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

Abstract

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 (r 2 > 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 (10 3 to 10 6 cells ml ؊1 ).

Effect of vitamins B1 and B12 on bloom dynamics of the harmful brown tide alga, Aureococcus anophagefferens (Pelagophyceae)

“…Abundance of heterotrophic bacteria (stained with Synergy Brands [SYBR TM ] Green I), phycoerythrin-containing picocyanobacteria, and photosynthetic picoeukaryotes was determined using a Fluorescence Activated Cell Scan (Becton, Dickinson and Company) flow cytometer using fluorescence patterns and particle size from side angle light scatter (Olson et al 1991). To enumerate A. anophagefferens, whole water was preserved with filter-sterilized 10% gluteraldehyde solution (1% final v : v), stored in glass tubes at 4uC, and later analyzed using an enzyme-linked immunosorbent assay with a flow cytometrically detected fluorescent, monoclonal antibody (Stauffer et al 2008). Nutrient samples were pre-filtered with acid-cleaned, polypropylene capsule filters (0.2 mm; General Electric Osmonics, DCP0200006) and stored frozen.…”

A Novel Immunofluorescence Flow Cytometry Technique Detects the Expansion of Brown Tides Caused by Aureoumbra lagunensis to the Caribbean Sea

“…While glutaraldehyde is the ideal preservative for enumeration of another brown tide-forming pelagophyte, A. anophagefferens (19), archived samples from monitoring programs are often preserved in buffered formalin or Lugol's iodine solution. To establish the efficacy of the new method for quantifying A. lagunensis in different preservatives, samples of a monoclonal culture of A. lagunensis (CCMP1530) and from water from Laguna Madre, TX (collected on 3 March 2013), were preserved in 1% (vol/vol, final concentration) glutaraldehyde, 1% phosphate-buffered formalin, and 1% Lugol's iodine solution (22) and stored in either glass or plastic scintillation vials at both 4°C and 25°C for a period of 3 months.…”

“…When the first brown tides occurred in New York during the late 20th century, Anderson et al (17) pioneered the use of a polyclonal, immunofluorescent antibody to quantify A. anophagefferens. Later, Caron et al (18) developed an enzyme-linked immunosorbent assay (ELISA)-based immunofluorescent approach by using a monoclonal antibody to quantify A. anophagefferens, and Stauffer et al (19) adapted the monoclonal antibody (18) for use on a flow cytometer (FCM), permitting even more accurate and rapid enumeration of A. anophagefferens. Lopez-Barrerio et al (20) developed a polyclonal antibody for A. lagunensis and used it in a microscopic format to detect and enumerate this alga, a method later used by Villareal et al (14) to reveal the distribution of A. lagunensis throughout the coastal waters of the Gulf of Mexico, from southern Texas through southern Florida.…”

Phytoplankton Community Structure Based on Photopigment Markers in a Mid-Atlantic U.S. Coastal Lagoon: Significance for Hard-Clam Production

“…The biovolume of each taxon was calculated based on microscopic measurements of cell dimensions and geometric models of phytoplankton (e.g., Hillebrand et al, 1999). Aureococcus anophagefferens cell concentrations were determined by immunofluorescence using a monoclonal antibody conjugated to fluorescein isothiocyanate and enumer-ated by flow cytometry following methods of Stauffer et al (2008).…”