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Appl. Environ. Microbiol. doi:10.1128/AEM.00996-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

An immunofluorescent flow cytometric technique for counting the brown tide alga, Aureococcus anophagefferens

Department of Biological Sciences, University of Southern California, Los Angeles, CA, 90089-0371, USA; Southern California Coastal Water Research Project, 3535 Harbor Blvd., Suite 110, Costa Mesa, CA 92626; Maryland Department of Natural Resources, 580 Taylor Avenue D2, Annapolis, Maryland 21401

^* To whom correspondence should be addressed. Email: stauffer{at}usc.edu.

	Abstract

A new immunologically-based flow cytometric (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 using a flow cytometer. Optimal conditions for FITC-MAb staining, including solution composition, incubation times, and FITC-MAb concentrations were investigated. The FITC-MAb was tested for cross-reactivity with non-target, 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 microscopical counts 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 10x 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 IFCM method. Application of the IFCM method to environmental ‘brown tide’ samples taken from coastal bays of Maryland demonstrated its efficacy in resolving A. anophagefferens abundances throughout the course of a bloom and over a large range of abundances. IFCM counts of the brown tide alga from natural samples were consistently lower than those obtained using the ELISA method and on parity with the polyclonal immuno-microscope 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 abundances (10³ – 10⁶ cells ml^-1).