Cryopreservation of fluorescent marker‐labeled algae (Selenastrum capricornutum) for toxicity testing using flow cytometry

Abstract

Abstract-A rapid, two-stain (fluorescein diacetate and ethidium homodimer-1) flow cytometric assay to evaluate viability and cytotoxicity of the alga Selenastrum capricornutum in preserved samples is described. For storage, stained cells were fixed in glutaraldehyde, flash frozen in liquid nitrogen, and stored frozen (Ϫ20ЊC) for assessment at a later date. Weekly analysis of frozen samples showed that fluorescence was stable for 7 weeks. A mixture of 50% healthy and 50% heat-killed cells of S. capricornutum showed 36.1% healthy cells, 13.2% compromised cells, 12% nonstained cells, and 38.7% dead cells. This technique was tested using sodium dodecyl sulfate (SDS) and phenol as toxicants. Threshold concentrations for toxicant impact were in the range of 1 to 10 mg/L for SDS and 10 to 100 mg/L for phenol. Estimates of mortality showed 96-h median lethal concentration (LC50) values of 2,340 mg/L and 6,970 mg/L for SDS and phenol, respectively. This two-stain flow cytometric procedure has proven to be a reliable, sensitive assay for determining viability of S. capricornutum in preserved samples. The sensitivity of this dual-color assay and the ability to store samples for later analysis are significant improvements over current techniques.

Optimization of a Microplate-Based Assay to Assess Esterase Activity in the Alga Pseudokirchneriella subcapitata

“…FDA has been used as a technique for determining the viability of algal cells by flow cytometry (Gala and Giesy 1994;Caux et al 1996;Faber et al 1997;Franklin et al 2001;Lage et al 2001;Peperzak and Brussaard 2011) or by fluorescence microscopy (Labra et al 2007;Nancharaiah et al 2007). Another approach for the use of FDA consists in the measurement of esterase activity.…”

Development of flow cytometry‐based algal bioassays for assessing toxicity of copper in natural waters

“…This technique has the ability to measure thousands of cells per second, and it can perform multiparameter analysis on a wide range of cellular properties by measurement of algal light-scatter signals and autofluorescence [5]. Through use of biochemically specific fluorescent dyes, flow cytometry can also provide additional information regarding the physiological condition of cells [6]. Flow cytometry has sufficient sensitivity to analyze cell densities as low as 10 2 cells/ml, and it has an advantage over conventional counting techniques in that it can differentiate live from dead algal cells and other particles on the basis of chlorophyll a fluorescence (i.e., autofluorescence).…”

“…More recently, flow cytometry has been applied to physiological studies with algae, bacteria, and yeasts. Examples include cell viability analysis using fluorescein diacetate (FDA) to measure esterase activity [8] and membrane potential analysis using the cationic dye 3,3Ј-dihexyloxacarbocyanine (DiOC 6 [3]) [9,10]. Flow cytometric techniques have important advantages over conventional biochemical assays, because cell functions can be quickly determined at conditions close to the in vivo state [9].…”

Acute Toxicity of LAS Homologues in Marine Microalgae: Esterase Activity and Inhibition Growth as Endpoints of Toxicity

“…A FACSCalibur #ow cytometry (Becton-Dickinson) equipped with a 488 nm excitation argon-laser and a three color photomultiplier with #uorescent emission "lters (FL1, 530 nm, FL2, 585 nm; FL3, 607 nm; forward light scatter (FSC) and side scatter (SSC)) was used in this study. Fluorescein acetate (FDA) has been used as a probe to assess esterase activity as an indicator of the viability of algal cells (Faber et al, 1997). A solution of FDA was prepared in acetone to yield a "nal FDA solution concentration of 1 mg/mL.…”