Request a copy)


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 μL 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.

Item Type: Publication - Article
Digital Object Identifier (DOI):
ISSN: 15415856
Date made live: 22 Mar 2012 10:40 +0 (UTC)

Actions (login required)

View Item View Item

Document Downloads

Downloads for past 30 days

Downloads per month over past year

More statistics for this item...