Propidium Iodide Staining and Flow Cytometry-Based Assessment of Heavy Metal Impact on Marine Phytoplankton

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Abstract
Seafloor resource development in the future is expected to be accompanied by the mining of metal ores, as these sulfide ores contain valuable metals. However, the mining process is detrimental to marine oceanic environments. As a precautionary approach, innovations in the collection of environmental baseline data and new onboard assessment methods for marine environmental impacts are required. Due to the low cell density in open ocean water, techniques for rapid impact assessment of seawater without sample concentration are required. The purpose of this study was to establish a heavy metal impact assessment system for seawater samples of marine microbes using a portable flow cytometer, On-chip Sort. We established a protocol for detecting heavy metal-induced damage to cells via propidium iodide (PI) staining using algal culture strains (Bathycoccus prasinos NIES-2670, Synechococcus sp. NIES-969, Prochlorococcus sp. NIES-2885, and Cyanobium sp. NIES-981) obtained from the marine environment maintained at the Microbial Culture Collection of the National Institute for Environmental Studies, Tsukuba, Japan. Results showed that PI staining could detect the effects of heavy metals on cells. The proportion of PI-positive cells increased with an increase in the concentration of heavy metal mixture or copper exposure. Compared to cyanobacteria, damaged cells of eukaryotic algae were detected. Therefore, the effects of heavy metals on both eukaryotic and prokaryotic algae can be rapidly assessed via PI-based flow cytometry using samples containing low cell densities.

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