Optimization of FDA–PI method using flow cytometry to measure metabolic activity of the cyanobacteria, Microcystis aeruginosa

Abstract

A rapid toxicity test based on inhibition of esterase activity in the harmful freshwater microalgae – Microcystis aeruginosa was developed using flow cytometry. The hydrolysis rate of fluorescein diacetate (FDA) by intracellular esterase to fluorescein was used to indicate the metabolic activity of algae. Uptake of FDA was optimized at different concentrations and incubation times. Propidium iodide (PI) was utilized to assess cell membrane integrity. The optimized FDA/PI staining dosages were 10 mg/L and 10 μM, respectively, lower than the reported concentrations. Correspondingly, the proper incubation time was 14–21 min at the optimal FDA dosage determined in this study. A new procedure based on optimized FDA/PI condition, called “whole algal culture flow cytometry with fluorescence triggering”, was developed for short-term bioassays. This new procedure, taking account of working conditions such as pH and impure cultures, is able to avoid algal cell damages in sample preparation and separate algal cells from non-algal particles by fluorescence triggering. This newly-developed procedure was then used to assess the toxicity of copper on M. aeruginosa in a short-term exposure (36 h). As copper concentrations increased, it was found that the esterase activity decreased in a concentration-dependent manner with increased membrane fragments. Moreover, esterase activity was a good indicator of copper toxicity in M. aeruginosa. The EC 50 value based on mean fluorescence intensity (MFI) was 123.3 μg/L (95% confidence limits 101.5–146.2 μg/L). Therefore, the new-developed procedure could be used for sublethal endpoints detection, and has the potential to be a rapid and cost-effective bioassay for selecting M. aeruginosa control methods or exploring the M. aeruginosa activity inhibition mechanism.