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L. Peperzak & S. Gollasch. A workshop to compare flow cytometers for the rapid counting of phytoplankton in the 2-10 μm and 10-50 μm IMO organism size classes was held in 2013 at the Royal Netherlands Institute of Sea Research (NIOZ) with 29 participants from 9 countries. Intercomparisons were made between five different flow cytometers and with a...
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Fig. 1 OVIZIO’s oLine D HM (left), and the OsOne software (right) showing its interface after cells have been ...
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... foundation of the OVIZIO organism detection system is digital holographic microscopy. Simpler optical configurations of in-line digital holography have frequently been developed for the marine sciences which have inherent problems with insufficient image quality compared to classical light microscopy. Professor Frank Dubois and his team at the Microgravity Research Center (MRC) of the Université Libre de Bruxelles (ULB) in Belgium, developed and patented their technological advancements, particularly, in off-axis digital holographic microscopy (DHM). They are subsequently able to obtain images of quality comparable to those of classical microscopy in a variety of settings by using spatial partial coherent optical sources in their DHM configuration. The continued development of the technology and the wish to set up further environmental applications for the system, led to the creation of a spin-off company, Ovizio Imaging Systems NV/SA (hereafter referred to as OVIZIO). OVIZIO was founded in December 2009 and since its foundation has rapidly grown and progressed in the commercialization of DHM technology with its first commercial Digital Holographic Microscopy (DHM) instrument being released in September 2010: the ‘ oLine ’ along with an easy to use software product ‘OsOne’ (Fig. 1). The oLine is designed to detect, visualize and quantify particles in either static or continuous fluid flow, as required in many environmental applications. The associated OsOne software provides the necessary hologram capturing and storage capabilities but also browsing, analysis and visualization ...
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... workshop was held at NIOZ, Texel, The Netherlands with 29 participants from Belgium, France, Germany, Italy, The Netherlands, Norway, Portugal, United Kingdom and the United States of America (see Annex 1, Agenda and Annex 2, List of Participants and Figure 1). The workshop took the form of a series of plenary sessions followed by demonstration exercises of the organism detection instruments using beads, cultured phytoplankton and a Wadden Sea sample. The Next, workshop the Ballast was Water opened Opportunity at noon on project Monday and 11 its February overall objectives 2013 with were welcoming introduced remarks by Jan from Boon. Prof. He explained Dr. Herman how Ridderinkhof important it and is to Louis avoid Peperzak. species introductions Ridderinkhof and stated how that many NIOZ likes stakeholders to link its are work potentially more with affected. societal A rapid benefits analysis so that of it samples becomes is known important how to the respond tax-payers money quickly was in case spent. of non-compliance The ballast water with issue ballast seems water a perfect management topic to address standards. this. With The this NIOZ he field of research has a long-lasting history regarding flow cytometry, with several decades of expertise. Next, the Ballast Water Opportunity project and its overall objectives were introduced by Jan Boon. He explained how important it is to avoid species introductions and how many stakeholders are potentially affected. A rapid analysis of samples is important to respond quickly in case of non-compliance with ballast water management standards. With this he highlighted the workshop objectives as flow cytometry instruments are candidate technologies for such ...
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... The flow cytometers were tested simultaneously at the Royal Netherlands Institute for Sea Research (NIOZ, Texel, The Netherlands) on 12-14 February 2013 with samples of an increasing complexity: (1) calibration beads, (2) algal monocultures, and (3) a natural Wadden Sea plankton sample, and compared to the microscope counts (Peperzak and Gollasch 2014). The purpose of counting size calibration beads with the flow cytometers was to determine the precision as coefficient of variation (CV) of the bead size, which was measured by FS as well as FBG. ...
The ability to quantify vital aquatic organisms in the 2–50 µm size range was compared between five different flow cytometers and several different microscopes. Counts of calibration beads, algal monocultures of different sizes as well as organisms in a Wadden Sea sample were compared. Flow cytometers and microscopes delivered different bead concentrations. These differences between the instruments became larger for algal monocultures and were even higher for the Wadden Sea sample. It was observed that the concentration differences were significant between flow cytometer and microscope counts, and that this difference increased with the size of the objects counted. Microscope counts were more accurate for larger (50 µm) objects because cytometers struggled with bigger particles that clogged the instruments. Contrary to microscopy, the flow cytometers were capable of accurately enumerating cultured cells in the 2–10 µm size range and cells in the lower size range of the 10–50 µm size class. Flow cytometers were also well-suited to assess low abundance samples due to their ability to process larger volumes than microscopes. The results were used to indicate which tools are suitable for ballast water monitoring: flow cytometry is a suitable technology for an indicative and real time analysis of ballast water samples whilst only microscopy would be robust enough for detailed taxonomical analyses.
... A framework has been proposed for the development and validation of official monitoring tools based on proof-of-concept pilot studies with increasing complexity to evaluate a series of items such as the data quality, physical characteristics, maintenance, costs, ease of use, and the technical expertise that is required . Instrumental analyzers are based on different approaches for obtaining information about size, concentration, and viability of organisms such as direct microscopy observation and enumeration, flow cytometry, and indirect biochemical measurements (Bakalar, 2014;Olsen et al., 2016;Peperzak and Gollasch, 2013;Poulton and Martin, 2010;Stehouwer et al., 2013;van Slooten et al., 2015). This study focuses on the assessment of the FlowCAM™ (Flow Cytometer and Microscope; Fluid Imaging Technologies, ME, USA) as an instrument to be used for systematic ballast water monitoring in the context of the BWMC. ...
Assessing the disinfection of ballast water and its compliance with international standards requires determining the size, viability, and concentration of planktonic organisms. The FlowCAM (Flow Cytometer and Microscope) is an Imaging Flow Cytometry designed to obtain the particle concentration, images, and quantitative morphologic information. The objective in this paper is to establish the basis for transforming the FlowCAM from being a laboratory analyzer into a tool for systematic monitoring of ballast water. The capacity of the FlowCAM was evaluated by analyzing artificial microbeads, phytoplankton monocultures, and real seawater samples. Microbead analyses reported high accuracy and precision in size and concentration measurements. Monoculture analyses showed the effect of disinfection treatments in cell appearance and growth. Low concentration and heterogeneity of particles in real seawater analyses require the comprehensive observation of images by experts. Additionally, some physical characteristics of the device must be improved. The optimization of device configuration enables the quick transferring of files and information between parties involved in ballast water management. FlowCAM may become a feasible technology for this after the device and protocols are adapted.
... The fluorescent product is retained in cells with intact membranes, and the fluorescence intensity is correlated with the metabolic activity. Esterase substrates are commonly used in FCM studies of phytoplankton (Garvey et al. 2007, Steinberg et al. 2011, Gorokhova et al. 2012, Peperzak & Gollasch 2013, Olsen et al. 2015. ...
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.
... Quantification of live bacteria traditionally relies on cultivation methods, which is time-consuming and may give false negatives as several species are uncultivable although viable (Roszak and Colwell, 1987;Staley and Konopka, 1985). Flow cytometry (FCM) has been suggested as a promising method for detailed analysis (International Maritime Organization, 2013;Peperzak and Gollasch, 2013). FCM facilitates rapid detection, enumeration and characterization of organisms in combination with fluorescent dyes, and enables to study populations and communities indirectly (Peperzak and Brussaard, 2011;Shapiro, 2000). ...
This study investigates different UV doses (mJ/cm(2)) and the effect of dark incubation on the survival of the algae Tetraselmis suecica, to simulate ballast water treatment and subsequent transport. Samples were UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of ≥400mJ/cm(2) rendered inactivation after 1day as measured by all analytical methods, and are recommended for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100-200mJ/cm(2)) gave considerable differences of inactivation between experiments and analytical methods. Nevertheless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100mJ/cm(2) and ≤200mJ/cm(2) can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable results demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation when algae are treated with low UV doses.
Disinfection of microbes is of importance to prevent the spread of pathogens and non-indigenous species in the environment. Here we test the applicability of using flow cytometry (FCM) to evaluate inactivation of the phytoplankter Tetraselmis suecica after UV irradiation and labeling with the esterase substrate 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). Non-irradiated and UV irradiated samples were analyzed with the plate count technique and FCM for 24days. The numbers of colony forming units were used as a standard to develop a FCM protocol. Our protocol readily distinguishes live and dead cells, but challenges were encountered when determining whether UV damaged cells are dying or repairable. As damaged cells can represent a risk to aquatic organisms and/or humans, this was taken into account when developing the FCM protocol. In spite of the above mentioned challenges we argue that FCM represents an accurate and rapid method to analyze T. suecica samples.
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