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 Stauffer, B. A.
Right arrow Articles by Caron, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stauffer, B. A.
Right arrow Articles by Caron, D. A.
Agricola
Right arrow Articles by Stauffer, B. A.
Right arrow Articles by Caron, D. A.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, November 2008, p. 6931-6940, Vol. 74, No. 22
0099-2240/08/$08.00+0     doi:10.1128/AEM.00996-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Immunofluorescence Flow Cytometry Technique for Enumeration of the Brown-Tide Alga, Aureococcus anophagefferens{triangledown}

Beth A. Stauffer,1* Rebecca A. Schaffner,2 Catherine Wazniak,3 and David A. Caron1

Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0371,1 Southern California Coastal Water Research Project, 3535 Harbor Boulevard, Suite 110, Costa Mesa, California 92626,2 Maryland Department of Natural Resources, 580 Taylor Avenue D2, Annapolis, Maryland 214013

Received 2 May 2008/ Accepted 22 September 2008

A new immunologically based flow cytometry (IFCM) technique was developed to enumerate Aureococcus anophagefferens, a small pelagophyte alga that is the cause of "brown tides" in bays and estuaries of the mid-Atlantic states along the U.S. coast. The method utilizes a monoclonal antibody conjugated to fluorescein isothiocyanate (FITC-MAb) to label the surface of A. anophagefferens cells which are then detected and enumerated by using a flow cytometer. Optimal conditions for FITC-MAb staining, including solution composition, incubation times, and FITC-MAb concentrations, were determined. The FITC-MAb method was tested for cross-reactivity with nontarget, similarly sized, photoautotrophic protists, and the method was compared to an enzyme-linked immunosorbent assay (ELISA) using the same MAb. Comparisons of the IFCM technique to traditional microscopy enumeration of cultures and spiked environmental samples showed consistent agreement over several orders of magnitude (r2 > 0.99). Comparisons of the IFCM and ELISA techniques for enumerating cells from a predation experiment showed a substantial overestimation (up to 10 times higher) of the ELISA in the presence of consumers of A. anophagefferens, presumably due to egested cell fragments that retained antigenicity, using the ELISA method, but were not characterized as whole algal cells by the IFCM method. Application of the IFCM method to environmental "brown-tide" samples taken from the coastal bays of Maryland demonstrated its efficacy in resolving A. anophagefferens abundance levels throughout the course of a bloom and over a large range of abundance values. IFCM counts of the brown-tide alga from natural samples were consistently lower than those obtained using the ELISA method and were equivalent to those of the polyclonal immunofluorescence microscopy technique, since both methods discriminate intact cells. Overall, the IFCM approach was an accurate and relatively simple technique for the rapid enumeration of A. anophagefferens in natural samples over a wide range of abundance values (103 to 106 cells ml–1).

* Corresponding author. Mailing Address: 3616 Trousdale Parkway, AHF 301, Los Angeles, CA 90089-0371. Phone: (213) 821-2123. Fax: (213) 740-8123. E-mail: stauffer{at}usc.edu

{triangledown} Published ahead of print on 26 September 2008.

Applied and Environmental Microbiology, November 2008, p. 6931-6940, Vol. 74, No. 22
0099-2240/08/$08.00+0     doi:10.1128/AEM.00996-08
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»