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

• Horticultural Science & Technology
• /
• v.17 no.3
• /
• pp.337-340
• /
• 1999

In order to evaluate stored apple pollen viability, in vitro germination test was performed on a microscope slide coated with the culture medium containing fluorescein diacetate (FDA). However, the inclusion of FDA to the culture medium declined pollen germination. Alternatively, the fluorochromatic reaction procedure was tested. The procedure involved dusting pollen grains onto drops of 10% sucrose solution containing 0.002% FDA and allowing them to accumulate fluorescein. Within 30 min after the fluorochromatic reaction, viable pollen grains clearly fluoresced under ultraviolet light. Both the in vitro germination test and the fluorochromatic reaction procedure revealed that stored apple pollen viability was not considerably decreased over storage up to at least 39 months. Of the cultivars examined by both methods, 'Fuji' and 'Senshu' pollen viability was highest, 'Tsugaru' was intermediate, and 'Jonagold' was lowest. The fluorescing percentages appeared approximately comparable to the germination percentages except for the 'Senshu' pollens stored for 3 months, although the fluorescing percentages was slightly higher than the germination percentages. Strong and highly significant correlations were found between the two methods. It can thus be concluded that the fluorochromatic reaction procedure provides a convenient and reliable evaluation of stored apple pollen viability.

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

• Korean Journal of Microbiology
• /
• v.24 no.2
• /
• pp.147-153
• /
• 1986

Viable potential of Mycobacterium smegmatis, a slow grower in vitro cultivation and of M. leprae, an obligate intracellular parasitic bacterium, which can not be cultured yet in vitro was assessed by fluorospectrophotometry. Bacterial cells in different numbers and under various physiological status were incubater with fluorescein diacetate(FDA). After an incubation of the bacterial preparations with FDA at specified conditions, amount of fluorescein inside bacteria was measured by a fluorospectrophotometer at 470nm and 510nm of excitation and emission wavelengths, respectively. Fluorounit given by such bacteria showed a correlation with assessment of viability of the same preparations made by other methods, such as optical density and colony forming units of M. smegmatis and intracellular ATP content of M. leprae. The possible use of fluorospectrophotometry in assessing viability or physiological potential of bacteria, particularly intracellular parasites and fastidious organisms to culture in vitro is discussed in relation to other methods.

• Clinical and Experimental Reproductive Medicine
• /
• v.19 no.1
• /
• pp.1-8
• /
• 1992

A fluorescence microscopy technique using flurescein diacetateCFDA) as a substract has been tested for the evaluation of the viability of early mouse embryos. Embryos were incubated in T6 containing FDA concentrations of 2.5 to $50{\mu}g/ml$ for 1 to 5min. Embryos were then examined by reflected light fluorescence using a KP 490 and 520 barrier filter in a Nicon Diaphot microscopy. The results were as follow. 1. The rate of fluorescein accumulation increased on the concentration on FDA from $2.5{\times}10^{-6}M$ to $20{\times}10^{-6}M$ 2. The rate at which intracellular fluorescein was lost from embryos was depended on the temperature at which are stored. 3. Embryos with 3 min exposure to FDA have the most intensity of fluorescence. 4. Exposure of 2 cell embryos to FDA ($2.5-5{\mu}g/ml$) for 1 min did not alter their ability to delope normally in vitro.

• Korean Journal of Animal Reproduction
• /
• v.21 no.3
• /
• pp.211-217
• /
• 1997

This experiment was carried out to determine the possibility of using FDA on the selection of viable bovine immature oocytes. The results obtained in these experiments were summarized as follows; 1. The rates of nuclear maturation (metaphase II) of bovine oocytes according to FDA concentration were 92.5% (37/40) in the control and those rates in the dilution on FDA to 1:400,000, 1:800,000 and 1:1,600,000 were 74.4% (67/90), 80.3% (38/46) and 71.1% (32/45), respectively. 2. The fertilization rate ( 2-cell) in the control was 72.2% (39/54) and those rates in the dilution of FDA to 1:400,000, 1:800,000 and 1:1,600,000 were 28.8% (23/80), 66.7% (32/48) and 62.2% (28/45), respectively. In these results, a, pp.opriate concentration of FDA to bovine immature oocytes was 1:800,000. 3. Effect of FDA treatment to the blastocyst development of bovine oocytes was indicated that control was 22.2% (18/81) and FDA treatment groups which were classified to strong, partial and weak were 21.4% (36/168), 14.5% (9/62) and 0% (0/15), respectively. This result suggested that in vitro development to the blastocyst was severely reduced except strong group according to the FDA fluorescent level (P<0.05) and that using FDA is possible to select of bovine oocytes.

• Journal of Embryo Transfer
• /
• v.10 no.3
• /
• pp.193-202
• /
• 1995

In order to improve the cryopreservatory techniques of livestock embryos, the quick freezing method which is directly plunged in liquid nitrogen via prefreezing procedure without freezing machine was carried out for mouse embryos treated with permeable and nonpermeable cryoprotectants. The viability of frozen-thawed embryos were evaluated by FDA vital dye test. The results obtained was summaried as follows: 1. A total of 720 embryos were recovered from frozen embryos for viability test. Evalution of the fluorescein diacetate(FDA) vital dye test with mice embryos were resulted of 2.3 total mean score - evaluted in orderly higher mean grade of P3 453 (63%), P2 133(18%), P1 51(7%) and P0 83(12%). 2. An all-round evalution of these combination, the highest viability was showed in 3M ethylene glycol + 0. 25M trehalose treated with the copper prefreezing. 3. Effects of permeable and nonpermeable cryoprotectants combination were evaluated by means FDA score. 3M ethylene glycol + 0.25M trehalose showed the highest survival rates of 2.8 mean FDA score. 4. Effects of permeable cryoprotectants were evaluated by mean FDA score but the results were not significantly different each other. 5. In evalution of the nonpermeable cryoprotectants, 0. 25M trehalose obtalned higher mean FDA score than of 0.25M sucrose and it was significantly different(P<0.05). 6. There was no significantly difference between copper and stainless-steel in prefreezing procedures.

• Korean Journal of Soil Science and Fertilizer
• /
• v.42 no.4
• /
• pp.307-316
• /
• 2009

Soil microbial activity and diversity are affected by organic sources applied to improve soil quality and fluctuate seasonally. We investigated the effects of municipal compost (MC), poultry litter (PL), and cover crops of spring oats and red clover (RC) on soil enzyme activities, and soil bacterial community-level physiological profiling (CLPP) in a Mexico silt loam in North Central Missouri, USA. Temporal patterns of these parameters were observed by periodic five soil sampling from spring to fall over a two year period. MC increased soil dehydrogenase (DH) activity consistently beginning about three months after MC application; fluorescein diacetate (FDA) hydrolytic activity significantly began to increase by the September of the first year but fluctuated during the following period. DH activity responded more directly to the amount or properties of organic residues in soils while FDA hydrolysis and CLPP were generally influenced by composition of organic sources, and enzyme activities and CLPP showed seasonal variation, which depended on organic sources and soil moisture. MC and cover crops may be useful organic sources for enhancing general soil microbial activity and altering soil microbial diversity, respectively. Because microbial activities and diversity are dynamic and subject to seasonal changes, the effects of organic amendments on these parameters should be investigated frequently during a growing season.

• Korean Journal of Animal Reproduction
• /
• v.18 no.1
• /
• pp.55-62
• /
• 1994

This study was carried out to test the effects of fluorescein diacetate(FDA 0${mu}ell$/ml, 0.5${mu}ell$/ml, 5${mu}ell$/ml, 10${mu}ell$/ml or 0${mu}ell$/ml, 5${mu}ell$/ml in PBS) treated before culture on the survival of vitrified mouse morulae in vitrification solution(20% glycerol＋glycerol＋10% ethylene glycol＋30% ficoll＋10% sucrose). The results were summarized as follows; 1. The survival rate of FDA-tested fresh mouse morulae after 24 hours culture was over 96%((P4.8) in the control or treatment groups with various levels of FDA. Because the rate of mouse morulae which developed to hatched blastocysts was higher with the various levels of FDA treatment(67%) than control(50%), it was considered that toxicity of FDA did not affect the survival of mouse morulae. 2. When mouse morulae were FDA-tested in FDA 0(control), 0.5, 5, and 10${mu}ell$/ml treatment after vitrification, the development rate to expanded blastocyst were 66, 82, 64 and 76%, and FDA scores were P4.2(84%), P4.7(94%), P4.2(84%) and P3.9(78%), respectively. There were no significant differences between control and FDA treatments, but there were significant difference between 0.5${mu}ell$/ml)94%) and 10${mu}ell$/ml(78%) treatment(P<0.01). 3. The survival rates of cultured mouse morulae according to FDA-scores(P0=non-fluorescence; P1~P5=according to their fluorescence) after vitrification were P5;92%, P4;67%, P3;42% and P2.P0;0%, respectively. 4. Implantation rates of morulae stage embryos cultured into early blastocysts and implanted into uterine hornes vitrification were 14 and 11% embryos treated control(0${mu}ell$/ml) and FDA 5${mu}ell$/ml and the normal fetus development was 2% embryos for both treatments. Results of this percent study indicated that toxicity of FDA does not affect not only the survival of fresh and vitrified mouse morulae but also the development rate and implantation of fetus after transfer as well. The development rates of mouse morulae with the FDA score of P5, P4 and P3 were 92, 67 and 42%, respectively, it was considered that FDA-test was fit for the judge of survival.

• Environmental Engineering Research
• /
• v.22 no.2
• /
• pp.224-229
• /
• 2017

Zinc oxide nanoparticles (ZnO NPs) have been used as additives in a variety of consumer products. While these particles may enter the environment, only a limited number of studies have investigated the effects of ZnO NPs on soil exoenzymes. Here, we investigate the long-term effects of ZnO NPs at concentrations of 50 and 500 mg/kg on the activities of six soil exoenzymes in planted soils: Dehydrogenase, fluorescein diacetate (FDA) hydrolase, urease, acid phosphatase, arylsulfatase, and ${\beta}-glucosidase$. Significant effects were observed at one or more time points for all enzymes except for FDA hydrolase. These effects included both decreases and increases in enzyme activity. Our results suggest that ZnO NP treatments of 50 and 500 mg/kg can adversely affect soil enzymes, particularly acid phosphatase and urease, and thus, these data may have implications for phosphorous and nitrogen cycles in the soil.