• Ocean and Polar Research
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• v.31 no.4
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• pp.349-357
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• 2009

Ballast water is widely recognized as a serious environmental problem due to the risk of introducing non-indigenous aquatic species. In this study we aimed to investigate measures which can minimize the transfer of aquatic organisms from ballast water. Securing more reliable technologies to determine the viability of aquatic organisms is an important initiative in ballast water management systems. To evaluate the viability of marine phytoplankton, we designed the staining methods of fluorescein diacetate (FDA) and Calcein-AM assay on each target species belonging to different groups, such as bacillariphyceae, dinophyceae, raphidophyceae, chrysophyceae, haptophyceae and chlorophyceae. The FDA method, which is based on measurements of cell esterase activity using a fluorimetric stain, was the best dye for determining live cells of almost all phytoplankton species, except several diatoms tested in this study. On the other hand, although fluorescence of Calcein-AM was very clear for a comparatively longer time, green fluorescence per cell volume was lacking in most of the tested species. According to the Flow CAM method, which is a continuous imaging technique designed to characterize particles, green fluorescence values of stained cells by FDA were significantly higher than those of Calcein-AM treatments and control, implying that the Flow CAM using FDA assay could be adapted as an important tool for distinguishing living cells from dead cells. Our results suggest that the FDA and Calcein-AM methods can be adapted for use on phytoplankton, though species-specific characters are greatly different from one organism to another.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.19 no.4
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• pp.223-231
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• 2014

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.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.16 no.6
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• pp.4328-4334
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• 2015

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.

• Korean Journal of Environmental Agriculture
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• v.27 no.2
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• pp.139-144
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• 2008

A field experiment was conducted to investigate the influence of cultivars and compost on soil microbial activities and diversities in a red pepper-grown field. Compost was applied with 0, 30, and 60M/T $ha^{-1}$ in April and then red pepper seedlings of "Yong-go 4" and "Koeun" were transplanted in May 2007. Soil samples were collected in early August 2007. Measurement of microbial activities was based on a dehydrogenase assay and a fluorescein diacetate hydrolysis. Soil microbial community was characterized with Biolog $EcoPlate^{TM}$ and phospholipid fatty acid(PLFA). Red pepper cultivars did not differentiate the selected soil chemical and microbial properties. Soil pH and soil microbial community changed by amending the soil with 30 and 60 M/T $ha^{-1}$ of compost, and the soil organic matter and potassium content, and soil microbial activities increased in soils amended with 60 M/T $ha^{-1}$ of compost. Red pepper cultivar induced a little different soil chemical properties and microbial activity in soils amended with 60 M/T $ha^{-1}$ of compost even though significant differences were not found in those properties. In conclusion the effects of compost on soil chemical and microbial properties were much higher than red pepper cultivars in short-term period but the effects of red pepper cultivars should be investigated in long-term field test.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.334-341
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• 2004

Polysaccharides were prepared from Orostachys japonicus by extration with hot steam water (OJPl). The OIPl fraction was further purified by Sephadex G-50 gel filtration chromatography to produce FI (polysaccharides) and FII (oligosaccharides) fraction. The average molecular masses o fFI and FII fraction were determined to be 3050 kDa and 13 kDa, respectively. The antimicrobial activity of OIPl was tested against 8 strains of bacteria and one strain of yeast by the disc diffusion method, fluorescein diacetate (FDA) method and broth dilution method. The OIPl exhibited a very strong growth inhibition to Candida albicans. The OIPl remarkably sup­pressed the growth of Salmonella typhimurium and Staphylococcus aureus. The OIPl showed higher growth inhibition to Escherichia coli and Pseudomonas aeruginosa than propolis, positive control. When the anticancer activity of the OIPl, FI or FII was examined against human cancer cell lines and the Sarcoma 180 cells, these widely suppressed the proliferation of cell lines in the MTT assay and morphology study. Especially, they remarkably inhibited the growth of A549, HeLa and AGS cells. Also treatment of cancer cells with OJPl, FI or FII induced apoptotic cell death characterized by DNA fragmentation. The OJPl, FI or FII exhibiting various biological activities such as antimicrobial activity and anticancer activity is expected to be developed as new biohealth products.

• Korean Journal of Soil Science and Fertilizer
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• v.40 no.4
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• pp.234-241
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• 2007

Biological amendments consisting of suspensions of selected microorganisms are often used in conjunction with various organic materials for amending soils to improve soil quality and plant growth. The effects of the biological amendment on chemical and biological properties of soil were investigated for a biological amendmentalone and when combined with different organic materials includingmunicipal compost (MC), poultry litter (PL), and cover crops (red clover (RC) and spring oats). A liquid preparation of a biological amendment called Effective Microorganisms was sprayed on the tested plots three times over a two-year period. Effective Microorganisms alone did not influence pH, K, or organic matter content in soil. However, increases in P in PL-treated soils in fall of both years andCa in MC-treated soil in fall 2001, and decreases in Ca, Mg, and cation exchange capacity (CEC) in RC-planted soil were associated with EM. Increased dehydrogenase(DH) activitiesassociated with Effective Microorganismswere only detected in July (P=0.0222) and October (P=0.0834) for RC-planted soils in the first year. Fluorescein diacetate (FDA) hydrolysisappeared to be enhanced by Effective Microorganisms in soils untreated or treated with MC and oatsbut only sporadically during the sampling period. FDA hydrolysis in both PL- and RC-treated soils as well as DH activity in PL-treated soils decreased with Effective Microorganisms treatment. Effective Microorganisms did not influence substrate utilization patterns expressed by the BIOLOG assay. We conclude that Effective Microorganisms effects on soil chemical and biological properties varied depending on the added organic materials. Effective Microorganisms periodically increased soil DH activity and FDA hydrolysis with RC and with MC plus oats, respectively.

• Development and Reproduction
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• v.22 no.3
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• pp.245-252
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• 2018

To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal stabilizer on an appropriate time is one of the possible approaches. However, the exact treatment timing and effect of cytoskeletal stabilizer such as cytochalasin B (CB) is not well known during oocyte vitrification procedures. Thus, the present study was conducted to determine optimal treatment timing of CB during vitrification and warming procedures. In experiment 1, the survival rates of the postwarming pig oocytes were analyzed by fluorescein diacetate (FDA) assays with 4 classifications. In results, post-warming oocytes showed significantly (p<0.05) decreased number of alive oocytes (31.8% vs. 86.4%) compared to fresh control. In detail, the significant difference (p<0.05) was found only in strong fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6% vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after (Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly (p<0.05) higher (91.6%) survival rates compared to group of CB-Vitri (83.7%), significantly (p<0.05) and comparable with group of Vitri Control (88.7%) by morphological inspection. In FDA assay results, group of Vitri-CB showed significantly (p<0.05) higher (44.2%) survival rates compared to groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the increased survival rates of post-warming pig oocyte treated with Vitri-CB method are firstly described here. The main finding of present study is that the CB treatment during recovery could be helpful to refresh the post-warming pig oocyte resulting its improved survival rates.

• Osong Public Health and Research Perspectives
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• v.8 no.6
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• pp.389-396
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• 2017

Objectives: To circumvent the limitations of the current golden standard method, colony-forming unit (CFU) assay, for viability of Bacille Calmette-$Gu{\acute{e}}rin$ (BCG) vaccines, we developed a new method to rapidly and accurately determine the potency of BCG vaccines. Methods: Based on flow cytometry (FACS) and fluorescein diacetate (FDA) as the most appropriate fluorescent staining reagent, 17 lots of BCG vaccines for percutaneous administration and 5 lots of BCG vaccines for intradermal administration were analyzed in this study. The percentage of viable cells measured by flow cytometry along with the total number of organisms in BCG vaccines, as determined on a cell counter, was used to quantify the number of viable cells. Results: Pearson correlation coefficients of FACS and CFU assays for percutaneous and intradermal BCG vaccines were 0.6962 and 0.7428, respectively, indicating a high correlation. The coefficient of variation value of the FACS assay was less than 7%, which was 11 times lower than that of the CFU assay. Conclusion: This study contributes to the evaluation of new potency test method for FACS-based determination of viable cells in BCG vaccines. Accordingly, quality control of BCG vaccines can be significantly improved.