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AQUATIC BIOLOGY
Aquat Biol
Vol. 25: 39– 52, 2016
doi: 10.3354/ab00662 Published August 10
INTRODUCTION
Live/dead analysis of organisms in disinfected
water, such as from ballast water treatment systems
(BWTS) or aquaculture-, drinking-, and wastewater
applications, is crucial to determine the efficiency of
the treatment technique. Disinfection can be per-
formed with chemical (e.g. chlorination, ozonation,
and electrolysis) and/or physical (e.g. ultraviolet
[UV] irradiation, heat, and cavitation) treatment
technologies (Shannon et al. 2008, Werschkun et al.
2012, 2014). Traditionally, water analysis used to
assess most treatment technologies has depended on
cultivation, such as the plate count or the most prob-
able number (MPN) technique. Cultivation methods
measure viability of organisms present, i.e. the abil-
ity of a cell to reproduce. Vital (live) cells can be
either viable or non-viable, whereas non-vital (dead)
© The authors 2016. Open Access under Creative Commons by
Attribution Licence. Use, distribution and reproduction are un -
restricted. Authors and original publication must be credited.
Publisher: Inter-Research · www.int-res.com
*Corresponding author: ingunn.hoell@hsh.no
Dual staining with CFDA-AM and SYTOX Blue
in flow cytometry analysis of UV-irradiated
Tetraselmis suecica to evaluate vitality
Ranveig Ottoey Olsen1, Ole-Kristian Hess-Erga2, Aud Larsen3,
Friederike Hoffmann4, 5, Gunnar Thuestad1, Ingunn Alne Hoell1,*
1Stord/Haugesund University College, Bjoernsonsgt. 45, 5528 Haugesund, Norway
2Norwegian Institute for Water Research, Thormoehlensgt. 53 D, 5006 Bergen, Norway
3Uni Research Environment and Hjort Centre for Marine Ecosystem Dynamics, 5006 Bergen, Norway
4Uni Research Environment, Thormoehlensgt. 49 B, 5006 Bergen, Norway
5University of Bergen, PO Box 7800, 5020 Bergen, Norway
ABSTRACT: After disinfection of ballast water, it is crucial to detect organisms and determine
their vitality to assess the performance of the chosen treatment technique. Ultraviolet (UV) irradi-
ation is a treatment technology commonly used for water disinfection. In this study, the phyto-
plankter Tetraselmis suecica was UV irradiated and subsequently stained with both 5-carboxyflu-
orescein diacetate acetoxymethyl ester (CFDA-AM) and SYTOX Blue, staining metabolically
active and membrane-permeable cells, respectively. This dual staining protocol can be used to
assess samples during type approval of UV-based treatment systems. Non-irradiated and UV-irra-
diated samples were incubated in darkness, to simulate a ballast water transport, after which the
vitality and viability T. suecica were monitored regularly over a period of 15 d. Flow cytometry
(FCM) analysis separated the cells into 4 FCM populations (=single cells grouped together based
on their fluorescence signals) according to differences in esterase activity and membrane
integrity. UV-irradiated samples followed a different staining pattern compared to non-irradiated
samples, where 1 specific FCM population of cells expressed esterase activity, but at the same
time gave signals for disrupted membranes. This is useful as a sign of future death and is inter-
preted as an ‘early warning’ FCM population. FCM results were also compared to corresponding
plate count results, differentiating vital, viable cells from vital, non-viable cells. We argue that
dual staining with SYTOX Blue and CFDA-AM facilitates and improves FCM analysis when
evaluating the performance of UV-based water treatment systems.
KEY WORDS: Phytoplankton · Ballast water · Water treatment · Live/dead analysis · Viability ·
Water analysis
O
PEN
PEN
A
CCESS
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Aquat Biol 25: 39– 52, 2016
cells are non-viable. Cultivation methods can be
time-consuming and may give false negatives, as
several species are unable to grow under laboratory
conditions, or they can be in a viable but non-cultur-
able state (Staley & Konopka 1985, Roszak & Colwell
1987). Problems related to cultivation methodology
can be circumvented by studying populations and
communities indirectly, e.g. by determination of bio-
logical material or activity using flow cytometry
(FCM), epifluorescent microscopy, photometer, lumi-
nometer, or DNA/RNA based methods. FCM facili-
tates rapid detection, enumeration, and characteriza-
tion of organisms when combined with fluorescent
dyes. Fluorescent dyes are molecules that label
microbes or cellular constituents according to their
biochemical, physiological, or taxonomic characteris-
tics, and that can fluoresce upon light excitation
(Shapiro 1983, 2000, Spence & Johnson 2010).
Staining technology for live/dead evaluation of
cells is largely based on 2 physiological characteris-
tics: membrane integrity and metabolic activity.
Commonly used stains to assess membrane integrity
are propidium iodide (PI) and SYTOX Green (Veld-
huis et al. 1997, Berney et al. 2007, Peperzak & Brus-
saard 2011). These non-fluorescent stains enter cells
when membrane integrity is lost and fluoresce when
bound to nucleic acids, while they are excluded from
cells with intact membranes. Permeabilized cells are
usually characterized as dead because they are un -
able to maintain the membrane potential and hence
their intracellular environment (Joux & Lebaron
2000, Kroemer et al. 2009, Hammes et al. 2011). The
Nomenclature Committee on Cell Death has pro-
posed criteria to evaluate a cell as dead; one of these
is loss of cell membrane integrity (Kroemer et al.
2009). Esterases are cellular housekeeping enzymes
indicative of the presence of metabolic activity.
Esterase substrates, like fluorescein diacetate (FDA)
and carboxyfluorescein diacetate (CFDA), can be
used to evaluate this activity (Bentley-Mowat 1982,
Dorsey et al. 1989). The non-fluorescent substrate
freely diffuses across membranes and is hydrolyzed
by unspecific intracellular esterases. The fluorescent
product is retained in cells with intact membranes,
and the fluorescence intensity is correlated with the
metabolic activity. Esterase substrates are commonly
used in FCM studies of phytoplankton (Garvey et al.
2007, Steinberg et al. 2011, Gorokhova et al. 2012,
Peperzak & Gollasch 2013, Olsen et al. 2015).
Previously, we developed an FCM protocol to
discriminate dead Tetraselmis suecica from live and
UV damaged cells (Olsen et al. 2015). Prior to FCM
analysis, the alga was stained with CFDA-acetoxy -
methyl ester (CFDA-AM), which is hydrolyzed to
carboxyfluorescein in esterase active cells. When UV
irradiated, some T. suecica cells still remained
esterase active even though their viability was lost,
as has also been observed for bacteria and yeast
(Schenk et al. 2011, Kramer & Muranyi 2014).
Staining with 2 or more fluorescent dyes can facili-
tate measurements of multiple cellular characteris-
tics simultaneously. When combining FDA and PI,
cells are stained according to their enzyme activity
and their permeability, respectively. For UV-C treated
Listeria innocua, combining FDA and PI stains
revealed a subpopulation that retained esterase
activity at the same time that the membrane integrity
was lost (Schenk et al. 2011). PI has an emission max-
imum (636 nm) in the same area as chlorophyll when
excited with a blue (488 nm) laser; hence PI fluores-
cence cannot be detected in the same detector as
chlorophyll-containing organisms (phytoplankton).
Yet, high concentrations of PI (7.5−120 µM) have
been applied to Microcystis aeruginosa and diverse
algal species in some studies, detecting orange fluo-
rescence in the 560−600 nm area (Franklin et al.
2001, Xiao et al. 2011). However, it is not feasible to
use PI and esterase substrates simultaneously, since
emission partly overlaps. SYTOX Green (emission
maximum 523 nm) has previously been used to
assess UV-irradiated phytoplankton (Martínez et al.
2012, Steinberg et al. 2012), but its fluorescence
interferes with fluorescence from CFDA-AM (emis-
sion maximum 535 nm) and combining the two is thus
not useful. Instead, SYTOX Blue (emission maximum
480 nm), which enters cells with permeabilized mem-
branes and binds to nucleic acids just like SYTOX
Green, was combined with CFDA-AM. Together
they facilitate separation of the fluorescence signals
based on esterase activity and membrane integrity.
Using UV irradiation as a disinfection technology
can induce structural changes to the genetic material
in cells and cause impairment of replication (Sinha &
Häder 2002). UV irradiation can also affect other bio-
logical molecules and induce formation of reactive
oxygen species (ROS) in bacteria and algae, causing
oxidative damage to lipids, proteins, and DNA (Kalis-
vaart 2001, Bosshard et al. 2010, Kottuparambil et al.
2012, Santos et al. 2012, 2013, Kramer & Muranyi
2014). As a response, organisms have various mech-
anisms to prevent and counteract the damage, in -
cluding DNA repair mechanisms (Sinha & Häder
2002). Such responses, which occasionally cause
damaged cells to recover after treatment, lead to
challenges for viability evaluations. Moreover, when
analyzing disinfected water, the terms describing
40
Olsen et al.: Flow cytometry analysis of Tetraselmis suecica
cellular status can cause confusion, e.g. ‘active’/
’inactive’, ‘live’/’dead,’ and ‘viability.’ For instance,
since metabolic activity in a cell varies depending on
cellular condition, the metabolic activity in a cell
is correlated with the fluorescence intensity from
esterase substrates, and the fluorescence signal will
vary accordingly (Bentley-Mowat 1982, Dorsey et al.
1989, Murphy & Cowles 1997, Brookes et al. 2000).
Membrane permeability can be reversed or may just
occur as a temporary condition (Shi et al. 2007),
which can cause difficulties for vitality indications
when using stains to assess membrane integrity, like
PI and SYTOX Blue. Moreover, for ballast water
discharge, the International Maritime Organization
(IMO) Convention refers to the ‘viable’ organisms
(IMO 2004); however, the guidelines for approval
of ballast water management systems (G8) define
viable organisms as ‘organisms and any life stages
thereof that are living’ (IMO 2008, p.7). The US Coast
Guard (USCG) also make use of the term ‘living’
(USCG 2012).
The aim of the study was to improve our previously
developed FCM protocol (Olsen et al. 2015) to evalu-
ate the performance of UV-based water treatment
techniques and overcome the challenges described
above. In order to do so, we aimed at answering the
following questions:
(1) Will dual staining of T. suecica using CFDA-AM
and SYTOX Blue yield fluorescent signals based on
esterase activity and membrane integrity suitable for
differentiating cells into FCM populations reflecting
cellular vitality?
(2) Can the fluorescent signals from dual-stained
cells (SYTOX Blue and CFDA-AM) be used to pre-
dict future death?
(3) Is dual staining with SYTOX Blue and CFDA-
AM recommendable for evaluation of live and viable
T. suecica for ballast water monitoring in compliance
with the IMO and USCG regulations?
MATERIALS AND METHODS
Experimental set-up
The phytoplankter Tetraselmis suecica (Strain K-
0297) was obtained from the Scandinavian Culture
Collection of Algae and Protozoa (University of Copen -
hagen, Denmark) and cultured in 36 g kg−1 artificial
sea water (ASW) (Marine SeaSalt) supplemented
with 0.12% Substral (The Scotts Company [Nordic]).
Incubation was performed in light at 36 W m−2 (corre-
sponding to 173 µmol m−2 s of photosynthetically
active radiation photons) at 15°C and 1.7 cm orbital
shaking at 100 rpm. The culture was further diluted
with growth medium to a concentration of 104cells
ml−1 prior to UV treatment. The cell concentration
was detected by FCM.
To inactivate the algae, a collimated medium pres-
sure (MP; 800 W) UV lamp (BestUV) with a polychro-
matic (200−400 nm) mercury lamp was used (Olsen
et al. 2015). The UV-C intensity was 0.525 mW cm−2,
and the weighted average germicidal factor, based
on the absorbance spectrum of DNA from Bacillus
subtilis (Chen 2009), was 0.5799.
A 45 ml aliquot of diluted T. suecica was irradiated
in a petri dish while being mixed with a magnetic stir
bar (150 rpm) at room temperature (RT). The expo-
sure times were 180, 359, 539, 718, and 1436 s for UV
doses 100, 200, 300, 400, and 800 mJ cm−2, respec-
tively. The lowest UV dose is comparable to those
used in BWTS, but ballast water is treated twice (on
uptake and discharge). UV doses between 400 and
800 mJ cm−2 were not applied, since previous FCM
studies showed that CFDA-AM-stained T. suecica
UV irradiated with 400 mJ cm−2 were permanently
inactivated (Olsen et al. 2016). Doses of 400 and
800 mJ cm−2 were both included in the study to
examine whether and how esterase activity and mem -
brane integrity differed in these samples. Three sub-
samples were prepared for each UV dose, irradiated
in 2 h intervals, and subsequently transferred to
50 ml Falcon conical centrifuge tubes (Fisher Scien-
tific). Non-irradiated cells (controls, 2 × 45 ml) and
dead cells (8.5 ml) fixed with formaldehyde (5% final
concentration; 36.5−38%, Sigma-Aldrich) were also
transferred to tubes. All tubes were wrapped in alu-
minum foil and incubated with loosened lids at 15°C
in the dark to simulate a dark ballast water transport
up to 15 d, with samples collected at several time
points.
Analysis
The cells were stained with the esterase substrate
CFDA-AM (C1354, Thermo Fisher Scientific) and
SYTOX Blue Dead Cell Stain (S34857, Thermo
Fisher Scientific) prior to FCM analyses. Aliquots of
10 and 1 mM CFDA-AM and SYTOX Blue, respec-
tively, in dimethyl sulfoxide were kept at −20°C.
Work solutions of CFDA-AM (1 mM) and SYTOX
Blue (0.3 mM) were prepared fresh each day by
diluting with Milli-Q water. The samples were first
stained with CFDA-AM to a final concentration of
2000 nM, and incubated for 50 min at RT under dark
41
Aquat Biol 25: 39– 52, 2016
conditions (Ganassin et al. 2000, Olsen et al. 2015).
Although no data addressing the toxicity of SYTOX
Blue was available from the manufacturer (Molecular
Probes), they recommended staining in the range of
625 to 10 000 nM. Optimal concentration and incuba-
tion time for SYTOX Blue was therefore determined
in advance; SYTOX Blue was added to the samples
at a final concentration of 2000 nM and further incu-
bated for 10 min at RT in the dark. Note that SYTOX
Blue was added last (after CFDA-AM) to avoid po -
tential harmful effects. The samples were analyzed
by FCM immediately after the incubation period
(60 min).
FCM analysis was performed on an Attune Acoustic
Focusing Cytometer (Thermo Fisher Scientific) equip -
ped with a 20 mW 488 nm (blue) laser exciting
CFDA-AM and chlorophyll a (chl a), and a 50 mW
405 nm (violet) laser exciting SYTOX Blue. The BL1
(530/30), VL1 (450/40), and BL3 (640LP) detectors
were used for detection of green, blue, and red fluo-
rescence from CFDA-AM, SYTOX Blue, and chl a,
respectively. Additionally, forward and side scatter
from the blue laser were detected. The trigger was
set to red fluorescence (BL3 detector), the threshold
to 60 000, and the voltage of the forward and side
scatter, BL1, VL1, and BL3 detectors to 300, 1300,
1100, 1200, and 1300, respectively. Compensation is
the mathematical method used to correct the overlap
of one fluorophore’s emission into another fluoro -
phore’s emission channel. However, emission of
CFDA-AM into the VL1 channel, and SYTOX-Blue
into the BL1 channel, were both low, and compensa-
tion corrections were therefore not performed. Emis-
sion overlap did not occur because the stains were
excited with different lasers and have separate emis-
sion areas. One ml of each sample was analyzed at
a flow rate of 1000 µl min−1 at standard sensitivity.
Control samples included were: (1) non-irradiated
cells both unstained and dual stained and (2) dead
cells killed using formaldehyde followed by the dual
staining procedure. The dead stained cells were not
expected to fluoresce either green or blue, but red.
The cells were not esterase active or permeabilized,
but were previously shown to retain their red fluores-
cence 24 d after formaldehyde treatment (Olsen et al.
2015).
In order to determine culturability (i.e. viable cells)
by the number of colony-forming units (cfu), samples
were cultured on 1% Bacto™ Agar (Difco Laborato-
ries, Becton-Dickinson) in 24 ppt ASW supplemented
with 0.12% Substral. The agar plates were incubated
in light at 36 W m−2 at 15°C for 3 wk. The number
of cfu for each plate was determined, and the mean
values of the 3 replicates were calculated. Regression
analysis for plate counts and FCM results were per-
formed in Microsoft Excel 2010.
FCM and plate counts were performed on Days 0
(4 h), 1, 2, 3, 4, 7, 10, and 15 after dark incubation. At
the beginning of the experiment, the volume of each
sample was 45 ml. On each analysis day, a total of
3.6 ml from each sample were removed for FCM ana -
lysis and plate counts. At the end of sampling, the
volumes of each sample were reduced from 45 to 16 ml.
RESULTS
FCM analysis of dual-stained Tetraselmis suecica
The FCM signals from T. suecica cells unstained
and dual stained with CFDA-AM and SYTOX Blue
are presented as dot plots and separated into quad-
rants (Q1, Q2, Q3, Q4) by a vertical and a horizontal
line that was set by eye, based on the blue and green
fluorescence intensity from the cells (Fig. 1). Cell
signals with low green fluorescence (no CFDA-AM
fluorescence) remained below the horizontal line, as
seen in the non-irradiated, unstained cells (Fig. 1b),
and the sample UV irradiated with 800 mJ cm−2
(Fig. 1e). In contrast, CFDA-AM-stained cells with
elevated green fluorescence appeared above the
horizontal line, as shown in the non-irradiated,
stained cells (Fig. 1a), and the sample UV irradiated
with 200 mJ cm−2 (Fig. 1d). Similarly, the vertical line
separates cells with low blue fluorescence (no SYTOX
Blue fluorescence; Fig. 1a,b) from cells with elevated
blue fluorescence (Fig. 1d). Interestingly, the sample
UV irradiated with 200 mJ cm−2 appeared as 2 FCM
populations located above the horizontal line, but on
both sides of the vertical line (Fig. 1c)
In general, cell signals from the non-irradiated,
stained cells appeared in Q1 (Fig. 1a), whereas the
non-irradiated, unstained cells appeared in Q3
(Fig. 1b). Also, UV-irradiated stained cells with
remaining esterase activity appeared in Q1 and Q2
(Fig. 1c), while UV-irradiated stained cells without
esterase activity appeared in Q4 and Q3 (Fig. 1d).
Table 1 summarizes how intersections in the dot plots
separate signals into 4 quadrants based on the stain-
ing characteristics of the cells.
From live via damaged to dead cells
Dot plots (Fig. 2) show a systematic progression of
FCM signals as a function of time spent in the dark of
42
Olsen et al.: Flow cytometry analysis of Tetraselmis suecica
non-irradiated, stained cells and cells irradiated with
increasing UV doses. For the non-irradiated, stained
cells most signals initially appeared in Q1 (live),
gradually appearing in Q3 (dead) and further to Q4
(dead) during incubation, as a general trend. A few
signals from single cells were observed in Q2 as well;
however, these signals did not form a separate FCM
population. Signals from the UV-irradiated samples
followed a different pattern. This was already evi-
dent on Day 0, where signals appeared in Q2 after
having been treated with the low UV doses and in Q4
after having irradiated the cells with the highest UV
doses. The cells displayed a similar response to dark
incubation; initially signals appeared in either Q1 or
Q2 (depending on UV dose). During incubation, the
signals disappeared from Q1/Q2 and appeared in
Q2/Q4, respectively, and then signals entered into
Q4. These trends are quantified in Fig. 3, showing
the percentage of the FCM populations located in the
different quadrants in the dot plots for the non-
irradiated, stained cells and each of the UV-irradi-
ated samples, as a function of time. The total amount
of signals detected was 100%. FCM signals from for-
ward and side scatter remained in the same position
in dot plots throughout the incubation period for all
controls and UV-irradiated samples (data not shown).
Red autofluorescence from chlorophyll was not re -
duced by high UV doses. However, chlorophyll fluo-
rescence decreased during dark incubation, both for
the non-irradiated and UV-irradiated samples. As the
trigger on the flow cytometer was set to red fluores-
cence, the autofluorescence was still high enough so
the cells could be detected and analyzed.
Some interesting observations were made for the
UV-irradiated samples. (1) During incubation, a FCM
population emerged in Q3, and was most prominent
for samples treated with 300 and 400 mJ cm−2 (Fig. 2).
This FCM population increased during dark incuba-
tion. (2) Some of the cells treated with low UV doses
(100−200 mJ cm−2) exhibited elevated green fluores-
cence intensity in Q1 compared to the non-irradiated,
stained cells (Fig. 2). (3) The most noteworthy obser-
vation is that the FCM population in Q2 (severely
damaged) observed in UV-treated samples, was hardly
present in the non-irradiated, stained cells. This FCM
population (Q2) increased as a function of UV dose
until reaching a dose of 800 mJ cm−2, where cells died
almost immediately and appeared in Q4. For lower
43
CFDA-AM
a) Non-irradiated,
stained, day 0
b) Non-irradiated,
unstained, day 0
d) 800 mJ cm–2,
stained, day 7
c) 200 mJ cm–2,
stained, day 0
102
104
106
10210410210410 210 410 210 4
SYTOX Blue
Q1 Q2
Q3 Q4
Q1 Q2
Q3 Q4
Q1 Q2
Q3 Q4
Q1 Q2
Q3 Q4
Fig. 1. Flow cytometry dot plots of Tetraselmis suecica cell signals plotted as coordinates of blue (SYTOX Blue) and green
(CFDA-AM) fluorescence intensity for (a) non-irradiated, stained cells analyzed after 4 h of dark incubation, (b) non-irradi-
ated, unstained cells analyzed after 4 h of dark incubation, (c) UV dose 200 mJ cm−2 analyzed 4 h after UV irradiation and dark
incubation, and (d) UV dose 800 mJ cm−2 analyzed 7 d after UV irradiation and dark incubation. Intersections in the dot plots
separate signals into 4 quadrants (Q1−Q4) based on the staining characteristics of the cells
Quadrant CFDA-AM SYTOX Blue Physiological characteristics Vitality
Q1 + − Esterase active; membrane intact Live
Q2 + + Esterase active; membrane damaged Severely damaged
Q3 − − Esterase inactive; membrane intact or DNA/RNA degraded Dead
Q4 − + Esterase inactive; membrane damaged Dead
Table 1. Overview of the quadrants (Q1−Q4) in response to elevated (+) or low (−) fluorescence intensity when cells were
stained with CFDA-AM and SYTOX Blue. The quadrants reflect various physiological characteristics and vitality
Aquat Biol 25: 39– 52, 2016
44
100 mJ cm–2 200 mJ cm–2 300 mJ cm–2 400 mJ cm–2 800 mJ cm–2
0
1
3
4
7
15
2
SYTOX Blue
CFDA-AM
Samples:
10
Day:
Non-irradiated
102
104
106
102
104
106
102
104
106
102
104
106
102
104
106
102
104
106
102
104
106
102
104
106
102104102104102104102104102104102104
Fig. 2. Flow cytometry dot plots of Tetraselmis suecica stained with SYTOX Blue and CFDA-AM. Treatments (non-irradiated
and various UV doses) are shown in the vertical columns and time of dark incubation in the horizontal rows. Intersections in
the dot plots separate signals into 4 quadrants (see Fig. 1)
Olsen et al.: Flow cytometry analysis of Tetraselmis suecica
UV doses, a similar pattern was observed during dark
incubation; initially, the Q2 population increased,
and when the cells died, the Q4 population increased
(and Q2 was reduced accordingly).
Plate count versus FCM
Parallel to the FCM analysis, all samples were ana-
lyzed by plate count, as shown in Table 2. Although
the number of reproducible cells (i.e. viable by defi-
nition) in the non-irradiated samples was reduced by
96% after 15 d of dark incubation, some cells
remained viable during the entire dark incubation
period. The logarithmic concentration of culturable
cells (as determined by cfu) was plotted versus time
of dark incubation, and regression analyses were
performed (Fig. 4a).
FCM signals appearing in Q1 are described as live
(Table 1), although live cells can either be viable or
non-viable. Therefore, in an attempt to quantify the
number of viable cells in the FCM dot plot, the FCM
45
Days of dark incubation
Percentage of cell signals in each quadrant
a) Non-irradiated
0%
20%
40%
60%
80%
100%
0 2 4 6 8 10 12 14
0%
20%
40%
60%
80%
100%
0 2 4 6 8 10 12 14
0%
20%
40%
60%
80%
100%
02468101214
0%
20%
40%
60%
80%
100%
02468101214
0%
20%
40%
60%
80%
100%
02468101214
0%
20%
40%
60%
80%
100%
02468101214
b) 100 mJ cm–2
c) 200 mJ cm–2 d) 300 mJ cm–2
e) 400 mJ cm–2 f) 800 mJ cm–2
Q1
Q2
Q3
Q4
Fig. 3. Percentage of cell signals after dark incubation from each quadrant for (a) the non-irradiated samples and (b−f) the
samples UV treated with (b) 100, (c) 200, (d) 300, (e) 400, and (f) 800 mJ cm−2. Data are means of 3 replicates and error bars
indicate ±1 SD. Q1−Q4 refer to the quadrants described in Fig. 1
Aquat Biol 25: 39– 52, 2016
cell signals were compared with the number of cfu
(Fig. 4a). As previously described (Olsen et al. 2015),
we defined a gate (= a collection of single cell signals)
in the FCM dot plot based on the non-irradiated,
stained sample (Fig. 5). The gate (Fig. 5) corresponds
to the number of culturable cells determined by plate
counts, and signals outside the gate originate from
non-culturable cells. The logarithmic concentration
of gated signals from FCM were plotted against time
of dark incubation. Regression analyses were per-
formed (Fig. 4b), and plate count regression (Fig. 4a)
corresponded with FCM regression.
46
–——Non-irradiated—–— ————–—100—–———— —————200 ———— ———300——— ——400 —— ——800——
Day FCM Cfu FCM Cfu FCM Cfu FCM Cfu FCM Cfu FCM Cfu
0 12016 (±824) 14133 (±2309) 15039 (±1254) 13600 (±608) 7562 (±1894) 1800 (±173) 136 (± 31) 13 (±15) 19 (± 20) <10 <10 <10
1 12768 (±705) 15067 (±2695) 15244 (± 360) 1160 (±259) 4010 (±233) 167 (±115) 9 (± 6) <10 <10 <10 <10 <10
2 13011 (±1184) 12467 (±2074) 14678 (± 749) 20 (±10) 3132 (±983) 33 (± 58) <10 <10 <10 <10 <10 <10
3 13454 (±191) 6400 (±1709) 12179 (±1583) 7 (±6) 1932 (±1219) <10 <10 <10 <10 <10 <10 <10
4 12001 (±628) 2667 (±379) 8517 (± 4171) <10 751 (±757) <10 <10 <10 <10 <10 <10 <10
7 1463 (± 237) 600 (± 361) 572 (±498) <10 18 (±14) <10 <10 <10 <10 <10 <10 < 10
10 579 (±163) 400 (±173) 51 (± 23) <10 <10 <10 <10 <10 <10 <10 <10 <10
15 343 (±113) 533 (±351) <10 <10 <10 <10 <10 <10 <10 <10 <10 <10
Table 2. Numbers of flow cytometry (FCM) gated signals and cfu for the non-irradiated and UV-irradiated samples (100−800 mJ cm−2)
analyzed during dark incubation. Results are in cells ml−1. Data are the means (±1 SD) of 3 replicates
a) Plate count
b) FCM
–1
0
1
2
3
4
5
0 2 4 6 8 10 12 14
–1
0
1
2
3
4
5
02468101214
Non-irradiated: y = –0.1193x + 4.0968
R² = 0.8026
P-value = 2.6 x 10-3
100 mJ cm–2 : y = –1.0508x + 3.9661
R² = 0.963
P-value = 2.01 x 10-2
200 mJ cm–2 : y = –0.9943x + 3.2007
R² = 0.9685
P-value = 2.85 x 10-2
Non-irradiated y = –0.1267x + 4.2866
R² = 0.898
P-value = 1.3 x 10-4
100 mJ cm–2 y = –0.2713x + 4.6095
R² = 0.9634
P-value = 1.6 x 10-5
200 mJ cm–2 y = –0.3264x + 3.9681
R² = 0.9558
P-value = 2.7 x 10-5
Days of dark incubation
Log (cfu ml–1) Log (gated signals ml–1)
Fig. 4. Logarithmic declines in cell concentration of cfu ml−1 from (a) plate counts and (b) the number of gated signals from flow
cytometry, for the non-irradiated samples (n) and samples that were UV irradiated with 100 (h) and 200 (s) mJ cm−2, during
dark incubation. Data are the means of 3 replicates. The regression equations, R2, and p-values for the non-irradiated and UV-
irradiated samples are given. The blue line indicates when the concentration of Tetraselmis suecica is <10 cells ml−1 and in
compliance with Regulation D-2 (IMO 2004)
Olsen et al.: Flow cytometry analysis of Tetraselmis suecica
The gate was also applied to UV-irradiated sam-
ples to evaluate whether the FCM results correlated
with the number of reproducible cells (cfu). For the
samples UV irradiated with 100 and 200 mJ cm−2, the
gated FCM signals were always higher than the cfu
(Fig. 4a,b, respectively, and Table 2). For samples
treated with UV doses ≥300 mJ cm−2, few or no cfu or
gated signals were detected during the entire dark
incubation period (Table 2).
DISCUSSION
UV irradiation can cause cell damage and change
biochemical and/or physiological cellular character-
istics, which can be studied with fluorescent dyes.
We expected the FCM results from dual-stained
(CFDA-AM and SYTOX Blue), non-irradiated cells to
differ from dual-stained, UV-irradiated cells, since
inactivation with UV light can affect both the
esterase activity and membrane permeability in cells
(Schenk et al. 2011). The FCM results did, indeed,
demonstrate changes in these 2 physiological charac-
teristics, which allowed separation of Tetraselmis
suecica into 4 FCM populations after dual staining of
non-irradiated and UV-irradiated samples. The
quadrants and physiological characteristics reflect
the vitality, i.e. whether the cell is live, severely dam-
aged, or dead.
Similar observations have been de scribed for bacte-
rial populations dual stained with esterase substrates
(CFDA/ FDA) and PI, viz. in bile-salt-stressed bifi-
dobacterial cells (Ben Amor et al. 2002), ethanol-
stressed malolactic cells (da Silveira et al. 2002),
pressure-stressed Lactobacillus rhamnosus (Ananta
et al. 2004, 2005, Ananta & Knorr 2009), UV-C-irradi-
ated Listeria innocua (Schenk et al.
2011), and antimicrobial peptide-
exposed, pulsed electric field-
treated or ultrasound-treated E. coli
(Zhao et al. 2011, Hong et al. 2015).
In order to judge the usefulness of
such FCM results for BWTS treat-
ment assessment, it was necessary
to examine how various UV doses
and subsequent dark incubation
affected T. suecica.
UV irradiation can cause loss of
membrane in tegrity (Sobrino et al.
2004, Berney et al. 2006, Bosshard et
al. 2009), and permeabilized cells
are usually characterized as dead
(Joux & Lebaron 2000, Kroemer et
al. 2009, Hammes et al. 2011). The membranes of
Saccharomyces cerevisiae developed a transient per-
meability to PI during and immediately following
exposure to physical (heat) and chemical (ethanol)
stress, but then repaired the damage after a short
incubation period (Davey & Hexley 2011). However,
since yeast cells do not use light as an energy source,
light/dark conditions do not affect repair. Hence, it
was important to examine the further development of
the cell membrane damaged FCM populations (Q2)
in UV-irradiated T. suecica samples.
During dark incubation, the esterase activity of
the non-irradiated (live) cells decreased, and this
was later followed by a loss of membrane integrity.
Darkness affects the vitality/viability of phytoplank-
ton over time (Jochem 1999), since light limitation
de prives cells of the principal energy source for
maintenance, including the support of the integrity
of the plasma membrane (Berges & Falkowski
1998). Further, phytoplankton can undergo pro-
grammed cell death in response to environmental
stress (Bidle & Falkowski 2004, Franklin et al. 2006,
Bidle 2015), such as light deprivation (Berges &
Falkowski 1998, Segovia et al. 2003, Segovia &
Berges 2009). Loss of membrane integrity during
incubation can also be caused by natural degrada-
tion processes (Davey 2011). However, phytoplank-
ton have survival strategies when exposed to unfa-
vorable conditions (Jochem 1999, 2000, Hallegraeff
2010). For ballast water, the voyage duration affects
organism survival, although photosynthetic organ-
isms can survive several weeks in darkness (Peters
1996, Jochem 1999, Carney et al. 2011). Therefore,
ballast water needs to be treated.
The T. suecica samples treated with UV irradia-
tion showed a different pattern in FCM dot plots, as
47
CFDA-AM
a) Day 0 b) Day 3 c) Day 10
102
104
106
102104
SYTOX Blue
102104102104
Fig. 5. Flow cytometry dot plot for non-irradiated samples after (a) 0 d (4 h),
(b) 3 d, and (c) 10 d of dark incubation. The gate (box in quadrant 1) is based on
the number of cfu. Therefore, signals inside the gate correspond to live, viable
cells, whereas signals outside the gate correspond to live, non-viable cells
Aquat Biol 25: 39– 52, 2016
the cells lost their membrane integrity immediately
after treatment and as a response to the UV dose
applied. This indicates that even low UV doses
cause membrane disruption. High UV irradiation
may increase membrane permeability (Sobrino et
al. 2004), but also target plant membrane lipids,
proteins, and their complexes (Murphy 1983), affect
human cell membrane receptors inducing pro-
grammed cell death (Schwarz 1998, Kulms et al.
1999, Franklin et al. 2006), and/or enhance ROS
production, potentially causing oxidative damage to
lipids and impair membrane transport in bacteria
(Santos et al. 2012, 2013).
Reduction in esterase activity during dark incuba-
tion was greater for the UV-irradiated cells than non-
irradiated ones, as previously demonstrated (Olsen
et al. 2015). For the samples UV irradiated with 100
and 200 mJ cm−2, 2 FCM populations of cells with
esterase activity were observed (in Q1 and Q2), indi-
cating heterogeneity within the samples. It has been
argued that a decrease in fluorescence from carboxy-
fluorescein (CF, hydrolyzed esterase substrate) may
occur as a result of leakage due to a damaged mem-
brane rather than inactivated enzymes (Kramer &
Muranyi 2014), and that cells with severely damaged
membranes thus would reduce their cell size (Ou et
al. 2012). As CF has negative charges at physiologi-
cal pH (Breeuwer & Abee 2000), this may partially
inhibit its leakage from damaged cells, unless a cer-
tain degree of membrane degradation is exceeded.
However, our forward and side scatter results im -
plied that the cells maintained their size during the
analysis period. This indicates that membrane dam-
age did not cause leakage, and the observed de -
crease in green fluorescence is therefore most likely
caused by inactivated enzymes. CFDA-AM is there-
fore a good indicator to evaluate metabolic activity in
UV-irradiated T. suecica.
Interestingly, some cells irradiated with low UV
doses (100−200 mJ cm−2) showed higher green
fluorescence intensity (Q1) compared to the non-
irradiated cells. Stains can be actively secreted from
active organisms by an energy-dependent process,
as shown for efflux of fluorescein and fluorescein
derivatives in Lactococcus lactis and S. cerevisiae
(Molenaar et al. 1991, 1992, Breeuwer et al. 1994).
Because of the negatively charged CF, passive trans-
port is unlikely (Martin & Lindqvist 1975). UV light
can cause loss of membrane potential, often occur-
ring before loss of membrane integrity, and this can
cause loss of efflux pump activity, as shown in UV-A
irradiated and/or pulsed light-treated (UV) E. coli,
Salmonella typhimurium, and Shigella flexneri (Berney
et al. 2006, Bosshard et al. 2009, Kramer & Muranyi
2014). If efflux pumps are damaged by UV, CF might
accumulate to higher concentrations in UV-irradiated
cells compared to non-irradiated ones. Elevated green
fluorescence intensity from dual-stained cells treated
with low UV doses can thus be a sign of cellular dam-
age and reduced membrane potential.
An FCM population of UV-irradiated cells with low
green and blue fluorescence (Q3) emerged after
prolonged incubation. These cells demonstrated ele-
vated blue fluorescence initially, indicating that the
cells were already dead. The loss of blue fluores-
cence can be explained by degraded DNA/RNA
as part of the cellular degradation process, which
results in fewer/no possible binding sites for SYTOX
Blue (Davey 2011). Therefore, cells in Q3 and Q4 are
considered dead with damaged membranes in both
FCM populations. This further demonstrates that
SYTOX Blue is not an ideal stain to use as a single
dye in long-term studies of UV-irradiated cells, as
dead cells are not always detectable.
Our results show that UV irradiation caused a loss
of membrane integrity prior to the loss of esterase
activity. This is evident in the FCM population with
elevated green and blue fluorescence (Q2), as also
demonstrated in other studies with bacteria as target
organisms (Ben Amor et al. 2002, da Silveira et al.
2002, Ananta et al. 2004, 2005, Ananta & Knorr 2009,
Schenk et al. 2011, Zhao et al. 2011, Hong et al.
2015). The esterase substrate CFDA-AM was selected
since both IMO and USCG have performance stan-
dards for concentrations of living cells at ballast water
discharge (IMO 2004, USCG 2012). The esterase
activity in the severely damaged cells (Q2) was fur-
ther reduced during incubation, i.e. the cells were
injured to such a degree that they would eventually
die. Some studies have denoted FCM populations
containing esterase activity but with damaged mem-
branes, as sub-lethally injured cells (Ananta et al.
2004, Zhao et al. 2011, Hong et al. 2015). Conse-
quently, simultaneous green and blue fluorescent
signals can detect these sub-lethal/dying cells and by
this indicate future death.
The MPN method was recently rejected by the
USCG. It is not considered equivalent to the USCG
preferred vital stain method for the 10−50 µm size
class since it assesses the ability of an organism to
recolonize after treatment, and BWTS must be evalu-
ated based on their ability to kill certain organisms.
Growth-based methods, like plate counts and MPN,
can be time consuming, especially for slow-growing
species. The number of organisms in a sample can be
underestimated due to selective growth media and
48
Olsen et al.: Flow cytometry analysis of Tetraselmis suecica
individual growth requirements, and many species
cannot even be grown in a laboratory. The FCM
protocol presented here does not rely on cultiva-
tion, gives information of the vitality of the cells in a
sample as preferred by the USCG, and allows for
faster analysis (and hence reduced fading of the
staining) and analysis of larger volumes compared to
microscopy.
To investigate the interrelationship between live
cells (esterase active cells with intact membranes)
and reproducibility, the FCM results were compared
with plate count results, as cfu represents cells with
the ability to grow and reproduce. Reasons explain-
ing the discrepancies between reproducibility and
physiological characteristics, as well as the possibil-
ity that the gate includes some non-culturable cells
for the UV-irradiated samples, have previously been
discussed (Olsen et al. 2015). Plate count and the
gated FCM results for the non-irradiated samples
showed that there were viable cells left after 15 d of
dark incubation. This indicates that some cells sur-
vive in darkness, explained by cells adapting to pro-
longed periods of darkness (Jochem 1999, Carney et
al. 2011). When using FCM and the dual staining
protocol (CFDA-AM and SYTOX Blue) developed in
this study, cells with severe membrane damage after
UV irradiation are excluded from the reproducible
cells gate. This is an improvement compared to our
previously developed FCM protocol, where staining
with only CFDA-AM did not exclude cells with mem-
brane damage. Consequently, quantification of repro -
ducible cells with the FCM dual-staining protocol is
more in accordance with results from the plate count
method. In this study, the T. suecica culture was
irradiated with an MP UV lamp, representing a
small-scale BWTS. Our study was conducted with this
organism only, as a representative of the ≥10−50 µm
size categories of marine organisms in Regulation
D-2 for fulfilling the biological water quality criteria
for approval of BWTS (IMO 2008). The species is not
as common as diatoms, dinoflagellates, and prymne-
siophytes in coastal waters, and although the dual-
staining protocol proved well suited for type ap -
proval, its full potential remains to be examined on
the diverse community of organisms that will be
encountered in ships’ ballasting practices. One dis-
advantage of using FCM in compliance testing is the
difficulty of minimum dimension (the smallest part of
the body) measurements, since FCM can more easily
detect maximum dimension of organisms. The T. sue-
cica culture used in this study consisted of live cells
with high enzymatic activity. In natural water, the
phytoplankton community is diverse and will vary
with location, season, and environmental conditions.
The level of activity can vary between species, and
they may have different tolerances to environmental
changes and disinfection treatments (Jochem 1999,
2000, Olsen et al. 2015). Additionally, algae can
develop resting stages, like cysts, with low activity
levels (Hallegraeff 2010). Even though the majority
of phytoplankton species are detectable by the
esterase substrates 5-chloromethylfluorescein di -
acetate and fluorescein diacetate, we are aware that
fluorescence intensity from esterase substrates can
vary over a range in living cells (Peperzak & Brus-
saard 2011). Therefore, the esterase activity in organ-
isms in natural water samples should be examined
further. Moreover, biotic and abiotic particles in sea-
water can influence the inactivation efficiency by
protecting the microbes during UV irradiation (Hess-
Erga et al. 2008, Tang et al. 2011). Normally, a com-
mercial BWTS comprises 2 or more treatment stages,
in contrast to the sole UV lamp used in our experi-
ment, possibly enhancing the inactivation efficiency
(Lloyd’s Register Marine 2015). However, UV irradi-
ation is a disinfection method that can cause delayed
mortality in cells, and our FCM detection method is
well suited for analysis of T. suecica processed by a
UV-based BWTS.
CONCLUSIONS
Combining SYTOX Blue and CFDA-AM facilitates
and improves FCM analysis to evaluate the perform-
ance of UV-based water treatment systems, and the
FCM protocol allows differentiation into live, dam-
aged, and dead Tetraselmis suecica cells, not only
live and dead. The damaged cells are prone to die
under dark incubation. The protocol can be used for
type approval of UV-based BWTS when T. suecica is
included in the test water as a representative for the
10−50 µm size group. Further, the dual-staining FCM
protocol has the potential to be used for detailed
compliance testing, although further research is
required.
Acknowledgements. This research was funded by the Nor-
wegian Research Council (project BallastFlow, project no.
208653) and Knutsen OAS Shipping AS, and supported by
Solstad Shipping, Stord/Haugesund University College,
VRI Rogaland, UH-nett Vest, and TeknoVest. We thank
Stephanie Delacroix, August Tobiesen (Norwegian Insti-
tute for Water Research, Oslo, Norway), and Per Lothe
(Knutsen OAS Shipping AS, Haugesund, Norway) for help-
ful discussions.
49
Aquat Biol 25: 39– 52, 2016
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Editorial responsibility: Brant Touchette,
Elon, North Carolina, USA
Submitted: March 2, 2016; Accepted: July 14, 2016
Proofs received from author(s): August 5, 2016
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