NIOZ Flow Cytometer Workshop. Comparing organism detection instruments in measuring 2-10 μm and 10-50 μm plankton cells

Abstract

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 number of different microscopes. A report summarizes the main results of the measurements that were made.

Full text

NIOZ Flow Cytometer Workshop Report
Comparing organism detection instruments in measuring
2-10 µm and 10-50 µm plankton cells
Louis Peperzak
NIOZ
Stephan Gollasch
GoConsult
July 2014
Royal Netherlands Institute for Sea Research

Workshop Report
NIOZ Flow Cytometer Workshop
Comparing organism detection instruments in measuring
2-10 µm and 10-50 µm plankton cells
Prepared for Interreg IVB Project
Ballast Water Opportunity
Reporting Period 9
(Mar 2013 to Aug 2013)
Louis Peperzak
Royal Netherlands Institute for Sea Research (NIOZ)
Landsdiep 4
1797 SZ Den Hoorn
The Netherlands
Stephan Gollasch
GoConsult
Grosse Brunnenstrasse 61
22763 Hamburg
Germany
4-07-2014
Version 2.0
This report should be quoted as follows:
Peperzak L & S Gollasch (eds) 2013. NIOZ Flow Cytometer Workshop, Comparing organism
detection instruments in measuring 2-10 µm and 10-50 µm plankton cells. Final report,
prepared for Interreg IVB Project Ballast Water Opportunity. 66 pp.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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TABLE OF CONTENTS
1.....INTRODUCTION..........................................................................................................................6
2.....WORKSHOP OBJECTIVES............................................................................................................7
3.....WORKSHOP ATTENDEES AND STRUCTURE..................................................................................7
4.....WORKSHOP DETAILS............................................................................................................10
4.1..DAY 1 SYSTEM INTRODUCTIONS...............................................................................................13
4.1.1. FACSCANTO II.............................................................................................................13
4.1.2. ATTUNE ACOUSTICS FOCUSING......................................................................................14
4.1.3. CYTOSENSE GROUP........................................................................................................16
4.1.4. DYMAPHY PROJECT......................................................................................................17
4.1.5. OVIZIO OLINE & ILINE...................................................................................................17
4.1.6. BD ACCURI, FACSCANTO, FACSVERSE.........................................................................18
4.1.7. MICROSCOPY GSI..........................................................................................................21
4.2..DAY 2: TESTS WITH CALIBRATION BEADS............................................................................22
4.2.1. TRUCOUNT BEADS AND FLOW RATES.......................................................................23
4.2.2. SIZE CALIBRATION BEADS........................................................................................24
4.2.2.1. MICROSCOPE AND FLOWCYTOMETER COUNTS..............................................................25
4.2.2.2. FLOW CYTOMETRY RESULTS.........................................................................................26
4.3..DAY 3: TESTS WITH CULTURED PHYTOPLANKTON AND WADDEN SEA WATER............................32
4.3.1. MICROSCOPY RESULTS..................................................................................................32
4.3.2. FLOW CYTOMETRY RESULTS.........................................................................................34
4.3.3. WADDEN SEA SAMPLE...................................................................................................36
4.3.4. DATA ANALYSIS.......................................................................................................38
4.4..DAY 4 SUMMING UP..................................................................................................................38
4.4.1. EASYCLUS SOFTWARE...................................................................................................38
4.4.2. DISCUSSION OF RESULTS................................................................................................41
OVERALL CONCLUSIONS...................................................................................................................44
ACKNOWLEDGEMENTS.....................................................................................................................45
APPENDIX 1 AGENDA OF THE WORKSHOP......................................................................................... 46

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A
PPENDIX 2 LIST OF PARTICIPANTS..................................................................................................49
APPENDIX 3 PROTOCOLS..................................................................................................................49
APPENDIX 4 SYSTEM DESCRIPTIONS..................................................................................................55
ACOUSTIC FOCUSING TECHNOLOGY OVERVIEW..........................................................................57
WHAT IS ACOUSTIC FOCUSING CYTOMETRY?................................................................................... 57
HYDRODYNAMIC FOCUSING VS. ACOUSTIC FOCUSING......................................................................57
APPENDIX 5 QUESTIONNAIRE BASED DETAILED SYSTEM CHARACTERISTICS.....................................64

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ABSTRACT
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 number of different
microscopes. This report summarizes the main results of the measurements that were made. A
complete dataset is available on request.
Flow cytometers and microscopes delivered varying counts of calibration beads, and the
differences between the different techniques was even larger for phytoplankton cultures and
increased further when a natural sample was counted.
One flow cytometer obtained a maximum flow rate of 0.9 mL/minute and at this rate
approximately 10,000 cells per minute could be counted and sized. Other flow cytometers had
10x lower flow rates. This means that flow cytometry surpasses the speed of microscope
counting by a factor of 100 to 1,000.
The average flow rate precision at high flow rates was good at <10% variation. On the other
hand, at the higher flow rates the precision of forward scatter (size) and green fluorescence (a
potential vitality indicator) was reduced by 5-10% to approximately 25%. It was also found
that at higher flow rates the count of beads was reduced indicating a lower counting precision
at higher fow rates.
Overall, bead counts made by the different instruments, microscopes as well as flow
cytometers, were mostly in the same order or magnitude but did show noticeable differences.
Although the flow cytometer counts and holographic microscope counts were similar for the 2
µm range, microscope counts were clearly higher. Microscopy counts were made at low
magnifications which would make the distinction of beads to other particles present, such as
debris, more difficult and may explain the higher counting rate. It was further noted that the
flow cytometers delivered higher counts when using 44 µm beads compared to the
microscopes, whilst at 50 µm beads it is vice versa. Therefore, it was assumed that the
microscope counts are more accurate for the 50 µm beads as the flow cytometers struggled
with this size and results are a bit closer to the D
3
HM which also does well with larger beads.
Contrary to microscopy, the flow cytometers were very capable of measuring the cultured
algal cells in the 2-10 µm size range. The flow cytometers also managed to count cells in the
lower size range of the 10-50 µm size objects. However, at object sizes of 50 µm most flow
cytometers had counting difficulties as well as sample handling problems because the large
objects clogged the instruments. The differences in absolute concentrations were significant
between the flow cytometer and the microscope counts, increasing with the size of the algae
counted. Therefore, multiple measurements of a sample, at least three replicates, are needed to
establish a confidence interval that enables the conclusion of the average cell concentration.
The workshop participants noted the limitations, but agreed that flow cytometry is capable of
rapidly counting organisms in the 2-10 µm and almost the full range of 10-50 µm size objects.

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However, there is a need for more microscope-flow cytometer comparative counting and
sizing analyses of ballast water samples because the viability of the organisms also needs to
be assessed. Therefore comparisons between microscopy and flow cytometry using vitality
stains need to be performed, which was not done during this exercise.
In general, careful validation of both flow cytometric and microscopic methods to assess the
concentration of vital organisms in ballast water should be carried out, preferably by
comparing several techniques simultaneously and by using a variety of samples that have an
increasing complexity.

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1. INTRODUCTION
The IMO Ballast Water Management Convention (BWMC) indicates in its Regulation D-2,
i.e., the Ballast Water Performance Standard, maximum allowable living organism
concentrations in ballast water to be discharged:
Regulation D-2 Ballast Water Performance Standard (D-2 standard)
1 Ships conducting Ballast Water Management in accordance with this
regulation shall discharge less than 10 viable organisms per cubic
meter greater than or equal to 50 micrometres in minimum dimension
and less than 10 viable organisms per millilitre less than 50
micrometres in minimum dimension and greater than or equal to 10
micrometres in minimum dimension; and discharge of the indicator
microbes shall not exceed the specified concentrations described in
paragraph 2.
2 Indicator microbes, as a human health standard, shall include:
.1 Toxicogenic Vibrio cholerae (O1 and O139) with less than 1
colony forming unit (cfu) per 100 millilitres or less than 1 cfu per
1 gram (wet weight) zooplankton samples ;
.2 Escherichia coli less than 250 cfu per 100 millilitres;
.3 Intestinal Enterococci less than 100 cfu per 100 millilitres.
As a result, reliable organism detection methods need to be developed to effectively and
uniformly prove compliance (or non-compliance) with this standard for each organism size
category. Organism detection technologies for two compliance control tests with the D-2
standard are needed, i.e. an “indicative” analysis to detect gross exceedance, and a “detailed”,
in-depth analysis.
This report addresses the organism detection options for cells in the size class 2-10 µm and
10-50 µm. The workshop report addresses the following BWO deliverables of WP2, WP3 and
WP4:
 D3-9 Implementation of procedure and evaluation of Ballast Water Protocol BWP:
Demonstration center and workshop on site (in collaboration with WP4),
 D4-7 Demonstration of size discrimination in flow cytometry technology, feasibility
and requirements for compliance control (in collaboration with WP2),
 D4-10 Feasibility and limitations of flow cytometry detection of larger organisms,
opportunities for future compliance control (in collaboration with WP2).

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2. WORKSHOP OBJECTIVES
The overall objectives of the workshop were:
 To compare newly developed as well as commercially available flow cytometry
instruments for the rapid counting of low abundance aquatic organisms in the 2-10
µm and 10-50 µm size classes (minimum dimension).
Flow cytometers are being used for decades to count eukaryotic cells. In the aquatic sciences
these instruments provide data on the abundance of plankton with a speed that is unparalleled
by microscopy. In the nearby future, ballast water that ships discharge, needs to be treated in
order to reduce the risk of spreading aquatic organisms. Ballast water treatment systems
should reduce the concentration of viable plankton cells in the 10-50 µm size class (in
minimum dimension) to <10 cells per ml (see D-2 standard above). However, the
phytoplankton organisms <10 µm in minimum dimension are not addressed by the D-2
standard. A similar low concentration standard may be chosen for the cells in the 2-10 µm
size range.
The demonstrations and practical exercises during the workshop will answer the question
which cytometers are presently capable of counting 2-10 µm and 10-50 µm cells accurately,
precisely and rapidly.
3. WORKSHOP ATTENDEES AND STRUCTURE
The workshop was organized by the NIOZ ballast water team, namely Louis Peperzak,
Marieke Vloemans, Anna Noordeloos, Josje Snoek, Eveline Garritsen, Alex Blin, Eva
Immler, Dennis Mosk and the organizing committee was assisted by Stephan Gollasch,
GoConsult, Hamburg, Germany.
In preparing the workshop, scientific institutes, companies that develop or sell flow
cytometers and other interested parties were invited to participate with a working instrument.
It was expected that all participants were willing to share their results. Flow cytometers are
common instruments for these tasks but also operators of other organism detection systems
which, for instance, use holography or microscope lenses for particle imaging, were
contacted.

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The 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.
Figure 1. Group photo of workshop participants, taken Monday 11
th
February 2013.
The workshop was opened at noon on Monday 11 February 2013 with welcoming remarks
from Prof. Dr. Herman Ridderinkhof and Louis Peperzak. Ridderinkhof stated that NIOZ
likes to link its work more with societal benefits so that it becomes known how the tax-payers
money was spent. The ballast water issue seems a perfect topic to address this. The NIOZ
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

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highlighted the workshop objectives as flow cytometry instruments are candidate technologies
for such analysis.
After this introduction, the workshop objectives and Agenda were presented by Louis
Peperzak.
Apologies were received from Harry Nelson (Fluid Imaging, USA), who was unable to attend
because of a snow storm in the USA which affected air travel; other participants would arrive
with a slight delay on Tuesday 12 February 2013. Peperzak discussed with the participants
that standard protocols on how to use the instruments are essential to later enable accurate
result comparison (see Appendix 3). The protocols were evaluated and adjusted to fit
everybody’s needs. For instance, a discussion was needed on how to calibrate the instruments
using standard solutions of beads in terms of the volumes and concentrations of beads and the
number of replicates needed.
When the experiments were running, all flow cytometry system operators were asked to first
analyze the data with their own software. Then, a “standard” data analysis software (FCS
Express) would be used, and thirdly a new software program (EasyClus) to automatically
analyze all flow cytometry results. For this purpose, Peperzak collected all data for
subsequent analysis. He highlighted that all systems likely have strengths and weaknesses and
that the workshop outcome was a comparison of systems rather than a competition.
After the participants introduced themselves by briefly mentioning their field of work and
affiliation, the presentation of systems began (see below).
On Tuesday 12 February 2013 the practical work started. The second day’s agenda focused
on hands-on tests of different flow cytometry systems and the holographic microscope with
size calibration beads.
This was followed the next day on Wednesday 13 February 2013 with cultured
phytoplankton.
To challenge the systems, on Thursday 14 February 2013 a sample from the North Sea,
taken earlier on from the NIOZ jetty (Wadden Sea sample), which contained phytoplankton,
smaller zooplankton, detritus and sediments, was used for testing.

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4. WORKSHOP DETAILS
The workshop addressed plankton cells in the 10-50 µm size class (in minimum dimension)
and organisms 2-10 µm in minimum dimension which were not addressed in the D-2
standard. This was done with several mono-algal cultures of the three size classes: 2, 10 and
50 µm in minimum dimension. The species Micromonas pusilla, Prorocentrum minimum and
Prorocentrum belizeanum that widely differ in cell size (2 to 50 µm) were used and these
cultures were provided in different cell concentrations to examine accuracy and precision of
the different instruments (Figure 2). Thereafter, a natural Wadden Sea sample containing
different sizes and shapes of organisms was tested (Figure 2). As the natural organism
concentration is low in winter, this sample was collected already a week before the workshop
started and was cultured to enhance the cell concentrations.
Figure 2. Preparation of algal cell cultures.

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During the workshop, flow rates and particle size limits were also measured using size
calibration beads provided by NIOZ.
Further, a comparison of counts by the systems (flow cytometers and digital holographic
microscope) and the traditional microscopes was done. The traditional microscope counts
were performed by August Tobiesen (NIVA) (Figure 3) and Euan Reavie (GSI) (Figure 4)
using a magnification up to 400x. Cell concentrations were determined by standard bright
field and fluorescence (as a proxy for a vitality probe) microscopy for reference purposes.
Figure 3. Preparing for microscope counts.

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Figure 4. Microscope counts using bright field microscopy.
As the FluidImaging representative was unable to join, the FlowCAM method could not be
considered in greater detail.
To challenge the flow cytometers, two flow rates, a maximum and one lower rate were
applied to determine sample analysis time and the effect of flow rate on accuracy and
precision. Firstly, a ‘general’ species (Phaeocystis globosa) was measured in a
commissioning run, followed by three species at two concentrations: one high concentration
(resembling a test water or control sample) and one low concentration (resembling a treated
ballast water sample).
Finally, planktonic organisms were counted in natural Wadden Sea samples. For flow
cytometrs, which are triggered to count on the red fluorescence of phytoplankton, this may
lead to un underestimation of the total (phyto- and non-phytoplankton) concentration of
organisms.
All data were shared and discussed amongst the participants and a scientific publication of the
results was planned.
Viability assessments were conducted as much as possible and the instrument developers
were interviewed individually by Gollasch to describe their systems and assess performance
limitations for Port State Control of ballast water management standards (Appendix 5).
Possibly a future workshop will further address the viability aspect.

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4.1. DAY 1 SYSTEM INTRODUCTIONS
The presentations given highlighted the characteristics, abilities and performance strengths
and weaknesses of individual flow cytometers. System descriptions provided are summarized
in Appendix 4 and more detailed system characteristics, based upon a questionnaire, are
shown in Appendices 5.
The following sections (4.1.1. – 4.1.7) summarize the presentations given on Monday
afternoon, 11 February 2013.
At the end of day 1, Louis Peperzak introduced the working principles for the next days and
explained the protocols (see Appendix 3). Minor adjustments were made to meet the
participant’s needs.
4.1.1. FACSCANTO II
Presentation given by Louis Peperzak

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Fig. 5. The FACSCanto II system in operation during the workshop.
4.1.2. ATTUNE ACOUSTICS FOCUSING
Presentation given by Sara Monteiro, Pieter De Vre
Most conventional flow cytometers use hydrodynamic focusing by using a sheath fluid. Uses
acoustic waves which do not damage cells to concentrate cells in a fluid. Cells become
exposed to piezoelectric ultrasonic devise for acoustic focusing and a capillary which results
in cells being sorted in the capillary center. Because no sheath fluid needed, fluid speed to be
manipulated to address different cell concentrations. Results in better precision compared to
conventional flow cytometry. No sample concentration needed as fluid speed can be
manipulated.
Presentation given by Sandra Schöttner
Project “Ballast FLOW”, real time monitoring with flow cytometry. KBAL BWTS (Shipping
company Knutsen Ballast Water Treatment System consisting of UV and rapid pressure drop,
tested at NIVA). Next step was to identify methods for compliance control with BWM
standards. Uses Attune cytometer because acoustic focusing, higher sensitivity and flow rate
(up to 1 ml per minute), violet and blue lasers as wider spectrum be analysed, very compact
instrument, portable (big as a kitchen micro wave).
Aud Larsen
Works with flow cytometry, flow sorting, ballast water samples, live/dead analysis.
Ole-Kristian Hess-Erga
Main focus on bacteria including livee/dead staining.

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Fig. 6. The Attune systems (At) in operation during the workshop.

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4.1.3. CYTOSENSE GROUP
Presentation given by George Dubelaar
CytoBuoy, optical particle analysis based upon images taken, uses sheath fluid, quantitative
laser excited fluorescence emission profiles to identify different organism types, works
onboard, measures, shape, length, chloroplast, Chl a.
Involved in Bio-monitored BWTS project.

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Fig. 7. The Cytobuoy systems (CS) in operation during the workshop.
4.1.4. DYMAPHY PROJECT
Presented by Felipe Artigas
Interreg project to combine different technologies to assess water quality based on
phytoplankton analysis. In-situ real-time measurements, comparison of systems performance
in real world scenario, uses CytoBuoy bench-top system, another approach is carbon per cell
measurements by flow cytometry, application of DNA staining in natural sample, worked
with Sytox green, works on species grouping/analysis by shape of signal detected, multi-
spectral fluorometers, applies optical characterization, including satellite imaging, ferry box,
etc.
4.1.5. OVIZIO OLINE & ILINE
Presentation given by Joël Henneghien and Eva-Maria Zetsche
OVIZIO, a spin-off company started in 2009, presented their 4D quantitative imaging
platforms based on digital holographic microscopy. Holography provides light intensity
images but also phase images – giving a so-called ‘holographic fingerprint’. The technology
has undergone several developments with the recent move (Autumn 2012) to differential
digital holographic microscopy. This allows for a much smaller more robust instrument, no
beam alignment is necessary and any transparent sample container material may be used. The
oLine and iLine instruments provide post acquisition refocusing capabilities (up to 100x
larger depth of focus compared to a light microscope) as well as 3D phase image
visualization. The counting of cells is an implemented feature of the specialized software
(OsOne), along with using differences in optical density to differentiate live from dead cells.
This allows for label-free non-invasive fast imaging of samples without the need for
calibration with beads. This has not been fully developed yet for algal cells and so still

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requires further calibration and validation. Future developments will include a fluorescence
module which will be more suited to follow current standard protocols of fluorescence
staining (FDA) for viability determination of algal cells.
Fig. 8. The OVIZIO oLine system in operation during the workshop.
4.1.6. BD ACCURI, FACSCANTO, FACSVERSE
Presentation given by “Kristien Raschert”, Albert Mosselaar, and Brian Maurer (works with
Nick Welschmeyer)
Research and clinical flow cytometry.
BD FACSVerse: absolute counting without beads, quicker time to result compared to other
flow cytometers, different lasers, high sensitivity, vacuum based, i.e. sucks up the sample,
compact design (not as small as Accuri), sample flow to manipulate, no flow speed calibration

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needed – works with heat and temperature detection before and after heater to calibrate flow,
remote support.
BD Accuri: portable, easy to use, two lasers, peristaltic pump driven, flow rate adjustable core
diameter adjustable, up to 10.000 events per second, selectable laser module, 8 peak beads for
system calibration/validation.
Fig. 9. The FACSVerse (FV) system in operation during the workshop.

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Fig. 10. The Accuri (C6) system in operation during the workshop.
Counting Live Organisms in Ballast Water Using Flow Cytometry
Presentation given by Brian Maurer
Nick Welschmeyer (Principal Investigator)
Moss Landing Marine Laboratories, Golden Bear Facility
The following issues pertaining to the use of flow cytometry to enumerate live organisms in
ballast water are addressed: 1) viability over time in stored samples, 2) the comparability of
flow cytometry and epifluorescence microscopy to enumerate live cells, and 3) the usefulness

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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of the flow cytometer's forward scatter signal as an indicator of cell size. Concentrated
zooplankton samples show rapid die-off in the hours following sample collection, while
untreated whole water samples (phytoplankton), if stored at in situ temperature and in the
dark, do not exhibit die-off over a 4 day period when counted by flow cytometry or
epifluorescence microscopy. Flow cyometry and epifluorescence microscopy generate similar
live counts for organisms 10-50µm (sample collected from Moss Landing harbor, California,
USA). Many bead standards of known size were used to build a regression between bead
volume (µm3) and forward scatter, which can be used to convert the forward scatter from any
particle to particle volume. This relationship was tested using algal cultures. The cell volume
of each culture was measured by Coulter Counter and by flow cytometry; the two methods
generate very similar estimates of cell volume.
4.1.7. MICROSCOPY GSI
Presentation given by Euan Reavie, GSI
Samples are concentrated by vacuum pump with 7 µm mesh (diagonal dimension), FDA
staining (live stain), problem is with colony forming phytoplankton, multicellular occurs very
often in freshwater, therefore no flow cytometry used, bright field and fluorescence
microscopy.

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4.2. DAY 2: TESTS WITH CALIBRATION BEADS
Day 2 started with additional instructions regarding the working protocols by Louis Peperzak.
Thereafter all participants were given samples with different sized calibration beads of a
known concentration (Figure 11). The analysis of these samples was performed by the system
experts. For reasons of simplicity we only present the total average calculated numbers for all
beads used in the experiments.
In parallel Gollasch interviewed the expert teams (Figure 12) to gather additional system
details, which are shown in the questionnaires attached as Appendix 5.
Figure 11. Preparation of bead suspensions.

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Figure 12. Interviews with flow cytometer expert teams.
4.2.1. TRUCOUNT BEADS AND FLOW RATES
In ballast water samples with low plankton concentrations a fast analysis requires a high flow
rate.
To be able to calculate actual organism concentrations from the number of organisms
counted, the flow rate i.e. the volume of sample that was analysed in a certain amount of time,
needs to be known. Flow rates can be measured in different ways but a simple and reliable
method is to make use of a known number of beads that is suspended in a known volume of
water. For this purpose TruCount
®
beads (Becton Dickinson, Franklin Lakes, USA) were
used in the flow cytometry workshop.
TruCount
®
beads with a concentration of 25,099 beads/mL were distributed in order to
calibrate the flow rate of the systems. An independent count made by holographic microscopy
(oLine D
3
HM) resulted in 24,901 beads/mL, i.e. within 1% of the calibration value.
The flow rates of the flow cytometers, i.e. the volume of sample being analysed per minute,
ranged from ca. 9 µL/min at low settings to 869 µL/min at high settings (Table 1). At a low
flow rate setting only one instrument had a relatively large variation (>25%), expressed as the

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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relative 95% confidence interval of the mean flow rate. At high settings most instruments
attained a good flow rate precision (<10%; Table 1).
Table 1. Mean flow rates in µL/min of the flow cytometers at low and high settings ± relative
precision. The precision was calculated by dividing the 95% confidence interval of the flow
rate by the mean. The mean flow rates are averages of 4 measurements (n = 4, unless
otherwise indicated) performed on two different days.
Flowcytometer
Flowrate
Low(µL/min)
Flowrate
High(µL/min) n
FACSCanto 10±26 107±6 4
CytoSense1 77±12 254±10 4
CytoSense2 77±33 276±0 2
AccuriC6 9±7 84±12 4
FACSVerse 16±5 103±3 4
Attune1 22±13 869±8 3
Attune2 24±5 843±8 4
Conclusions:
1. At the high settings the maximum flow rates of the instruments tested ranged nearly
hundredfold: from 9 to 900 µL/minute. In other words, the analysis time of 1 mL
sample is 1-100 minutes depending on the instrument.
2. In general the average precision of the flow rates at the high settings was <10%.
4.2.2. SIZE CALIBRATION BEADS
In ballast water research the concentration of viable organisms in certain size ranges needs to
be established. In the present workshop the emphasis is not only on the 10-50 µm size fraction
(as required by IMO) but also on the neglected 2-10 µm fraction.
Size calibration beads of different sizes (2, 10 and 51 µm) at an approximate concentration
were handed out to the system operators for the measurement of forward scatter (size), green
fluorescence and bead concentration. The 10 µm beads had a nominal value of 9.9 µm. For
flow cytometers not able to measure the large 51 µm beads a substitute of 44 µm was
available.
Unfortunately, the CytoSense 2 had a recently adapted configuration that the operators were
unfamiliar with, leading to inaccurate measurements. In addition, the 552 nm (green) laser
was not suited for measuring the size calibration beads: these are normally excited by a blue
laser. Furthermore, the operators made some errors in following the workshop protocols.
Therefore the operator M. Rijkeboer decided to retract the data from the data analysis (e-mail

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to Louis Peperzak 25-2-2013). Therefore, the CytoSense 2 data or not part of the ensuing
analyses.
For the oLine D
3
HM microscope the automated software detection and sizing algorithm was
not capable of accurately sizing the calibration beads due to their refractive and non-
transparent properties. Manual sizing using the optical density profiles was therefore carried
out. The system was, however, able to provide the concentration of the beads (Table 2). An
objective of 20x was used in the system set-up for this workshop.
Table 2. Mean and 95% confidence intervals in µm of three size calibration beads measured
with the oLine D
3
HM holographic microscope. The concentration was estimated as
beads/mL.
Size
[µm]
Mean
[µm] 95%c.i.
beads/mL
measured
2 2.6 0.4 8,833
10 9.9 1.5 21,691
51 48.0 4.9 17,952
Conclusion:
The oLine D
3
HM measurements show that the size calibration bead concentrations ranged
from 10,000 to 20,000/mL. According to the protocol, the intended concentrations were
10,000 per mL. The mean size of the smallest (2 µm) beads was higher than reported by the
manufacturer but the mean sizes of the other beads were not significantly different from
expected.
4.2.2.1. MICROSCOPE AND FLOWCYTOMETER COUNTS
Results from the three methods (brightfield and epifluorescence microscopy, flow cytometry
and holographic microscopy) used were averaged and compared. There were some substantial
differences between the three methods in counting beads (Table 3). The relatively low 51 µm
bead count by the flow cytometers is probably related to the difficulty some of these
instruments had in processing such large particles. Despite this, the Kruskal-Wallis one-way
ANOVA test statistic was 2.3 (P > 0.05); and the Dwass-Steel-Chritchlow-Fligner Test for
All Pairwise Comparisons did not reveal significant differences between the three methods.
Table 3. Averaged concentrations in beads/mL of the size calibration beads counted by three
different methods: the oLine D
3
HM holographic microscope, two microscopes and five flow
cytometers (at low and high flow rates). 44 µm beads were not counted with the D
3
HM. For

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reasons of simplicity we only present the total average calculated numbers for all four beads
here.
Beadsize(µm)
Brightfieldand
fluorescence
microscopes
Flow
cytometers
Holographic
microscope
2 11,500 7,386 8,833
10 16,275 15,616 21,691
44 8,563 13,962‐
51 14,347 8,784 17,952
Conclusion:
Overall, counts made by the different instruments, microscopes as well as flow cytometers,
were mostly in the same order or magnitude but did show noticeable differences. Although
the flow cytometer counts and holographic microscope counts were similar for the 2 µm
beads, microscope counts were clearly higher. Microscope counts were made at low
magnifications which would make the distinction of beads to other particles present, such as
debris, more difficult and may explain the higher counts. It was further noted that the flow
cytometers delivered higher counts when using 44 µm beads compared to the microscopes,
whilst at 50 µm beads it is vice versa. Therefore, it was assumed that the microscope counts
are more accurate for the 50 µm beads as the flow cytometers struggled with this size and
results are a bit closer to the D
3
HM which also does well with larger beads.
4.2.2.2. FLOW CYTOMETRY RESULTS
In ballast water research, forward scatter is important for determining the size of the
organisms and green fluorescence is often used as a vitality marker, i.e. are the organisms
dead or alive after applying a vital stain such as fluorescein diacetate (FDA) or SYTOX
®
Green Stain?
The objective to count size calibration beads was to measure the precision (% coefficient of
variation (CV)) of the size (forward scatter or FS) and fluorescence (green or FBG) at two
flow rates. In addition, the concentrations of beads between the two flow rates can be
compared (Figure 13).

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Figure 13. Size calibration bead concentrations measured by flow cytometry at two different
flow rates. The dashed line is the 1:1 ratio. The solid line is the linear regression line.
The comparison between the low and high flow rate indicates that at high flow rates relatively
fewer size calibration beads were counted. The slope (0.84, Figure 13) is significantly
different from 1 (t-test, degree of freedom (df) = 16) indicating a significant underestimation.
No further analyses were performed to investigate the cause of this phenomenon (e.g.
coincidence).

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Figure 14. Forward Scatter (FS) precision as coefficient of variation (CV) of size calibration
beads measured by flow cytometry at two different flow rates. The dashed line is the 1:1 ratio.
The solid line is the linear regression line.
The Forward Scatter (FS) precision is generally below 25% although some values exceeded
50% at a high flow rate (Figure 14). The comparison between the low and high flow rates
indicates that at higher flow rates the coefficient of variation increased, hence precision
decreased (Figure 14). The slope (1.55) is significantly different from 1 (t-test, df = 18)
indicating a significant increase of the %CV.

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Figure 15. Fluorescence from Blue (excitation) to Green (emission) or FBG precision as
coefficient of variation (CV) of size calibration beads measured by flow cytometry at two
different flow rates. The dashed line is the 1:1 ratio. The solid line is the linear regression line.
The FBG precision is generally below 25% although some values up to or exceeding 50%
were measured (Figure 15). The comparison of FBG precision between the low and high flow
rates indicates that at higher flow rates the coefficient of variation is systematically 8%
higher; hence precision is 8% lower (Figure 15).
Except for the difference in slopes between FS and FBG precisions at low and high flow
rates, the averages for all beads together were not much different between FS and FBG and
between low and high flow rates (Table 4).

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Table 4. Precision of Forward Scatter and Green Fluorescence expressed as % coefficient of
variation. The precision were measured for differently sized calibration beads at low and high
flow rates. For reasons of simplicity we only present the total average calculated numbers for
all four beads here.
 ForwardScatter GreenFluorescence
Beadsize(µm)
%CVatlow
flowrate
%CVathigh
flowrate
%CVatlow
flowrate
%CVathigh
flowrate
2 13 23 11 27
10 6 13 5 8
44 26 35 25 34
51 23 35 26 27
totalaverage: 17 27 17 24
In general, the precision of both FS and FBG at higher flow rates decreased (Table 4).
To examine how the flow rate influenced precision of both FS and FBG the %CV data were
plotted against the average flow rates from Table 1. The resulting Figure 16 shows that a
considerable variation in %CV was encountered at all flow rates.
Figure 16. Precision of Forward Scatter (FS) and Green Fluorescence (FBG) of four
differently sized calibration beads as a function of flow rate.
0
25
50
75
0 200 400 600 800 1000
CV(%)
Flowrate(µL/minute)
CoefficientofVariationandflowrate

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Figure 17. Precision of Forward Scatter (FS) and Green Fluorescence (FBG) measured at
different flow rates as a function of bead size.
To examine how the bead size influenced precision of both FS and FBG the %CV data were
plotted against the bead sizes as reported by the manufacturer. The resulting Figure 17 shows
that a considerable variation in %CV was encountered at all bead sizes, except perhaps for the
10 µm beads that had an average precision of 8% (Table 4).
Conclusions:
1. The comparison between the low and high flow rates indicates that at high flow rates
relatively fewer size calibration beads were counted.
2. The precision of measurement of the Forward Scatter and the Green Fluorescence of
size calibration beads was comparable (15-25%).
3. At higher flow rates a 5-10% lower precision in the measurement of the Forward
Scatter and the Green Fluorescence was attained (ca. 25%) than at lower flow rates
(ca. 15%).
4. The 10 µm size calibration beads had the highest precision (<10%), the 2, 44 and 51
µm beads the lowest precision (20-30%) in both Forward Scatter and the Green
Fluorescence.
0
25
50
75
0 102030405060
CV(%)
Beadsize(µm)
CoefficientofVariationandbeadsize

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4.3. DAY 3: TESTS WITH CULTURED PHYTOPLANKTON AND
WADDEN SEA WATER
The next step in the workshop, after calibrating the instruments and a first basic comparison
with calibration beads, was counting phytoplankton cells in unicellular cultures. This step is
an intermediate between the analysis of beads and the analysis of real world samples that
contain a plethora of differently sized, fluorescent and non-fluorescent particles. Because the
cultures were diluted with fresh medium two days prior to the workshop, and because the
incubated Wadden Sea winter sample was assumed to contain a surplus of nutrients (see
protocols), it was assumed that the organisms in these samples were predominantly viable, i.e.
able to reproduce and hence vital (as would be measured by a vital stain such as FDA).
The participants were provided with diluted phytoplankton cultures with cells of different
sizes (Table 5). Before measuring these three species, a culture of Phaeocystis globosa (ca. 4
µm) was handed out in order to perform a commissioning run.
The comparison between the flow cytometers, and the (holographic) microscope techniques
was firstly based on the cell concentration estimates. NIOZ tried to dilute the cultures so that
relevant IMO concentrations were obtained, i.e. 10 cells/mL (D-2 Ballast Water Performance
Standard) and 1000 cells/ml (G8 Guideline for ballast water treatment system challenge water
in land-based tests). Secondly, Forward Scatter and the Fluorescence from Blue to Red
(chlorophyll-a) or FBR precision data in the form of %CVs were to be calculated and
compared.
Table 5. The three phytoplankton cultures and their respective sizes used by three different
methods: the oLine D
3
HM holographic microscope, two microscopes and five flow
cytometers (at low and high flow rates).
Name Abbreviation Approximatesize(µm)
Micromonaspusilla Mp 2
Prorocentrumminimum Pmin 10
Prorocentrumbelizeanum Pbel 50
Finally, at the end of the day a Wadden Sea (real world) sample was analyzed.
4.3.1. MICROSCOPY RESULTS
For the 2 µm alga (M. pusilla) the microscopists reported that this size is below the limit to
enable accurate counts with the microscopy settings (400x magnification) used, either by
Sedgwick Rafter chamber, Bürker haemocytometer or the oLine D
3
HM. In fact, the D
3
HM
gave an overestimation because the black & white camera had difficulty distinguishing
between cells and other small particles. Therefore, only one estimate for this species was
made (Table 6).

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The 10 µm high abundance P. minimum had a nearly identical concentrations when counted
by brightfield and holographic microscopy (Table 6).
A good agreement was also achieved for the 50 µm P. belizeanum between fluorescence and
holographic microscopy (Table 6). The counts from brightfield microscopy were higher.
Table 6. Average concentrations (duplicate counts) in cells/mL of the three cultures with low
and high concentrations of organisms. Blanks mean that no counts were made. (Mp =
Micromonas pusilla, Pmin = Prorocentrum minimum, Pbel = Prorocentrum belizeanum).
  Microscopes
Species Concentration Brightfield Fluorescence Holographic
   
Mp high 11,023
Pmin low12
Pmin high 3,963 3,934
Pbel low121
Pbel high 1,174 715 814
Conclusions:
1. Small cells such as M. pusilla (2 µm) are very difficult to count by bright field
microscopy. Especially holographic microscopy overestimates by far total numbers as
specks of dust or debris are difficult to distinguish at a magnification of 20x and were
thus not counted.
2. For the concentration of cells ≥ 10 µm the different microscope techniques were in
relative agreement.

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4.3.2. FLOW CYTOMETRY RESULTS
Table 7. Concentrations of M. pusilla (Mp), P. minimum (Pmin) and P. belizeanum (Pbel) in
cells/mL at ‘low’ and ‘high’ concentration cultures, counted by six flow cytometers at high
and low flow rates respectively. The numbers are means of duplicate measurements. The
overall average is the mean of all flow cytometers with ± 95% confidence intervals in
absolute (no. of cells/mL) or relative numbers (%). The microscope counts are averages of the
concentrations in Table 6. (At 1 and 2 = Attune systems, C6 = Accuri C6, Ca = FACSCanto,
CS1 = Cytobuoy system, FV = FACSVerse).
The concentrations of cells in the three cultures were not as originally planned (10 and 1000
cells/mL). The M. pusilla concentrations were 10x higher and the P. minimum concentrations
were 3x higher. Only the P. belizeanum concentrations resembled, on average, the planned
concentrations (Table 7).
The differences between the flow cytometer and the microscope counts were relatively small
for M. pusilla: despite the difficulty in counting M. pusilla with the microscope, there was
excellent agreement with the flow cytometer count. The differences between microscope and
flow cytometer were significant for P. minimum at both low and high concentrations and the
highest difference was found for the largest algae (Pbel).
The smallest 95% confidence interval (14%) for flow cytometry counts was achieved for the
small M. pusilla cells at the highest concentration. The average confidence interval of all six
estimates was 55%. The highest confidence interval was for the large P. belizeanum (145%)
at the high concentration due to the widely varying estimates of the different flow cytometers.
The large signal made it difficult to count accurately and some operators reported clogging of
the instrument. This might be related to the formation of large amounts of extracellular carbon
by the P. belizeanum cells that make the cells sticky. Apparently, clogging was less
problematic at the low P. belizeanum concentration, in diluted sample, where the confidence
interval was reduced to 59%.
A practical example of the interpretation of such flow cytometry data is provided by P.
belizeanum at the low concentration. According to instruments At1 and At2 this sample
Spe cies
Concentration low high low hi gh l ow high
At1 40 9,465 30 2,625 10 515
At2 55 10,470 40 2,505 10 80
C6 105 11,822 28 3,620 9 1,323
Ca 116 12,770 notcalculated 2,914 1 56
CS1 143 11,100 43 2,670 8 266
FV notcalculated notcalculated notcalculated notcalculated notcalculated notcalculated
Overallaverage 92±54 11,125±1,570 35±12 2,867±555 8±5 448±650
Overallaverage 92±58% 11,125±14% 35±34% 2,867±19% 8±59% 448±145%
M icroscope notcounted 11,023 12 3,821 notcounted 901
Mp Pmin Pbel

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would fail the D-2 standard (measurement result was 10 cells/mL and D-2 requires less than
10 cells/ml) while the C6, Ca and CS1 would register a pass (<10 cells/mL; Table 7). Taking
the data of all five instruments the average 8 ± 5 cells/mL, i.e. with a 95% confidence the real
cell concentration in the sample was 3 – 13 cells/mL. This result also fails the D-2 standard
because this concentration is not significantly less than 10 cells/mL.
Conclusions:
1. The differences in absolute concentrations were significant between the flow
cytometer and the microscope counts and were increasing with the size of the algae
counted.
2. Small and intermediately sized cells (2 and 10 µm) are easily counted with flow
cytometry; cells of 50 µm are at the border of what is practically feasible.
3. Counting precision expressed as the 95% confidence interval increased from 14% at
high cell concentrations (>10,000 cells/mL) to 59% at low concentrations (<100
cells/mL) although cell-specific characteristics may interfere with counting and
decrease the counting precision.
4. Multiple measurements of a sample, at least three, are needed to establish a confidence
interval that enables the conclusion that the average cell concentration is below a
standard limit.

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4.3.3. WADDEN SEA SAMPLE
The Wadden Sea sample, incubated prior to the workshop in the NIOZ climate room to
enhance the concentration of viable phytoplankton (see protocols) had to be diluted 10x in
order to achieve ca. 1000 cells/mL to be comparable to the high cell concentration that was
aimed at with the cultures.
This “real-world” sample was examined using bright field microscopy and flow cytometry
and the phytoplankton in both the 2-10 and the 10-50 µm size classes were counted. For the
flow cytometers both low and high flow rates were used.
A single cell is the reproductive unit that can start a new community and therefore is also the
unit that has to be counted in ballast water research. In other words, a single viable cell in
discharged ballast water can start an invasion. Therefore, according to the IMO G2 Guidelines
for Ballast Water Sampling, the single cells with a minimum dimension >10 µm that make up
a colony should be counted. This may be difficult to achieve by a flow cytometer that
probably will count a colony as a single particle of a larger size than that of the individual
cells.
For microscopy the sample was Lugol-fixed and also assuming most intact cells in the sample
were living at the point of preservation. The two microscopists made a differentiation of the
phytoplankton entities that were counted. One microscopist counted “entities” (i.e. a colony
of multiple cells was counted as one object) which is comparable to flow cytometer counting,
while the second microscopist counted the single cells (also in a colony).
Table 8. Concentrations of phytoplankton in the 2-10 and 10-50 µm size classes in cells/mL at
‘low’ and ‘high’ concentration cultures, counted by six flow cytometers: the numbers are
means of duplicate measurements. Bright field 1 is the estimate of “entities” and bright field 2
is the estimate of “single cells”. For the 10-50 µm size class two “single cell” counts were
made. FR = Flow rate. (At 1 and 2 = Attune systems, C6 = Accuri C6, Ca = FACSCanto, CS1
= Cytobuoy system, FV = FACSVerse).
 2‐10µm 10‐50µm
 lowFR highFR low FR  highFR
Brightfield"entities"(1) 1,133 579
Brightfield"singlecells"(2) 6,646 1,088;4,185
At1 1,570 1,110 1,250 1,410
At2 980 785 1,180 1,340
Ca‐2,262‐2,127
C6 10,027 13,349 1,496 1,539
CS1 12,195 13,390 715 901
FV 93‐ 7‐

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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Two microscopists (NIVA and GSI) examined the difference between counting single cells
and counting of “entities”, which are the combination of single cells and colonies. This lead to
factors of 4x (10-50 µm) and 6x (2-10 µm) higher concentrations compared to when single
cells were counted (Table 8).
Most flow cytometers cannot discern the single cells of which a colony is made of. Therefore,
it was expected that their counts would be more in accordance with the “entity”-microscope
counts. Indeed, for the 2-10 µm sized organisms the two At and the Ca flow cytometers
produce numbers that are comparable to that of “entities” by the microscope. On the other
hand, the C6 and CS1 reached concentrations a factor of ca. 10x higher than the microscope
counts, while the FV was a factor of ca. 100x lower compared to C6 and CS1. It is unclear at
this moment how to explain these factors of differences.
For the 10-50 µm sized organisms a different result was found to that of the 2-10 µm fraction.
The microscopic estimate of the “entities” concentration was surpassed by a factor of 2 to 4x
by the flow cytometers. In fact, the At’s, C6 and Ca flow cytometers obtained concentrations
that were in the range of the microscope “single cell” estimate. It is not clear what caused this
difference with the results for the 2-10 µm fraction, but it might be related to differences in
species composition. The CS1 produced a concentration that was inbetween the microscopic
“entities” and lower “singe cell” estimates. The FV finally again had a very low estimate of
the organism concentration, i.e. >100x lower than the C6 and CS1.
Finally, from those flow cytometers that made counts at both low and high flow rate the
estimate of the organism concentrations were significantly (P < 0.05) 21% higher at higher
flow rates (Table 8; t-test, df = 7).
A note of caution: the estimates of organism concentrations were obtained from a single
Wadden Sea sample only. In addition, the counts were made in duplicate at most. Therefore,
care must be taken to extend the present findings to the general performance of microscopy
and flow cytometry and their comparability.
The holographic microscope was also used to count the 10-50 µm range. A total count of
3,376 objects was obtained. Given the color and fluorescence restrictions of the D
3
HM, this
total count would also include debris. An initial manual check of what was clearly identifiable
as phytoplankton led to a count of 422 organisms. Although the D
3
HM’s software is currently
restrictive for plankton analysis, the total count is in alignment with the microscopy counts
(Table 8).
Conclusions:
1. By microscope a factor of 4 to 6 times higher “single cell” organism concentrations
were counted when compared to “entity” counts (single cells + colonies).
2. In the 2-10 µm size range, three flow cytometers deliver organism concentrations that
were in the same order of magnitude as the “entity” microscope counts. The other

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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flow cytometers deliver 10 x or more higher counts compared to the “entity”
microscope count.
3. In the 10-50 µm size range, most flow cytometers deliver organism concentrations that
were in the same order of magnitude as the microscope “single cell” counts.
4. At high flow rates of cell counting the flow cytometers counted 21% higher
concentrations.
4.3.4. DATA ANALYSIS
The phytoplankton concentrations that were presented in the previous paragraphs were
calculated by the participants themselves from particle selection and counts and from flow
rate estimates that were made in their own software. These data, as well as the original flow
cytometer files, were collected during the workshop. In addition, to avoid any personal bias or
errors in the process of obtaining the particle concentrations, two extra data analysis methods
were proposed at the start of the workshop.
The first extra method of data analysis would be to redo all analyses in one software analysis
package (FCS Express™) by NIOZ from the original flow cytometer files that were delivered
to the central database by each participant during the workshop. Although this would not
completely eradicate personal bias in particle selection, all data would be analyzed by the
same person. Unfortunately, there was insufficient time to perform this extra data analysis. In
addition, the CS data (.cyz files) could not be read by the FCS Express™ software, making it
impossible to complete a full comparison.
The second method of data analysis was to have all analyses redone by an independent
observer. For this workshop, Thomas Rutten (Thomas Rutten Projects, The Netherlands) used
EasyClus™ software to carry out such an analysis. The description of this software and the
results that were obtained by this analysis are noted in section 4.4.1..
4.4. DAY 4 SUMMING UP
4.4.1. EASYCLUS SOFTWARE
The second method of data analysis was a recalculation by an independent observer: Thomas
Rutten (Thomas Rutten Projects, The Netherlands) using EasyClus™ software (Figure 18).
This software is designed to automatically cluster the particles thus rendering the data
analysis as objective as possible. The data were analysed in EasyClus™ after the workshop
and the results of the size calibration beads were reported. Unfortunately, data was lacking on

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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the volumes counted making it impossible to calculate cell concentrations per mL.
Furthermore, some of the files contained negative values (probably baseline corrections) for
which it was unclear how they could be analyzed by EasyClus™. Therefore, eventually only
the precisions of the size calibration beads were analyzed EasyClus™ and statistically
processed to be able to make a comparison with the precision calculation that were made on
the basis of data that was processed by the individual participants.
Figure 18. The EasyClus™ software developer Thomas Rutten.
Again, the objective to count size calibration beads was to measure the precision (%
coefficient of variation) of the size (forward scatter or FS) and fluorescence (green or FBG) at
two flow rates. In addition, the precision of the two methods of analysis, individual and by
EasyClus™, can be compared to examine if these two methods give comparable results.

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Table 9. Precision of Forward Scatter expressed as % coefficient of variation. The precision
was measured for differently sized calibration beads at low and high flow rates by either the
workshop participants or by using the automated software: EasyClus™.
Forward
Scatter
Lowflowrate
%coefficientofvariation
Highflowrate
%coefficientofvariation
Beadsize(µm) Participants EasyClus Participants EasyClus
2 13 17 23 72
10 6 6 13 14
44 26 21 35 32
51 23 38 35 54
totalaverage: 17 21 27 43
Table 10. Precision of Green Fluorescence expressed as % coefficient of variation. The
precisions were measured for differently sized calibration beads at low and high flow rates by
either the workshop participants or by using automated software: EasyClus™.
Fluorescence
Lowflowrate
%coefficientofvariation
Highflowrate
%coefficientofvariation
Beadsize(µm) Participants EasyClus Participants EasyClus
2 11 11 27 29
10 5 5 8 9
44 25 21 34 34
51 26 26 27 36
totalaverage: 17 16 24 27
In general, the calibration bead coefficients of variation as determined by EasyClus™ were
slightly higher than the precision estimates of the individual participants (Tables 9 and 10).
Only in the 2 µm beads forward scatter at a high flow rate EasyClus™ found a substantially
lower precision. Inspection of the underlying data revealed that this increase in %CV was
primarily caused in the analyses of At1 and At2 data. Apparently, the automatic software
made a wider cluster selection of the data compared to a human analyst.
Conclusions:
1. Automatic cluster analysis software, EasyClus™, enabled an objective analysis of the
precision of size calibration beads measurements.
2. When using automatic cluster analysis the software performance must be validated.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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4.4.2. DISCUSSION OF RESULTS
On the last day of the workshop Louis Peperzak summarized the data from the practical
exercises, for as far they had been analysed, and presented this to the participants. During the
discussion, the following observations were made.
1. Beads. High flow rates show a decrease in the number of beads counted, likely due to
unstable flow streams. Microscope bead counts seem to be lower compared to flow
cytometry counts.
2. Phytoplankton cultures. The bigger the algae the more difficult it is to deliver accurate
cell counts for flow cytometers, the microscope counts were closer to the real
organism concentration compared to several flow cytometers.
3. Wadden Sea sample. Most flow cytometers deliver organism concentrations for larger
organisms that were in the same order of magnitude as the “single cell” microscope
counts. For organisms below 10 µm the difference is much higher.
In addition, a number of questions that the workshop posed to the participants or that arose
during the work could (partly) be answered:
1. Are all instruments capable of measuring 2-10 µm particles? Yes.
2. Are all instruments capable of measuring 10-50 µm particles? The Canto and
Cytobuoy, especially on low flow rates, had problems, all others were able to measure
the 50 µm well. The Accuri had clogging problems with the 50 µm algae.
3. Are all instruments capable of measuring >50 µm particles? In general, flow
cytometry has a limited capacity to handle the larger organisms. The use of a
FlowCAM should be investigated, or a conventional scanner to scan a sample that is
on top of a large transparent plate (e.g. ZooScan: http://www.obs-
vlfr.fr/LOV/ZooPart/ZooScan/).
4. Are flow cytometers capable of live/dead analysis? This is limited to the use of probes
or stains. Stains may also be needed to include living heterotrophic (non-fluorescent)
cells in the total plankton count.
5. Are beads useful as proxies for phytoplankton cells? For round shaped cells yes, but
for non-spherical cells it is difficult to assume that beads represent the minimum
dimension. Size calibration could be performed with plankton meshes, e.g. measure
sample, filter out small ones, measure again and establish the cut-off limit. However,
filtration of phytoplankton can create other problems such as cell damage.
6. Is there a flow rate effect? It seems that at higher flow rates you measure fewer
particles. For larger particles this could be reversed. On the other hand, in the wadden
Sea sample more cells were counted at higher flow rates.
7. Wadden Sea sample, problems with red fluorescence? The Accuri had problems, but
also the Attunes faced non-optimal conditions.
8. Can low phytoplankton concentrations be measured by all flow cytometers? Yes.
9. In general phytoplankton concentration counts mismatched between different
machines.
10. Also with machine versus microscope counts. Particles compare well, but not
necessarily the cell counts.

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11. Can flow cytometers prove compliance with D-2 and at which level of accuracy and
precision?
Based on the detailed data analysis in this report a summary that is more quantitative follows
here:
Flow rates:
1. The maximum flow rates of the instruments tested ranged nearly tenfold: from 0.1 to
0.9 mL/minute. In other words, the analysis time of 1 mL sample is 1-10 minutes
depending on the instrument.
2. In general the average precision of the flow rates at the high setting was <10%.
Beads:
3. Overall, bead counts made by the different instruments, microscopes as well as flow
cytometers, were mostly in the same order or magnitude but did show noticeable
differences.
4. Although the flow cytometer counts and holographic microscope counts were similar
for the 2 µm range, microscope counts were clearly higher.
5. Microscopy counts were made at low magnifications which would make the
distinction of beads to other particles present, such as debris, more difficult and may
explain the higher counting rate.
6. It was noted that the flow cytometers delivered higher counts when using 44 µm beads
compared to the microscopes, whilst at 50 µm beads it is vice versa. Therefore, it was
assumed that the microscope counts are more accurate for the 50 µm beads as the flow
cytometers struggled with this size and results are a bit closer to the D
3
HM which also
does well with larger beads..
7. The comparison between the low and high flow rate indicates that at high flow rates
relatively less size calibration beads were counted.
8. The precision of measurement of the Forward Scatter and the Green Fluorescence of
size calibration beads was comparable (15-25%).
9. At higher flow rates a 5-10% lower precision in the measurement of the Forward
Scatter and the Green Fluorescence was attained (ca. 25%) than at lower flow rates
(ca. 15%).
10. The 10 µm size calibration beads had the highest precision (<10%), the 2, 44 and 51
µm beads the lowest precision (20-30%) in both Forward Scatter and the Green
Fluorescence.
Microscopy of phytoplankton cultures:
11. Small cells such as M. pusilla (2 µm) are very difficult to count by bright field or
holographic microscopy.

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12. For the concentration of cells ≥ 10 µm the different microscope techniques were in
relative agreement. The high abundance P. minimum (10 µm) had a nearly identical
concentrations when counted by brightfield and holographic microscopy.
13. A good agreement was also achieved for the 50 µm P. belizeanum between
fluorescence and holographic microscopy. The counts from brightfield microscopy
were higher.
Flow cytometry of phytoplankton cultures:
14. Contrary to microscopy, the flow cytometers were very capable of measuring the
cultured algal cells in the 2-10 µm size range.
15. The flow cytometers also managed to count cells in the lower size range of the 10-50
µm size objects. However, at object sizes of 50 µm most flow cytometers had
counting difficulties as well as sample handling problems because the large objects
clogged the instruments. However, the differences in absolute concentrations were
significant between the flow cytometer and the microscope counts, increasing with the
size of the algae counted.
16. Counting precision expressed as the 95% confidence interval increased from 14% at
high cell concentrations (>10,000 cells/mL) to 59% at low concentrations (<100
cells/mL) although cell-specific characteristics may interfere with counting and
increase the counting precision.
17. Multiple measurements of a sample, at least three, are needed to establish a confidence
interval that enables the conclusion of the average cell concentration .
Wadden Sea sample:
18. By microscope a factor of 4 to 6 times higher “single cell” organism concentrations
were counted when compared to “entity” counts (single cells + colonies).
19. In the 2-10 µm size range, three flow cytometers deliver organism concentrations that
were in the same order of magnitude as the “entity” microscope counts. The other
flow cytometers deliver 10 x or more higher counts compared to the “entity”
microscope count.
20. In the 10-50 µm size range, most flow cytometers deliver organisms concentrations
that were comparable to the microscopic “single cell” counts.
21. Higher flow rates for cell counting resulted in a higher organism concentration
estimates of the flow cytometers.
Data analysis:
22. Automatic cluster analysis software, EasyClus™, enabled an objective analysis of the
precision of size calibration beads measurements.
23. When using automatic cluster analysis the software performance must be validated.
24. The comparison of the results generated by the two microscopists is biased as one
microscopist counted “entities” (i.e. a colony of multiple cells was counted as one

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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object) which is likely more comparable to flow cytometer counting, while the second
microscopist counted the single cells (also in a colony) which always delivered higher
cell counts.
OVERALL CONCLUSIONS
The participants felt that, with today´s organism detection technologies, ballast water analysis
can only be performed to an acceptable level of precision and confidence when the samples
are processed by a trained biologist.
Because a single cell is the reproductive unit that can start a new community this is also the
unit that has to be counted in ballast water research. In other words, theoretically a single
viable cell in discharged ballast water can start an invasion. Therefore, according to the IMO
G2 Guidelines for Ballast Water Sampling, the single cells with a minimum dimension >10
µm that make up a colony should be counted.
As a workshop result, flow cytometry based organism detection technologies are readily
available to meet the requirements for compliance monitoring and enforcement with ballast
water management standards for indicative analysis. Some new approaches, such as the
digital holographic microscope, are at a developmental stage. To prove compliance with
ballast water management standards a precise enumeration of cells according to their
minimum dimension and at the same time a separation of living and dead organisms is
needed. Several flow cytometry based systems are able to provide the enumeration and
living/dead assessment analysis for phytoplankton cells for indicative ballast water sample
analysis. Given the large variation in cell concentrations measured in the Wadden Sea sample,
a thorough validation should be carried out between microscopic and flow cytometric
methods for a detailed ballast water sample analysis.
Furthermore, additional developments are required to document the minimum dimension of
organisms. This measurement can with today´s flow cytometers only be achieved for
spherical objects, but for organisms with elongated or other non-spherical shapes, this seems
to be difficult. This is the case for all flow cytometers tested so that the results obtained by
these systems should be interpreted carefully. The same carefulness is needed in microscopy:
it would also be impossible to always identify properly the minimum dimension of, for
example, pennate diatoms. These “coin-shaped” organisms settle in the observation chamber
and usually “land” flat so that they cannot be inspected from the side, which is the minimum
dimension for these organisms.
It may further be considered to undertake experiments using different probes or stains as these
seem to be the most appropriate method to assess viability in automated organism detection
technologies.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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The test organisms used during the workshop were “perfect cultures” with healthy organisms
grown in optimal conditions. Additional tests may be undertaken with treated ballast water
samples or organisms treated otherwise, so that detection technologies are challenged to
identify living cells in sub-optimal conditions.
ACKNOWLEDGEMENTS
We express our grateful thanks to endless helping hands at NIOZ to make this workshop a
success. Especially the practical comparative test of the organism detection technologies
required intensive preparations, which were undertaken by NIOZ.
The participants thanked NIOZ for having sponsored the coffee and lunch breaks.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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APPENDIX 1 AGENDA OF THE WORKSHOP
NIOZ Flow Cytometer Workshop
Comparing instruments in measuring 2-10 µm and 10-50 µm plankton cells
Agenda
11. Monday 2013
9:00: registration in the Beril meeting room (Marieke Vloemans, phone 350).
Installation of instruments in the labs and test runs (help provided by Eva Immler, Dennis
Mosk and Louis Peperzak).
12:00: lunch in the NIOZ canteen
Beril meeting room
13:00: Welcome: NIOZ director Prof. Dr. Herman Ridderinkhof
13:05: The North Sea Ballast Water Opportunity (NSBWO) project: dr. Jan Boon
13:20: Introduction to the workshop: Louis Peperzak
Introduction of participants. Where do you work and what do you do? How do you use flow
cytometry/microscopy? If one of you wants to give a power point presentation, please limit it
to 10 minutes per instrument:
13:30: Attune: Ole-Kristian Hess-Erga, Aud Larsen, Sandra Schöttner
13:50: CytoSense: Machteld Rijkeboer, Veronique Creach, George Dubelaar, Luis Felipe
Artigas, Simon Bonato
14:10: FACS Canto II: Anna Noordeloos, Eveline Garritsen, Josje Snoek
15:00: Coffee break
15:20: oLine D
3
HM: Eva-Maria Zetsche and Joël Henneghien
15:40: FACSVerse: Albert Mosselaar and Roland Langelaar
16:00: Accuri C6: Brian Maurer, Kristien Rasschaert, Paola Bruno

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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16:20: Bright Field and Fluorescence Microscopy: Alex Blin, August Tobiesen, Euan Reavie
16:40: EasyClus software: Thomas Rutten
17:00: Final agreement for Tuesday to Thursday: schedule and protocols
17:30: Walk to bus station and return to hotels (bus leaves near ferry at 17:50)
19:00: Dinner at the “12 Balcken” restaurant in Den Burg
12. Tuesday 2013
09:00: arrive at NIOZ
10:00: start flow rate calibrations with TruCount
®
beads
11:30: discussion of results (Beril meeting room)
12:00: lunch
13:00: size calibration 50, 10 and 2 µm beads
16:00: discussion of results (Beril meeting room)
13. Wednesday 2013
09:00: arrive at NIOZ and instrument calibrations
09:30: commissioning run with Phaeocystis globosa (size 4 µm) single cells
10:30: counting Micromonas pusilla (high + low sample, size 2 µm)
11:30: discussion of results (Beril meeting room)
12:00: lunch
13:00: counting Prorocentrum minimum (high + low sample, size 10 µm)
14:00: counting Prorocentrum belizeanum (high + low sample, size 50 µm)
15:00: counting natural Wadden Sea sample
16:00: discussion of results (Beril meeting room)

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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14. Thursday 2013
09:00: arrive at NIOZ and discussion of all workshop results (Beril meeting room)
10:00: break
10:30: draft report (Beril meeting room)
12:00: lunch and end of workshop.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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APPENDIX 2 LIST OF PARTICIPANTS
Name Affiliation instrument email
AlbertMosselaar BectonDickinson FACSVerse Albert_MOSSELAAR@europe.bd.com
AlexBlin NIOZ
BrightField‐
Microscope Alex.Blin@nioz.nl
AnnaNoordeloos NIOZ FACSCantoII Anna.Noorde loos@nioz.nl
AudLarsen
UniResearch&
UniversityofBergen Attune2 Aud.Larsen@bio.uib.no
AugustTobiessen NIVA
Fluorescence‐
Microscope
august.tobiesen@niva.no
BrianMaurer
MossLandingMarine
Labs AccuriC6 bmaurer@mlml.calstate.edu
EuanReavie
Universityof
MinnesotaDuluth
Fluorescence‐
Microscope ereavie@d.umn.edu
Eva‐MariaZetsche VUB,Ovizio oLineD
3
HM ezetsche@vub.ac.be
EvelineGarritsen NIOZ FACSCantoII Eveline.Garritsen@nioz.nl
GeorgeDubelaar
Cytobuoy CytoSense2 dubelaar@cytobuoy.com
JoëlHenneghien
OvizioImaging
Systems oLineD
3
HM joel.henneghien@ovizio.com
JosjeSnoek NIOZ FACSCantoII Josje.Snoek@nioz.nl
KristienRasschaert BectonDickinson AccuriC6 kristien_rasschaert@europe.bd.com
LouisPeperzak NIOZ none Louis.Peperzak@nioz.nl
LuisFelipeArtigas
UniLittoral(LOG),
Wimereux CytoSense3 Felipe.Artigas@univ‐littoral.fr
MachteldRijkeboer RWS CytoSense1 machteld.rijkeboer@rws.nl
Ole‐KristianHess‐Erga
NIVABergen
Attune1 Ole‐Kristian.Hess‐Erga@niva.no
PaolaBruno BectonDickinson AccuriC6
Paola_Bruno@europe.bd.com
PieterdeVre
Attune®Acoustic
FocusingCytometry&
FlowSolutions Attune1 pieter.devre@lifetech.com
RolandLangelaar BectonDickinson FACSVerse
Roland_Langelaar@europe.bd.com
SandraSchöttner
UniResearch&
UniversityofBergen Attune2 Sandra.Schoettner@bio.uib.no

SimonBonato
UniLittoral(LOG),
Wimereux CytoSense3 
StephanGollasch GoConsult none sgollasch@aol.com
ThomasRutten ThomasRuttenProjects
Easyclus
software
thomasruttenprojects@gmail.com
VeroniqueCreach CEFAS CytoSense1 veronique.creach@cefas.co.uk
APPENDIX 3 PROTOCOLS

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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Abbreviations used:
CV coefficients of variation
FBG fluorescence blue -> green
FBO fluorescence blue -> orange
FBR fluorescence blue -> red
FS forward scatter
SS side scatter
General data acquisition and analysis protocol
1. TruCount beads are used on both days for flow rate calibrations.
2. Size calibration beads are used to determine 2-10-50 µm size classes in Forward
Scatter and to compare counts among instruments and techniques.
3. Phytoplankton cultures (approximately 2-10-50 µm in minimum dimension) are used
to check size calibration and to compare counts among instruments and techniques.
4. A Wadden Sea sample is counted to check the abilities to measure a ‘real world’
sample.
5. Different flow rates (size beads, cultures) are used to compare accuracy (particle
concentrations) and precision (%CV of FS, FBR, FBG) (abbreviations see below).
6. Data are acquired and stored according to each instruments’ regular settings.
7. .FCS file names are coded:
 Instrument code (see participant list)_TC (TruCount)_d1/d2_lo/hi _1/2
 Instrument code_SC (Size calibration)_2/10/50_lo/hi_1/2
 Instrument code_AC (Algal culture)_Pb/Pm/Mp _G8/D2_1/2
 Instrument code_WS (Wadden Sea)_hi_1/2 (e.g. Ca_WS_hi_2.fcs)
8. Data are also copied and stored in a NIOZ database (Louis, Beril room).
9. The first data analysis is by each team itself using the instrument data analysis
software (or other, see specs list in the workshop Excel file). These data are filled out
in an Excel file that will be supplied at the start of the workshop.
10. Second data analysis is by NIOZ using FCS Express version 4 (De Novo Software,
Los Angeles, USA). This is to ensure uniform data analysis.
11. Third data analysis is by the automatic clustering software Easyclus version 1.17
(Thomas Rutten Projects, Middelburg, The Netherlands). This is to ensure uniform
“automatic” data analysis.\
12. The flow rates and the relative measures (CV% of Forward Scatter) at each flow rate,
from each data analysis step, and for both size calibration as for the phytoplankton
cultures, will be used in a multivariate plot.
TruCount bead protocol
1. The aim of this protocol is to establish comparable flow rate data for all instruments.
2. TruCount™ (Becton Dickinson, Franklin Lakes, USA) beads are suspended in 2 mL
SDS 0.1% by Josje Snoek (NIOZ), capped and stored at room temperature.
3. The bead concentration per mL will be calculated.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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4. TruCount™ beads are made fresh daily. The bead solutions are not vortexed as this
may damage the beads, but are mixed well by gently shaking the solution.
5. Trigger and threshold are on FBR (red fluorescence).
6. PMT’s are set in such a way that the beads appear near the middle of a FBG (green
fluorescence) versus FBR (red fluorescence) bi-plot with logarithmic scales.
7. Flow rates: the maximum flow rate and the minimum flow rate (typically 100 and 10
µL/min for a Canto II).
8. The number of beads to be counted is approximately >1000 and counting time should
not be less than 1 minute.
9. The beads are gated into a histogram of FBR (red fluorescence) and the number of
single, double and triple beads (2x and 3x the FS of single beads) are calculated using
the instrument software.
10. The total number of beads is 1x single + 1x double + 1x triple beads.
11. The flow rates are calculated from the time it took to count the total number of beads
(≥ 1000) and the bead concentration.
12. Measurements are made with duplicate tubes at both flow rates on Tuesday (n = 4).
13. Measurements are made with single tubes at both flow rates on Wednesday at the
beginning and at the end of the phytoplankton counts (n = 4).
14. Data to report: two flow rates (low, high) in duplicate. Both on Tuesday and
Wednesday.
Size calibration protocol
1. The aim of this protocol is to establish comparable instruments settings for the 2-50
µm size range, so that size calibration beads as well as phytoplankton in the 2-50 µm
size range can be counted with one setting. In addition, the mean forward scatter (FS)
and green fluorescence (FBG) %CV values are measured in order to compare
instruments.
2. Green Fluorescent Polymer (polystyrene ) microspheres of 2.0, 9.9 and 51 µm
(Thermo-Scientific, Freemont, USA, catalog numbers G0200, G1000 and 35-8) are
diluted in 2 mL SDS 0.1% in standard flow cytometer tubes by Josje Snoek (NIOZ),
capped and stored at room temperature. The dry 50 µm beads are suspended in a 0.1 %
SDS solution by NIOZ. The excitation maximum for these beads is 468 nm; the
emission maximum is 508 nm (DSC). Participants may need to pour the sample in a
tube that is specific to their instrument. Note added after the workshop: the
manufacturer had become Thermo-Scientific (Freemont, USA). The name of the
microspheres was Fluoro-Max™; the catalogue numbers had remained identical. The
sizes were: 2.0 (<5% uniformity), 9.9 µm (<5% uniformity) and 51 µm (12% CV).
3. PMT’s are set in such a way that the 50 µm beads appear in the right hand corner of
FBG (green fluorescence) versus FS (Forward Scatter) bi-plot with logarithmic scales.
4. In case 50 µm beads cannot be measured because of their large size (out of scale or
clogging) they will be replaced by 44.2 µm polystyrene Dragon Green beads (Bangs
Laboratories, Fishers, USA, catalog code FS08F). The excitation maximum for these
beads is 480 nm; the emission maximum is 520 nm (BL).
5. Trigger and threshold is set on green fluorescence.
6. Flow rates: the maximum flow rate and the minimum flow rate (typically 100 and 10
µL/min for a Canto II).
7. The number of beads to be counted is approximately 1000.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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8. The beads are gated into histograms of FS (Forward Scatter) and FBG (green
fluorescence) and the geometric means, medians and the coefficients of variation
(%CV) of single beads are calculated using the instrument software.
9. The actual mean diameters according to DSC and BL are: 1.9, 10.1, 44.2 and 48 µm)
which means that exact FS values at 2, 10 and 50 µm should be calculated from a
linear regression equation of measured FS geometric means versus actual sizes.
10. Measurements are to be taken in duplicate.
11. Data to report: three mean and median bead FS and FBG values with three CV’s at
two flow rates (low, high) in duplicate. Concentrations in beads per mL, in duplicate.
12. Data to report: Flow Cytometry Standard (.FCS) files.
Phytoplankton culture protocol
1. The aim of this protocol is to measure the cell concentration in phytoplankton cultures
in the 2-50 µm size range. In addition, the mean forward scatter (FS) and red
fluorescence (FBR) %CV values are measured in order to compare instruments.
2. Phytoplankton species from the NIOZ plankton culture collection are red fluorescent
particles with a size ranging from 2 µm (Micromonas pusilla) to 10 µm
(Prorocentrum minimum) to 50 µm (Prorocentrum belizeanum). One to two days
prior to measurement cultures of these species were diluted with fresh culture medium
in order to have cells with a good physiological condition. The approximate cell
concentrations will be approximately 1000 per mL (IMO G8 guideline) and 10 per mL
(IMO D-2 Ballast Water performance Standard). The low concentration sample is a
100x dilution of the high concentration sample with fresh culture medium.
3. On the day of measurement the cultures will be dispensed in 2 mL quantities in in
standard flow cytometer tubes by Josje Snoek and Alex Blin (NIOZ), capped and
stored in a refrigerator. Participants may need to pour the sample in a tube that is
specific to their instrument.
4. A high concentration culture of 4 µm Phaeocystis globosa cells will be used in a
commissioning run that is intended to first find phytoplankton cells with each
instrument by adjusting the red fluorescence PMT.
5. Trigger and threshold is set on red fluorescence.
6. Next, the red fluorescence PMT is set in such a way that the 50 µm plankton cells
appear in the right hand corner of an FBR (red fluorescence) versus FS (Forward
Scatter) bi-plot with logarithmic scales. Note that the FS settings should not be
changed; these settings should be the same as determined the previous day using the 2-
10-50 µm size calibration beads. Ideally, this red fluorescence PMT setting can also be
used for the smaller phytoplankton species.
7. Flow rates: the maximum flow rate and the minimum flow rate (typically 100 and 10
µL/min for a Canto II).
8. The number of cells to be counted is preferably 1000. However, because at 10 cells
per mL this would lead to an impractical counting time and sample volume (100 mL),
a minimum of 25 counted cells is taken as the minimum (count error = 20%). This
means that a minimum of 2.5 mL needs to be counted.
9. The count time: 5 minutes at high and 10 minutes at low flow rate respectively. On a
Canto II these settings would lead to counting of 500 and 100 cells respectively, i.e.
less than 1000. However, the counting times should be practical in view of analysis
time and the reduction of the physiological condition of the cells at room temperature.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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10. The cell clusters are gated into histograms of FS (Forward Scatter) and FBR (Red
fluorescence) and the geometric mean and median size/red fluorescence of the cells
and the coefficients of variation (%CV) are calculated using the instrument software.
A pre-selection to remove debris and detritus may be needed.
11. Measurements are in duplicate (i.e. 30 minutes on a Canto II).
12. Data to report: three mean and median plankton FS and FBR values with three CV’s at
two flow rates (low, high) in duplicate. Phytoplankton concentrations in cells per mL,
in duplicate.
13. Data to report: Flow Cytometry Standard (.FCS) files.
Wadden Sea sample protocol
1. The aim of this protocol is to measure the cell concentration of phytoplankton in a
natural but relatively dilute sample in the 2-10 and 10-50 µm size ranges, comparable
to ballast water treatment (challenge) test water.
2. A Wadden Sea (winter) sample that has been incubated at 15°C and 50 µmol photons
s
-1
in a 16:8 L:D cycle prior to the workshop to ensure sufficient plankton cell
concentrations will be dispensed in 2 mL quantities in standard flow cytometer tubes
by Josje Snoek and Alex Blin (NIOZ), capped and stored in a refrigerator. Participants
may need to pour the sample in a tube that is specific to their instrument.
3. The sample will be counted using the FS and FBR PMT settings that were achieved
with the phytoplankton cultures.
4. Maximum and minimum flow rate, with the aim of measuring approximately 10
3
– 10
4
cells.
5. The number of cells between 2 and 10, and between 10 and 50 µm are counted. A pre-
selection to remove debris, detritus and cyanobacteria may be needed. The
measurement is made in duplicate.
6. Data to report: cell concentrations per mL of size classes 2-10 and 10-50 µm, in
duplicate.
7. Data to report: Flow Cytometry Standard (.FCS) files.
Microscopic analyses
1. The aim of this protocol is to measure the bead and cell concentrations so that a
comparison with the concentrations obtained by the instruments can be made.
2. Size calibration beads of 10 and 50 µm will be counted using brightfield illumination
in 1 mL Sedgewick-Rafter chambers.
3. Size calibration beads of 2 µm will be counted using brightfield illumination in a
Bürker haemocytometer.
4. Size calibration beads of 2, 10 and 50 µm will be counted on 0.2 µm polycarbonate
filters using fluorescence microscopy (green fluorescence).
5. Plankton cultures will be counted using brightfield illumination in 1 mL Sedgewick-
Rafter chambers or in a Bürker haemocytometer.
6. Plankton cultures will be counted in 1 mL Sedgewick-Rafter chambers or on 0.2 µm
polycarbonate filters after adding a vitality stain (FDA, CMFDA or a combination)
using fluorescence microscopy (green fluorescence).

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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7. Plankton cells of 2-10 and 10-50 µm in the Wadden Sea sample will be counted in 1
mL Sedgewick-Rafter chambers or on 0.2 µm polycarbonate filters after adding a
vitality stain (FDA or CMFDA) using fluorescence microscopy (green fluorescence).
8. Plankton cells of 2-10 and 10-50 µm in the Wadden Sea sample will be counted in two
Lugol-fixed aliquots using the Utermöhl sedimentation method and an inverted
microscope (after the workshop).
9. Data to report: bead (n = 3) and cell culture (n = 3) concentrations per mL from
brightfield and fluorescence microscopy, Wadden Sea sample cell concentrations of 2-
10 and 10-50 µm from inverted (Lugol) and fluorescence microscopy, in duplicate.
Data analyses
1. The aim of this protocol is to provide a means to comprehensively analyze the data
obtained by all instruments and techniques.
2. To compare the flow rates, the minimum and maximum values as measured with
TruCount™ beads will be listed in a table.
3. To compare the ability to measure 2, 10 and 50 µm beads using one set of PMT
settings, these abilities will be listed in a check table (, ).
4. The concentration of beads and cells, as counted by microscopy and by the
instruments, will be compared in a regression diagram.
5. The differences in performance between low and high flow rates will be compared in
regression diagrams. Performance is accuracy (100% cell count at the lowest flow
rate) and precision (%CV).
6. For the size calibration beads (2, 10 and 50 µm) all duplicates (concentration per mL
and %CV of FS and FBG at both low and high flow rate) will be averaged before
multivariate analysis.
7. The average data will be analyzed in a non-metrical multi-dimensional scaling (MDS)
diagram (Primer 6, version 6.1.13) in which the ‘position’ of an instrument is relative
to others depending on measurement performance (instruments that have near equal
performance will cluster together).
8. The first data analysis will take place with data computed directly by each participant;
the second after using FCS Express and the third using EasyClus.
Reporting
1. A NSBWO report will be written by Louis Peperzak and Stephan Gollasch.
2. A joint paper will be sent to a journal.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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APPENDIX 4 SYSTEM DESCRIPTIONS
The following pages show detailed system descriptions as provided by the manufacturers. The
list is arranged in alphabetical order.
Accuri C6
BDACCURIC6
Supportscellanalysisforuptosixparameters
TheBDAccuri™C6makestheanalyticalpowerofflow
cytometrymoreaccessiblewithease‐of‐useand
affordability.Itscompactfootprintandportableweight
makeitavaluablepersonalusetoolforbothnoviceandexperiencedresearcherswhowant
acytometerto
beeasilyavailablewhenandwheretheyneedit.
Thesystemfeaturesanintuitivesoftwareinterface,softwaretemplates,andreagentkits
thatguideusersnewtoflowcytometrythroughworkflowsforpopularapplications.
SampleFlexibilitywithOptionalWalkawaySampleLoading
Auniquelow‐pressurepumpingsystemdrivesthe
fluidics.Asheath‐focusedcoreenableseventratesofup
to10,000eventspersecondandasampleconcentration
ofover5x106cellspermL.Inaddition,thesystem
derivessamplevolumeandcancalculateabsolutecounts
orsampleconcentrationpermicroliter.
MinimizedSetupTime
Thesystemisequippedwithablueandaredlaser,twolightscatterdetectors,andfour
fluorescencedetectorswithopticalfiltersoptimizedforthedetectionoffluorochromessuch
asFITC,PE,PerCP,andAPC.Acompactopticaldesign,
fixedalignment,andpre‐optimizeddetectorsettings
makethesystemeasiertouse.
Duringmanufacture,laserandopticalalignmentsareset
andlockeddown.TheresultisthateachBDAccuri™C6
cytometerismanufacturedwithstandardized
fluorescenceperformancesothatusersdonotneedto

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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adjustdetectorvoltages.
Applicationsinkineticanalysisofcellularresponses
TheBDAccuriC6employsnon‐pressurizedperistalticpumpsinanopenfluidicssystem.
Opentubesallowconvenientadditionoftestcompoundstothecellsuspensionwithout
interruptingsampling.This“continuous‐flow”methodenablesnon‐stopanalysisofcalcium
fluxandotherkineticcellularresponses,suchaspH,reactiveoxygenandnitrogenspecies,
mitochondrialmembranepotential,andnanoparticleuptake.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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Attune
The Attune® Acoustic Focusing Flow Cytometer gives you both high precision and sensitivity at
any rate of throughput. You can control your sample concentration, the flow rate, the number of
photons you detect, the length of your experiment, the number of samples you run, and more. All this
translates to dependable results―faster and easier than ever before.
ACOUSTIC FOCUSING TECHNOLOGY OVERVIEW
The Attune® Acoustic Focusing Flow Cytometer is the firs
t
cytometer that uses ultrasonic waves (over 2 MHz, similar to those
used in medical imaging), rather than hydrodynamic forces, to
position cells into a single, focused line along the central axis of a
capillary.
WHAT IS ACOUSTIC FOCUSING CYTOMETRY?
Acoustic focusing cytometry is a technology that uses ultrasonic waves (over 2 MHz, similar to those
used in medical imaging), rather than hydrodynamic forces, to position cells into a single, focused line
along the central axis of a capillary (Video 1). Acoustic focusing is largely independent of the sample
input rate, enabling cells to be tightly focused at the point of laser interrogation regardless of the
sample-to-sheath ratio. This, in turn, allows slower cell velocities to collect more photons for high-
precision analysis at unprecedented volumetric sample throughput.
 Video1.Acousticfocusinginaction.Thisvideodemonstrateshowsamplesarealignedwhentheacoustic
focusingisonoroff.
The Attune® cytometer accomplishes all this without high velocity or high volumetric sheath fluid,
which can damage cells. In addition, volumetric syringe pumps enable absolute cell counting without
beads―minimizing cost and sample preparation time.
HYDRODYNAMIC FOCUSING VS. ACOUSTIC FOCUSING
Manipulating cells in a conventional flow cytometer is accomplished using hydrodynamic forces. A
suspension of cells (the sample stream) is injected into the center of a rapidly flowing sheath fluid, and
the forces of the surrounding sheath fluid confine the sample stream to a narrow “core” that carries
cells through the path of a laser that excites the associated fluorophores and creates a scatter
pattern.
Keeping cells within a confined focal point is important for consistent excitation of the associated
fluorophores as they pass through the tightly focused laser beam. Cytometers that use hydrodynamic
focusing maintain the same sample speed at all flow rates, causing cells to lose focus as the sample
core widens to accommodate the increase in flow rate (Figure 2).

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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Figure2.Awidersamplecoreresultsinabroaderdistributionofcells as
theytransitthroughthelaser,meaningfewercellsareaccuratelyaligned
withthelaserfocalpoint.Toobtainoptimaldatafromaconventionalflow
cytometer,withthelowestvariabilityinsignaldetection,theinstrument
mustberunatthelowestsamplerate,whichistypically10�20μL/min.
Highersampleratesresultingreatervariabilityandlessprecise
measurements.Acousticfocusingavoidsthiscompromiseindataand
sampleratesbyuncouplingcellalignmentfromsheathflow.
clicktoenlarge
Due to many limitations of hydrodynamic cytometers, users often have to sacrifice:
 Throughput for sensitivity
 Features for ease of use
 Performance for price
 Power for footprint
Acoustic focusing technology keeps cells within a confined focal point, so these tradeoffs are not
required.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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BDFACSCanto™II
Cellanalyzerwithprovenreliability
TheBDFACSCanto™familyofbenchtopanalyzersfeatures
reliableperformanceandaccurateresultsforexperiments
requiringupto10parameters.Numerousinnovations
makethesystemeasytouse,powerful,andproductive:
HighPerformance,InnovativeSystem
Attheheartofthecytometer,thefluidicssystemfeaturesafixed‐alignmentflowcellto
minimizestartuptimeandimprovereproducibility.TheBDFACSCantoIIfluidicssystemis
designedtostreamlinework,savetime,andimproveperformance.
ExcitationandEmission
Theexcitationopticsconsistofmultiplefixed‐wavelength
lasers,fiberopticsuptothebeam‐shapingprisms,and
achromaticfocusinglensesthatproducespatially
separatedbeamspotsintheflowcell.Eachlensfocusesthe
laserlightintothegel‐coupledcuvetteflowcell.Sincethe
opticalpathwayandthesamplecorestreamarefixed,
alignmentisfixedfromdaytodayandfromexperimentto
experimentwithnoneedforuserintervention.
Theemissionsignalsaretransmittedfromtheflowcelltothedetectorarrays:anoctagonfor
theblueandatrigoneachfortheredandthevioletlasersignals.Theoctagoncontainsfive
PMTsanddetectslightfromthe488‐nmbluelaser.APMTintheoctagoncollectsside
scattersignals.ThetrigonscontaintwoPMTseachanddetectlightfromthe633‐nm(red)
andthe405‐nm(violet)lasers.
Collectionoptics
TheoctagonandtrigonareBD‐patenteddetectorarraysthatuseseriallightreflectionsto
guidesignalstotheirtargetdetectors,resultinginhighlyefficientlightcollectionand
providingmaximumsignalretentionatthedetectorlevel.ThisBDserialreflectivedesign
furtherenhancesinstrumentsensitivitybycollectingthedimmestemissionsignalsfirst,
movingfromthelongestwavelengths(typicallyPE‐Cy™7)totheshortest(FITC).
OptionsandRelatedToolstoSupportLabRequirements
SampleintroductionproductivitycanbegainedwiththeoptionalBDFACS™Loader(for40
tubes)ortheBD™HighThroughputSampler(HTS).TheBDFACSLoaderisaninstrument
optionthatallowswalkawaysampleintroductiontofurtherimproveproductivity.
Cytobuoy:

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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Cytobuoy,theNetherlands(www.cytobuoy.com).Cytobuoy´sCytoSenseisaportable
flowmeterwhichworksasanimageanalyser.Viablephytoplanktonmaybedistinguished
fromotherparticlesbyanexpertusingthetool.Althoughaphytoplanktonviability
assessmentispossible,itseemsdifficulttoenableaviabilityassessmentofzooplanktonwith
thistool.
AsubmersibleversionoftheCytoSenseflowcytometer(theCytoSub)wasdesignedforin‐
situautonomoussampleanalysis,whichmaybeusedtopromptlymonitorphytoplankton
andtoobtainaccurateinformationaboutphytoplanktondynamics.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells

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FACSVerse
BDFACSVerse
Supportscellanalysisforupto10parameters
TheBDFACSVerse™flowcytomerisdesignedtosupport
cellanalysisforresearchapplicationsusingupto10
parameters.Hardwareinnovationsensurethereliabilityof
resultsandsignalresolution.BDFACSuite™software
deliversaseamlessworkflowfromsystemsetup,through
dataacquisition,analysis,andshutdown.

Thecompactopticalsystemusesfree‐spacelaserstoconcentrateintensityattheflowcell.It
isdesignedtominimizelightlossandmaximizeresolutionformulticolorapplications.A
numberofinnovationsbuiltintotheopticalsystem—includingpatentedautomatedlaser
alignment,smartfilter‐mirrorunitsforthedetector
arrays,andastainlesssteelflowcell—
aredesignedtomaximizereliabilityandimprovesystemperformance.

Best‐in‐ClassDesignwithExceptionalFlexibility
Throughtheprecisecoordinationoftheopticalandfluidicssubsystems,theBDFACSVerse™
analyzeroffersbest‐in‐classopticaldetection,sensitivity,andflexibilityformulticolor
applications.

Vacuum‐Based
SystemEnablesFlexibility
TheBDFACSVersefluidicssystemisvacuum‐basedforexceptional
flexibility.Thisallowsuserscompletefreedomtoacquiresamples
manuallyfromvirtuallyanytubeformatandstreamlinesworkflow.
Userssimplyclickthesampletubeinplacetobeginacquisition.The
tubeholderisbuiltintothecytometer’schassis
tomakeitmore
robustandtoprotecttheSampleInjectionTube(SIT)fromdailywear.

OptionalVolumetricFlowSensor
TheBDFlowsensoroptionforvolumetricmeasurementmeasuresthevolumeoverthe
entireacquisitiontime.Itallowstheusertoreproduciblyobtainaprecisemeasureofcellsor
particles
inavolumeofsampleacquiredbythesystem.Itdirectlydeterminesthevolumeof
thesamplesasitpassesthroughthesampleinjectiontubingforaccuratecounts.

TrulyUniversalWalkawayProcessing
TheoptionalBDFACS™UniversalLoader
automatessamplehandling,enabling
rapidprocessingofmultituberacksand
microtiterplatesinthissingleunit.
Designedtobeeasytouseandflexible,
thisoptionsupportswalkaway
operationforbusyresearchlaboratories.

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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OVIZIO’s oLine D
3
HM
The 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
tools.
Fig. 1 OVIZIO’s oLine D
3
HM (left), and the OsOne software (right) showing its interface
after cells have been counted.
The OVIZIO oLine instrument and associated OsOne software are based on off-axis
digital holographic microscopy (DHM). In this approach, a source of partially coherent laser
light is split into an undisturbed light beam (the reference beam) and a light beam that passes
through the object/sample (the object beam). These two beams are then recombined to record
an interference image or so-called hologram by a digital camera (F. Dubois and C.
Yourassowsky, Patent No. US 7,463,366, US 7,009,700). DHM technology has several
advantages over classical light microscope technology, where image formation is based on
light intensity only and particles need to be imaged in a narrow focus plane. DHM counters
these disadvantages by (1) strongly increasing the depth of focus and (2) acquiring both light
intensity as well as phase information, which are combined in the digital hologram (Fig. 3).
Out-of-focus objects can be refocused by computational means and without a mechanical

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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refocusing stage. Using holography, the depth of investigation can therefore be considerably
extended.
By having the phase information of the object of interest, the captured image can also
be visualized in 3- D (Fig. 3), and, if taken over a length of time, also in 4-D, in contrast to
images captured with classical microscopy only. In addition, DHM technology is quantitative
– optical path changes can be quantitatively detected with nanometric precision. And such
changes in optical density can also be utilized for viability assays. In their life sciences
applications, OVIZIO are able to use differences in the phase information of cells to screen
for viability of cells, or, for example, to screen cervical cells for cancer.OVIZIO has recently
progressed in its implementation of newer, more powerful holographic technology using an
approach of differential digital holographic microscopy (the ‘D
3
HM’ technology) within its
current systems, including the ‘oLine’. This allows the instrument to maintain its advantages
such as increased depth of focus, ability to refocus post acquisition as well as quantitative
phase imaging capabilities, but additionally dramatically improves the stability and flexibility
of the system. For example, a larger variety of sampling vessels may now be used with the
D
3
HM technology since laser alignment has become obsolete. For future devices, the ‘D
3
HM’
technology will enable us to downsize the size and weight of the device by a factor of three.
In the context of cell enumeration, the D
3
HM allows automated analysis providing not only
information on total cell numbers but additionally capturing actual images of the cells. Further
features such as morphologic parameters including diameter, perimeter and circularity, to
name a few, are also provided.
These improvements have dominated OVIZIO’s recent developments so that the
implementation of fluorescence needed for ballast water compliance testing by allowing live-
dead differentiation with fluorescent staining has not yet been included.

APPENDIX 5 QUESTIONNAIRE BASED DETAILED SYSTEM CHARACTERISTICS

Characteristics AccuriC6 Attune CantoII Cytobuoy FACSVerse OVIZIO oLineD
3
HM
Briefdescriptionof
workingprinciple
Flowcytometry,fixed
opticalbench,blue
laser
Flowcytometry
withacousticfocus,
twolasers,
measuresupto6
colours
Flowcytometry,488
nm,bluelaser
Flowcytometry,blue
laser,images
Flowcytometry,up
tothreelasers
Differentialdigital
holographic
microscopy
Imagestaken? No No No Yes No Yes
Weight 14kg 27 kg Ca.100kg 20kg 40kg 35kg
Sizeoftool
(approx.widthxheightx
depth)
Instrument28x38x
42cm,Fluidicsbottle
tray25x32x11cm
Instrument60x50
x50cm
Instrument60x60x
100cm
Instrument30x80x
30cm
Instrument70x70x

70cm
33x70x55cm
Sizewhenpackedfor
shipment(approx.length
xheightxdepth)
55x65x80cm 80 x 85 x100 cm Notportable 50x100x50cm Notportable 45x85x70cm
Robustnessfortransport +++ ++ Notportable ++ Notportable ++
Timetoset‐upsystem 30mins 120mins 60mins‐ 10mins 20mins 20mins
Timetoaresultonce
sampleprocessinghas
started
10minsfor1ml
sample
10minsforupto1
mlsample
<1minute,10
–
100µl
perminute
10minsfor1ml
sample
10minsfor1ml
sample
5‐10 minsfor1ml
sample
Externalpowersupply
needed?
Yes Yes Yes No Yes Yes
Externalcomputer
needed?
Yes Yes Yes Yes Yes Yes
Maximumwatervolume
analysedperminute
100µlperminute 1 ml perminute 100µlperminute 600µlperminute 120µlperminute 200µlperminutein
staticmode,500µl
perminuteinflow
mode
Requiredoperator
education
Technician Technician Technician Technician Technician Technician
Requiredresult
interpretereducation
Technicianwithspecial
training
Technicianwith
specialtraining
Technicianwithspecial
training
Technicianwith
specialtraining
Technicianwith
specialtraining
Technicianwith
specialtraining
Canitbeoperatedon
boardavessel(ordoesit
Yes Yes Yes Yes Yes Yes

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells
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Characteristics AccuriC6 Attune CantoII Cytobuoy FACSVerse OVIZIO oLineD
3
HM
needacleanlaboratory
environment)?
Particlesizelimitfor
analysis(upperandlower)
0.5µm
–
45µm 1µm
–
45µm 0.1µm
–
50µm 0.5µm
–
100µmand
above
0.1µm
–
50µmand
above
Lowerlimit2
–
10
µm,upperlimitas
perflowcell
dimensions,upto
200µm
Minimumdimension
measurement
Needfurther
developmentfornon‐
sphericalobjects
Needfurther
developmentfor
non‐spherical
objects
Needfurther
developmentfornon‐
sphericalobjects
Needfurther
developmentfor
non‐sphericalobjects
Needfurther
developmentfor
non‐sphericalobjects
Needfurther
developmentfor
non‐sphericalobjects
Abilitytodistinguish
betweenbiologicaland
sedimentordetritus
(fractionsofdeadplants)
material
Yes,withstains,FDA Yes,e.g.withstains Yes,withoutstaining
forphytoplankton,
withstainsviruses
(SybrGreen),bacteria
(PicoGreen),
zooplankton(FDA)
Yes,
autofluorescence
Yes,withstains,e.g.
FDA
Yes toacertain
extentwithphase
information
scattering
Abilitytoidentifybacteria Yes,withstain Yes Yes,withstain
(PicoGreen)
Yes Yes Yes(notinwork
focus)
Abilitytodistinguish
live/deadbacteria
Yes,withPropedium
Iodite(PI)deadstain,
optimizationneeded
Yes,e.g.with
CMFDA,Syto9/PI
(PropipiumIodite)
Yes,(deadbacteria
withSytoxGreen)
Yes,withastaine.g.
PicoGreen
Yes,withviability
stain
Yes,withstaine.g.
Acridineorange
(differentinstrument
model)
Abilitytoidentifybacteria
colonyformingunits
No No No No No No(butcan
documentgrowthof
bacteriaduring
culture)
AbilitytoidentifyIMO
indicator“microbes”
(Enterococci,E.coliand
Vibriocholerae)
Yes,FISHwithspecies
specificmarkers
Yes,FISHwith
speciesspecific
markersordies
Yes,FISHwithspecies
specificVibriosp.stain
Yes,FISHwith
speciesspecific
markers
Yes,FISHwith
speciesspecific
markers
No
Abilitytoidentify
phytoplankton
Yes Yes Yes Yes Yes Yes
Abilitytodistinguish Yes,stainingwithFDA, Yes,stainingwith Yes,stainingwith Yes, Yes,stainingwith Yes,stainingwith

NIOZFlowCytometerWorkshop,Comparinginstrumentsinmeasuring2‐10µmand10‐50µmplanktoncells

Page66of66
Characteristics AccuriC6 Attune CantoII Cytobuoy FACSVerse OVIZIO oLineD
3
HM
live/deadphytoplankton CMFDA CMFDA SytoxGreen
(FDA/CMFDAmethod
indevelopment)
autofluorescense,
CytoxGreen
FDA,CMFDA FDA,CMFDA
(differentinstrument
model)
Sizelimitfor
phytoplanktonanalysis
Yes,0.5
–
45µm Yes,1µm
–
45µm Below50µm Yes,0.5
–
100and
above
Yes,0.5
–
50µm Yes,2
–
200µm
Abilitytoidentify
zooplankton
Yes Yes Yes Yes Yes No
Abilitytodistinguish
live/deadzooplankton
Yes,withFDAstain Yes,withusinga
dye
Yes,withFDAstain No Yes,withFDAstain No
Sizelimitforzooplankton
analysis
Below45µm Below45µm Below50µm Below100µm Below50µm No
Adjustmentprocedureto
changetooltoenable
bacteria,phyto‐and
zooplanktonanalysis
<1min <1min Ca.1min <1min <1min Forbacteriaanalysis
withdarkfield,notin
workfocus
Capitalcost 35,000€ 88,200€ 170,000€ 55,000€ >84,000€withone
laserandwithout
loader,upto186,000
€includingthree
lasers,loaderand
flowcensor
65,000€(new
development
plannedfor25,000€)
Costpersample <1€(consumables,
stain,poweretc)
Peristaliticpumpneeds
replacementevery2
months=1000€/year
(costsincludefilters
andsolutions).
<1€(consumables,
stain,poweretc)
<5€(consumables,
stain,poweretc)
<5€(consumables,
stain,poweretc)
<5€
(consumables,
stain,power,daily
QCwithbeadsetc)
<1€(consumables,
stain,poweretc)
Didyouprocessballast
watersamplesyet?
Yes No Yes No Yes Yes
