Dual staining with CFDA-AM and SYTOX Blue in flow cytometry analysis of UV-irradiated Tetraselmis suecica to evaluate vitality

After disinfection of ballast water, it is crucial to detect organisms and determine their vitality to assess the performance of the chosen treatment technique. Ultraviolet (UV) irradiation is a treatment technology commonly used for water disinfection. In this study, the phytoplankter Tetraselmis suecica was UV irradiated and subsequently stained with both 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and SYTOX Blue, staining metabolically active and membrane-permeable cells, respectively. This dual staining protocol can be used to assess samples during type approval of UV-based treatment systems. Non-irradiated and UV-irradiated samples were incubated in darkness, to simulate a ballast water transport, after which the vitality and viability T. suecica were monitored regularly over a period of 15 d. Flow cytometry (FCM) analysis separated the cells into 4 FCM populations (=single cells grouped together based on their fluorescence signals) according to differences in esterase activity and membrane integrity. UV-irradiated samples followed a different staining pattern compared to non-irradiated samples, where 1 specific FCM population of cells expressed esterase activity, but at the same time gave signals for disrupted membranes. This is useful as a sign of future death and is interpreted as an ‘early warning’ FCM population. FCM results were also compared to corresponding plate count results, differentiating vital, viable cells from vital, non-viable cells. We argue that dual staining with SYTOX Blue and CFDA-AM facilitates and improves FCM analysis when evaluating the performance of UV-based water treatment systems.publishedVersio

Application of flow cytometry in ballast water analysis - biological aspects

Ballast water may, when discharged, cause the spread of nonindigenous and potentially invasive species. International ballast water treatment regulations have accelerated the development of new methods to detect, enumerate and assess the status of organisms in the water to be discharged. Flow cytometry (FCM) is a powerful technique with a broad range of applications with the possibility for multi-parametric analysis and the potential of combining it with other techniques being two strong advantages. This review will discuss whether FCM is suitable for ballast water analysis according to international ballast water regulations, and sum up the advantages and disadvantages. It will also give an overview of available labeling techniques. Finally, a discussion on the knowledge gaps and future potential for FCM within ballast water analysis is presented.publishedVersio

Preliminary Results From Detection of Microplastics in Liquid Samples Using Flow Cytometry

Microplastics are globally recognized as contaminants in freshwater and marine aquatic systems. To date there is no universally accepted protocol for isolation and quantification of microplastics from aqueous media. Various methodologies exist, many of which are time consuming and have the potential to introduce contaminants into samples, thereby obscuring characterization of the environmental microplastic load. Here, we present first steps in the detection of microplastics in liquid samples, based on their fluorescent staining followed by high throughput analysis and quantification using Flow Cytometry. Using controlled laboratory settings nine polymer types [polystyrene (PS); polyethylene (PE); polyethylene terephthalate (PET/PETE); high density polyethylene (HDPE); low density polyethylene (LDPE); polyvinyl chloride (PVC); polypropylene (PP); nylon (PA); polycarbonate (PC)] were tested for identification and quantification in freshwater. All nine plastic types were stained with 10 μg/mL Nile Red in 10% dimethyl sulfoxide with a 10 min incubation time. The lowest spatial detectable limit for plastic particles was 200 nm. Out of the nine polymer types chosen for the study PS, PE, PET, and PC were well-identified; however, results for other plastic types (PVC, PP, PA, LDPE, and HDPE) were masked to certain extent by Nile Red aggregation and precipitation. The methodology presented here permits identification of a range of particle sizes and types. It represents a significant step in the quantification of microplastics by replacing visual data interpretation with a sensitive and automated method

Dual staining with CFDA-AM and SYTOX Blue in flow cytometry analysis of UV-irradiated Tetraselmis suecica to evaluate vitality

After disinfection of ballast water, it is crucial to detect organisms and determine their vitality to assess the performance of the chosen treatment technique. Ultraviolet (UV) irradiation is a treatment technology commonly used for water disinfection. In this study, the phytoplankter Tetraselmis suecica was UV irradiated and subsequently stained with both 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and SYTOX Blue, staining metabolically active and membrane-permeable cells, respectively. This dual staining protocol can be used to assess samples during type approval of UV-based treatment systems. Non-irradiated and UV-irradiated samples were incubated in darkness, to simulate a ballast water transport, after which the vitality and viability T. suecica were monitored regularly over a period of 15 d. Flow cytometry (FCM) analysis separated the cells into 4 FCM populations (=single cells grouped together based on their fluorescence signals) according to differences in esterase activity and membrane integrity. UV-irradiated samples followed a different staining pattern compared to non-irradiated samples, where 1 specific FCM population of cells expressed esterase activity, but at the same time gave signals for disrupted membranes. This is useful as a sign of future death and is interpreted as an ‘early warning’ FCM population. FCM results were also compared to corresponding plate count results, differentiating vital, viable cells from vital, non-viable cells. We argue that dual staining with SYTOX Blue and CFDA-AM facilitates and improves FCM analysis when evaluating the performance of UV-based water treatment systems.publishedVersio

Effects on inactivation of Tetraselmis suecica following treatment by KBAL: a UV-based ballast water treatment system with an in-line vacuum drop

The transfer of non-native, possibly invasive species in ship’s ballast water is of global concern, and the International Maritime Organization and U.S. Coast Guard have adopted standards to minimize the environmental footprint caused by the maritime industry. In this study, seawater spiked with the phytoplankter Tetraselmis suecica, was treated with Knutsen Ballast Water Treatment Technology (KBAL), combining UV irradiation with an in-line vacuum drop. The test water was subsequently incubated in dark tanks, simulating what happens onboard a ship, where ballast water is treated at intake, stored in dark ballast tanks during the voyage, and then treated at discharge. Our results of the test water treated with KBAL and stored 5 days in the dark showed  10 living cells ml−1 when assessing metabolism. This highlights the challenge UV-based BWTS can encounter when meeting testing regimes assessing different characteristics of life. By comparing the effects caused by KBAL treatment with effects caused by UV irradiation only, we demonstrated that the pressure/vacuum technology seems to improve the disinfection effect. In addition, our investigations point out possible challenges with in situ conditions getting representative ballast water samples

Effects on inactivation of Tetraselmis suecica following treatment by KBAL: a UV-based ballast water treatment system with an in-line vacuum drop

The transfer of non-native, possibly invasive species in ship’s ballast water is of global concern, and the International Maritime Organization and U.S. Coast Guard have adopted standards to minimize the environmental footprint caused by the maritime industry. In this study, seawater spiked with the phytoplankter Tetraselmis suecica, was treated with Knutsen Ballast Water Treatment Technology (KBAL), combining UV irradiation with an in-line vacuum drop. The test water was subsequently incubated in dark tanks, simulating what happens onboard a ship, where ballast water is treated at intake, stored in dark ballast tanks during the voyage, and then treated at discharge. Our results of the test water treated with KBAL and stored 5 days in the dark showed  10 living cells ml−1 when assessing metabolism. This highlights the challenge UV-based BWTS can encounter when meeting testing regimes assessing different characteristics of life. By comparing the effects caused by KBAL treatment with effects caused by UV irradiation only, we demonstrated that the pressure/vacuum technology seems to improve the disinfection effect. In addition, our investigations point out possible challenges with in situ conditions getting representative ballast water samples

Dual staining with CFDA-AM and SYTOX Blue in flow cytometry analysis of UV-irradiated Tetraselmis suecica to evaluate vitality

After disinfection of ballast water, it is crucial to detect organisms and determine their vitality to assess the performance of the chosen treatment technique. Ultraviolet (UV) irradiation is a treatment technology commonly used for water disinfection. In this study, the phytoplankter Tetraselmis suecica was UV irradiated and subsequently stained with both 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and SYTOX Blue, staining metabolically active and membrane-permeable cells, respectively. This dual staining protocol can be used to assess samples during type approval of UV-based treatment systems. Non-irradiated and UV-irradiated samples were incubated in darkness, to simulate a ballast water transport, after which the vitality and viability T. suecica were monitored regularly over a period of 15 d. Flow cytometry (FCM) analysis separated the cells into 4 FCM populations (=single cells grouped together based on their fluorescence signals) according to differences in esterase activity and membrane integrity. UV-irradiated samples followed a different staining pattern compared to non-irradiated samples, where 1 specific FCM population of cells expressed esterase activity, but at the same time gave signals for disrupted membranes. This is useful as a sign of future death and is interpreted as an ‘early warning’ FCM population. FCM results were also compared to corresponding plate count results, differentiating vital, viable cells from vital, non-viable cells. We argue that dual staining with SYTOX Blue and CFDA-AM facilitates and improves FCM analysis when evaluating the performance of UV-based water treatment systems.publishedVersio

Preliminary results from detection of microplastics in liquid samples using flow cytometry

Microplastics are globally recognized as contaminants in freshwater and marine aquatic systems. To date there is no universally accepted protocol for isolation and quantification of microplastics from aqueous media. Various methodologies exist, many of which are time consuming and have the potential to introduce contaminants into samples, thereby obscuring characterization of the environmental microplastic load. Here, we present first steps in the detection of microplastics in liquid samples, based on their fluorescent staining followed by high throughput analysis and quantification using Flow Cytometry. Using controlled laboratory settings nine polymer types [polystyrene (PS); polyethylene (PE); polyethylene terephthalate (PET/PETE); high density polyethylene (HDPE); low density polyethylene (LDPE); polyvinyl chloride (PVC); polypropylene (PP); nylon (PA); polycarbonate (PC)] were tested for identification and quantification in freshwater. All nine plastic types were stained with 10 μg/mL Nile Red in 10% dimethyl sulfoxide with a 10 min incubation time. The lowest spatial detectable limit for plastic particles was 200 nm. Out of the nine polymer types chosen for the study PS, PE, PET, and PC were well-identified; however, results for other plastic types (PVC, PP, PA, LDPE, and HDPE) were masked to certain extent by Nile Red aggregation and precipitation. The methodology presented here permits identification of a range of particle sizes and types. It represents a significant step in the quantification of microplastics by replacing visual data interpretation with a sensitive and automated method