A staining method to determine marine microplanktonic organism viability and investigate the ef cacy of a ship’s ballast water treatment system

Abstract

We determined a method to determine marine planktonic organism viability using Evan’s blue, Aniline blue, and 5-choromethy uorescein diacetate (CMFDA). The Evan’s blue and Aniline blue methods produced bright blue light for dead phytoplankton and zooplankton and were the best dyes to detect dead cells. The staining ef ciency of Evan’s blue and Aniline blue were ≥ 90% of the original eld sample. However, it was dif cult to test the ef ciency of a ship’s ballast water treatment system because detection of living cells. In contrast, the CMFDA method, which is based on measuring cell esterase activity using a uorimetric stain, was the best dye to detect live cells of almost all phytoplankton species, and staining ef ciency was 70%. The CMFDA method is similar to the uorescein diacetate (FDA) staining method. Therefore, we estimated viability of phytoplankton species using a double- staining method by combining CMFDA and FDA to determine optimum staining ef ciency. As a result, the frequency of dying cells based on the double-staining method was 95%, which was signi cantly higher than that of single CMDFA staining. Our results suggest that a CMDFA + FDA assay is more effective to determine survival of marine plankton and that this method was applicable to investigate the ef cacy of a ship’s ballast water treatment system.kton and zooplankton and were the best dyes to detect dead cells. The staining ef ciency of Evan’s blue and Aniline blue were ≥ 90% of the original eld sample. However, it was dif cult to test the ef ciency of a ship’s ballast water treatment system because detection of living cells. In contrast, the CMFDA method, which is based on measuring cell esterase activity using a uorimetric stain, was the best dye to detect live cells of almost all phytoplankton species, and staining ef ciency was 70%. The CMFDA method is similar to the uorescein diacetate (FDA) staining method. Therefore, we estimated viability of phytoplankton species using a double- staining method by combining CMFDA and FDA to determine optimum staining ef ciency. As a result, the frequency of dying cells based on the double-staining method was 95%, which was signi cantly higher than that of single CMDFA staining. Our results suggest that a CMDFA + FDA assay is more effective to determine survival of marine plankton and that this method was applicable to investigate the ef cacy of a ship’s ballast water treatment system.