This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Falcioni, T.
Right arrow Articles by Gasol, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Falcioni, T.
Right arrow Articles by Gasol, J. M.
Agricola
Right arrow Articles by Falcioni, T.
Right arrow Articles by Gasol, J. M.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, March 2008, p. 1767-1779, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.01668-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evaluating the Flow-Cytometric Nucleic Acid Double-Staining Protocol in Realistic Situations of Planktonic Bacterial Death{triangledown}

Tania Falcioni,1,2,{dagger} Stefano Papa,1 and Josep M. Gasol2*

Center of Cytometry and Cytomorphology, University Carlo Bo, Urbino, Italy,1 Institut de Ciències del Mar, CSIC, Barcelona, Spain2

Received 20 July 2007/ Accepted 11 January 2008

Since heterotrophic prokaryotes play an important biogeochemical role in aquatic ecosystems and have a high capacity to survive in extreme environments, easy-to-perform protocols that probe their physiological states and the effects of environmental variables on those states are highly desired. Some methodologies combine a general nucleic acid stain with a membrane integrity probe. We calibrated one of these, the nucleic acid double-staining (NADS) protocol (G. Grégori, S. Citterio, A. Ghiani, M. Labra, S. Sgorbati, S. Brown, and M. Denis, Appl. Environ. Microbiol. 67:4662-4670, 2001), determining the optimal stain concentrations in seawater and the response to conditions that generate prokaryote death (such as heat) and to conditions that are known to produce death in plankton, such as nutrient limitation or flagellate grazing. The protocol was validated by comparison to two methods used to detect viability: active respiration by 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and incorporation of tritiated leucine. We show that concentrations in the range of 5 to 20 µg ml–1 of propidium iodide, simultaneous to a 10x concentration of Sybr green I, are best for detecting two separated populations of "live" (green cells) and "dead" (red cells) organisms. During exposure to heat and UVC, we observed that the number of live cells declined concurrently with that of actively respiring cells (CTC positive) and with total leucine incorporation. In seawater mesocosms, the NADS protocol allowed detection of bacterioplankton starvation-related death and flagellate predation. The protocol was also tested in deep profiles in the northwest Atlantic, demonstrating its potential for routine characterization of this fraction of the physiological diversity of marine heterotrophic prokaryotic plankton.

* Corresponding author. Mailing address: Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, CMIMA-CSIC, Passeig Marítim de la Barceloneta, 37-49, E-08003 Barcelona, Catalunya, Spain. Phone: 349322309500. Fax: 34932309555. E-mail: pepgasol{at}icm.csic.es

{triangledown} Published ahead of print on 25 January 2008.

{dagger} Present address: Servei de Microscòpia Electrònica, Universitat de Lleida, c. Rovira Roure 44, 25198 Lleida, Spain.

Applied and Environmental Microbiology, March 2008, p. 1767-1779, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.01668-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»