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Journal of Oceanography, Vol. 62, pp. 903 to 908, 2006
Short Contribution
Effects of Long-Term Sample Preservation on Flow
Cytometric Analysis of Natural Populations of Pico- and
Nanophytoplankton
M ITSUHIDE SATO*, SHIGENOBU TAKEDA and KEN FURUYA
Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan
(Received 20 January 2006; in revised form 6 July 2006; accepted 6 July 2006)
The effects of long-term preservation on flow cytometric parameters of natural oceanic populations of pico- and nanophytoplankton have been examined. Populations
collected at oligotrophic subtropical and subarctic locations in the North Pacific were
fixed with glutaraldehyde and frozen in liquid nitrogen, according to Vaulot et al.
(1989). During six months’ storage, chlorophyll red fluorescence declined in all the
groups examined, while forward light scatter was enhanced in Synechococcus and
Prochlorococcus, and weakened in nanoeucaryotes. Cell loss was not significant except for Synechococcus. Caution is required when analyzing flow cytometric data of
samples stored for more than a month.
Keywords:
⋅ Flow cytometry,
⋅ phytoplankton,
⋅ preservation,
⋅ natural community.
However, we have only limited knowledge of how
samples change after preservation by this method, particularly in regard to natural phytoplankton assemblages
collected in the open oceans. There are therefore uncertainties about how flow cytometric parameters and cell
counts change during preservation and the relationship
of the changes to the values obtained for unfixed samples. Vaulot et al. (1989) applied their preservation method
to two natural samples, one from coastal water and the
other from subtropical oceanic water, and confirmed that
it is also efficient for natural samples. However, they made
little mention of temporal changes in flow cytometric
parameters during time spent in storage. Troussellier et
al. (1995) examined the change of flow cytometric parameters of natural populations fixed with formaldehyde,
paraformaldehyde, or glutaraldehyde over a period of sixteen weeks. However, their study mainly focused on
changes in membrane permeability, with only little reference to changes in autofluorescence. Moreover, their samples were collected only in temperate coastal lagoons.
Cavender-Bares (1999) reported drastic changes in flow
cytometric parameters after chemical fixation of natural
phytoplankton populations in the open ocean, but without examining the effect of preservation period. Recently,
Pan et al. (2005) reported as much as 70% decline in cell
counts of Prochlorococcus taken from the East China Sea
after 3 months’ preservation with 1% paraformaldehyde,
1. Introduction
Flow cytometry is a powerful tool for analyzing microbial communities and has been used in biological oceanographic studies for nearly thirty years (Paau et al.,
1978), in particular with phytoplankton which exhibits
autofluorescence from chlorophyll or phycoerythrin
(Veldhuis and Kraay, 2000). However, portability constraints restrict the ease with which flow cytometric analyses can be performed on a research vessel. Moreover,
rough weather or time limitations often do not permit
analysis immediately after collection. In such cases, samples should be fixed and frozen for storage.
Vaulot et al. (1989) proposed a simple preservation
method, in which samples are fixed with glutaraldehyde
at a final concentration of 1%, and frozen in liquid nitrogen. Lepesteur et al. (1993) proposed a more complicated
method, in which samples are fixed by different methods
according to the community structures or purposes. The
method reported by Vaulot et al. (1989) and its variations,
e.g. substitution of glutaraldehyde with paraformaldehyde
(Campbell and Vaulot, 1993), are currently widely used.
* Corresponding author. E-mail: [email protected].
ac.jp
Copyright©The Oceanographic Society of Japan/TERRAPUB/Springer
903
A
B
Red fluorescence
Nanoeucaryotes
Nanoeucaryotes
Synechococcus
C
Synechococcus
D
E
6-µm beads
Nanoeucaryotes
Nanoeucaryotes
2-µm beads
Synechococcus
Synechococcus
Orange fluorescence
Forward light scatter
Fig. 1. Cytograms of the unfixed samples (A and B) and the same samples 6 months after fixation (C and D), collected from St.
7, and calibration fluorescent beads (E). The cytograms A and C are those of red fluorescence (FL3) vs. orange fluorescence
(FL2), while the other three are of FL3 vs. forward light scatter (FSC).
challenging the reliability of flow cytometric cell counts
of samples cryopreserved after chemical fixation. These
two observations suggest that the effects of chemical fixation and preservation on flow cytometric analysis of natural phytoplankton populations in the open oceans are different from those of cultured or coastal populations, probably due to differences in their species composition or
physiological status. However, there is only scarce knowledge on how flow cytometric parameters of natural
phytoplankton in the open oceans, such as light scatter
and autofluorescence, change during storage, even though
such parameters are very important in probing cell size
or physiological status of the phytoplankton.
In the present study we aimed at elucidating how flow
cytometric parameters and cell counts of natural
phytoplankton populations in the open oceans change
during preservation due to shifts in optical properties,
using samples collected from two oceanic locations.
2. Materials and Methods
Seawater samples were collected from 10 m depth
at St. 7 (48.5°N, 165°E) in the subarctic water and from
100 m depth at St. 10 (28°N, 165°E) in the subtropical
water, using a CTD-carousel system equipped with 12liter Niskin bottles during the KH-03-2 cruise on board
the R/V Hakuho-Maru (September 30–October 17, 2003).
The sample was introduced into a 250 mL opaque black
polyethylene bottle and then dispensed into twenty-five
4.5 mL cryogenic vials. Five replicates were immediately
subjected to flow cytometric analysis without fixation,
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M. Sato et al.
while the remaining twenty replicates were fixed with
glutaraldehyde (50% aqueous solution, Wako, Japan) at
a final concentration of 1% in a refrigerator (4°C) for 10
minutes and five of them were analyzed immediately after fixation. The rest of the samples were frozen in liquid
nitrogen and stored at –80°C until analysis (Vaulot et al.,
1989). After five days, one month, and 6 months of preservation, five samples were thawed at room temperature
in the dark and analyzed with a flow cytometer. The samples stored for one month and 6 months after preservation were analyzed on land.
Flow cytometry was carried out using a PAS-III flow
cytometer (Partec, Germany) equipped with a 488-nm
argon-ion laser. The power was adjusted to 10 and 20 mW
for samples taken at St. 7 and St. 10, respectively.
Seawater filtered through a 0.2-µm Nuclepore filter and
diluted with distilled water to adjust salinity to 33 was
used as a sheath fluid (Cucci and Sieracki, 2001). Sample flow rate was set to approximately 10 µL s–1 and calculated linearly by weight of water aspirated during measurement. Sample flow rate was calibrated using the suspension of a known concentration of 2-µm fluorescent
polystyrene beads (Fluoresbrite® YG, Polysciences,
USA). Forward light scatter (FSC), side light scatter
(SSC), orange fluorescence (570–610 nm, FL2), and red
fluorescence (>630 nm, FL3) were recorded in list mode
using FL3 as a trigger parameter and processed with
FloMax® (Partec, Germany). Instrumental settings were
standardized for all parameters using the 2-µm fluorescent polystyrene beads. The same gain settings were ap-
1.2
Prochlorococcus
B
Synechococcus (St. 7)
Nanoeucaryotes (St. 7)
Prochlorococcus (St. 10)
Nanoeucaryotes (St. 10)
1.0
Red fluorescence
(relative intensity to unfixed)
Red fluorescence
A
Nanoeucaryotes
Nanoeucaryotes
C
6-µm beads
0.8
0.6
0.4
0.2
0.0
Just fixed
5 days
1 month
6 months
Fig. 3. Changes in intensity of cellular red fluorescence (FL3)
of the four phytoplankton groups after fixation. All the values are normalized to the average intensity of the unfixed
samples. Dotted line indicates level of the unfixed sample.
Error bars are standard deviations of five replicates.
2-µm beads
Prochlorococcus
Forward light scatter
Fig. 2. Red fluorescence (FL3) vs. forward light scatter (FSC)
cytograms of the unfixed sample (A) and the same sample
6 months after fixation (B), collected from St. 10, and calibration fluorescent beads (C).
plied to all samples from the same station, regardless of
preservation periods. Phytoplankton groups were identified on the basis of FSC, FL2 and FL3 parameters, as
described in Marie et al. (1999). All data were statistically analyzed using Microsoft Excel®.
3. Results and Discussion
At St. 7, Synechococcus was evidently distinguished
by its orange fluorescence (Fig. 1A). On the FL3 vs. FSC
cytogram, two clusters of eucaryotic phytoplankton were
observed, although the counts of the smaller one were
<200, which is insufficient to give enough counts for statistical analysis (Fig. 1B). From comparison with the
cytogram of fluorescent beads (Fig. 1E), the smaller and
larger clusters were determined to be picoeucaryotes and
nanoeucaryotes, respectively. At St. 10, we observed a
distinct cluster of Prochlorococcus, which have dim red
autofluorescence (Fig. 2A). Another cluster on the FL3
vs. FSC cytogram was considered to be composed of
nanoeucaryotes (Figs. 2A and C). The concentration of
Synechococcus was too low to form a distinct cluster.
Thus, Synechococcus and nanoeucaryotes were analyzed
at St. 7, and Prochlorococcus and nanoeucaryotes at St.
10.
FL3 of all the four populations showed similar trends,
with two abrupt declines over six months (Fig. 3). The
first decline was observed immediately after fixation but
prior to freezing, and was especially remarkable for the
two subarctic water groups. The second decline occurred
between five days and one month after fixation. During
the last five months, FL3 was relatively stable, except
for Synechococcus, which showed another 10% decline.
The decline just after fixation contradicts the observations reported by Vaulot et al. (1989), who observed an
increase in chlorophyll red fluorescence in many
phytoplankton culture strains, especially Synechococcus.
They ascribed this to fluorescence enhancement due to
the cessation of electron transfer in the light reaction of
photosynthesis. However, Neale et al. (1989) pointed out
that chlorophyll fluorescence measured in flow cytometry
is intermediate between the minimum and maximum fluorescence yields, and the degree of enhancement above the
minimum fluorescence could depend on the cytometer’s
flow rate, the diameter of the laser beam, and the laser
power of the flow cytometer used. Since chlorophyll fluorescence enhancement after cessation of electron transfer was unclear in the present study, the red fluorescence
measured by our flow cytometer may be closer to the
maximum fluorescence yield, and presumably was influenced more by the degradation of photosynthetic pigments
due to chemical fixation than by cessation of electron
transfer subsequent to cell death. Although the cause of
the second decline is unclear, deterioration of photosynthetic pigments or leakage of the pigments through compromised membrane during storage may have occurred.
FL2 of Synechococcus fluctuated in a different manner from FL3 (Fig. 4). It increased just after fixation, more
than doubled after one month, and subsequently decreased
to 1.5 times that of the unfixed samples after six months.
This pattern is in good agreement with the observation of
cultures of Synechococcus reported by Vaulot et al.
Effects of Long-Term Sample Preservation on Flow Cytometric Analysis
905
2
2.0
Side light scatter
(relative value to unfixed)
Orange fluorescence
(relative intensity to unfixed)
2.5
1.5
1.0
0.5
0.0
1
0
Just fixed
5 days
1 month
6 months
Fig. 4. Changes in intensity of cellular orange fluorescence
(FL2) of Synechococcus collected from St. 7 after fixation.
All the values are normalized to the average intensity of
the unfixed samples. Dotted line indicates level of the
unfixed sample. Error bars are standard deviations of five
replicates.
Synechococcus (St. 7)
Nanoeucaryotes (St. 7)
Prochlorococcus (St. 10)
Nanoeucaryotes (St. 10)
Just fixed
5 days
1 month
6 months
Fig. 6. Changes in intensity of side light scatter (SSC) of the
four phytoplankton groups after fixation. All the values are
normalized to the average intensity of the unfixed samples.
Dotted line indicates level of the unfixed sample. Error bars
are standard deviations of five replicates.
2.0
3
Synechococcus (St. 7)
Nanoeucaryotes (St. 7)
Prochlorococcus (St. 10)
Nanoeucaryotes (St. 10)
Cell counts
(relative value to unfixed)
Forward light scatter
(relative value to unfixed)
4
2
1.5
*
Synechococcus (St. 7)
Nanoeucaryotes (St. 7)
Prochlorococcus (St. 10)
Nanoeucaryotes (St. 10)
1.0
**
*
*
0.5
1
0.0
0
Just fixed
5 days
1 month
6 months
Just fixed
5 days
1 month
6 months
Fig. 5. Changes in intensity of forward light scatter (FSC) of
the four phytoplankton groups after fixation. All the values
are normalized to the average intensity of the unfixed samples. Dotted line indicates level of the unfixed sample. Error bars are standard deviations of five replicates.
Fig. 7. Changes in cell count of the four phytoplankton groups
determined by flow cytometry after fixation. All the values
are normalized to the average count of the unfixed samples. Dotted line indicates level of the unfixed sample. Error bars are standard deviations of five replicates. Star above
a bar indicates statistically significant difference (Student’s
t-test, p < 0.01) from the unfixed samples.
(1989). They attributed the increase in green-orange fluorescence immediately after fixation to the decoupling of
electron transfer between phycoerythrin and chlorophyll.
The greenish/yellow fluorescence emitted from glutaraldehyde present in algal cells observed by Vaulot et al.
(1989) may be partly responsible for the increase. Leakage of phycoerythrin across compromised cell membranes
as well as deterioration of the pigment may account for a
subsequent decrease in FL2, since phycoerythrin is water soluble.
Procaryotic picoplankton and eucaryotic
nanoplankton showed obviously different trends in FSC
(Fig. 5). Just after fixation, FSC changed little, but the
procaryotic group showed a constant increase, while the
eucaryotes showed a small fluctuation. These different
fluctuations are consistent with the findings of CavenderBares (1999), although these were not reported for cultured or coastal phytoplankton populations (Vaulot et al.,
1989). Cavender-Bares (1999) reported that
nanophytoplankton and picophytoplankton show different responses of FSC to chemical fixation and suggested
that the refractive index of nanophytoplankton cells in-
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M. Sato et al.
creased more than that of the other groups. For
phytoplankton, an increase in FSC is ascribed to an increase in a cell diameter or a decrease in cell refractive
index (Ackleson and Spinrad, 1988). Since a chemical
fixation method widely employed for phytoplankton microscopic samples causes cell shrinkage (Choi and
Stoecker, 1989; Verity et al., 1992) and a subsequent rise
in cell refractive index due to the concentration of solid
materials in the cell, it is expected that FSC of
phytoplankton cells decreases after chemical fixation.
However, in the present study, the samples were frozen
abruptly after fixation for 10 min, which is very different
from a chemical fixation method used for microscopy,
where samples are filtered onto a membrane filter and
frozen in a freezer within one day after chemical fixation. This may have caused the apparently strange changes
in FSC. To explain the increase in FSC, it is possible that
cell membrane and/or organelles were compromised during preservation, resulting in a lowered refractive index.
However, a gradual increase in SSC of Prochlorococcus
was observed during storage (Fig. 6), which demonstrates
that the decrease in the cellular refractive index is not
sufficient to explain the observed increase in FSC. This
observation implies that the cellular swelling occurred
during the storage. Although it is not clear why the increase in FSC did not occur for algal cultures (Vaulot et
al., 1989), there is a possibility that flow cytometric analysis of long-preserved samples from oceanic locations
could lead to an overestimate of biovolume and biomass
of procaryotic picoplankton.
Changes were observed in flow cytometric cell
counts during storage (Fig. 7). For five days after fixation, no groups showed a statistically significant difference from the unfixed samples (Student’s t-test, p > 0.01).
However, one month after fixation, all groups except
nanoeucaryotes from St. 7 showed a significant difference (p < 0.01), and this trend persisted until six months
after fixation. Most importantly, Prochlorococcus and
nanoeucaryotes from St. 10 showed a consecutive increase
in their cell counts, while those of Synechococcus declined
gradually. For Prochlorococcus, the increase is statistically significant only for the sample analyzed after one
month. The unexpected observation at St. 10, where both
Prochlorococcus and nanoeucaryotes showed consecutive
increases in cell counts despite their contrast changes in
FSC, is partly explained by the detection range (Figs. 2A
and B). In the case of Prochlorococcus, an increase in
FSC (Fig. 5) likely led to a rise in the apparent cell concentration, because particles below the lower detection
limit came within the detection range. By contrast, for
nanoeucaryotes from St. 10, a drop in FSC shifted particles that had existed above the upper limit into the detection range. This indicates that the preservation method
adopted in the present study can prevent significant cell
loss, as reported by Pan et al. (2005). Tolerance to chemical fixation may depend on the physiological status of
phytoplankton, because it was observed that membrane
permeability was enhanced in physiologically deteriorated
cells due to nutrient limitation or viral infection
(Brussaard et al., 2001; Veldhuis et al., 2001). Although
cell loss due to chemical fixation, freezing and thawing
probably also occurred, the possible influence of the loss
relative to an apparent increase in cell count due to a
change in FSC intensity was unclear from the present
study. The changes in cell counts may be reduced by an
adjustment of the detection range. For Synechococcus,
the decline in FL3 (Figs. 1A and C) is thought to have
been a main cause of the decrease in cell concentration.
This problem cannot be solved merely by adjusting the
detection range, because the high sensitivity in FL3 could
lead to too much noise overlapping the signals from
picophytoplankton. However, alteration of a trigger parameter from FL3 to FL2 may help to solve the problem,
because Synechococcus cells have high FL2 from
phycoerythrin, and moreover, FL2 signals were enhanced
after the fixation and remained relatively high during six
months (Fig. 4).
4. Conclusion
Long-term storage as described by Vaulot et al.
(1989) produced a considerable change in flow cytometric
parameters of phytoplankton collected in the open oceans,
especially if the storage period was longer than one month,
while no significant cell loss occurred. These changes
proceeded continuously during 6 months’ preservation and
were most conspicuous in FSC. Moreover, the direction
or magnitude of the shift varied according to
phytoplankton groups. For procaryotic phytoplankton,
cellular size estimated from light scatter is smaller in preserved samples than in live ones, while the opposite holds
for eucaryotic nanoplankton. The magnitude of the shifts
in FSC became greater over time of preservation, which
may cause over/underestimation of phytoplankton
biomass in natural populations. Since cellular chlorophyll
fluorescence continuously fades, it is strongly recommended to preserved all samples for the same length of
time when comparing chlorophyll contents between samples, e.g. in an enrichment experiment. Although cell loss
during preservation is considered to be relatively insignificant, inadequate instrumentation of a flow cytometer
could lead to an apparent change in cell concentration.
For accurate enumeration, it may be helpful to measure
the same sample twice at different gain settings when it
is preserved for a long period.
Acknowledgements
We wish to thank the scientific party of the KH-03-2
cruise, the captain and all the crew of R/V Hakuho-Maru
Effects of Long-Term Sample Preservation on Flow Cytometric Analysis
907
for seawater sample collection. We greatly appreciate
excellent FCM technical support of Junichi Iijima. We
also thank two anonymous reviewers for their helpful
comments and suggestions which greatly improved our
original manuscript.
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