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

선박평형수 처리장치 효율 검증을 위한 해양미소부유생물 생사판별기법

Baek, Seung Ho;Shin, kyoungsoon

  • Received : 2015.03.11
  • Accepted : 2015.06.11
  • Published : 2015.06.30


We determined a method to determine marine planktonic organism viability using Evan's blue, Aniline blue, and 5-choromethyfluorescein 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 efficiency of Evan's blue and Aniline blue were ${\geq}90%$ of the original field sample. However, it was difficult to test the efficiency 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 fluorimetric stain, was the best dye to detect live cells of almost all phytoplankton species, and staining efficiency was 70%. The CMFDA method is similar to the fluorescein 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 efficiency. As a result, the frequency of dying cells based on the double-staining method was 95%, which was significantly 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 efficacy of a ship's ballast water treatment system.


Ballast water;Evan blue;Aniline blue;CMFDA;cell viability


  1. IMO "Report on the ballast water treatment standards workshop". In 1st International ballast water treatment standards workshop, IMO London, 28-30 March., 2001.
  2. N. Bax, A. Williamson, M. Aguero, E. Gonzalez, W. Geeves, "Marine invasive alien species: a threat to global biodiversity". Mar. Policy, 27, 313-323, 2003. DOI:
  3. E. M. Zetsche, F. J. R. Meysman,"Dead or alive? Viability assessment of micro- and mesoplankton". J. Plankton Res., 34, 493-509, 2012. DOI:
  4. M. Garvey, B. Moriceau, U. Passow, "Applicability of the FDA assay to determine the viability of marine phytoplankton under different environmental conditions". Mar. Ecol. 352:17-26, 2007. DOI:
  5. M. Steinberg, E. Lemieux, L. Drake, "Determining the viability of marine protists using a combination of vital, fluorescent stains". Mar. Biol., 158, 1431-1437, 2011. DOI:
  6. S. H. Baek, K. Shin, "Applicability of fluorescein diacetate (FDA) and Calcein-AM to determine the viability of marine plankton". Ocean Polar Res., 31, 349-357, 2009. DOI:
  7. D. M. Dressel, D. R. Heinle, M. C. Grote, "Vital staining to sort dead and live copepods". Ches. Sci., 13, 156-159, 1972. DOI:
  8. R. W. Crippen, J. L. Perrier, "The use of neutral red and Evans blue for live-dead determinations of marine plankton (with comments on the use of rotenone for inhibition of grazing". Biotech. Histochem., 49, 97-104, 1974. DOI:
  9. S. L. Bickel, K. W. Tang, H. P. Grossart, " Use of anline blue to distinguish live and dead crustacean zooplankton composition in freshwaters". Freshwater Biol. 54, 971-981, 2009. DOI:


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