Application of Neutral Red Staining Method to Distinguishing Live and Dead Marine Plankton for the Investigation of Efficacy of Ship's Ballast Water Treatment System

선박평형수 처리 시스템 효율 검증을 위한 해양 플랑크톤 생사판별시 Neutral red 염색법 적용 가능성 연구

Hyun, Bonggil;Shin, Kyoungsoon;Chung, Hansik;Choi, Seo-Yeol;Jang, Min-Chul;Lee, Woo-Jin;Choi, Keun-Hyung

  • Received : 2014.03.06
  • Accepted : 2014.09.03
  • Published : 2014.11.28


In order to prevent the spread of non-indigenous aquatic species through the ballast water in commercial ships, International Maritime Organization (IMO) adopted in 2004 the International Convention for Control and Management of Ship's Ballast Water and Sediments. The Convention mandates treatment of ballast water for most transoceanic voyages and its confirmation of treatment is made with plankton live/dead assay. Fluorescein diacetate assay (FDA), which produces bright green light for live phytoplankton, has been a de facto standard method to determine the survival of marine plankton, but its staining efficacy has been in dispute. In the present study, we examined the limitation of FDA, and compared its efficacy with Neutral red (NR) staining, another promising assay and widely used especially for zooplankton mortality. For all phytoplankton species studied in the present study, except Ditylum brightwellii, the staining efficiency was <50% with FDA. The green FDA fluorescence interfered with phytoplankton autofluorescence in most samples. In contrast, NR assay stained over 90% of both phytoplankton and zooplankton species tested in this study. FDA assay also showed that green FDA fluorescence rapidly faded when phytoplankton cells were exposed to microscope light. Both FDA and NR assay were negative on formalin-killed individuals of both phytoplankton and zooplankton species. Our results suggest that NR assay is more effective for determining the survival of marine plankton and can be applied to test the efficacy of ballast water treatment.


ballast water;International Maritime Organization (IMO);marine plankton;Neutral red (NR);fluorescein diacetate (FDA)


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