TY - JOUR
T1 - An efficient and rapid method for the enumeration of heterotrophic prokaryotes in coastal sediments by flow cytometry
AU - Lavergne, Céline
AU - Beaugeard, Laureen
AU - Dupuy, Christine
AU - Courties, Claude
AU - Agogué, Hélène
N1 - Funding Information:
This research was supported by a PhD grant from the Charente Maritime Department , by the CPER 2006–2013 (Contrat Projet Etat Région) of Charente Maritime and by EC2CO project ( CAPABIOC , CNRS and INSU , 2013–2014). We are grateful to the cytometry and imaging platform that provided organization and service in our lab. We also appreciate the cytometry platform of Banyuls/Mer, which allows us to use cell-sorting cytometry. The authors are grateful to E. Pante (LIENSs, La Rochelle) for his help. In our study, we analyzed various sediments and the authors would like to thank all those who provided the environmental samples: P. Bocher (LIENSs, La Rochelle) in collaboration with D.S. Mizrahi (project supporting by the US Fish and Wildlife Service and the Western Hemisphere Shorebird Reserve Network, New Jersey Audubon Society), K. Guizien and S. Lucas (DynDiagHyd project, EC2CO - LECOB, Banyuls/Mer), H. Montanie (Biofilms Project, FREDD - LIENSs, La Rochelle) and F. Rossi (CHARM project, EC2CO - ECOSYM, Montpellier). The authors are grateful to the anonymous reviewers for their thorough evaluations which help us to improve the quality of manuscript.
PY - 2014/10
Y1 - 2014/10
N2 - Flow cytometry offers an easy and powerful way to assess multi-parametric data in different domains, notably in the environmental sciences. Because evaluating heterotrophic prokaryotic abundance is crucial to understand an ecosystem's functioning, we propose a quick and efficient protocol for (1) cell's detachment in muddy coastal sediments followed by (2) enumeration of prokaryotes by flow cytometry compared to epifluorescence microscopy and (3) a type of storage adapted for benthic samples. First, sample preparation by incubation in a detergent mix containing sodium pyrophosphate (0.01M final concentration) and Tween 80 (0.1% final concentration) drastically increased cell detachment from sediment particles (+130.40%) compared to extraction with sodium pyrophosphate only. Cell sorting allowed to control the efficiency of the extraction as few cells were found attached to sediment particles in epifluorescence microscopy after sorting. Flow cytometry gave consistent results with strong reliability by counting 1.81 times more cells compared to epifluorescence microscopy. Thirdly, results revealed that sediment samples fixed with formaldehyde and then liquid-N2 frozen and directly stored at -80°C can be analyzed within 3months. In routine, our method of extraction and counting allowed to evaluate 83.67% of the real abundance in a sediment sample. Finally, this optimized technique was applied on sandy and muddy coastal and freshwater sediments and allowed us to prove the high efficiency of this new method. Flow cytometry is a fast, replicable and low-cost method for counting heterotrophic prokaryotes, even for sediment samples. The two-step method that we developed enables high frequency analyses (30 samples in less than 4h).
AB - Flow cytometry offers an easy and powerful way to assess multi-parametric data in different domains, notably in the environmental sciences. Because evaluating heterotrophic prokaryotic abundance is crucial to understand an ecosystem's functioning, we propose a quick and efficient protocol for (1) cell's detachment in muddy coastal sediments followed by (2) enumeration of prokaryotes by flow cytometry compared to epifluorescence microscopy and (3) a type of storage adapted for benthic samples. First, sample preparation by incubation in a detergent mix containing sodium pyrophosphate (0.01M final concentration) and Tween 80 (0.1% final concentration) drastically increased cell detachment from sediment particles (+130.40%) compared to extraction with sodium pyrophosphate only. Cell sorting allowed to control the efficiency of the extraction as few cells were found attached to sediment particles in epifluorescence microscopy after sorting. Flow cytometry gave consistent results with strong reliability by counting 1.81 times more cells compared to epifluorescence microscopy. Thirdly, results revealed that sediment samples fixed with formaldehyde and then liquid-N2 frozen and directly stored at -80°C can be analyzed within 3months. In routine, our method of extraction and counting allowed to evaluate 83.67% of the real abundance in a sediment sample. Finally, this optimized technique was applied on sandy and muddy coastal and freshwater sediments and allowed us to prove the high efficiency of this new method. Flow cytometry is a fast, replicable and low-cost method for counting heterotrophic prokaryotes, even for sediment samples. The two-step method that we developed enables high frequency analyses (30 samples in less than 4h).
KW - Cell enumeration
KW - Coastal sediments
KW - Flow cytometry
KW - Prokaryotes
UR - http://www.scopus.com/inward/record.url?scp=84904762725&partnerID=8YFLogxK
U2 - 10.1016/j.mimet.2014.07.002
DO - 10.1016/j.mimet.2014.07.002
M3 - Article
C2 - 25017902
AN - SCOPUS:84904762725
SN - 0167-7012
VL - 105
SP - 31
EP - 38
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
ER -